PEPTIDE COMPOSITIONS THAT DOWNREGULATE TLR-4 SIGNALING PATHWAY AND METHODS OF PRODUCING AND USING SAME
Compositions that include isolated peptides that inhibit TLR-4 signaling pathways and inflammation are disclosed. Methods of producing and using the compositions to inhibit TLR-4 signaling and/or inflammation are also disclosed herein.
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The present application is a divisional of U.S. Ser. No. 13/289,820, filed Nov. 4, 2011; which claims benefit under 35 U.S.C. 119(e) of provisional patent applications U.S. Ser. No. 61/410,077, filed Nov. 4, 2010; and U.S. Ser. No. 61/469,202, filed Mar. 30, 2011. The entire contents of the above-referenced patent applications are hereby expressly incorporated herein by reference.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENTNot Applicable.
BACKGROUND OF THE INVENTION1. Field of the Inventive concept(s)
The presently disclosed and claimed inventive concept(s) relates generally to peptide compositions, and in particular, but not by way of limitation, to Surfactant-A-protein peptide compositions and derivatives thereof, and methods of producing and using same.
2. Description of the Background Art
The pathogen-pattern recognition receptors (PPRRs) are important components of innate immunity that sense the pathogenic stimuli and regulate host immune responses (Pastva et al., 2007; Wright, 1997). Surfactant protein-A (SP-A) and Toll-like receptor-4 (TLR4) have been identified as important PPRRs (Barton, 2007; Brinker et al., 2003; Hoebe et al., 2006; Kawai and Akira, 2007; Kuroki et al., 2007; Miyake, 2007). TLR4 is expressed as a transmembrane receptor and is known as a “Signalling-PPRR” (Kawai and Akira, 2007). On the other hand, SP-A is synthesized by type II lung epithelial cells and secreted in the alveoli as a component of surfactant. SP-A is known as a “Secretory-PPRR” (Pastva et al., 2007). It has been demonstrated by the inventor and others that SP-A constitutes the majority of surfactant proteins (SPs) (Johansson and Curstedt, 1997) and plays a critical role in the clearance of pathogens and downregulation of the inflammatory response (see also, Awasthi, 2010). On the other hand, TLR4 recognizes pathogen or pathogen-derived ligands and endogenous stress proteins, and induces inflammatory and adaptive immune responses. In a number of diseases, including but not limited to lung inflammatory conditions, an exaggerated activation of TLR4 has been found associated with NF-κB and pro-inflammatory cytokine response (Guillot et al., 2004; He et al., 2009; Lv et al., 2009; Maes et al., 2006; Villar et al., 2010).
Published reports suggest that the bronchoalveolar lavage pools (extracellular pools) of SP-A are significantly reduced in lungs of infected patients and animal models (Alcorn et al., 2005; Awasthi et al., 1999; Awasthi et al., 2001; Awasthi et al., 2004; Chang et al., 2006; Kajikawa et al., 2005). In contrast, TLR4 expression is increased (Awasthi et al., 2008; Chang et al., 2006; Gagro et al., 2004; Kajikawa et al., 2005). The reduction in the amounts of SP-A, and simultaneous increase in TLR4 expression corroborates well with the clinical condition of patients having fulminant infection and inflammation, respectively. In these clinical scenarios, the introduction of SP-A should facilitate clearance of pathogens and attenuate inflammation. However, currently-available clinical surfactants (used for improving lung function and maturity in pre-term infants) do not contain SP-A or SP-D because it is difficult to mix large hydrophilic SP-A proteins with lipids. As with any large protein, rapid clearance of large proteins, degradation and a non-specific immune response have also hampered the development of clinical surfactant having SP-A.
Inflammatory Bowel Disease (IBD) causes chronic inflammation in the intestine and accounts for a huge economic cost associated with multiple clinic visits and hospitalizations. Therapeutic efficacy with currently recommended drugs has been limited because of toxic effects, nonspecific downregulation of overall immunity and increased risk of infection. Contemporary understanding suggests that activation of Toll-like receptor-4 (TLR4) and TLR4-nuclear factor (NF)-kappa B signaling in the gut causes an overproduction of inflammatory cytokines and trafficking of leukocytes, thus leading to uncontrolled intestinal inflammation. Moreover, persistent inflammation can lead to carcinogenesis. Thus, new therapies targeting TLR4 may be of clinical utility in these conditions.
Interestingly, recently published reports suggested that SP-A directly binds to TLR4 (Guillot et al., 2002; Yamada et al., 2006). However, the in vivo evidence of such an interaction has been lacking, and its functional relevance has not been fully elucidated.
Therefore, there is a need in the art for an understanding of the functional relationship of TLR4 and SP-A, as well as compositions that interact in and/or inhibit said interaction and thereby block TLR4 signaling. It is to said compositions, as well as methods of producing and using same, that the presently disclosed and claimed inventive concept(s) is directed.
This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Before explaining at least one embodiment of the inventive concept(s) in detail by way of exemplary drawings, experimentation, results, and laboratory procedures, it is to be understood that the inventive concept(s) is not limited in its application to the details of construction and the arrangement of the components set forth in the following description or illustrated in the drawings, experimentation and/or results. The inventive concept(s) is capable of other embodiments or of being practiced or carried out in various ways. As such, the language used herein is intended to be given the broadest possible scope and meaning; and the embodiments are meant to be exemplary—not exhaustive. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.
Unless otherwise defined herein, scientific and technical terms used in connection with the presently disclosed and claimed inventive concept(s) shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and Coligan et al. Current Protocols in Immunology (Current Protocols, Wiley Interscience (1994)), which are incorporated herein by reference. The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.
All patents, published patent applications, and non-patent publications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this presently disclosed and claimed inventive concept(s) pertains. All patents, published patent applications, and non-patent publications referenced in any portion of this application are herein expressly incorporated by reference in their entirety to the same extent as if each individual patent or publication was specifically and individually indicated to be incorporated by reference.
All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the inventive concept(s) as defined by the appended claims.
As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:
The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or”. Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects. The use of the term “at least one” will be understood to include one as well as any quantity more than one, including but not limited to, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term “at least one” may extend up to 100 or 1000 or more, depending on the term to which it is attached; in addition, the quantities of 100/1000 are not to be considered limiting, as higher limits may also produce satisfactory results. In addition, the use of the term “at least one of X, Y and Z” will be understood to include X alone, Y alone, and Z alone, as well as any combination of X, Y and Z.
The term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value and/or the variation that exists among study subjects.
As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
The term “or combinations thereof” as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof” is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, MB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.
The term “naturally-occurring” as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory or otherwise is naturally-occurring.
The term “pharmaceutically acceptable” refers to compounds and compositions which are suitable for administration to humans and/or animals without undue adverse side effects such as toxicity, irritation and/or allergic response commensurate with a reasonable benefit/risk ratio.
By “biologically active” is meant the ability to modify the physiological system of an organism. A molecule can be biologically active through its own functionalities, or may be biologically active based on its ability to activate or inhibit molecules having their own biological activity.
As used herein, “substantially pure” means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition), and preferably a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will comprise more than about 80 percent of all macromolecular species present in the composition, more preferably more than about 85%, 90%, 95%, and 99%. Most preferably, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.
The term “patient” as used herein includes human and veterinary subjects. “Mammal” for purposes of treatment refers to any animal classified as a mammal, including human, domestic and farm animals, nonhuman primates, and any other animal that has mammary tissue.
“Treatment” refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include, but are not limited to, individuals already having a particular condition or disorder as well as individuals who are at risk of acquiring a particular condition or disorder (e.g., those needing prophylactic/preventative measures). The term “treating” refers to administering an agent to a patient for therapeutic and/or prophylactic/preventative purposes.
A “therapeutic composition” or “pharmaceutical composition” refers to an agent that may be administered in vivo to bring about a therapeutic and/or prophylactic/preventative effect.
Administering a therapeutically effective amount or prophylactically effective amount is intended to provide a therapeutic benefit in the treatment, prevention, or management of a disease and/or cancer. The specific amount that is therapeutically effective can be readily determined by the ordinary medical practitioner, and can vary depending on factors known in the art, such as the type of disease/cancer, the patient's history and age, the stage of disease/cancer, and the co-administration of other agents.
A “disorder” is any condition that would benefit from treatment with the polypeptide. This includes chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question.
The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer, renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, and various types of head and neck cancer.
The term “effective amount” refers to an amount of a biologically active molecule or conjugate or derivative thereof sufficient to exhibit a detectable therapeutic effect without undue adverse side effects (such as toxicity, irritation and allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of the inventive concept(s). The therapeutic effect may include, for example but not by way of limitation, inhibiting the growth of undesired tissue or malignant cells. The effective amount for a subject will depend upon the type of subject, the subject's size and health, the nature and severity of the condition to be treated, the method of administration, the duration of treatment, the nature of concurrent therapy (if any), the specific formulations employed, and the like. Thus, it is not possible to specify an exact effective amount in advance. However, the effective amount for a given situation can be determined by one of ordinary skill in the art using routine experimentation based on the information provided herein.
As used herein, the term “concurrent therapy” is used interchangeably with the terms “combination therapy” and “adjunct therapy”, and will be understood to mean that the patient in need of treatment is treated or given another drug for the disease in conjunction with the pharmaceutical compositions of the presently disclosed and claimed inventive concept(s). This concurrent therapy can be sequential therapy, where the patient is treated first with one drug and then the other, or the two drugs are given simultaneously.
The terms “administration” and “administering”, as used herein will be understood to include all routes of administration known in the art, including but not limited to, oral, topical, transdermal, parenteral, subcutaneous, intranasal, mucosal, intramuscular, intraperitoneal, intravitreal and intravenous routes, including both local and systemic applications. In addition, the compositions of the presently disclosed and claimed inventive concept(s) (and/or the methods of administration of same) may be designed to provide delayed, controlled or sustained release using formulation techniques which are well-known in the art.
The presently disclosed and claimed inventive concept(s) also includes a pharmaceutical composition comprising a therapeutically effective amount of at least one of the compositions described herein in combination with a pharmaceutically acceptable carrier. As used herein, a “pharmaceutically acceptable carrier” is a pharmaceutically acceptable solvent, suspending agent or vehicle for delivering the compositions of the presently disclosed and claimed inventive concept(s) to the human or animal. The carrier may be liquid or solid and is selected with the planned manner of administration in mind. Examples of pharmaceutically acceptable carriers that may be utilized in accordance with the presently disclosed and claimed inventive concept(s) include, but are not limited to, PEG, liposomes, ethanol, DMSO, aqueous buffers, oils, DPPC, lipids, other biologically-active molecules, vaccine-adjuvants, and combinations thereof.
The terms “liposome”, “lipid nanostructure” and “vesicle” may be used interchangeably herein and will be understood to refer to an assembled structure constructed of molecules such as lipids and/or proteins, for example, not through covalent bonds but through interactions (such as but not limited to, hydrophobic interactions, electrostatic interactions and hydrogen bonds) acting between the molecules in an aqueous medium.
The terms “aqueous solution” and “aqueous medium” will be used interchangeably herein and will be understood to refer to water as well as any kind of solution which is physiologically acceptable and solvent in water.
The presently disclosed and claimed inventive concept(s) is directed to a composition comprising an isolated peptide that comprises a portion of Surfactant protein A (SEQ ID NO:1 or any of the sequences shown in
Said compositions of the presently disclosed and claimed inventive concept(s) are capable of binding to Toll-like receptor-4 (TLR-4) and inhibiting TLR-4 signaling pathway(s).
In one embodiment, the isolated peptide may comprise any of SEQ ID NOS:3-7, or a fragment thereof. For example but not by way of limitation, Table 1 lists SEQ ID NOS:8-246, which are exemplary fragments of SEQ ID NOS:3 and 5.
The presently disclosed and claimed inventive concept(s) is also directed to a composition that includes an isolated peptide that is a mutant or derivative of a portion of Surfactant-A-protein and which still retains the ability to bind TLR-4 and inhibit TLR-4 signaling pathway(s). In certain embodiments, the isolated peptide may comprise an amino acid sequence that is at least 80% identical to a portion of SEQ ID NO:1 or 80% identical to any of SEQ ID NOS:3-7. In other embodiments, the isolated peptide may comprise an amino acid sequence that is at least 90% identical to a portion of SEQ ID NO:1 or 90% identical to any of SEQ ID NOS:3-7. In yet other embodiments, the isolated peptide may comprise an amino acid sequence that has 1-5 amino acid changes when compared to a portion of SEQ ID NO:1 or any of SEQ ID NOS:3-7; for example, the isolated peptide may comprise an amino acid sequence that differs from any of SEQ ID NOS:3-7 by 5 amino acids or less, by 4 amino acids or less, by 3 amino acids or less, by two amino acids or less, or by one amino acid or less.
In certain embodiments of the presently disclosed and claimed inventive concept(s), the composition may include multiple isolated peptides as described herein above.
The presently disclosed and claimed inventive concept(s) further includes a method of producing any of the compositions described herein above. Said method may comprise any of the steps described herein or otherwise known in the art. The compositions of the presently disclosed and claimed inventive concept(s) may be prepared according to methods known in the art, particularly in light of the disclosure and examples set forth herein. The starting materials used to synthesize the compositions of the presently disclosed and claimed inventive concept(s) are commercially available or capable of preparation using methods known in the art.
The presently disclosed and claimed inventive concept(s) also includes an isolated nucleic acid segment encoding any of the compositions described herein above. In addition, a recombinant vector comprising said nucleic acid segment, as well as a recombinant host cell comprising said recombinant vector, are also contemplated within the scope of the presently disclosed and claimed inventive concept(s). In certain embodiments, the recombinant host cell produces the peptide composition.
The presently disclosed and claimed inventive concept(s) is further directed to a pharmaceutical composition comprising any of the isolated peptide compositions described herein above or otherwise contemplated herein, in combination with a pharmaceutically acceptable carrier (or biologically-active molecule or vaccine-adjuvant). In addition, a pharmaceutical composition comprising a nucleic acid segment encoding said peptide composition in combination with a pharmaceutically acceptable carrier is also contemplated in accordance with the presently disclosed and claimed inventive concept(s).
The presently disclosed and claimed inventive concept(s) is also directed to a method of using any of the pharmaceutical compositions described herein above. Said method includes the steps of providing the pharmaceutical composition and administering an effective amount of the pharmaceutical composition to a patient in need thereof.
The presently disclosed and claimed inventive concept(s) is also directed to a method of inhibiting TLR4 signaling. Said method comprises providing any of the isolated peptide compositions described herein above or otherwise contemplated herein, and contacting a cell expressing TLR4 on a surface thereof with said composition, wherein the peptide composition binds to TLR4 on the surface of the cell and inhibits TLR4 signaling by the cell.
The presently disclosed and claimed inventive concept(s) is further directed to a method of decreasing the occurrence and/or severity of inflammation associated with a disease condition. Said method comprises administering an effective amount of a composition (or pharmaceutical composition) as described in detail herein above to a subject suffering from or predisposed to the inflammation/disease condition, thereby decreasing the occurrence and/or severity of inflammation associated with the disease condition in the subject.
Any inflammatory conditions known in the art or otherwise contemplated herein may be treated in accordance with the presently disclosed and claimed inventive concept(s). Non-limiting examples of disease conditions having inflammation associated therewith include infection-related or non-infectious inflammatory conditions in the lung (i.e., sepsis, lung infections, Respiratory Distress Syndrome, bronchopulmonary dysplasia, etc.); infection-related or non-infectious inflammatory conditions in other organs (i.e., colitis, Inflammatory Bowel Disease, diabetic nephropathy, hemorrhagic shock); inflammation-induced cancer (i.e., cancer progression in patients with colitis or Inflammatory Bowel Disease); and the like.
The presently disclosed and claimed inventive concept(s) further include a method of promoting lung development and/or function in infants born pre-term (who are unable to make enough surfactant). Said method comprises administering an effective amount of a composition (or pharmaceutical composition) as described in detail herein above to a subject to promote lung development and/or function and/or maintain immune homeostasis. The composition (or pharmaceutical composition) may be administered alone or in combination with surfactant (i.e., currently available lipid-based clinical surfactants).
EXAMPLESExamples are provided hereinbelow. However, the present invention is to be understood to not be limited in its application to the specific experimentation, results and laboratory procedures. Rather, the Examples are simply provided as one of various embodiments and are meant to be exemplary, not exhaustive.
Example 1 Surfactant Protein-A and Toll-Like Receptor 4 Modulate Immune Functions of Lung Dendritic Precursor Cells Harvested from Preterm BaboonsPreterm infants are highly susceptible to infections; this increased susceptibility to infections is associated with perturbed development and extreme immaturity of the immune system. Antigen-presenting cells have an important role in pathogen-uptake and processing as well as in regulating inflammatory and adaptive host immune responses. Among various types of antigen-presenting cells, dendritic cells (DCs) have been recognized as the most potent (Fajardo-Moser et al., 2008; T. Ohteki, 2007; Sabatte et al., 2007; van Vliet et al., 2007). In the past, several studies have confirmed an immunomodulatory role of lung DCs against a variety of antigens in adult humans and animal models (Banks et al. 1999; Fach et al., 2007; Holt et al., 1995; Lammatzsch et al., 2007; Masten et al., 2006; Webb et al., 2005) with a mature immune system; however, until recently, the phenotypes and functions of lung DCs remained poorly known in preterm babies. The inventor was the first to isolate a unique low-density lung cell population from preterm baby baboons (Awasthi et al., 2009). The results of this study showed that the cells have a density similar to adult baboon lung DCs, are lineage-negative and defective in responding to infectious stimuli. Overall these results suggest that despite having similar isolation characteristics, this unique cell-population harvested from fetal lung does not belong to conventional immature or mature DC categories (Awasthi et al., 2009). Based on these unique properties, they were identified as DC-precursor cells (Awasthi et al., 2009). Recent results from the inventor further demonstrate that the fetal DC-precursor cells express low level of DC-markers, and incubation with DC-promoting cytokines (GM-CSF, IL-4 and TNF-α) induces differentiation of these fetal cells into typical DCs (unpublished results).
The DCs are well-known to coordinate innate and adaptive immunity via pathogen-pattern recognition receptors, such as Toll-like receptors (TLR), mannose receptors, scavenger receptors and collections (such as but not limited to, surfactant protein-A (SP-A)) (J. Wright, 2004). Deficiencies or functional defects of pathogen-pattern recognition receptors can negatively affect the DC functions, compromise the host defense and lead to serious consequences in early life-periods of preterm babies. The inventor's previous studies have mainly focused on SP-A, a major part of lung surfactant that lines the alveoli, and TLR4, a potent membrane-receptor that senses both pathogen-associated and damage-associated molecular patterns (PAMP and DAMPs) (Piccinini et al., 2010; and Brown et al., 2010).
The inventor's previous studies observed that the expression of SP-A and TLR4 is undetectable or negligible in lung tissues of fetuses (at 67%-75% of complete gestation term) under steady-state conditions, and increases to the levels equivalent to adult counterparts as the gestation period reaches closer to term (Awasthi et al., 1999; Awasthi et al., 2001; Awasthi et al., 2008). However, preterm birth, mechanical injury (ventilation-associated) and infection significantly influence lung-homeostasis and decrease the alveolar SP-A pools to significantly low levels (Awasthi et al., 1999; Awasthi et al., 2001). In contrast, the expression of TLR4 is increased in preterm babies with lung infections (Awasthi et al., 2008).
To this end, recent understanding in the field suggests that SP-A and TLR4 both enhance phagocytosis. The lack of SP-A in alveoli may compromise the uptake of pathogens. However, an exaggerated activation of TLR4 can lead to chronic inflammatory response or “cytokine storm” (Kramer et al., 2009; Kramer et al.-2, 2009; Sano et al., 2005; J. Blander, 2008; J. Blander, 2007). This pattern correlates well with fulminating infection (low SP-A=low phagocytic uptake) and inflammatory response (increased TLR4=increased amounts of pro-inflammatory cytokines) in preterm babies having lung infection. These results led the inventor to hypothesize that the introduction of SP-A-based clinical surfactants and TLR4-antagonists may compensate for the loss of SP-A and downregulate an exaggerated TLR4-mediated inflammatory response, respectively. It has also been learned recently that SP-A interacts with TLR4 in vitro (Yamada et al., 2006; and Ohya et al., 2006) and in lung (Awasthi et al., 2010). Thus, introduction of SP-A may have an effect on TLR4-mediated immune responses. In this Example, the immunomodulatory effects of native SP-A and recombinant TLR4-MD2 proteins were investigated on selected immune functions (phagocytosis and cytokine response) of fetal baboon lung DC-precursor cells and compared with those of adult baboon lung DCs. The results presented in this Example demonstrate that in both adult and fetal systems, pulsing of cells with SP-A and TLR4-MD2 proteins increases the phagocytic uptake of Escherichia coli bioparticles. When added together, no additive effect was demonstrated on phagocytic function of DCs. Co-incubation of cells with SP-A and TLR4-MD2 proteins, however, significantly inhibits the TLR4-MD2-induced release of TNF-α against E. coli.
Overall, the results presented in this Example support a significant role of SP-A in improving innate phagocytic function and in suppressing the TLR4-mediated deleterious inflammatory response against infectious stimuli.
Materials and Methods for Example 1:
Baboon lung tissues: The animal studies were approved by Institutional Animal Care and Use Committees, Environmental Health and Safety or Institutional Biosafety Committee of the University of Oklahoma Health Science Center, Oklahoma City, Okla. (OUHSC). Baboon (P. anubis) colonies were maintained at Baboon Resources, OUHSC, Oklahoma City, Okla. At the time of necropsy, whole fresh lung or a lobe of lung from fetal (delivered at 125 days of gestation (125 dGA); complete term is 185 dGA) and adult baboons (age range 10-22 years) was collected in RPMI 1640 medium containing 2 mM glutamine, 1 mM N-2-Hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), 10 μg/ml gentamicin, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% fetal bovine serum (low endotoxin <10 EU/ml, FBS; Invitrogen, Carlsbad, Calif.). None of the animals recruited in this study showed any clinical sign of infection or lung pathology. Gross and microscopic examinations of all major viscera and the placenta revealed no signs of inflammation or infection.
Purification of baboon lung SP-A: SP-A was purified from bronchoalveolar lavage fluid of a normal healthy adult baboon by a slight modification of the procedure described previously (Yang et al., 2005). The bronchoalveolar lavage fluid was collected from an adult baboon lung by instilling endotoxin-free, sterile normal saline (endotoxin-free 0.9% NaCl, 1.9-2 L with approximately 90% recovery). The lavage fluid was centrifuged, and the supernatant was concentrated using a tangential flow filtration technique (10 kDa hollow fiber filter; GE Healthcare Bio-Sciences Corp, NJ). The surfactant lipids were removed using isobutyl alcohol (1:5 ratio lavage:isobutyl alcohol). The delipidated protein was centrifuged at 5,000×g for 15 minutes at room temperature, dried under nitrogen gas, and subsequently completely dried in a lyophilizer (Labconco, MO). The dried lavage residue was rehydrated in extraction buffer (25 mM Tris (pH 7.5), 0.15 M NaCl, and 20 mM octyl-β-D-glucoside) overnight at 4° C. Rehydrated surfactant was extracted six times with extraction buffer by vortex mixing and centrifugation at 20,000×g for 30 minutes at 4° C. Insoluble SP-A was then suspended in solubilization buffer (5 mM HEPES (pH 7.5), 0.02% sodium azide) and dialyzed for 72 hours against four changes of the solubilization buffer. Insoluble protein was removed by centrifugation at 50,000×g for 30 minutes at 4° C., and supernatant was adjusted to 20 mM CaCl2 and 1 M NaCl to re-precipitate SP-A. Precipitated SP-A was pelleted by centrifugation at 50,000×g for 30 minutes at 4° C., and washed two times in 5 mM HEPES (pH 7.5), 20 mM CaCl2 and 1 M NaCl. The SP-A was suspended in 5 mM HEPES, 5 mM EDTA (pH 7.5) and dialyzed for 72 hours against four changes of the solubilization buffer to remove EDTA. The purified SP-A was dialyzed against four changes of the endotoxin-free, highly-purified water (Invitrogen, CA) for 72 hours to remove any remaining EDTA or salts (CaCl2 and NaCl). Finally, purified SP-A was lyophilized completely and resuspended in endotoxin-free Dulbecco's phosphate buffered saline. The purified protein was filter-sterilized using a 0.2 μm low-protein binding, HT Tuffryn membrane filter (Pall Life Sciences, NY) and stored frozen at −80° C. The protein concentration of purified SP-A was measured by microBCA method (Pierce, Ill.).
All the purification steps were performed under aseptic conditions using endotoxin-free solutions and reagents. The endotoxin concentration was measured using the End-point chromogenic Limulus Amebocyte Lysate (LAL) assay (Charles River Laboratories, MA). The purity of the SP-A protein was confirmed by SDS-PAGE and Western blotting using the procedures described earlier (Awasthi et al., 1999; Awasthi et al., 2001). The isolated protein was further characterized by high performance liquid chromatography (HPLC) on a Phenomenex C-18 reverse phase column using solvents acetonitrile/water/trifluoroacetic acid (60:40:1) at 1 ml/min, with the UV detector set at 280 nm. The retention time of SP-A was determined to be 1.3 minutes (
Culture of KG-1-derived DCs and isolation of primary lung DCs: KG-1 cells (Bone marrow myeloblast cells derived from a leukemia patient; ATCC, VA) were cultured in the presence of recombinant human-GM-CSF (100 ng/ml), IL-4 (100 ng/ml) and TNF-α (40 ng/ml) (all the cytokines were purchased from PeproTech, NJ) for a period of 5 days (Ackerman et al., 2003; Bharadwaj et al., 2005; Hulette et al., 2001). The phenotype and morphology of the KG-1-derived DCs were confirmed by flow cytometry and light microscopy, respectively (Awasthi and Cooper, 2006). The KG-1-derived DCs were included as model system to optimize the amounts of effector molecules (purified baboon lung SP-A and recombinant TLR4-MD2 proteins).
Isolation of Adult Baboon Lung DC or Fetal Baboon Lung DC-Precursor Population:
Freshly collected lobe of the lung or whole lung samples of adult and fetal baboons were transported on ice in RPMI 1640 medium containing 2 mM glutamine, 1 mM HEPES, 10 μg/ml gentamicin, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% FBS. After a mild mechanical disruption, the single cell suspension was seeded in a tissue culture flask (Nalge-Nunc International Corp, NY) at a density of 30-50×106 leukocytes/175 cm2 flask in RPMI 1640 medium containing 2 mM glutamine, 1 mM HEPES, 10 μg/ml gentamicin, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% FBS. The light-density DC-populations were harvested using OptiPrep cell-separation solution (density 1.32 g/ml, Accurate Chemicals, NY) (Awasthi and Cooper, 2006; Awasthi et al., 2009). The immunophenotype and basic characteristics of the lung DC-population isolated from fetal and adult baboons have been described earlier (Awasthi et al., 2009). Here, first the immunophenotype of KG-1-derived DCs and fetal baboon lung DC-precursors or adult baboon lung DCs were compared by flow cytometry. Briefly, cells were suspended in Dulbecco's phosphate buffered saline (DPBS) containing 1% FBS and 0.09% sodium azide and incubated with fluorochrome-conjugated antibodies to HLA-DP, DQ, DR, CD11c, CD40, CD80, CD86 (typical DC-markers) as described earlier.
Expression of endogenous TLR4 by flow-cytometry and western blotting: The harvested cells were suspended in 100 μl DPBS containing 1% FBS and 0.09% sodium azide. Previously titrated phycoerythrinin (PE)-conjugated anti-human TLR4 antibody (BD Biosciences, CA), was added at the ratio of 1 μg antibody per 1×106 cells. Cells and antibody were incubated for 30 minutes on ice in the dark. The cells were washed three times with PBS containing 1% FBS and 0.09% sodium azide and fixed in freshly prepared 0.5% paraformaldehyde. The cells were run through an automated dual laser excited FACS Calibur at the Flow and Imaging Core Facility (OUHSC, Oklahoma City). The histogram and dot-plot charts were obtained and analyzed using Summit V4.3 software (Dako Colorado Inc, CO). The isotype control antibody-stained cells served as controls for background staining.
For western immunoblotting, whole cell lysates were prepared in lysis buffer (50 mM Tris-HCl, pH 7.4) containing 1% Igepal, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM phenyl methyl sulfonyl fluoride (PMSF), and 1 μg/ml each of leupeptin and pepstatin. After protein estimation, about 15 μg of total proteins were fractionated by Novex 4-20% Tris-glycine gradient SDS-PAGE gel (Invitrogen, CA). Separated proteins were electro-transferred onto a nitrocellulose membrane using an iBlot gel transfer device (Invitrogen, CA). The non-specific sites were blocked by incubating the membrane with 7% skim milk in Tris-buffered saline with 0.4% Tween-20 (TBST). The blocked membrane was incubated overnight at 4° C. with monoclonal antibody against TLR4 (clone HTA125; ebioscience, CA) diluted 1:1000 in TBST. The membrane was washed and incubated with horseradish peroxidase (HRP)-conjugated-anti-rabbit-IgG antibody (Sigma-Aldrich, MO) diluted 1:1000 in TBST. The immunoreactive bands were detected by SuperSignal West Femto detection reagent (Thermo Fischer Scientific, IL).
Cellular distribution of exogenously added TLR4-MD2 protein: Since KG-1-derived-DCs, adult baboon lung-DCs and fetal baboon lung DC-precursor cells expressed negligible amounts of TLR4 protein; the cells were pulsed with recombinant human TLR4-MD2 proteins (RnD Systems, MN). The cellular distribution of exogenously-added recombinant TLR4-MD2 protein was investigated by confocal microscopy and flow cytometry.
Labeling of recombinant TLR4-MD2 protein with Alexa-fluor 594 fluorescent dye. Recombinant TLR4-MD2 protein was labeled with Alexa-fluor 594 fluorescent dye using a microscale protein labeling kit (Invitrogen-Molecular Probes, CA) optimized for labeling proteins with molecular weights between 12 and 150 kDa, as per the manufacturer's directions. Briefly, 40 μg of recombinant TLR4-MD2 protein (at the concentration of 1 mg/ml in DPBS) was labeled with Alexa-Fluor 594 carboxylic acid, succinimidyl ester (Excitation/Emission wavelengths: 590/617 nm). The pH of the protein was adjusted to 8.3 using 1/10 volume of 1M sodium bicarbonate, and Alexa-Fluor 594 reactive dye stock solution (12.2 nmol/μl) was added to the protein. The protein:dye mixture was incubated for 15 minutes at room temperature. Fluorochrome-conjugated protein was then separated from unconjugated dye using a spin filter conditioned with gel resin. The spin filter loaded with reaction mixture was centrifuged at 16,000×g for 1 minute. The absorbance of the Alexa-Fluor 594 dye-conjugated TLR4-MD2 protein was read at 280 nm and 590 nm using UV/Vis NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, DE) and degree of labeling (DOL) was determined using following formula:
Where A280 and A590 are the protein's absorbance at 280 nm and at 590 nm, respectively, the value of 0.56 is a correction factor for the fluorophore's contribution to the A280, and 90,000 cm−1M−1 is the approximate molar extinction coefficient of the Alexa-Fluor 594 dye.
Cellular distribution of Alexa-fluor 594-conjugated TLR4-MD2 protein by confocal microscopy. The KG-1-derived DCs were seeded at a density of 2.5×105 cells per well in an 8-well chamber slide (Nalge Nunc international, NY) in serum-free Opti-MEM medium (Invitrogen, CA). The cells were then incubated with 10 μg of Alexa fluor 594-conjugated recombinant TLR4-MD2 protein (DOL ˜4.0) for 1 hour and 4 hours at 37° C. in 5% CO2 atmosphere. Thirty minutes prior to the completion of incubation period, the Vybrant DiO cell labeling solution (5 nM final concentration, Invitrogen-Molecular Probes, CA) and Hoechst 33342 dye (0.3 μg/ml final concentration, Invitrogen-Molecular Probes, CA) were added to the cells for staining cell-cytoplasm and nucleus, respectively. Finally, the cells were washed twice in Opti-MEM medium, fixed using Vectashield-antifade mounting medium (Vector Laboratories, CA) and observed under Leica TCS SP2 AOBS (Acousto Optical Beam Splitter) multi-photon laser confocal microscope at the Flow and Image Cytometry laboratory (OUHSC, Oklahoma City). Images were acquired with 63× lense objective (at excitation/emission wavelengths: 405/410-550 nm for Hoechst 33342 dye, 488/500-550 nm for DiO dye, and 594/610-660 nm for Alexa-fluor 594 dye) and were analyzed using the Leica TCS software (Leica Microsystems CMS, Mannheim, Germany). Finally, the images acquired were merged and composite pictures were obtained.
Localization of Alexa-fluor 594-conjugated TLR4-MD2 protein by flow cytometry. The KG-1-derived DCs were suspended in Opti-MEM medium at the density of 2.5×105 cells per 100 μl and incubated with 10 μg Alexa-fluor 594-conjugated recombinant TLR4-MD2 protein. After 1 hour and 4 hours of incubation, cells were washed three times in fresh Opti-MEM medium, re-suspended in 500 μl of 37° C. pre-warmed DPBS, and run on Influx Cell Sorter (BD Biosciences, CA) at the Flow and Image Cytometry laboratory (OUHSC, Oklahoma City). The cells were gated to remove debris and histogram charts were obtained at 624-40 nm emission wavelengths. Cell-staining with Alexa-fluor 594-conjugated TLR4-MD2 protein was analyzed using Summit V4.3 software (Dako Colorado Inc, CO).
Phagocytosis assay: In this Example, pHrodo-conjugated, heat-killed E. coli K12 (encapsulated) bioparticles (Invitrogen-Molecular Probes, CA) were employed. The pHrodo-fluorescent label offers an advantage over other conventional methods, in that it fluoresces only in acidic conditions (i.e., after the bioparticles are taken inside the intracellular lysosomes) (Invitrogen-Molecular Probes, CA). To ensure that the fluorescence relates to phagocytosed pHrodo-conjugated E. coli bioparticles only, the phagocytosis reaction mix was imaged by confocal microscopy. Briefly, the KG-1-derived DCs were seeded at a density of 2.5×105 cells/well in an 8-well chamber slide and incubated with pHrodo-conjugated E. coli K12 bioparticles (one cell-to-˜300 bacterial bioparticles) for 3 hours. The Hoechst 33342 dye was added to the cells (0.3 μg/ml final concentration, Invitrogen-Molecular Probes, CA). The cells were washed once, re-suspended in Opti-MEM medium, fixed with Vectashield-antifade mounting medium (Vector Laboratories, CA), and observed under Leica TCS SP2 AOBS (Acousto Optical Beam Splitter) multi-photon laser confocal microscope (at excitation/emission wavelengths: 550/600 nm for pHrodo-labeled bioparticles, 405/410-550 nm for Hoechst 33342 dye) and under brightfield. Images taken at different wavelengths were merged, and composite pictures were obtained.
After confirming that the fluorescence is of phagocytosed bioparticles, comprehensive experiments were performed in presence and absence of effector molecules (SP-A and TLR4-MD2 proteins). The cells were pulsed with purified, native baboon lung SP-A and recombinant human TLR4-MD2 or MD2 proteins (RnD Systems, MN) for an hour prior to phagocytosis assay with pHrodo-conjugated, heat-killed E. coli K12 bioparticles (Invitrogen-Molecular Probes, CA). MD2 protein was also included, because SP-A is known to interact with MD2, and it was questioned if MD2 can influence the immune functions of DC-population. The assay was performed in serum-free Opti-MEM medium (Invitrogen, CA), as described by the manufacturer (Invitrogen-Molecular Probes, CA). The assay mixtures were incubated for another 3 hours at 37° C. in 5% CO2 incubator. The fluorescence readings were taken at 550 nm excitation and 600 nm emission wavelengths using SpectraMax M2 spectrofluorometer (Molecular Devices, CA).
The phagocytosis index was calculated by subtracting the average fluorescence intensity of the reaction with bioparticles alone from the control (basal without any effector molecules) and experimental wells. Finally, the percent effect was calculated using the following formula:
% effect=(Net experimental phagocytosis/Net basal phagocytosis)×100%
The percent phagocytosis of E. coli bioparticles was also confirmed by fluorescence microscopy in representative reaction wells. The cell-free supernatants were collected after taking the fluoremetric readings and stored at −80° C. for further analysis.
Cytokine (TNF-α) measurement: The TNF-α levels were measured in cell-free supernatants by enzyme linked immunosorbent assay (ELISA) using a commercially available kit (eBioscience, CA), as per the manufacturer's instructions. Briefly, the microwells of a 96 well plate were coated with diluted purified anti-human TNF-α antibody. The wells were washed, and nonspecific sites were blocked. Diluted recombinant human TNF-α (7.8-500 pg/ml standard) and cell-free-supernatant (1:10) were added to the antibody-coated wells, and the plate was incubated overnight at 4° C. The next day, the plate was washed and incubated with biotin-conjugated anti-human TNF-α antibody followed by avidin-horseradish peroxidase and substrate solution. The reaction was stopped by adding 2 N H2SO4 and read at 450 nm (Molecular Devices, CA).
Statistical Analysis: The results were analyzed by Student's t-test for statistical significance using Prism software (Graphpad, San Diego, Calif.). p<0.05 was considered significant.
Results of Example 1:
The fetal baboon lung DC-population collected from the top of the density gradient were unique to fetal baboons, and were not identified in adult baboons. The morphologic features and phenotypic characteristics have been described in the inventor's publication (Awasthi et al., 2009, the contents of which are expressly incorporated herein by reference).
Prior to conducting experiments with adult baboon lung DCs or fetal baboon lung DC-precursor cells, the KG-1-DCs (harvested on 6th day of culture) were used in the initial experiments to optimize the concentration of effector molecules. Morphologically, KG-1-derived DCs harvested on day 5 of culture were round and did not show any tentacles or dendrites (typical of immature DCs). However, after 13 days, the DCs developed dendrites and an irregular shape, characteristic features of mature DCs (
Characterization of purified native baboon lung SP-A. To elucidate the effects of SP-A, first native SP-A protein was first purified from bronchoalveolar lavage fluid specimens of a normal, healthy adult baboon (Awasthi et al., 1999; Awasthi et al., 2001). The purity and identity of the native baboon lung SP-A was confirmed by SDS-PAGE, western blotting and HPLC. Under partially-reducing conditions (heating and no DTT), SP-A separated as an oligomer, ˜100 kDa trimer, and a 66 kDa dimer on the SDS-PAGE gel. Under reducing condition (heating+DTT), SP-A ran as a 32-34 kDa monomer and 66 kDa partially reduced dimer (
Since TLR4 is a potent receptor for endotoxin, the presence of endotoxin can significantly influence the results. All of the solutions and reagents were prepared in endotoxin-free water, and all assays were performed in an aseptic environment. Endotoxin concentration was measured in the purified baboon SP-A, and in the reconstituted TLR4-MD2 and MD2 proteins by the chromogenic LAL method (Charles River Lab, MA). The endotoxin concentration was negligible in purified baboon SP-A (0.0003 ng/μg protein) and in recombinant TLR4-MD2 and MD2 protein suspensions (≦0.006 ng/μg protein).
KG-1-derived DCs and primary DCs express negligible TLR4 protein under normal conditions; exogenously added TLR4-MD2 protein localizes mainly on the cell surface. The basal cell-surface expression of TLR4 on primary adult baboon lung DCs and KG-1 derived DCs was negligible (
Exogenous addition of native SP-A and recombinant TLR4-MD2 proteins increases the phagocytic uptake of E. coli bioparticles. In this Example, pHrodo-labeled, heat-killed encapsulated E. coli K12 bioparticles were utilized for investigating the phagocytic ability of DCs. First, the fluorescence of phagocytosed bioparticles in KG-1-derived DCs was confirmed by confocal microscopy. The images showed that only the phagocytosed bioparticles fluoresce (
In comprehensive phagocytosis experiments, the fluorescence signal reflecting the red fluorescence emitted by phagocytosed pHrodo-labeled E. coli bioparticles, was measured by spectrofluorometry using identical wavelengths setting. Briefly, the KG-1-derived DCs were incubated with purified baboon lung SP-A protein±TLR4-MD2 protein. The % net effect on phagocytosis was calculated in the presence of effector molecules (TLR4-MD2, MD2 and SP-A) after normalizing with the basal phagocytosis in the absence of the effector molecules. The percent phagocytosis calculated by fluorescence microscopy (number of fluorescing cells/total number of cells in a composite of 5 different fields) correlated with the phagocytosis indices calculated by the spectrofluorometry methods. These data demonstrate that both SP-A and TLR4-MD2 proteins increase the phagocytic uptake of E. coli bioparticles by 1.5-2 fold in concentration-dependent manner (p<0.05,
SP-A reduces the TLR4-MD2 protein-induced TNF-α release against E. coli. TNF-α levels were measured in cell-free supernatants of primary adult baboon lung DCs and fetal baboon lung DC-precursor cells treated with SP-A±TLR4-MD2 proteins after 3 hours of phagocytosis reaction. Addition of purified lung SP-A did not induce the secretion of TNF-α by DCs in response to E. coli, but pulsing with TLR4-MD2 protein increased the TNF-α secretion significantly (p<0.05). However, when the SP-A and TLR4-MD2 proteins were added together to the cells and incubated further for another 3 hours with E. coli, the TNF-α levels were equivalent to those incubated without any exogenous protein or with SP-A only (
Discussion for Example 1
The inventor's results on fetal baboon bone marrow-derived DCs (Awasthi and Cooper, 2006) as well as the reports of others with monocytes (Kramer, 2003) provided evidence that DC functions (i.e., phagocytosis and cytokine secretion) are impaired during prenatal and neonatal periods (Awasthi and Cooper, 2006). However, recent understanding indicates that the tissue-resident DCs are different than the circulating or bone marrow-derived DCs (Diao et al., 2006). Results obtained by the inventor also demonstrate that fetal baboon lung cells are at precursor stage, express negligible levels of DC-markers, and are functionally defective in responding to infectious stimuli (Awasthi et al., 2009; Awasthi and Cooper, 2006). Although the developmental stage of the fetal lung DC-population remains to be completely elucidated in fetal baboons, they have been identified by the inventor as DC-precursor cells because they convert into typical DCs after incubation with DC-promoting cytokines (unpublished data).
One possibility is that since these cells are not fully equipped with TLR or other pathogen-pattern recognition receptors because of developmental immaturity, the DC-precursor cells are not capable of capturing the microorganisms. SP-A also serves as a pathogen-pattern recognition receptor and is known to stimulate DC-maturation and phagocytic uptake of infectious organisms (Kingma and Whitsett, 2006). However, at 125 days of gestation, SP-A is not detectable. NICU care and proper clinical management induce expression of both SP-A and TLR4 which reaches to optimal levels under normal conditions (Awasthi et al., 1999; Awasthi et al., 2001). However, despite an advanced and sophisticated clinical care, preterm babies are more prone to infection, and infection and ventilator-associated lung injury remarkably perturb the expression of SP-A and TLR4. Specifically, lavage pools of SP-A are decreased, and tissue expression of TLR4 is increased. These published results suggest that introduction of SP-A may help maintain the tissue homeostasis and exert anti-infective and anti-inflammatory effects (Lee et al., 2010; Goldmann et al., 2009; Madan et al., 2010; K. Hartshorn, 2010). The present Example was designed to determine if the introduction of SP-A will impact the functions of DCs in the lungs of preterm babies.
The present example was focused on studying selected immune functions: phagocytosis and cytokine secretion against infectious stimuli. Primary cells were pulsed with purified or recombinant protein preparations for two reasons: first, the genetic-transfection of primary DCs will require longer time for efficient protein expression, and longer incubation may induce phenotypic changes in DCs (Brinker et al., 2003); and second, the protein-pulsing mimics the physiological scenario, because both SP-A and TLR4 proteins are known to exist in soluble extracellular, cell surface as well as in intracellular forms under steady-state conditions (Iwami et al., 2000; Ochs et al., 2002). MD2 was also included in conjunction with TLR4, because it serves as an important adaptor molecule to TLR4 and binds to SP-A (Yamada et al., 2006). However, MD2 does not carry an intracellular signaling TIR domain and does not affect the phagocytic function of DCs on its own (
The present Example demonstrates that an exogenous addition of SP-A and TLR4-MD2 proteins in the DC population increases phagocytic uptake of encapsulated E. coli (
The results of this Example are of clinical importance because surfactant preparations currently-used in NICUs do not contain SP-A. Ultimately, this Example supports the idea of reformulating the currently-available clinical surfactant preparations to contain SP-A and their clinical usage in NICU.
Example 2 A Novel TLR4-interacting Surfactant Protein-A-Derived Peptide Suppresses LPS-Induced TLR4 Expression and TNF-α ReleasePublished reports suggest that the bronchoalveolar lavage pools (extracellular pools) of SP-A are significantly reduced in lungs of infected patients and animal models (Alcorn et al., 2005; Awasthi et al., 1999; Awasthi et al., 2001; Awasthi et al., 2004; Chang et al., 2006; Kajikawa et al., 2005). In contrast, the TLR4 expression is increased (Awasthi et al., 2008; Chang et al., 2006; Gagro et al., 2004; Kajikawa et al., 2005). The reduction in the amounts of SP-A, and simultaneous increase in TLR4 expression corroborates well with the clinical condition of patients having fulminant infection and inflammation, respectively. In these clinical scenarios, the introduction of SP-A should facilitate clearance of pathogens and attenuate inflammation. However, currently-available clinical surfactants do not contain SP-A or SP-D. Thus, a great need has been felt for designing a shorter fragment of SP-A as well as reformulating the surfactant.
Interestingly, recently published reports suggested that SP-A directly binds to TLR4 (Guillot et al., 2002; Yamada et al., 2006). The in vivo evidence of such an interaction has been lacking, and its functional relevance has not been fully elucidated. In this Example, the binding of SP-A to TLR4-MD2 in non-infected, normal baboon lung tissues was determined by co-immunoprecipitation/immunoblotting, and in vitro by a microwell-based method. Next, a bioinformatics approach was used to examine the interaction between SP-A and TLR4-MD2 proteins. In conjunction, potential binding regions were identified in an in silico model of the SP-A-TLR4-MD2 complex. Based on the information obtained from the bioinformatics analysis, an SP-A-derived peptide library was synthesized. Studies were further extended to investigate the functional relevance of SP-A-TLR4 interaction in a dendritic cell system. The present Example demonstrates that similar to native SP-A, an SP-A-derived peptide (SPA4; SEQ ID NO:3) binds to TLR4-MD2 protein, inhibits expression of TLR4 and reduces the release of TNF-α in response to the most potent TLR4-ligand: Gram-negative bacteria-derived lipopolysaccharide (LPS).
Materials and Methods of Example 2:
Animals: The animal studies were approved by the Institutional Animal Care and Use and Institutional Biosafety Committees at the University of Oklahoma Health Science Center (OUHSC), Oklahoma City, Okla. Baboons (Papio anubis) were maintained at the Baboon Research Resource, OUHSC, Oklahoma City, Okla. At the time of necropsy, lung tissue or bronchoalveolar lavage fluid specimens were obtained from normal healthy adult baboons. Gross and microscopic examinations of major viscera as well as the lung tissue specimens from these baboons showed no signs of inflammation or infection.
Preparation of baboon lung tissue homogenate: The frozen lung tissue samples were homogenized in a buffer containing 1% Igepal CA630, 0.1% sodium dodecyl sulfate, and protease inhibitors (1 μM leupeptin, 1 mM ethylenediamine tetraacetic acid, 0.7 mg/L pepstatin and 0.2 mM phenylmethyl sulphonyl fluoride; Sigma-Aldrich, MO) at a concentration of 100 mg tissue/ml buffer (Awasthi et al., 1999; Awasthi et al., 2001). The tissue homogenates were centrifuged to remove cell debris, and total protein concentration was measured in supernatants using the MicroBCA protein estimation kit (Pierce, Ill.).
Western blotting: The inventor has recognized the cross-reactivity of anti-human-SP-A- and anti-human-TLR4-antibodies with respective antigens in baboons, and studied the expression of SP-A and TLR4 in lung tissue homogenates of fetal and adult baboons, and neonate baboons having Bronchopulmonary dysplasia (Awasthi et al., 1999; Awasthi et al., 2008). Here, using western blotting, the immunoreactivity of these antibodies with respective antigens was first confirmed in baboon lung tissue homogenates to ensure the integrity of the antigens. Lysates of HEK-293 cells stably-transfected with human-TLR4-cDNA (provided by Invivogen, CA), and purified human- and baboon-lung SP-A proteins served as positive controls.
The protein samples were prepared in SDS-PAGE sample buffer without dithiothreitol (DTT)+no heating (non-reducing), without DTT+heating at 100° C. for 5 minutes (partially-reducing) or with DTT+heating at 100° C. for 5 minutes (reducing). The samples were loaded and separated on a SDS-PAGE gel (8% running and 5% stacking gel or Novex 4-20% Tris-glycine gel, Invitrogen, CA). Separated proteins were then electro-transferred overnight onto a nitrocellulose membrane. The nonspecific sites were blocked by incubating the membrane in 7% skim milk diluted in Tris-buffered saline containing 0.4% Tween 20 (TBST). The membranes were then incubated with anti-human-SP-A polyclonal antibody (Awasthi et al., 1999; Awasthi et al., 2001) or TLR4 antibody (eBioscience, CA) (Awasthi et al., 2008), diluted 1:1000 in TBST, for 1 hour at room temperature. The membrane was washed and then incubated with horseradish peroxidase (HRP)-conjugated-anti-mouse or anti-rabbit IgG antibody (1:1000 diluted in TBST; Sigma-Aldrich, MO). The immunoreactive bands were detected by Supersignal West Pico or Femto chemiluminescent substrate (Pierce, Ill.).
Immunoprecipitation of lung-SP-A or TLR4 and cross-immunoblotting: After confirming the reactivity of the antibodies and the integrity of TLR4 and SP-A proteins in baboon lung tissue homogenates, the physical binding between the two proteins was examined by immunoprecipitation/cross-immunoblotting. The SP-A and TLR4 proteins were immunoprecipitated from baboon lung tissue homogenates and cross-immunoblotted with anti-human-TLR4 and SP-A antibodies, respectively. The SP-A (IP-SP-A) and TLR4 (IP-TLR4) were immunoprecipitated using Primary Seize Immunoprecipitation kit (Pierce, Ill.) as per the manufacturer's instructions. Approximately 200 μg of anti-human-TLR4 or SP-A antibody (Awasthi et al., 2001; Awasthi et al., 2008) was conjugated to the AminoLink plus coupling gel column at 4° C. overnight. Five hundred μg to 1 mg of total lung tissue homogenate protein was loaded into the columns and the immunoprecipitation reaction was performed overnight at 4° C. The IP-SP-A and IP-TLR4 were eluted from the antibody-bound column using ImmunoPure elution buffer. No Calcium was added to the immunoprecipitation reaction at any step. Also, none of the buffers in the kit contained calcium.
Various amounts of IP-TLR4 and IP-SP-A were run on SDS-PAGE gels. The separated proteins were then transferred on nitrocellulose membrane using the i-Blot system (Invitrogen, CA). For cross-immunoblotting, IP-SP-A and IP-TLR4 were immunoblotted with anti-TLR4 and anti-SP-A antibodies, respectively, as described above. Positive controls included lung tissue homogenate protein, purified human SP-A and lysate-protein of HEK-293 cells-transfected with full-length, human-TLR4-cDNA. Negative controls included IP-SP-A and IP-TLR4 immunoblotted with a non-specific antibody, and immunoprecipitates from columns where the lung tissue homogenate or the primary antibody had been omitted.
Purification and characterization of native Baboon SP-A: SP-A was purified from bronchoalveolar lavage fluid of an adult baboon by a modification of the procedure described previously (Yang et al., 2005). The bronchoalveolar lavage fluid was collected from an adult baboon lung by instilling endotoxin-free, sterile normal saline (endotoxin-free 0.9% NaCl, 1.9-2 L with approximately 90% recovery). The lavage fluid was centrifuged, and the supernatant was concentrated using a tangential flow filtration technique (10 kDa hollow fiber filter; GE Healthcare Bio-Sciences Corp, NJ). The surfactant lipids were removed using isobutyl alcohol (1:5 ratio lavage:isobutyl alcohol). The delipidated protein was centrifuged at 5000×g for 15 minutes at room temperature, dried under nitrogen gas, and subsequently completely dried in a lyophilizer (Labconco, MO). The dried lavage residue was rehydrated in extraction buffer (25 mM Tris, pH 7.5, 0.15 M NaCl, and 20 mM octyl-β-D-glucoside) overnight at 4° C. Rehydrated surfactant was extracted six times with extraction buffer by vortex mixing and centrifugation at 20,000×g for 30 minutes at 4° C. Insoluble SP-A was then suspended in solubilization buffer (5 mM HEPES, pH 7.5, 0.02% sodium azide) and dialyzed for 72 h against four changes of the solubilization buffer. Insoluble protein was removed by centrifugation at 50,000×g for 30 minutes at 4° C., and supernatant was adjusted to 20 mM CaCl2 and 1 M NaCl to re-precipitate SP-A. Precipitated SP-A was pelleted by centrifugation at 50,000×g for 30 minutes at 4° C., and washed two times in 5 mM HEPES pH 7.5, 20 mM CaCl2 and 1 M NaCl. The SP-A was suspended in 5 mM HEPES, 5 mM EDTA, pH 7.5 and dialyzed for 72 h against four changes of the solubilization buffer to remove EDTA. The purified SP-A was dialyzed against four changes of endotoxin-free, highly-purified water (Invitrogen, CA) for 72 h to remove any remaining EDTA or salts (CaCl2 and NaCl). Finally, purified SP-A was lyophilized completely and resuspended in endotoxin-free Dulbecco's phosphate buffered saline. The purified protein was filter-sterilized using a 0.2 μm low-protein binding, HT Tuffryn membrane filter (Pall Life Sciences, NY) and stored frozen at −80° C. The protein concentration of purified SP-A was measured by microBCA method (Pierce, Ill.).
All the purification steps were performed under aseptic conditions using endotoxin-free solutions and reagents. The endotoxin concentration was measured using the End-point chromogenic Limulus Amebocyte Lysate (LAL) assay (Charles River Laboratories, MA). The purity of the SP-A protein was confirmed by SDS-PAGE and Western blotting using the procedures described above.
Interaction between purified baboon lung SP-A, SP-A-peptides and TLR4-MD2 proteins using a microwell-based method: The binding between the purified baboon lung SP-A, SP-A-peptides and recombinant TLR4-MD2 and MD2 proteins was studied in vitro using a microwell-based method (Awasthi et al., 2004). The soluble, recombinant TLR4-MD2 protein (R&D Systems, MN) consisted of a mixture of recombinant human-TLR4 and MD2 proteins. The recombinant extracellular domain of human TLR4 protein (Glu 24-Lys 631 amino acids), was joined with a DNA sequence encoding the signal peptide from human CD33 and a 10× histidine tag at the C-terminus (Accession #000206). For MD2 protein, a DNA sequence encoding the signal peptide from human CD33 was joined with the mature region of human MD-2 (mature region, Glu 17-Asn 160 amino acids) and a 10× histidine tag at the C-terminus (Accession # Q9Y6Y9). The chimeric proteins were expressed in a mouse myeloma cell line, NSO(R&D Systems, MN). The proteins were obtained from the manufacturer in carrier-free condition and reconstituted in PBS containing 0.1% low-endotoxin BSA (MP Biomedicals, OH). MD2, an adaptor molecule for TLR4, is expressed by immune cells, and is known to bind TLR4 in a non-covalent manner (Jain et al., 2008). Thus, the binding of SP-A to the recombinant MD2 protein (R&D Systems, MN) was also studied.
For the binding assay, microwell ultra-high-protein binding Immunolon 4HBX strips (Thermo Scientific, MA) were coated with soluble recombinant-TLR4-MD2 protein or MD2 protein (R&D Systems, 250 ng per well, diluted in 0.1 M NaHCO3, pH 9.6) overnight at room temperature. The plates were washed three times, and non-specific sites were blocked for 2 h at room temperature using phosphate buffered saline containing 0.1% triton-X 100 and 3% nonfat milk (Buffer A). The wells were washed and incubated for 3 h at 37° C. with purified baboon lung SP-A (0.125-10 μg), SPA4 peptide (2-20 μg; SEQ ID NO:3) or adult baboon lung tissue homogenate protein (10-100 μg) diluted in 20 mM Tris (pH 7.4) buffer containing 0.15 M NaCl, 5 mM CaCl2 or equal amount of bovine serum albumin (BSA) protein. The wells were washed with Buffer A and incubated with anti-human SP-A antibody (1:1000 diluted in Buffer A) for 2 h at room temperature followed by HRP-conjugated secondary antibody. The immune-complex was detected using 3,3′,5,5′-tetramethylbenzidine (TMB) substrate system (Sigma-Aldrich, MO). The reaction was stopped with 2 N H2SO4 and read at 405 and/or 450 nm spectrophotometrically (Molecular Devices, CA).
JAWS II dendritic cell culture: The JAWS II dendritic cell line is an immortalized cell line derived from bone marrow of C57BL/6 mice (ATCC, Manassas, Va.). The cells were maintained in Alpha-modified minimum essential medium (Sigma, St Louis, Mo.) supplemented with 20% fetal bovine serum (FBS), 4 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 50 μg/ml gentamicin (Invitrogen, Grand Island, N.Y.) and 5 ng/ml of recombinant murine granulocyte macrophage-colony stimulating factor (Peprotech, Rocky Hill, N.J.). (Awasthi et al., 2005). The culture medium was replaced with fresh medium every 48 h.
LPS treatment of JAWS II dendritic cells with and without SP-A peptides: Based on the results of the bioinformatics analysis (described in Results section), SP-A peptides (SPA1 (SEQ ID NO:258); SPA2 (SEQ ID NO:259); SPA3 (SEQ ID NO:4); SPA4 (SEQ ID NO:3); SPA5 (SEQ ID NO:5); SPA6 (SEQ ID NO:260); and SPA7 (SEQ ID NO:261)) were synthesized. The amino acid sequences were derived from the C-terminal Carbohydrate Recognition Domain (CRD) of human SP-A corresponding to the TLR4-interacting sites identified in the in silico model of SP-A-TLR4-MD2 complex (
In Example 1, it was demonstrated that short-pulsing of baboon lung dendritic cells with purified lung SP-A and recombinant TLR4-MD2 protein leads to TLR4-induced cytokine release against E. coli. Thus, it was questioned if the SP-A-derived peptides from the TLR4-interacting regions will demonstrate a similar effect. The JAWS II dendritic cells (1 million) were treated with SP-A peptides (1 or 10 μM) with or without E. coli derived LPS (75 ng/ml, highly purified, low protein, Calbiochem, CA). In order to observe the anti-inflammatory properties of the SP-A peptides, the cells were treated with peptides for 1 hour prior to addition of LPS (pre-LPS treatment) or after 4 hours incubation with LPS (Post-LPS treatment). The incubation was continued for a total period of 5 hours. Control cells were treated with vehicle, LPS (75 ng/ml), and/or SP-A peptides (1 and 10 μM) for a period of 5 hours. The cell-free supernatants were collected and stored at −80° C. for further analysis.
TLR4 expression by immunocytochemistry: The JAWS II-DCs were seeded at a density of 25,000 cells per well of an 8-well chamber slide (Nalge Nunc international, New York, USA). Post-LPS (100 ng/ml) treatment with SPA4 peptide (1 and 10 μM; SEQ ID NO:3) followed, and the cells were fixed for 20 minutes with 3.5% paraformaldehyde prepared in PBS on ice. Permeablization was carried out for 20 minutes on ice with Alpha-MEM medium containing 10% FBS and 0.05% saponin and 10 mM HEPES (Inaba et al., 1998). The cells were washed with PBS supplemented with 1% FBS and 0.05% saponin (wash buffer). Non-specific binding sites were blocked using PBS containing 10% normal mouse serum (Sigma, St Louis, Mo.) for 1 hour at room temperature in a humidified chamber. A rabbit polyclonal antibody to mouse TLR4 (Abcam, Cambridge, Mass.) was added to the cells at a dilution of 1:50 and incubated overnight at 4° C. in a humidified chamber. The cells were washed three times for 5 minutes each, followed by incubation with 10 μg/ml Alexa Fuor 488-labeled donkey anti-rabbit IgG antibody (Molecular Probes, Carlsbad, Calif.) for 1 hour at room temperature in humidified chamber protected from light. The cells were incubated with 100 nM rhodamine-phalloidin (Cytoskeleton Inc, Denver Colo.) for 30 minutes at room temperature. Finally, 1 μg/ml Hoechst 3342 (Molecular Probes, Carlsbad, Calif.) dye was added to the cells. Confocal microscopic images were acquired at Imaging and core facility of Oklahoma Medical Research Foundation using the Zeiss LSM-510META laser scanning confocal microscope. Images were acquired with lens objective of 63× with the x/y stack sizes being 146.2 μm using band pass filter specifications at 435-485, 560-615 and 505-530.
Cytokine (TNF-α) measurement: The TNF-α levels were measured in cell-free supernatants of JAWS II dendritic cells treated with LPS with or without SP-A peptides by enzyme linked immunosorbent assay (ELISA) as described earlier (Awasthi and Cox, 2003).
Statistical analysis: The results were analyzed by the Student t-test or ANOVA for statistical significance using Prism software (Graphpad, San Diego, Calif.). At p<0.05, the null hypothesis was rejected.
Results of Example 2
TLR4 and SP-A co-immunoprecipitated from baboon lung tissue homogenates. The inventor has previously shown that human-SP-A and TLR4-specific antibodies react with baboon-SP-A and TLR4 proteins, respectively (Awasthi et al., 1999; Awasthi et al., 2001; Awasthi et al., 2008). Using the same antibody clones, the integrity of SP-A and TLR4 was confirmed in baboon lung tissue homogenates. The immunoprecipitation of specific proteins was identified by immunoblotting the IP-SP-A and IP-TLR4 eluates from adult baboon lung tissue homogenates using SP-A- and TLR4-specific antibodies, respectively (
Next, it was hypothesized that if the SP-A and TLR4 proteins interact with each other, the two proteins may exist together in the lung and may be co-immunoprecipitated from lung tissue homogenates. The cross-immunoblotting results indicate that SP-A and TLR4 are co-immunoprecipitated from baboon lung specimens (
Characterization of purified native baboon lung SP-A: To further elucidate the interaction between SP-A and TLR4, native SP-A protein was first purified from bronchoalveolar lavage fluid specimens of a normal, healthy adult baboon (Awasthi et al., 1999; Awasthi et al., 2001). The purity and identity of the native baboon lung SP-A was confirmed by SDS-PAGE and western blotting. Under partially-reducing conditions (heating and no DTT), SP-A separated as an oligomer, a 90 kDa-100 kDa trimer, and a 66 kDa dimer on the SDS-PAGE gel. Under reducing conditions (heating+DTT), SP-A ran as a 32-34 kDa monomer and a 66 kDa partially reduced dimer. The purified baboon lung SP-A protein was also immunoblotted with SP-A antibody which identified the SP-A-specific protein bands. The solubility of purified baboon lung SP-A was 51% (Stenvall et al., 2005). Since the TLR4-MD2 proteins are less abundant in biological systems and distributed throughout, it is difficult to obtain native TLR4-MD2 protein in sufficient quantity. Thus, recombinant human-TLR4-MD2 protein (RnD Systems, MN) was included.
Since TLR4 is a potent receptor for endotoxin, the presence of endotoxin can significantly influence the results. Thus, all solutions and reagents were prepared in endotoxin-free water, and all assays were performed in an aseptic environment. Endotoxin concentration was measured in the purified baboon SP-A preparation and in the reconstituted TLR4-MD2 and MD2 proteins by the chromogenic LAL method. The endotoxin concentration was negligible in purified baboon SP-A (0.0003 ng/μg protein) and in recombinant TLR4-MD2 and MD2 protein suspensions (≦0.006 ng/μg protein).
Lung SP-A and recombinant TLR4-MD2 proteins interact in vitro. The surface-bound TLR4-MD2 and MD2 proteins showed binding with purified baboon lung SP-A and SP-A protein present in native form in lung tissue homogenate (
Protein-protein docking and prediction of interacting amino acids at the interface of SP-A-TLR4-MD2 protein complex. In previous sections, the interaction between SP-A and TLR4-MD2 proteins was experimentally characterized. In this section, the bioinformatics approaches used to examine the interaction are described. First it is described how data was obtained for bioinformatic analyses; then, the in silico docking of SP-A with TLR4-MD2 is described, followed by a description of the rendering of the docking interface.
SP-A structure: Under physiological conditions, SP-A exists as an octadecamer comprising 6× trimer units (Palaniyar et al., 2000), and TLR4-MD2 exists as a dimer (Park et al., 2009). The trimeric crystal structure of neither the human SP-A nor the baboon SP-A is available in the protein data bank (PDB, www.rcsb.org/pdb) (Berman et al., 2000). Head et al., (2003) solved the crystal structure of the trimeric carbohydrate recognition domain/neck domain of SP-A. However, the PDB file and X-ray structure in the protein data bank were available for the monomeric subunit of rat SP-A (PDB ID:1R13) (Head et al., 2003). Using bioinformatics approaches, it is possible to obtain the structure of trimer by docking three monomers to form a single complex. SymmDock (Schneidman-Duhovny et al., 2005a; b), an automated server that deduces the structure of homomultimer with cyclic symmetry when the structure of a monomeric subunit is available, was used for the above task. SymmDock server returned 100 possible trimer complexes that differed in the arrangement of monomers, accompanied by a priority score. Of all the configurations returned by the server, only the top scoring complex was identical to the structure of the trimer shown in the prior art (Head et al., 2003; Palaniyar et al., 2000), and the rest had different arrangements.
TLR4-MD2 structure: For TLR4-MD2 proteins, the amino acid sequences and dimer crystal structure of human TLR4-MD2 complex are available in PDB bank (PDB ID: 3FXI). Although the TLR4 and SP-A proteins are considered highly conserved proteins, SP-A, TLR4 and MD2 sequence homology was checked between the respective animal species using CLUSTALW multiple alignment program (Protein Information Resource, Georgetown University Medical Center, Washington D.C.). Only partial sequences were available for baboon SP-A and TLR4, and there was no information available on baboon MD2. The alignment results demonstrate that the SP-A, TLR4 and MD2 proteins are highly conserved among different species (including mouse, rat, macaca, baboon and human) (
Protein-protein docking: Next, the protein-protein docking was carried out using Global Range Molecular Matching (GRAMM-X) methodology (Tovchigrechko and Vakser, 2006) on a public web server by submitting the PDB files (trimer assembly of SP-A and dimer receptor-adaptor molecule complex of TLR4 and MD2). GRAMM-X represents a new implementation of original GRAMM methodology that uses a smoothed Lennard-Jones potential on a fine grid during the global search Fast Fourier Transformation stage, followed by refinement optimization in continuous coordinates and rescoring with several knowledge-based potential terms (Tovchigrechko and Vakser, 2006). The top 100 docked configurations were visually examined to select the most plausible configurations. Results from published studies (Yamada et al., 2006) and the inventor's data were considered to set the inclusion and exclusion criteria for the selection of the most plausible model of SP-A-TLR4-MD2 complex. First, 90 configurations that did not show the MD2 adaptor molecule interacting with SPA in the SP-A-TLR4-MD2 complex were discarded, because the microwell-based assay results indicated binding between SP-A and MD2 adaptor molecule (
Identification of amino acids at the interface of in silico model of SP-A-TLR4-MD2 protein complex: To examine the binding interface of the complex and identify the amino acids at the SP-A-TLR4 and SP-A-MD2 interfaces, the structures were input into another server called Knowledge-based FADE and Contacts (KFC; comprised of Fast Atomic Density Evaluator (K-Fade): shape specificity features and K-Con: biochemical contacts such as intermolecular hydrogen bonds and atomic contacts) (Darnell et al., 2007). The server predicts the binding hotspots and the associated prediction confidence based on the shape specificity features and biochemical contact features of the residues at the interface. The predicted docking configuration of the SP-A-TLR4-MD2 complex with high confidence (K-Fade>0.9 or K-Con>0.9) have been highlighted in
Functional Screening of SP-A library revealed a peptide (SPA4; SEQ ID NO:3) that reduces LPS-induced TNF-α secretion. Based on the in silico observations and homology to respective SP-A regions between rat and humans, the SP-A peptides derived from C-terminal CRD of human SP-A were synthesized. SP-A peptides were tested for purity by mass spectrometry (Genscript, CA) and for endotoxin contamination by LAL test.
Since an exaggerated activation of TLR4 is directly linked to secretion of pro-inflammatory cytokine (TNF-α) and SP-A in downregulating TLR4-induced TNF-α in lung dendritic cells (Example 1), SP-A peptides were screened for any changes in LPS-induced TNF-α cytokine secretion in JAWS II dendritic cells. Pre-LPS and post-LPS inflammation models were included in this study to investigate if the peptides affect the LPS-mediated responses, prophylactically or therapeutically. It was found that most of the peptides had no effect on LPS-induced TNF-α secretion in the pre-LPS model, except SPA2 (SEQ ID NO:248), SPA3 (SEQ ID NO:249) and SPA7 (SEQ ID NO:251), which stimulated a slight increase in TNF-α secretion. In the post-LPS model, two peptides (SPA4 and SPA5 peptides; SEQ ID NOS:3 and 5, respectively) were found that inhibited the secretion of TNF-α in post-LPS treated cells at both 1 and 10 μM concentrations (
SPA4 peptide binds to TLR4 and blocks the LPS-induced TLR4 expression. Next, the binding of the SPA4 peptide with recombinant TLR4-MD2 protein was confirmed by an in vitro microwell-based binding assay. The binding results showed that similar to purified native SP-A, the SPA4 peptide binds to TLR4-MD2 protein (
SPA4 peptide-induced changes in the expression of TLR4 were also investigated in dendritic cells. It was found that SPA4 peptide treatment reduced the basal TLR4 expression in JAWS II cells. These results further demonstrate that the LPS-induced TLR4 expression was also suppressed significantly after treatment with SPA4 peptide (* p<0.05,
Discussion of Example 2
In lung, SP-A is synthesized by type II lung epithelial cells, and is secreted in alveoli as a component of surfactant (King and Clements, 1972). SP-A plays a critical role in pathogen-opsonization, clearance, downregulation of inflammation, and maintenance of lung function. Earlier the inventor observed that the amounts of SP-A secreted in alveoli are significantly reduced in preterm baby baboon having bronchopulmonary dysplasia and in mouse models of lung infection (Awasthi et al., 1999; Awasthi et al., 2001). Thus, it is reasonable to imagine that administration of SP-A should enhance clearance of pathogens and inhibit inflammation. Unfortunately, currently available surfactants do not contain SP-A, because it is a large and hydrophilic protein and cannot be mixed efficiently with surfactant lipids. Therefore, it is important to search for smaller fragments of SP-A. Unavailability of such an SP-A-derived fragment has been associated with the lack of an appropriate model to mimic such a complex scenario.
Since the discovery of TLR4 as a pathogen-recognition receptor that is mainly expressed by the antigen-presenting cells (Armstrong et al., 2004; Awasthi and Cropper, 2006; Basu and Fenton, 2004), it is now established that an exaggerated expression and activity of TLR4 leads to a deleterious inflammatory response. However, basal activity is important for antigen-presentation and adaptive immunity. Subsequent to finding the reduced levels of SP-A, it was observed that the expression of TLR4 is significantly increased in lungs of baby baboons having bronchopulmonary dysplasia (Awasthi et al., 2008). Similar results (i.e., reduction in SP-A and increase in TLR4 expression) have also been reported in other models by other investigators (Alcorn et al., 2005; Chang et al., 2006; Gagro et al., 2004; Kajikawa et al., 2005). The reduction in SP-A amounts and concomitant increase in TLR4 expression corroborates with the clinical condition of patients with lung infection where reduced pathogen-clearance is observed with robust inflammation.
A number of SP-A-binding proteins and receptors have been recognized; however their functions and expression by cell type remain unexplored (Gil et al., 2009; Stevens et al., 1995; Strayer et al., 1993; Wissel et al., 1996). The binding of SP-A to the TLR4 protein has also been recently shown to occur under in vitro conditions (Yamada et al., 2006); however, the in vivo evidence had been lacking, and functional relevance remained largely unexplored. Example 1 demonstrates that simultaneous pulsing of dendritic cells with SP-A and TLR4-MD2 proteins maintains the increased phagocytic uptake, but downregulates the TLR4-MD2-induced inflammatory response against infectious stimuli. It is believed that downregulation of the inflammatory response may be via interaction between SP-A and TLR4. Thus, a smaller fragment of SP-A containing the TLR4-interacting region should inhibit the TLR4-mediated inflammatory response while maintaining the basic functions of antigen-presenting cells.
In this Example, it was demonstrated that SP-A and TLR4 proteins are co-immunoprecipitated from baboon lung tissue homogenates. This is the first report where such an interaction between SP-A and TLR4 has been shown to exist in the lung by immunoprecipitation/immunoblotting and microwell-based methods using lung tissue homogenates and purified lung SP-A. Earlier, interaction between SP-A and TLR4 was studied with purified or recombinant forms of proteins by ligand-blot, microwell-based binding assay and BIAcore methods (Chroneos et al., 2010; Yamada et al., 2006). Bioinformatics simulation studies further support the interaction between SP-A and TLR4-MD2 protein. Although several aspects of TLR4 and SP-A binding are not clearly understood, it is clear that the lung microenvironment may significantly influence their interaction. It should be noted that in the antibody-based methods employed here, the kinetics and characteristics of binding between the two proteins depend on the antigen-antibody affinity. The specific binding sites of both the SP-A and TLR4 proteins and the kinetic parameters of the native-SP-A-TLR4 interaction needed further investigation. It is important to note that the native SP-A molecule (ligand) is quite large (octadecamer) because of the oligomerization of trimers (Pastva et al., 2007), and TLR4 protein is a homomer and associates with other adaptor (MD2) and signaling receptors for its activity (Re and Strominger, 2002). Moreover, it was also found that SP-A can bind to the TLR4 adaptor molecule MD2 as well. Thus, computer modeling of the SP-A-TLR4-MD2 complex was considered; an in silico model of SPA-TLR4-MD2 complex was obtained where the binding features fitted best with the results from immunobiochemical assays (
As identified earlier, the functional significance of such an interaction is very difficult to assess in vivo. The functional relevance of such an interaction can be better examined under in vitro conditions in a controlled environment using cell culture systems and the appropriate dosage of effector molecules. Thus, the JAWS II dendritic cell system, established in the inventor's lab (Awasthi et al., 2005; Awasthi and Cox, 2003), was used to investigate the effects of SP-A peptides derived from the TLR4-interacting region on cytokine response against a well-known inflammatory stimuli: LPS. It was found that SPA4 peptide (1) encodes most of the amino acids belonging to TLR4-interacting region in in silico model, (2) binds to TLR4-MD2 protein, and (3) reduces LPS-induced TLR4 expression and cytokine response.
These results demonstrate that SP-A blocks the TLR4-MD2-mediated intracellular signaling and cytokine release against infectious stimuli. Recently in human monocytes culture system, Henning et al. found that SP-A did not affect TLR4 expression, but it downregulated the TLR4-mediated signaling against LPS (Henning et al., 2008). However, based on the information on the interacting amino acids at the SP-A-TLR4 interface in the computer-simulated SP-A-TLR4-MD2 complex model, and screening of the peptides, one peptide was identified (SPA4) that not only inhibits the LPS-stimulated TLR4 expression, but also suppresses LPS-induced TNF-α release.
Example 3 Use of SPA4 to Modulate TLR4 Signaling for Treatment of Intestinal InflammationAbout a million people are currently suffering from inflammatory bowel diseases (IBD) in the US alone, and new cases are being diagnosed at the rate of 10 cases per 100,000 people (American College of Gastroenterology). IBD causes chronic inflammation in the intestine with alternating periods of active and latent disease, and accounts for a huge economic cost associated with multiple clinic visits and hospitalizations. Chronic inflammation can lead to debilitating complications including colon cancer. Lifelong pharmacotherapy remains the mainstay of IBD management, whereas surgery is indicated for the treatment of refractory disease or specific complications.
Conventional IBD therapies include the use of aminosalicylates, corticosteroids and immunosuppressive drugs (e.g., methotrexate, cyclosporin A). These traditional treatment modalities provide symptomatic relief to some extent depending on the severity of the disease, but exert numerous side effects. The side effects can range from perturbed physiological functioning of important organ systems to potentially fatal opportunistic infections. However, recently a better understanding of the mucosal immune system and genetics involved in the pathogenesis of IBD led to development of biologic medications (Guidi et al., 2010; Bosani, et al., 2009; Melmed and Targan 2010; S. Hanauer, 2010). These medications include infliximab, adalimumab and certolizumab pegol, which are antibodies to block TNF-α, an inflammatory cytokine that is present in increased amounts in patients with IBD. Adverse reactions with these anti-TNF-α products include infusion or injection site reactions, upper respiratory infections and malignancies. Other new biologics that have recently entered into clinical trials include adhesion molecule inhibitor (Natalizumab), anti-IL-12, IFN-γ antibodies and growth factors (Gordon et al., 2002; Guan, et al., 2009). As the safety and toxic effects remain to be completely evaluated for these new biologics, Natalizumab has already been temporarily discontinued due to JC virus brain infections. At a molecular level, these inhibitors and antibodies target the pre-formed or secreted inflammatory cytokines or cell-surface molecules that can provide neither cure nor a durable effect after discontinuation. Well thought-out strategies are needed to design novel treatment modalities that can provide more sustained therapeutic effect without any significant toxicity or side effects.
Toll-like receptor-4 (TLR4) was first discovered in 1996 as an innate immune recognition receptor for Gram negative bacterial lipopolysaccharide (LPS). Besides LPS, TLR4 is now known to recognize endogenous inflammatory signals, such as heat-shock proteins, fibronectin etc. Over a period of the last 15 years, since the discovery of TLR4, a great deal about its critical role in inflammatory responses in infectious and non-infectious diseases has been determined. Since inflammation is a hallmark of IBD, TLR4 is thought to be important. It has been recently hypothesized that TLR4-signaling probably serves a dual role in the gut as a mediator of both inflammation and mucosal repair. A hypothetical model was provided suggesting that basal TLR4-signaling is required for normal functioning and intestinal homeostasis. It is the exaggerated TLR4-signaling in response to physiological stressors (e.g., hypoxia) and infectious stimuli (e.g., LPS), that leads to intestinal inflammation. Thus, the novel therapeutic agents that can block this exaggerated TLR4/TLR4-signaling may eventually suppress the inflammatory response and help alleviate the symptoms of IBD.
Results of Example 3
SPA4 peptide (SEQ ID NO:3) inhibits the LPS-induced TLR4 expression in dendritic cells and SW480 intestinal (colonic) epithelial cells. After the shorter SP-A fragment (SPA4) was designed that showed binding to TLR4-MD2 protein (Example 2), its immunomodulatory effects were studied using well-established dendritic (JAWS II; ATCC, VA) (Vilekar et al., 2010) and intestinal epithelial cells (SW480; ATCC, VA). Both of these cells constitutively express the TLR4 gene; the protein expression is low in dendritic cells under basal conditions. LPS treatment induced TLR4 expression in both cell types. First, it was determined if the SPA4 peptide inhibits LPS-induced expression of TLR4 in these cells. The cells were treated with 75 ng/ml or 1 μg/ml E. coli-derived LPS (Calbiochem, CA; highly purified, low protein, does not activate TLR2 signaling) for 4 hours prior to addition of SPA4 peptide. After a total of 5 hours, TLR4 expression was studied by confocal microscopy.
The results demonstrated that the SPA4 peptide reduced LPS-induced TLR4 expression to a basal level (
SPA4 peptide reduced serum TNF-α and inflammation in Dextran sodium sulfate (DSS)-colitis model in mice (
Therefore, this Example has demonstrated that the SPA4 peptide suppresses TLR4-signaling in intestinal epithelial and immune cells under inflammatory stress conditions. This suppression of TLR4 at the cellular level results in reducing intestinal inflammation in animals.
Example 4 SPA4 Inhibits Lipopolysaccharide-Induced NF-κB Signaling, Cytokines and Migration of Colon Cancer SW480 CellsColorectal cancer is the third most common cancer and leading cause of cancer-related mortality in the United States. As per a recent annual report, 141,210 new cases of colorectal cancer and 49,380-associated-deaths were reported in the US only (National Cancer Institute at the National Institute of Health, Washington D.C.). An exaggerated inflammatory response has been reported to increase the risk of colorectal cancer in patients with inflammatory bowel disease (IBD), ulcerative colitis (UC) or Crohn's colitis. This inflammation-induced progression of cancer can potentially be suppressed by anti-inflammatory agents. Common anti-inflammatory medications include the use of aminosalicylates, corticosteroids and immunosuppressive drugs (e.g., methotrexate, cyclosporin A). While it remains to be established whether conventional anti-inflammatory agents can have chemopreventive effects against cancer, an understanding of mucosal immune system and genetics has led to the recent advancements in development of biologic medications against IBD and cancer, specifically anti-TNF-α products (Bosani et al., 2009). A number of anti-TNF-α products (antibodies and receptor antagonists) have been approved by the FDA for reducing inflammation in patients with colitis. Traditional medications and biologics provide only transient relief and have significant side effects that include increased risk of infections and perturbed physiological functioning of important organ systems. These treatment strategies provide short-lived symptomatic relief, mainly because these products act against already secreted TNF-α cytokine or other chemical mediators.
Key molecules involved in inflammatory pathways include Toll-like receptors (TLRs), nuclear factor (NF)-kB, cytokines, growth factors, kinases, cyclooxygenases and nitric oxide synthases. TLRs are unique because they not only sense the “danger signals” in the form of infectious agents or stress-ligands, but by the virtue of their intracellular Toll/Interleukin-1 receptor (TIR) domain, the TLRs are associated with a complex intracellular signaling network, including NF-κB-inflammatory pathway. Thus, new therapies targeting TLR may be of benefit in suppressing inflammation in more sustained fashion. Among a number of TLRs, Toll-like receptor-4 (TLR4) was first discovered in 1996 as an innate immune recognition receptor for Gram-negative bacterial lipopolysaccharide (LPS). TLR4 is now well-recognized as pattern-recognition receptor against a diverse array of ligands including endogenous stress ligands such as but not limited to, heat-shock proteins, fibronectin, etc. A number of recent studies have reported the involvement of TLR4 in colitis and cancer progression. Constitutive activation of TLR4 augments inflammatory response in colitis-induced tumorigenesis. Colon cancer cell lines SW480 and SW620 constitutively express TLR4. In SW480 cells, LPS treatment induces cytokine synthesis/secretion, cell-migration and adhesion. Increased cell migration and adhesion are hallmarks of tumor growth and metastasis. Thus, the inventor postulated that suppression of LPS-stimulated TLR4-signaling will help control inflammation and inflammation-induced metastatic property of SW480 cells. Presumably, a therapeutic that inhibits intracellular inflammatory signaling is expected to exert sustained anti-inflammatory effects and help prevent inflammation-induced cancer.
The previous Examples describe the identification of the TLR4-interacting SPA4 peptide, as well as its ability to reduce secretion of TNF-α by a dendritic cell line in response to LPS stimuli. In this Example, the ability of SP-A4 peptide to inhibit the LPS-induced TLR4-NF-κB signaling and resulting inflammatory response in SW480 colon cancer cell line was studied. Simultaneously, the effects of SPA4 peptide on migration, viability and cell cycle progression of SW480 cells were also investigated.
Materials and Methods for Example 4
Cell culture: Human colorectal adenocarcinoma cells: SW480, derived from the colon of a cancer patient (original stock from ATCC, VA), were obtained from the laboratory of Dr. Shrikant Anant (University of Kansas Medical Center, Kansas City, Kans.). The cells were maintained in Dulbecco's minimum essential medium (D-MEM, Invitrogen, CA), supplemented with high glucose (4.5 g/l D-glucose), sodium pyruvate (1 mM), L-glutamine (4 mM), fetal bovine serum (10%) and antibiomyco (1%, Invitrogen, CA). Cells were maintained at 37° C. in a humidified 5% CO2 incubator.
SPA4 peptide: The 20-mer SPA4 peptide (amino acid sequence: GDFRYSDGT PVNYTNWYRGE; SEQ ID NO:3) derived from the C-terminal region of SP-A, was synthesized by Genscript (Piscataway, N.J.). The mass spectroscopy and high-performance liquid chromatography were performed on all the batch-preparations of the SPA4 peptide to confirm its purity (
Measurement of n-octanol/water partition coefficient (Ko/w) of SPA4 peptide: The n-octanol/water partition coefficient (Ko/w) is a measure of hydrophobicity/hydrophilicity. It is calculated as the ratio of the concentration of a chemical in n-octanol to that in water in a two-phase system at equilibrium. An equal volume of MiliQ water and n-Octanol was mixed in a microcentrifuge tube, and shaken for 4 hours at 25° C. Weighed amount of the SPA4 peptide was then added to this n-Octanol-water mixture and shaken overnight at 25° C. The SPA4 peptide-n-Octanol-water mixture was allowed to settle for 2 hours. The aqueous phase was separated by centrifugation at 16,000×g for 10 minutes. The concentration of SPA4 peptide in aqueous phase was measured by spectrophotometric absorbance readings at 280 nm. The concentration of the SPA4 peptide in n-Octanol phase was obtained after subtracting the amount of peptide in water phase from that of the total SPA4 peptide added. Finally, Ko/w of SPA4 peptide was determined using following formula:
Binding of SPA4 peptide to LPS: The binding of SPA4 peptide to LPS was studied by LAL assay as per the method described by Giacometti et al. (2004). Briefly, 0-20 μM SPA4 peptide or polymyxin B (positive control) solutions were added to 0.01 ng/ml Escherichia coli O111:B4 LPS (supplied with the kit, Charles River Laboratories International, Inc., Wilmington, Mass.) in the wells of a 96 well plate and incubated at 37° C. for 40 minutes. Fifty μl of LAL substrate solution was then added to each well, and the plate was incubated for another 10 minutes. Finally, substrate-buffer solution was added, and optical density readings (OD) were obtained at 405 nm after 0, 6 and 12 minutes of addition of substrate. AOD values for SPA4 peptide or polymyxin B incubated with LPS (ΔODtreatment), LPS alone (ΔODLPS) and blank (ΔODBlank) were calculated by subtracting the OD values obtained at 6 or 12 minutes from those obtained at 0 minutes. Percent binding of SPA4 peptide and polymyxin B was calculated at 6 and 12 minutes of reaction using following formula:
Expression of TLR4: Next, the effect of SPA4 peptide on the expression of TLR4 in SW480 cells was investigated by immunocytochemistry and laser confocal microscopy. Briefly, about 2.5×104 cells were seeded in an 8-well chamber slide (Thermo Fisher Scientific, Rochester, N.Y.) in complete medium. The cells were treated with E. coli O111:B4-derived, highly-purified, low-protein LPS (100 ng/ml or 1.0 μg/ml; Calbiochem, CA) for 4 hours following 1 hour incubation with SPA4 peptide (1, 10 and 100 μM). The cells were fixed in 3.5% paraformaldehyde in Dulbecco's PBS (DPBS) and permeabilized with 0.05% saponin solution (Inaba et al. 1998). The wells were washed with DPBS supplemented with 1% FBS and 0.05% saponin, and stained with TLR4-specific antibody (1:50 dilution, Abcam, MA) and 10 μg/ml Alexa-fluor 488-labeled secondary anti-rabbit IgG antibody (Invitrogen-Molecular Probes, CA). After washing, the cells were stained with 100 nM rhodamin-phallodin (Cytoskeleton Inc, CO) and 1 μg/ml Hoechst 33342 dyes (Invitrogen-Molecular Probes, CA). Confocal microscopic images were acquired at the Imaging core facility of the Oklahoma Medical Research Foundation, Oklahoma City, using the Zeiss LSM-510 META laser scanning confocal microscope. Images were acquired with lens objective of 63× with the x/y stack sizes being 146.2 μm using band pass filter specifications at 435-485, 560-615 and 505-530.
NF-κB activity: Since binding of LPS to TLR4 activates NF-κB through MYD88-dependent and independent pathways, the effects of SPA4 peptide on basal and LPS-induced NF-κB activity were investigated in SW480 cells transfected with a dominant negative construct of MYD88 (MYD88DN) and NF-κB firefly-luciferase reporter plasmid DNA. Both the short- (1 hour) and long-term (5 hours) effects of the SPA4 peptide on NF-κB activity were studied.
The SW480 cells were transiently-transfected with NF-κB firefly-luciferase reporter plasmid pGL4.32 (luc2P/NF-κB-RE/Hygro, Promega, WI; provided by Dr. Kelly Standifer, Department of Pharmaceutical Sciences, University of Oklahoma Health Sciences Center, Oklahoma City, Okla.) and MYD88-dominant negative plasmid construct (MYD88DN, provided by Dr. Ruslan Medzhitov, Yale University, CT). MYD88 dominant negative plasmid DNA construct lacked the death domain and intermediate domain (Medzhitov et al., 1998). Briefly, NF-κB-Luciferase reporter and MYD88-dominant negative plasmids (1.0 μg each) were mixed with 6 μl of Fugene HD reagent (Roche, Ind.) in 92 μl of serum-free low-glucose DMEM medium (Invitrogen, CA), and incubated for 20 minutesat room temperature. The transfection-mix was then added to the SW480 cells. The SW480 cells transfected with a plasmid DNA construct expressing enhanced green fluorescent protein (pHYG-EGFP; Clontech, CA) were observed under Leica DM4000B fluorescent microscope. The transfection efficiency was calculated as percent of cells expressing EGFP over total number of cells in the brightfield channel. An empty vector plasmid DNA (pcDNA 3.0; obtained from Dr. Brian Ceresa, Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Okla.) was used as negative control. The cells were incubated for 18-20 h at 37° C. in humidified 5% CO2 chamber. After the completion of incubation period, fresh complete medium was added to the cells. Cells were then challenged with LPS (1.0 μg/ml) for 4 hours following the treatment with SPA4 peptide (1, 10, 50 μM) for 1 hour (total period of 5 hours; short-term treatment model) and 5 hours (total period of 9 hours; long-term treatment model). The LPS remained in the medium throughout the incubation period.
After completion of the incubation period, the medium supernatants were removed and cells were washed with room-temperature DPBS. The cell extracts were prepared using the reporter assay cell-lysis buffer (Promega, Fitchburg, Wis.) and stored at −80° C. for further analysis. The firefly-luciferase activity (measurement of NF-κB activity) was measured using the luciferase reporter assay system (Promega, Fitchburg, Wis.). Briefly, 50 μl of luciferase assay reagent was added to the 20 μl cell lysate by automated dispenser of Synergy HT multi-mode microplate reader (Biotek, Winooski, Vt.), and luminescence was read within 10 seconds. Total protein content in cell lysates was estimated using BCA protein assay kit (Pierce, Rockford, Ill.). The luciferase activity units were finally normalized with the total protein content of cell lysates.
Expression of NF-κB pathway molecules by immunoblotting: The expression of NF-κB pathway molecules (inhibitor kappa-Bα: IKBα, phosphorylated IKBα, p65, phosphorylated p65, RelB and COX-2) in SW480 cell-lysates treated with LPS±SPA4 peptide was studied by immunoblotting. For the immunoblotting, 10 μg of total cell-lysate proteins were fractionated on Novex 4-20% Tris-glycine gradient SDS-PAGE gel (Invitrogen, Carlsbad, Calif.) by electrophoresis. Separated proteins were electro-transferred onto a nitrocellulose membrane using iBlot gel transfer device (Invitrogen, Carlsbad, Calif.). The non-specific sites were blocked by incubating the membrane with 7% skimmed milk in Tris-buffered saline with 0.4% TWEEN®-20 (TBST) for 1 hour at room temperature. The blocked membranes were incubated overnight at 4° C. with 1:1000 diluted anti-human antibodies against NF-κB canonical pathway molecules: phosphorylated inhibitor kappa-Bα (IKBα), total-IKBα, p65, RelB (Cell Signaling Technology, Inc., Danvers, Mass.), phosphorylated p65 (Santacruz Biotech, Inc., Santa Cruz, Calif.) and cyclooxygenase-2 (COX-2; Santacruz Biotech, Inc., Santa Cruz, Calif.). The membranes were washed with TBST and incubated at room temperature for 45 minutes with 1:3500 diluted horse-radish peroxidase (HRP)-conjugated-secondary antibody (Sigma-Aldrich, St. Louis, Mo.). The immunoreactive bands were detected by Super Signal West Femto detection reagent (Thermo Fisher Scientific, Barrington, Ill.). In order to ensure equal protein loading in the wells, the membranes were stripped of probing antibodies at 60° C. for 45 minutes using a stripping solution containing 10% SDS, 0.5 M Tris and 3-mercaptoethanol (35 μl/ml), and re-probed with anti-actin antibody (Sigma-Aldrich, MO; 1:1000 in TBST). The immunoblots were imaged using the Ultraquant image acquisition program (UltraLum Inc., Claremont, Calif.). The densitometric readings were obtained for immunoreactive bands with Image J 1.42q program (NIH, USA). Finally, arbitrary densitometric values for proteins of interest were normalized with those of β-actin.
Expression of IL-13 and IL-6: Two treatment models (post- and pre-LPS treatment) were utilized for assessing the effects of SPA4 peptide on LPS-induced cytokines: IL-13 and IL-6. Post-LPS treatment models (short-term and long-term) are described above. In pre-LPS treatment model, the SW480 cells were pretreated with SPA4 peptide (10 μM) for 2 hours, followed by LPS-challenge (1.0 μg/ml) for another 4 hours. The cell-lysates were prepared either in commercially available cell-culture lysis reagent (Promega, WI) or directly into SDS-PAGE sample buffer containing 50 mM dithithreitol (Cell Signalling Technology, MA). Ten μg of cell-lysate proteins were separated on 4-20% Novex Tris-glycine gradient SDS-PAGE gel (Invitrogen, CA) or 10% separating-5% stacking acrylamide gel. The expression levels of cytokines were measured by immunoblotting as described above using 1:1000 diluted antibodies against IL-13 and IL-6. Both antibodies were purchased from Santacruz Biotech, CA.
Cell migration: SW480 cells were plated in 30 mm tissue-culture-treated dishes at a density of 1.0×106 cells per plate. At 80-90% confluence, a “reference line” was drawn at the bottom of the plate. The cells were scratched off from one side of the reference line using a rubber policeman. A picture was taken at 0t that helped in marking the “start line”. Cells were then washed with complete medium and incubated with LPS (1.0 μg/ml) and/or SPA4 peptide (1, 10 and 50 μM). Photomicrographs of cells migrated across the “start line” were taken in different fields after each treatment at 24, 48, and 72 hours (+2 hours) following traceable inscriptions made under the plate at three different points, with a Canon digital camera. On 24, 48 and 72 hour images, a second line was drawn along the edge of cells to represent the migration of cells. Cell migration was calculated by measuring the distance cells migrated from the “start line”. Only the continuous migration of cells was considered for measurement. The islets of cells were disregarded. The cell migration was calculated using the following formula: (distance between “start line” at 0 h−“reference line”)−(distance between “72 hours line”−“reference line”).
Cell cycle analysis: The effect of SPA4 peptide on cell cycle progression was studied by flow-cytometry. About 500,000 cells were seeded per well into 6 well plate. The cells were challenged with LPS (100 ng/ml) for 4 hours. After the completion of 4 hours LPS-challenge period, SPA4 peptide (10, 50 and 100 μM) was added to the cells. Cells were further incubated for 20 and 40 hours. Vehicle-treated cells were also included. After 20 and 40 hours of total incubation period, both adherent and non-adherent cells were collected and centrifuged at 260×g for 5 minutes. The supernatant was discarded and cell pellet was washed with DPBS (Invitrogen-Gibco, NY). The cells were fixed in 70% ice-cold ethanol on ice for 1 hour and stained with a buffer containing 200 μg/ml DNase-free RNase A (Sigma, St Louis, Mo.), 0.1% v/v Triton-X 100 and 20 μg/ml propidium iodide (Molecular Probes, Carlsbad, Calif.). The cells were incubated at 4° C. for 30 minutes in the dark, before measuring cell fluorescence using Becton Dickson FACS Calibur flow cytometer. The single cells were selected by gating out the aggregates and the percent number of cell populations in different cell cycle phases were calculated by de-convoluting the results ModFIT software (Verity software house, Topsham, Me.).
Cell viability: The TLR4-NF-κB signaling can have multifaceted implications, including the effects on proliferation and viability of SW480 cells. Thus, the effect of SPA4 peptide on viability of SW480 cells was studied. The cells (250,000 and 500,000 per well) were seeded into 24 well plates and treated with SPA4 peptide (10, 50 and 100 μM) after LPS-challenge (100 ng/ml) for 4 hours. The LPS remained in the medium throughout the incubation period thereafter. The cells were collected after 3-5 days of treatment and stained with propidium iodide (1 μg/ml) for 20 minutes on ice. The stained cells were run on a flow-cytometer (Accuri flow cytometer, MI), and propidium iodide staining was assessed on FL2 channel. The histograms were obtained for each treatment using C Flow Plus software and compared between the groups. The % number of cells exhibiting positive (dead cells) and negative (live cells) propidium iodide staining were noted. Untreated cells and LPS-treated cells served as controls.
Statistics: The results were analyzed by one way Analysis of Variance (ANOVA) using a statistical analysis program (Graphpad Prism, CA). A p-value of <0.05 was noted, otherwise indicated.
Results of Example 4
Characteristics of SPA4 peptide: The SPA4 peptide batch preparations were always tested for purity by HPLC chromatography and Mass spectrometry (
As per the computer simulation Solvent AccesiBiLitiEs (SABLE) program (Division of Biomedical Informatics, Children Hospital Research Foundation, Cincinnati, Ohio), the SPA4 peptide is predicted to have coiled and beta strand structures. Relative solvent accessibility (RSA) of 11 amino acid residues is above 25% (
SPA4 peptide does not bind to LPS. Example 2 illustrated that the SPA4 peptide binds to recombinant TLR4-MD2 protein. In order to further validate that the anti-inflammatory effects of SPA4 peptide are not through the binding of SPA4 peptide to TLR4-ligand LPS, the binding of SPA4 peptide to LPS was studied in vitro. The results in
Additional evidence was provided by superimposing the predicted SPA4 peptide-binding site on a computer model exhibiting LPS-binding site within the TLR4-MD2 complex (Carpenter et al., 2009). Herein it was observed that the binding site of the SPA4 peptide to TLR4 remains farther away from the LPS-binding site (
SPA4 peptide reduces the expression of TLR4. Next, the effects of SPA4 peptide on the expression of TLR4 were investigated by confocal microscopy. The results show that SW480 cells constitutively express TLR4. As expected, the TLR4 expression is further increased in response to Gram-negative bacterial LPS. However, the SPA4 peptide treatment reduced the LPS-stimulated TLR4 expression to basal level (
SPA4 peptide inhibits the LPS-induced MYD88-dependent NF-κB activity: Myeloid differentiation primary response gene (88; MYD88) is a known adaptor molecule of TLR4 and serves as an important molecule downstream of LPS-TLR4-MD2 binding, but upstream of activation of transcription factors (AP-1 and NF-κB) and transcription of cytokine and chemokine genes. The MYD88 engages IL-1 receptor-associated kinase (IRAK) molecule through its death domain and transduces the signal. In this Example, SW480 cells were transfected with MYD88 dominant negative construct (MYD88DN) lacking the death domain. The transfection efficiency was observed as 63-68%. The NF-κB activity was measured using a reporter plasmid (pGL4.32, Promega, WI) that contained five copies of NF-κB-response element driving the expression of luciferase reporter gene. The results show that the SPA4 peptide (at 10 and 50 μM concentrations) reduces the LPS induced-NF-κB activity after 5 hours. At 9 hours of treatment, the LPS induced NF-κB activity was, however, not significantly inhibited by 1, 10 or 50 μM concentrations of SPA4 peptide. The MYD88DN-transfected cells exhibited reduced NF-κB activity against LPS stimuli as compared to that in pcDNA3.0 vector plasmid DNA-transfected cells. The SPA4 peptide-treatment did not further reduce the NF-κB activity in cells transfected with dominant negative construct of MYD88, illustrating that the SPA4 peptide treatment affects only the MYD88-dependent NF-κB activity and does not affect the MYD88-independent NF-κB activity (
Expression of NF-κB signaling molecules is affected by SPA4 peptide: The effects of SPA4 peptide on LPS-induced TLR4-NF-κB signaling were studied in cells treated with SPA4 peptide (1, 10 and 50 μM) for a short-term and a long-term basis. The expression of NF-κB-signaling molecules was measured in cell lysates by immunoblotting. It was found that the SPA4 peptide alone did not affect the expression of any of the NF-κB signaling molecules studied, except for COX-2.
As expected, an oscillating expression pattern was observed for the total IKBα, total p65 and COX-2 molecules in short- and long-term LPS-challenged SW480 cells. The SPA4 peptide treatment on short-term basis stimulated an increase in expression of total IKBα, but decrease in phosphorylated p65 and COX-2 expression. The long-term treatment with SPA4 peptide, on the other hand, decreased the total IKBα expression. The expression of phosphorylated p65 and COX-2 remained low. No effect was observed on the expression of phosphorylated IKBα or RelB molecules (
SPA4 peptide inhibits the intracellular expression of IL-1β and IL-6. Immunoblotting with anti-human IL-1β antibody recognized three major precursor forms (identified as numerals 1, 2 and 3 in
Next, the expression of IL-6 in cell-lysates of SW480 cells treated with LPS±SPA4 peptide was studied. Three major precursor forms of IL-6 (identified as numeral 1 for two reactive bands together and numeral 2 in
SPA4 peptide treatment inhibits the LPS-induced migration of SW480 cells. Increased cell migration is a known characteristic of tumor metastasis. As expected, LPS was found to induce the metastatic property (i.e., cell migration) of SW480 cells. Treatment with the SPA4 peptide inhibited the LPS-induced migration of SW480 cells (
SPA4 peptide treatment does not affect the cell cycle progression, but inhibits cell viability. It was found that LPS-treatment did not affect cell cycle progression or viability. Treatment with SPA4 peptide (10, 50 and 100 μM concentrations) did not affect the cell cycle progression of SW480 cells over a period of 40 h (
Discussion of Example 4
Toll-like receptor 4 (TLR4) has been well-recognized for its critical role in sensing of pathogens or pathogen-derived signals and immune regulation. Although its involvement in cancer has not been fully-established, an increased expression of TLR4 is associated with inflammation induced cancer. Correspondingly, reduced TLR4 activity was found to inhibit inflammatory cytokine secretion, cancer cell proliferation and cancer-associated pain. On the basis of these initial findings, it is proposed herein that TLR4-blocking novel therapeutics will help reduce the inflammation and inflammation-induced cancer-progression. SW480 colorectal cells were utilized as the model system for inflammation-induced cancer progression; the SW480 cells constitutively express TLR4. This Example was focused on studying the effects of a TLR4-interacting SPA4 peptide on TLR4-signaling, inflammatory response, cell migration, cell cycle and viability of SW480 cells.
The SPA4 peptide is derived from an endogenous host-defense protein: surfactant protein-A (SP-A). SP-A is mainly expressed as a component of lung surfactant; its expression has been noted at other mucosal surfaces, such as intestine, skin, eye, and urinogenitary systems. In lung, SP-A maintains normal lung function and exerts anti-microbial and anti-inflammatory effects against pathogens and stress-ligands. Since during infection and inflammation, the amounts of SP-A are reduced significantly, an SP-A based surfactant or therapeutic may be of clinical value. It has not been possible to formulate an SP-A-based therapeutic because of its large size and its amenability to degradation. Using computer-modeling and functional screening of a small peptide library, a shorter region of SP-A was identified from the TLR4-interacting region and referred to herein as the SPA4 peptide. Example 2 illustrates that the SPA4 peptide inhibits the LPS-induced inflammatory response in JAWS II dendritic cell line.
TLR4-signaling is induced in response to a number of endogenous and exogenous ligands, including bacterial LPS. LPS binds to TLR4-MD2 complex and induces inflammatory response via activation of a complex intracellular signaling network. The SW480 cells constitutively express TLR4, and LPS is known to induce inflammatory response, adhesion and migration. Thus, this system was utilized to investigate the effects of SPA4 peptide on LPS-induced TLR4-NF-κB signaling, inflammation and cancer cell properties. In order to understand the mechanism of action of SPA4 peptide, first it was confirmed that the SPA4 peptide does not directly bind to stress-ligand LPS, and the binding site of LPS onto the TLR4-MD2 complex is farther away from the binding site of SPA4 peptide. (
LPS is known to induce inflammation via activation of TLR4-NF-κB signaling in MYD88-dependent as well as MYD88-independent manner. Full-length SP-A has been identified to inhibit NF-κB activity, but upstream molecular events occurring after SP-A-TLR4 interaction remain unknown. As such, the LPS-TLR4-MD2-signaling is quite complex. Nothing is known in particular for SPA4 peptide. This Example shows that the SPA4 peptide inhibits NF-κB-activity in a MYD88-dependent manner. Furthermore, SPA4 peptide induces the expression of IKBα, an inhibitor of NF-κB, but does not affect the Ser 32-phosphorylated IKBα. The increase in the expression of IKBα is in accordance with the published data on full-length SP-A (Wu et al., 2004). Since NF-κB is composed of five different subunits, c-Rel, RelB, p65, p50 and p52, it was investigated if the stimulation of IKBα translates into the inhibition of these downstream factors. Inhibition of phosphorylation of p65 by SPA4 peptide translates into the inhibition of NF-κB activity (
An oscillating pattern of expression of signaling molecules were observed in 5 hour and 9 hour treatment-models; it is possible that different mechanisms in conjunction with NF-κB are affected downstream of the binding of SPA4 peptide to TLR4. In conjunction with the inhibition of NF-κB signaling by SPA4 peptide, a significant decrease in the amounts of LPS-induced IL-1β and IL-6 cytokines were observed. This reduction in inflammatory signaling and cytokine response correlated with inhibition of LPS-induced cell migration. Although a change in cell cycle progression was not observed, significant reduction in cell viability was observed upon treatment with SPA4 peptide. The percent cell-death increased as per the increase in duration and treatment dose of SPA4 peptide. These results will facilitate development of a novel chemopreventive immunotherapeutic to control inflammation-induced cancer progression in patients with colitis.
Example 5In Example 5, the efficacy of SPA4 peptide in inhibiting LPS-induced acute inflammation was investigated in a mouse model of LPS-inflammation. Balb/c Mice were challenged with E. coli O111:B4 highly purified, low protein-derived LPS (0.1 or 1 or 15 μg/g body wt) via an intraperitoneal route, one hour prior to treatment with SPA4 peptide (2.5 μg/g body weight) or full-length purified SP-A (0.5 μg/g body weight) via an intraperitoneal route. The mice were monitored for endotoxic-shock like clinical symptoms (including, but not limited to, ruffled fur, eye exudates, diarrhea, prostration, and lack of reactivity), and these symptoms were scored on the basis of severity from 0 to 3, and average clinical symptom indices were calculated for each animal based on the symptom grades (Metkar et al., 2007). Endotoxic-shock like clinical symptoms were noted (at 5 or 7 hours), and mice were sacrificed at different time-intervals after LPS challenge. Major organs and blood specimens were collected. Secreted amounts of TNF-α cytokine were measured in serum and lung tissue homogenates by ELISA method.
At the molecular level, the effects of SPA4 peptide on LPS-induced NF-κB activity were studied in dendritic cells (JAWS II cells) co-transfected with NFκB luciferase reporter plasmid construct and dominant negative construct of MYD88 (an adaptor protein downstream of TLR4; MYD88DN). Briefly, JAWS II dendritic cells (0.5×106) were transfected with NF-κB luciferase reporter and/or MYD88DN using TransIT-TKO transfection reagent as described earlier (Awasthi S et al., 2003). After 20-24 hours of incubation, the transfected cells were challenged with LPS (100 ng/ml) for 4 hours and treated with SPA4 peptide (10 μM) for 1 hour. After the completion of 5 hours incubation, the cell lysates were prepared in cell lysis buffer and NF-κB-luciferase reporter activity was measured as per the manufacturer's instructions (Promega, WI).
SPA4 peptide inhibits LPS-induced NF-κB activity upstream of MYD88 in dendritic cells. The MYD88 is an intracellular adaptor protein which transduces the LPS-TLR4-signal to NF-κB. Although LPS-TLR4 binding is known to induce NF-κB in MYD88-independent manner also, the inhibition of the NF-κB activity in MYD88−/−-transfected cells by SPA4 peptide was equivalent to that in untreated MYD88−/−-transfected cells against LPS challenge (
The endotoxic shock-like symptoms induced after LPS challenge were measured (Metkar et al., Infect Immun 2007). Results presented in
LPS is known to induce the secretion of TNF-α, and a significant amount is detected in the blood. In
SPA4 peptide treatment also reduced LPS-induced TNF-α levels in lung tissue homogenates.
In summary, the results show that the treatment with SPA4 peptide inhibited NF-κB luciferase activity in dendritic cells, inflammatory symptoms, and TNFα levels in serum and lung tissue homogenates of mice against LPS stimuli. Moreover, the inhibition of LPS-induced NF-κB activity by SPA4 peptide was indistinguishable from those challenged with LPS alone, in absence of functional MYD88. Taken together, the results indicate that the SPA4 peptide exerts its inhibitory effect on the TLR-NF-κB pathway upstream of MYD88 adaptor protein and thereby regulates the TNFα response.
Thus, in accordance with the present invention, there have been provided compositions, as well as methods of producing and using same, that fully satisfy the objectives and advantages set forth hereinabove. Although the invention has been described in conjunction with the specific drawings, experimentation, results and language set forth hereinabove, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the presently disclosed and claimed inventive concept(s).
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Claims
1. A method of inhibiting TLR4 signaling, the method comprising the step of:
- contacting a cell expressing TLR4 on a surface thereof with a composition wherein the composition binds to TLR4 on the surface of the cell and inhibits TLR4 signaling by the cell, and wherein the composition comprises at least one of:
- (a) an isolated peptide fragment of SEQ ID NO:1, wherein the peptide fragment is less than 50 amino acids in length and comprises the amino acid sequence as set forth in SEQ ID NO:3;
- (b) an isolated peptide having an amino acid sequence of at least one of SEQ ID NOS:3-7 and 8-246;
- (c) an isolated peptide having an amino acid sequence that is at least 90% identical to at least one of SEQ ID NOS:3 and 5; and
- (d) an isolated peptide having an amino acid sequence that differs by two amino acids or less from SEQ ID NO:3 or 5.
2. The method of claim 1(a), wherein the isolated peptide fragment comprises a portion of the C-terminal carbohydrate recognition domain of SEQ ID NO:1.
3. The method of claim 1(a), wherein the isolated peptide fragment is less than 40 amino acids in length.
4. The method of claim 1(a), wherein the isolated peptide fragment is less than 30 amino acids in length.
5. The method of claim 1(a), wherein the isolated peptide fragment is less than 25 amino acids in length.
6. The method of claim 1(a), wherein the isolated peptide fragment is 20 amino acids in length.
7. A method of decreasing the occurrence and/or severity of inflammation associated with a disease condition, the method comprising the step of:
- administering an effective amount of a composition to a subject suffering from or predisposed to the disease condition, thereby decreasing the occurrence and/or severity of inflammation associated with the disease condition in the subject, wherein the composition comprises at least one of: (a) an isolated peptide fragment of SEQ ID NO:1, wherein the peptide fragment comprises the motif of SEQ ID NO:2 and is less than 50 amino acids in length; (b) an isolated peptide having an amino acid sequence of at least one of SEQ ID NOS:3-7 and 8-246; (c) an isolated peptide having an amino acid sequence that is at least 90% identical to at least one of SEQ ID NOS:3 and 5; and (d) an isolated peptide having an amino acid sequence that differs by two amino acids or less from SEQ ID NO:3 or 5.
8. The method of claim 7, wherein the disease condition is further defined as a disease condition of the lung.
9. The method of claim 7, wherein the disease condition is further defined as an intestinal disease condition having inflammation associated therewith.
10. The method of claim 7, wherein the disease condition is further defined as a cancer.
11. The method of claim 7(a), wherein the isolated peptide fragment comprises a portion of the C-terminal carbohydrate recognition domain of SEQ ID NO:1.
12. The method of claim 7(a), wherein the isolated peptide fragment is less than 40 amino acids in length.
13. The method of claim 7(a), wherein the isolated peptide fragment is less than 30 amino acids in length.
14. The method of claim 7(a), wherein the isolated peptide fragment is less than 25 amino acids in length.
15. The method of claim 7(a), wherein the isolated peptide fragment is 20 amino acids in length.
16. A method of promoting lung development and/or function in an infant born pre-term, the method comprising the step of:
- administering an effective amount of a composition to the infant, wherein the composition comprises at least one of: (a) an isolated peptide fragment of SEQ ID NO:1, wherein the peptide fragment comprises the motif of SEQ ID NO:2 and is less than 50 amino acids in length; (b) an isolated peptide having an amino acid sequence of at least one of SEQ ID NOS:3-7 and 8-246; (c) an isolated peptide having an amino acid sequence that is at least 90% identical to at least one of SEQ ID NOS:3 and 5; and (d) an isolated peptide having an amino acid sequence that differs by two amino acids or less from SEQ ID NO:3 or 5.
17. The method of claim 16(a), wherein the isolated peptide fragment comprises a portion of the C-terminal carbohydrate recognition domain of SEQ ID NO:1.
18. The method of claim 16(a), wherein the isolated peptide fragment is less than 40 amino acids in length.
19. The method of claim 16(a), wherein the isolated peptide fragment is less than 30 amino acids in length.
20. The method of claim 16(a), wherein the isolated peptide fragment is less than 25 amino acids in length.
Type: Application
Filed: Aug 30, 2013
Publication Date: Feb 13, 2014
Applicant: The Board of Regents of the University of Oklahoma (Norman, OK)
Inventor: Shanjana Awasthi (Edmond, OK)
Application Number: 14/015,144
International Classification: C07K 14/78 (20060101);
