To study the mechanism of immunomodulation caused by peptides at cellular and molecular level

Abstract

Immune system is a complex network of interdependent cells that collectively protect the body from bacterial, parasitic, fungal, viral infections and from the growth of tumor cells. Sometimes the immune system fails to protect the host from pathogens (immune deficiency) or misdirects its activities to cause discomfort, debilitating disease, or even death (Abbas, 2001). Immune deficiency diseases (congenital or acquired) decrease the body's ability to fight invaders, causing vulnerability to infections on the other hand overactive immune system leads to manifestation of several disorders like allergy, asthma, autoimmune diseases and graft-versus-host disease. Advances made in recent years in the understanding of cellular and molecular basis of immune response and also the unfolding of the intricate networking of the immune system with other body systems point to important role of immunomodulators in maintaining healthy state. Immunomodulators are any biological or synthetic agents that can modulate the immune response to bring homeostasis in over activated or suppressed immune system. Immunosuppressants subsides the over activated immune system and are used for treatment of allergy, asthma, autoimmune disorders. Immunosuppressants are also used in organ transplantation to prevent graft versus host rejections; some of them which are in use these days include calcineurin inhibitors (cyclosporine A, Tacrolimus); Glucorticoids (prednisolone and others); antibodies (Muromonab CD3, Antithymocyte globin (ATG), Rho (D) immunoglobin) and antiproliferative compounds (Azathioprine, Cyclophosphamide, Methotrexate, Chlorambucil, Mycophenolate mofetil). Immunostimulating agents or immune adjuvants restore the normal immune response in immunocompromised conditions and can also boost the immune status of the subjects susceptible to infective invasions due to environmental factors. Immunomostimulators which are currently available or under development for use; include cytokines, Toll-like receptor (TLR) agonists, cytokine receptors, transcription factor modulators, bacterial cell surface molecules, antimicrobial and immunostimulatory peptides and microbes such as probiotics (Ballas, 2008; Pirofski & Casadevall, 2006). The immunostimulatory therapy is a preventive therapy and if judiciously used in combination with antibiotics it can help in the treatment of various infectious diseases. It has been found to be valuable in patients who are in immunocompromised state either due to infection or surgical trauma, irradiation, cancer, severe burns etc. (Turanek et. al., 1997). In combination therapy the antibiotics/antiviral agents reduces the magnitude of the infection, while the immunomodulatory agents stimulate the natural immune response of the host for better fight. This eventually helps in complete elimination of infection and also reduces the chances of recurrence of infection. Since the non-specific defense mechanisms are also important in controlling tumor growth and reducing the probability of metastasis, the immunomodulators that can enhance the cytotoxic activity of natural killer cells and macrophages, can become promising candidate in cancer therapeutics (Chernysh et. al., 2012). Thymostimulin and thymic extract, for example, are found to reduce the chemotherapy induced toxicity and prolong the survival of cancer patients (Macchiarini et. al., 1989). Thymopentin (TP5), an immunomodulatory peptide, also suppressed proliferation of human promyelocyte leukemia cell line, HL-60 cells (Fan et. al., 2006) thus a judicious immunomodulation can also assist in the treatment of diseases other than immunological disorders. Immunomodulators are of different natures and can be derived from various sources ranging from bacterial cell wall, protein, nucleotides, vitamins et.c. A number of small peptides capable of modulating the natural immune response either specifically or non-specifically have been identified till date of which many of them like cyclosporine A, muramyl dipeptide (MDP) and thymic peptide analogues are already being used for therapeutic purpose (Morales, 2005; Junko et. al., 2004; Govorin and Stupina, 1990). Like other immunomodulators there can be a remarkable scope for the use of immunomodulating peptides depending upon their specific and non-specific responses. Immunostimulatory peptides can provide clinical help in cases of malaria and Leishmania where macrophages, the prime immune cells are impaired by the parasites and also in common viral infections which generally surface due to low immunity. They can also provide clinical help in some of the immunologically related neural disorders. Similar to vitamins and minerals, the immunomodulatory peptides can be used as supplement for the maintenance of adequate level of immunity, especially in the epidemic situations. Immunomodulating peptides origin can be diverse ranging from bacterial cell wall peptidoglycans, microbial metabolites, peptide hormone secretions from different cells, fragments of immunoglobulins to other milk and plasma proteins. Immunomodulatory peptides derived from milk are of particular interest these days due to lack of unwanted side effects associated with peptides of bacterial origin. A large number of immunomodulatory peptides, derived from milk protein have been reported till date (Samuel et. al., 2010). Peptides derived from caseins and major whey proteins have been shown to have immunostimulatory activity, measured as lymphocyte proliferation, antibody synthesis and cytokine regulation (Gill et. al., 2000). Immunomodulatory peptides formed during milk fermentation have been shown to contribute to anti tumor effects (Matar et. al., 2003) inhibit cancer cell growth or stimulate the activity of immuno-competent cells and neonatal intestinal cells and diminished colicky symptoms in infants (Hartmann & Meisel, 2007). A proline-rich polypeptide was recently found to improve or stabilize the condition of Alzheimer’s disease patients (Zimecki & Kruzel, 2007). Glycomacropeptide (GMP), derived from kappa casein was found to be a potent immunoenhancer at low concentrations, significantly enhancing the proliferation and phagocytic activities of U937 cells (Li & Mine, 2004). GMP also exhibits antibacterial (Zimecki & Kruzel, 2007) and bacterial toxin binding effects (Daddaoua et. al., 2005). A commercially available caseinophosphopeptide (CPP) preparation CPP-III, consisting mainly of f1-32 of bovine αs2-casein and f1-28 of β-casein enhances the proliferative response induced by lipopolysaccharide, phytohaemagglutinin and concavalinA stimulation and immunoglobulin production in mouse spleen cultures (Otani et. al., 2000). Most of the milk derived immunomodulatory peptides were obtained from bovine milk beside these peptides, a potent immunostimulatory peptide was identified in tryptic digest of human milk casein, this peptide having sequence Val-Glu-Pro-Ile-Pro-Tyr was found to correspond to 54-59 amino acid residue of β-casein protein (Parker et. al., 1984). This hexapeptide was shown to stimulate the in vitro phagocytosis of sheep RBC by murine macrophages and it significantly enhanced the resistance of mice to normally lethal infection with Klebsiella pneumoniae. It also exerted a significant stimulating effect on binding and phagocytosis of in vivo aged human RBC by human monocytic-macrophagic cells (Gattegno et. al., 1988). This peptide has also been reported to increase nitric oxide release from neutrophils (Rysz et. al., 2000) and to reduce Leishmania donovani burden in host when used prophylactically by stimulating the host’s resistance to parasite (Sharma et. al., 2004). However, the mechanisms by which these peptides exert their immunomodulatory effects or influence cell proliferation are not currently fully elucidated. There are a number of probable sites during the differentiation and maturation events in developing immune cells where an immunomodulatory peptide can intervene and modulate its function therefore judicious selection is needed before using them for therapeutic purpose. For example, peptides which can stimulate B-cell differentiation and antibody secretion would be appropriate for patients with hypo or agammaglobulinemia but not for patients with T-cell immunodeficiency. There are reports that a peptide can have variable effect on different arm of immune system for example peptide related to the immunosuppressive loop of lactoferrin having sequence RKPVD exhibit lack of suppressive activity regarding the humoral immune response, but show quite strong immunosuppressive activity with respect to the cellular response (Wieczorek et. al., 1995). Similarly, the wild type octapeptide GMNRRPIL of DNA-binding domain of P53 protein showed immunostimulative activity with regard to the humoral immune response but was found to be inactive on the cellular immune response. Literature also suggests a possible conversion of an immunostimulating peptide to immunosuppressive ones simply by single substitution, addition or deletion; e.g. RKPVD, the lactoferrin derived pentapeptide is immunosuppressive (Wieczorek et al., 1995) whereas RKDVP, a thymopentin analogue in which only the positions of P and D are exchanged is immunostimulative (Cheido et. al., 1997) similarly RKDVY (thymopentin) and RPDVY from IgM are immunostimulating but RGDVYT from HLA-DQ-β2 loop is immunosuppressive (Szewczuk et. al., 1997). Substitution of Val2 and/or Leu4 residue in a hexapeptide YVPLFP transforms this immunostimulant into immunosuppressive peptides, here the VPL tripeptide seems to be important for the stimulant activity as the YVPGFP shows strong suppressive activity (Siemion, et. al., 1991). The difference of immune activity may be attributed to the difference in overall charge and functionality of the sequence. The charge and conformational requirements for the suppression of the cellular and humoral immune response appear to be different. Another plausible explanation, however, could be the individual's immune status that may govern the expression of receptors thereby altering the response of similar peptides. Besides charge and conformational requirements the immunomodualtaory response of these peptides also depends on concentration as revealed by the in-vitro studies with a number of immunomodulating peptides (Siemion et. al., 1991 and Babcock et. al., 1983). A complete understanding of the systematic chain reaction and/or combined molecular effects of immunopeptides, neurotransmitters and hormones before and after the disease, may open up a new therapeutic approach, essentially to bring back the physiological balance. So there is need to study the cellular and molecular target of every peptide before using them for therapeutic purpose. With the use of proteomics, identification of molecular targets of peptides will not be incompressible task in the future. Although a large number of immunomodulatory peptides have been identified till date out of which many are being used for therapeutic purpose, the list can be increased further by screening biological and synthetic peptide libraries available these days. Beside identification of novel immunomodulatory peptides there is also need to screen drugs and other compounds for unintended imunosuppression; hypersensitivity or autoimmune reactions (immunotoxicity) caused by them. Most of the existing methodologies for testing immunomodulatory activity of compounds is animal based but there is need to develop alternative in vitro methods in order to reduce, refine and replace use of laboratory animal, beside this testing on animal model is costly and time consuming so testing of large number of compounds will be very difficult so in order to screen peptide libraries and compounds for their immunomodulatory activity there is a need to develop high throughput in vitro screening platform. Keeping all these points in mind in present work an endeavor was taken to identify the cellular and molecular target of an immunostimulatory hexapeptide derived from human beta casein (fragment 54-59) and some of its analogues. Different immunomodulatory activities of these peptides were studied. Proteomics profiling was done in order to identify the molecular target of these peptides. Lastly a cytokine based reporter system was developed for fast high throughput in vitro screening of peptides and other compounds for their immunomodulatory activity. In broad respect the present work was undertaken with following objectives: (a) To study the effect of peptides on immune cells. (b) To study the mode of action of these peptides at the molecular level following proteomics approach. (c) To develop an in vitro model for identifying the type of cellular immune response in context to peptides and other compounds.