A TNFSF15 disease-risk polymorphism increases pattern-recognition receptor-induced signaling through caspase-8-induced IL-1.

Inflammatory diseases are characterized by dysregulated cytokine production. Altered functions for most risk loci, including the inflammatory bowel disease and leprosy-associated tumor necrosis factor ligand superfamily member 15 (TNFSF15) region, are unclear. Regulation of pattern-recognition-receptor (PRR)-induced signaling and cytokines is crucial for immune homeostasis; TNFSF15:death receptor 3 (DR3) contributions to PRR responses have not been described. We found that human macrophages expressed DR3 and that TNFSF15:DR3 interactions were critical for amplifying PRR-initiated MAPK/NF-κB/PI3K signaling and cytokine secretion in macrophages. Mechanisms mediating TNFSF15:DR3 contributions to PRR outcomes included TACE-induced TNFSF15 cleavage to soluble TNFSF15; soluble TNFSF15 then led to TRADD/FADD/MALT-1- and caspase-8-mediated autocrine IL-1 secretion. Notably, TNFSF15 treatment also induced cytokine secretion through a caspase-8-dependent pathway in intestinal myeloid cells. Importantly, rs6478108 A disease risk-carrier macrophages demonstrated increased TNFSF15 expression and PRR-induced signaling and cytokines. Taken together, TNFSF15:DR3 interactions amplify PRR-induced signaling and cytokines, and the rs6478108 TNFSF15 disease-risk polymorphism results in a gain of function.

Murabutide revisited: a review of its pleiotropic biological effects.

Despite the great efforts put into their development, the list of clinically approved immunological adjuvants is still very short. Evolution of the knowledge of the immune system has enabled for rational design of novel adjuvants and has led to the conclusion that more than one type of adjuvant will be required. Derivatives of muramyl dipeptide (MDP), the minimal immunomodulatory structure of bacterial cell wall peptidoglycan, have gained considerable attention in the past decades, because of their potent adjuvant effects. Murabutide is a safe derivative of MDP, which interacts with cells of the immune system, both innate and adaptive, and exerts its effect through activation of Nod2. The transcriptional response of murabutide-stimulated macrophages revealed enhanced expression of genes coding for various proteins such as immune mediators and their receptors, transcription factors and kinases, ion channels/transporters and proteins involved in cell metabolic activity, thus reflecting a broad spectrum of biological effects. In addition to its well recognized adjuvant effect, murabutide has also been shown to enhance the host's resistance against microbial infections, nonspecific resistance against tumors and the induction of cytokines and chemokines implicated in enhancing the immune response and hematopoesis. This article provides an insight into the mechanism of action of murabutide and its interactions with the cells of the immune system in vitro and in vivo. On account of its numerous biological effects, murabutide has been the subject of several clinical studies. Many of these have confirmed its potential to synergize with cytokines of therapeutic interest in potentiating the tumoricidal activity of macrophages or targeting chronic viral diseases, as well as reducing the cytokine dosage needed to achieve a therapeutic effect. This review covers the findings of all relevant studies and focuses on the role of murabutide and its potential in the treatment of several microbial diseases.

Inhibition of apoptosis, activation of NKT cell and upregulation of CD40 and CD40L mediated by M. leprae antigen(s) combined with Murabutide and Trat peptide in leprosy patients.

Protective immunity against intracellular pathogen Mycobacterium leprae is dependent on the activation of T cells. Repeated stimulation of T cells by M. leprae antigens MLCwA (M. leprae total cell wall antigen) and ManLAM (mannose-capped lipoarabinomannan), may lead to apoptosis in leprosy patients. In the present study, inhibition of the Fas-induced apoptosis of peripheral blood mononuclear cells of leprosy patients was investigated using above M. leprae antigen(s), in combination with immunomodulators murabutide (MB) and a Trat peptide in particulate form (liposome). Incubation of the cells with antigen containing the two immunomodulators in particulate form (liposomes) led to decrease in percentage of propidium iodide positive cells and T cells expressing Fas-FasL as well as decreased caspase-8/-3 activities in lepromatous patients, thereby inhibiting apoptosis, while converse was true upon stimulation with soluble antigen. Concurrently, there was an upregulation of antiapoptotic protein Bcl-xL in lepromatous patients, leading to the inhibition of apoptosis. It was also observed that same formulation upregulated the expression of CD40 on B cells and monocytes-macrophages and CD40L on T cells of lepromatous leprosy patients. The same liposomal formulation significantly increased the expression of CD1b and CD1d on monocytes-macrophages as well as percentage of NKT cells secreting IFN-gamma in lepromatous leprosy patients. Thus, the liposomal formulation of antigen with the immunomodulators in vitro promoted the activation of CD40:CD40L pathways and NKT cell function involved in providing cell-mediated immunity to these patients. The same formulation also caused reversal of T cell anergy by inhibiting apoptosis through decreased expression of death receptors (Fas-FasL) and caspase activities (3 and 8) and increased expression of antiapoptotic protein Bcl-xL in these patients.

Expression of costimulatory molecules (CD80, CD86, CD28, CD152), accessory molecules (TCR alphabeta, TCR gammadelta) and T cell lineage molecules (CD4+, CD8+) in PBMC of leprosy patients using Mycobacterium leprae antigen (MLCWA) with murabutide and T cell peptide of Trat protein.

In leprosy, cell-mediated immunity (CMI) is more significant than humoral response to eliminate intracellular pathogen. T cell defect is a common feature in lepromatous leprosy (LL) patients as compared to tuberculoid type (TT) patients. For efficient initiation of CD4+, T cell response requires T cell receptor (TCR) activation and costimulation provided by molecules on antigen-presenting cells (APC) and their counter receptors on T cells. In our previous study, the defective T cell function in LL patients was restored to a proliferating state with the release of TH1 type cytokines using mycobacterial antigen(s) with two immunomodulators (Murabutide (MDP-BE) and T cell epitope of Trat protein of Escherichia coli) by presenting the antigen in particulate form in vitro to PBMC derived from leprosy patients. This observation prompted us to study the expression of the costimulatory molecules (CD80, CD86, CD28, CD152), other accessory molecules (TCR alphabeta/gammadelta) and T cell lineage molecules (CD4+ and CD8+) during constitutive and activated state of peripheral blood mononuclear cells (PBMC) derived from normal and leprosy individuals using different formulations of Mycobacterium leprae total cell wall antigen (MLCWA), Trat and MDP-BE using flow cytometric analysis. An increased surface expression of CD80, CD86 and CD28 but decreased CD152 expression was observed when PBMC of normal, BT/TT (tuberculoid) and BL/LL (lepromatous) patients were stimulated in vitro with MLCWA+MDP-BE+Trat peptide using liposomal mode of antigen delivery, while opposite results were obtained with the antigen alone. Antibody inhibition study using antihuman CD80 or CD86 completely abolished the T cell lymphoproliferation, thereby reconfirming the importance of these costimulatory molecules during T cell activation/differentiation. Though the liposome-entrapped antigen formulation has no effect on expression of alphabeta/gammadelta T cell receptor, the constitutive levels of TCR gammadelta were high in lepromatous patients. Thus, TCR bearing gammadelta appears to have a negligible regulatory role in peripheral blood of leprosy patients. The percentage of cells positive for CD4+ are increased in inducible state in all the three groups, while CD8+-positive cells were decreased in LL patients, thereby reconfirming the fact that priming of CD4+ cells are necessary for producing final effector functions. Lastly, intracellular cytokine staining experiment indicated that CD4+ cells are the major producers of IFN-gamma but not NK cells. The study highlights the reversal of T cell anergy especially in lepromatous patients through the modulation of costimulatory molecule expression under the influence of Th1 cytokines, i.e., IL-2 and IFNgamma.

Reversal of T cell anergy in leprosy patients: in vitro presentation with Mycobacterium leprae antigens using murabutide and Trat peptide in liposomal delivery.

Mycobacterium leprae, the causative agent of leprosy resides and multiplies within the host monocytes and macrophages, thereby evading host immune system. Cell-mediated immune response (CMI) plays a vital role as evidenced from the high CMI in BT/TT (borderline and tuberculoid) patients and conversely low in BL/LL (borderline and lepromatous) patients. In the present study, an attempt was made to immunomodulate the anergized T cells of lepromatous leprosy patients by presenting the mycobacterial antigen in combination with T cell adjuvant, murabutide (active analog of muramyl' dipeptide, MDP-BE) and a Trat peptide (T cell epitope of Integral membrane protein (Trat) from Escherichia coli) in particulate form (liposomes) or soluble form (media). PBMNC of normal, BT/TT and BL/LL were stimulated in vitro with five mycobacterial antigens (Ag) in the following formulations, Ag, Ag+murabutide, Ag+murabutide+Trat peptide either in liposomes or in medium. All the five antigen(s) when delivered in liposomes containing murabutide and Trat peptide showed a very high lymphoproliferative response (p<0.001) in all the three groups. IFN-gamma and IL-2 were significantly (p<0.001) high in these culture supernatants compared to IL-10 and IL-4 confirming a shift from CD4+Th2 to Th1 response in leprosy patients with particulate mode of antigen presentation. Interestingly, PBMNC derived from lepromatous patients also showed consistent T cell proliferation with all the formulations. Further, the mechanism of liposomal processing of antigens was studied using different inhibitors that interfere at different stages of antigen presentation. Results indicate that this study may pave way for an immunotherapeutic approach for reverting the anergic T cells of lepromatous patients to proliferating T cells with the release of Th1 cytokines thereby restoring the CMI response in these patients.

Amyloidosis and the serum amyloid A protein response to muramyl dipeptide analogs and different mycobacterial species.

Serum amyloid A protein (SAA) elevation accompanies induction of secondary amyloidosis in mice given Mycobacterium butyricum in Freund adjuvant. The synthesis of SAA by cultured hepatocytes is induced by a macrophage-derived mediator, which has been identified as interleukin 1. In these studies, SAA synthesis has been used as an index of macrophage activation to examine the in vivo response of mice to challenge with seven different mycobacteria and with synthetic analogs of the immunoadjuvant N-acetylmuramyl-L-alanyl-D-isoglutamine [MDP(L-D)]. SAA synthesis was stimulated by administration (by the intraperitoneal route) of the mycobacteria dissolved in saline, with Mycobacterium vaccae being the most active and Mycobacterium leprae being the least stimulatory. MDP(L-D), which is the minimal structure (molecular weight, 492) able to substitute for mycobacteria in Freund adjuvant, stimulated SAA synthesis, whereas the MDP(D-D) isomer was inactive. The butyl ester of MDP, which induces no detectable pyrogenicity but retains adjuvanticity, required a 100-fold greater dosage than MDP(L-D) in stimulating SAA synthesis. Amyloidosis was detected histologically only when active SAA inducers MDP(L-D), M. vaccae, and M. butyricum, were administered in incomplete Freund adjuvant, with amyloid-enhancing factor. These studies demonstrated that SAA elevation was a sensitive in vivo marker of the capacity of antigens to stimulate macrophages to produce interleukin 1. A point of considerable relevance to the human use of MDP was the observation that repeated injections of the adjuvant MDP in saline did not induce secondary amyloidosis.