Arunika Mukhopadhaya

Selective delivery of beta cell antigen to dendritic cells in vivo leads to deletion and tolerance of autoreactive CD8+ T cells in NOD mice.

Type 1 diabetes (T1D) is an autoimmune disease resulting from defects in central and peripheral tolerance and characterized by T cell-mediated destruction of islet β cells. Cytotoxic CD8+ T cells, reactive to β cell antigens, are required for T1D development in the NOD mouse model of the disease, and CD8+ T cells specific for β cell antigens can be detected in the peripheral blood of T1D patients. It has been evident that in nonautoimmune-prone mice, dendritic cells (DCs) present model antigens in a tolerogenic manner in the steady state, e.g., in the absence of infection, and cause T cells to proliferate initially but then to be deleted or rendered unresponsive. However, this fundamental concept has not been evaluated in the setting of a spontaneous autoimmune disease. To do so, we delivered a mimotope peptide, recognized by the diabetogenic CD8+ T cell clone AI4, to DCs in NOD mice via the endocytic receptor DEC-205. Proliferation of transferred antigen-specific T cells was initially observed, but this was followed by deletion. Tolerance was achieved because rechallenge of mice with the mimotope peptide in adjuvant did not induce an immune response. Thus, targeting of DCs with β cell antigens leads to deletion of autoreactive CD8+ T cells even in the context of ongoing autoimmunity in NOD mice with known tolerance defects. Our results provide support for the development of DC targeting of self antigens for treatment of chronic T cell-mediated autoimmune diseases.

Localized Hyperthermia Combined with Intratumoral Dendritic Cells Induces Systemic Antitumor Immunity

Prostate adenocarcinoma, treated with localized tumor hyperthermia (LTH), can potentially serve as a source of tumor antigen, where dying apoptotic/necrotic cells release tumor peptides slowly over time. In addition, LTH-treated cells can release heat shock proteins that can chaperone antigenic peptides to antigen-presenting cells, such as dendritic cells. We attempted to discern whether sequential LTH and intratumoral dendritic cell and/or systemic granulocyte macrophage colony-stimulating factor (GM-CSF) would activate antitumor immune response in a syngeneic murine model of prostate cancer (RM-1). Palpable RM-1 tumors, grown in the distal appendage of C57BL/6 male mice, were subjected to LTH (43.7 degrees C for 1 h) x 2, separated by 5 days. Following the second LTH treatment, animals received either PBS or dendritic cells (2 x 10(6)) intratumorally (every 3 days for three injections). Separate cohorts also received i.v. injection of recombinant adenovirus-expressing murine GM-CSF (AdGMCSF), 1 day after LTH. Control animals received AdenoLacZ or AdenoGFP. Intratumoral dendritic cell injection induced tumor-specific T-helper cell activity (IFNgamma ELISPOTS) and CTL activity, which was further augmented by AdGMCSF, indicating amplification of tumor-specific TH1 immunity. The combination of LTH, AdGMCSF, and intratumoral dendritic cell injection resulted in significant tumor growth delays when compared with animal cohorts that received LTH alone. These results support an in situ autovaccination strategy where systemic administration of GM-CSF and/or intratumoral injection of autologous dendritic cells, when combined with LTH, could be an effective treatment for local and systemic recurrence of prostate cancer.

Assembly and structural properties of glucocorticoid-induced TNF receptor ligand: Implications for function

Glucocorticoid-induced TNF receptor ligand (GITRL), a recently identified member of the TNF family, binds to its receptor GITR on both effector and regulatory T cells and generates positive costimulatory signals implicated in a wide range of T cell functions. Structural analysis reveals that the human GITRL (hGITRL) ectodomain self-assembles into an atypical expanded homotrimer with sparse monomer–monomer interfaces. Consistent with the small intersubunit interfaces, hGITRL exhibits a relatively weak tendency to trimerize in solution and displays a monomer–trimer equilibrium not reported for other TNF family members. This unique assembly behavior has direct implications for hGITRL–GITR signaling, because enforced trimerization of soluble hGITRL ectodomain results in an ≈100-fold increase in its receptor binding affinity and also in enhanced costimulatory activity. The apparent reduction in affinity that is the consequence of this dynamic equilibrium may represent a mechanism to realize the biologically optimal level of signaling through the hGITRL–GITR pathway, as opposed to the maximal achievable level.

Vibrio cholerae Porin OmpU Induces Pro-Inflammatory Responses, but Down-Regulates LPS-Mediated Effects in RAW 264.7, THP-1 and Human PBMCs

Vibrio cholerae porin OmpU plays a crucial role in the survival of the organism in the human gut. Various observations suggest critical involvement of OmpU in V. cholerae pathogenesis. However, OmpU is poorly characterized in terms of its ability to evoke cellular responses, particularly in the context of host immune system. Therefore, towards characterizing V. cholerae OmpU for its host immunomodulatory functions, we have studied the ability of OmpU to elicit pro-inflammatory responses in a range of immune cells which include, mouse RAW 264.7 macrophages, human THP-1 monocytes and human PBMCs. We have observed that purified OmpU induces pro-inflammatory responses in terms of production of NO, TNFα and IL-6. Interestingly, pre-treatment of the cells with OmpU suppresses the production of NO, TNFα, IL-6 as well as IL-12 upon subsequent activation with LPS. Our results therefore suggest that V. cholerae OmpU may have a differential regulatory role in terms of host immunomodulatory function: it can induce pro-inflammatory responses in target host immune cells, whereas it can also exert suppressive effect on LPS-induced pro-inflammatory responses. In addition, our study indicates that purified OmpU may have the ability to skew the Th1 response towards the Th2 response, presumably via suppression of IL-12 production.

Transmembrane oligomeric form of Vibrio cholerae cytolysin triggers TLR2/TLR6-dependent proinflammatory responses in monocytes and macrophages.

Vibrio cholerae cytolysin (VCC) kills target eukaryotic cells by forming transmembrane oligomeric β-barrel pores. Once irreversibly converted into the transmembrane oligomeric form, VCC acquires an unusual structural stability and loses its cytotoxic property. It is therefore possible that, on exertion of its cytotoxic activity, the oligomeric form of VCC retained in the disintegrated membrane fractions of the lysed cells would survive within the host cellular milieu for a long period, without causing any further cytotoxicity. Under such circumstances, VCC oligomers may potentially be recognized by the host immune cells. Based on such a hypothesis, in the present study we explored the interaction of the transmembrane oligomeric form of VCC with the monocytes and macrophages of the innate immune system. Our study shows that the VCC oligomers assembled in the liposome membranes elicit potent proinflammatory responses in monocytes and macrophages, via stimulation of the toll-like receptor (TLR)2/TLR6-dependent signalling cascades that involve myeloid differentiation factor 88 (MyD88)/interleukin-1-receptor-associated kinase (IRAK)1/tumour-necrosis-factor-receptor-associated factor (TRAF)6. VCC oligomer-mediated proinflammatory responses critically depend on the activation of the transcription factor nuclear factor-κB. Proinflammatory responses induced by the VCC oligomers also require activation of the mitogen-activated protein kinase (MAPK) family member c-Jun N-terminal kinase, which presumably acts via stimulation of the transcription factor activator protein-1. Notably, the role of the MAPK p38 could not be documented in the process.

Vibrio cholerae Porin OmpU Induces Caspase-independent Programmed Cell Death upon Translocation to the Host Cell Mitochondria

Porins, a major class of outer membrane proteins in Gram-negative bacteria, primarily act as transport channels. OmpU is one of the major porins of human pathogen, Vibrio cholerae. In the present study, we show that V. cholerae OmpU has the ability to induce target cell death. Although OmpU-mediated cell death shows some characteristics of apoptosis, such as flipping of phosphatidylserine in the membrane as well as cell size shrinkage and increased cell granularity, it does not show the caspase-3 activation and DNA laddering pattern typical of apoptotic cells. Increased release of lactate dehydrogenase in OmpU-treated cells indicates that the OmpU-mediated cell death also has characteristics of necrosis. Further, we show that the mechanism of OmpU-mediated cell death involves major mitochondrial changes in the target cells. We observe that OmpU treatment leads to the disruption of mitochondrial membrane potential, resulting in the release of cytochrome c and apoptosis-inducing factor (AIF). AIF translocates to the host cell nucleus, implying that it has a crucial role in OmpU-mediated cell death. Finally, we observe that OmpU translocates to the target cell mitochondria, where it directly initiates mitochondrial changes leading to mitochondrial membrane permeability transition and AIF release. Partial blocking of AIF release by cyclosporine A in OmpU-treated cells further suggests that OmpU may be inducing the opening of the mitochondrial permeability transition pore. All of these results lead us to the conclusion that OmpU induces cell death in target cells in a programmed manner in which mitochondria play a central role.

Poly(l-lysine)-Coated Liquid Crystal Droplets for Cell-Based Sensing Applications

Exploring intermolecular interactions in the presence of biomolecules that dictate director configurations of liquid crystals (LCs) enables new techniques for optically probing complex biological phenomena and realizing new classes of sensors and actuators. However, the design of a new approach by probing direct protein-LC interactions (in aqueous media) that can mimic chemico-biological interactions at the cellular level remains elusive. Here, we present a simple method to produce biocompatible LC droplets through poly(l-lysine) (PLL)-LC interactions in situ for reporting the presence of cells and monitoring the real-time interaction of cells with their environments that are mediated by topological defects in those droplets. In addition, responsive PLL droplets have been found to be useful as a template for reporting Annexin V-phosphatidylserine interactions, providing a simple measure of the harmful effect on cell health.

Vibrio cholerae porin OmpU induces LPS tolerance by attenuating TLR-mediated signaling.

Porins can act as pathogen-associated molecular patterns, can be recognized by the host immune system and modulate immune responses. Vibrio choleraeporin OmpU aids in bacterial survival in the human gut by increasing resistance against bile acids and anti-microbial peptides. V. choleraeOmpU is pro-inflammatory in nature. However, interestingly, it can also down-regulate LPS-mediated pro-inflammatory responses. In this study, we have explored how OmpU-pretreatment affects LPS-mediated responses. Our study indicates that OmpU-pretreatment followed by LPS-activation does not induce M2-polarization of macrophages/monocytes. Further, OmpU attenuates LPS-mediated TLR2/TLR6 signaling by decreasing the association of TLRs along with recruitment of MyD88 and IRAKs to the receptor complex. This results in decreased translocation of NFκB in the nucleus. Additionally, OmpU-pretreatment up-regulates expression of IRAK-M, a negative regulator of TLR signaling, in RAW 264.7 mouse macrophage cells upon LPS-stimulation. Suppressor cytokine IL-10 is partially involved in OmpU-induced down-regulation of LPS-mediated TNFα production in human PBMCs. Furthermore, OmpU-pretreatment also affects macrophage function, by enhancing phagocytosis in LPS-treated RAW 264.7 cells, and down-regulates LPS-induced cell surface expression of co-stimulatory molecules. Altogether, OmpU causes suppression of LPS-mediated responses by attenuating the LPS-mediated TLR signaling pathway.

Immuno-Modulatory Role of Porins: Host Immune Responses, Signaling Mechanisms and Vaccine Potential

The outer membrane of gram-negative bacteria allows the bacteria to survive amidst harsh conditions and is crucial for its interaction with the environment. The outer membrane consists of proteins and LPS. The protein content of the outer membrane contributes up to 50 % of the total mass of the whole bacterium. Further, about one-third of the genome of gram-negative bacteria encodes for outer membrane proteins. These proteins are beta barrel in structure and perform various roles apart from providing structural stability. They can help in transport of substances, signal transduction, adherence and invasion, enzymatic reactions, as well as act as PAMPs (pathogen associated molecular patterns) which can be recognized by PRRs (pattern recognition receptors) present on host immune cells. Porins are a class of outer membrane proteins that aid in transport of substances across the outer membrane. This review highlights the role of porins in various host immuno-modulatory processes, the signaling mechanisms by which they activate host immune cells and their use as vaccines against various gram-negative bacterial infections.