Vishal Khairnar

Involvement of Toso in activation of monocytes, macrophages, and granulocytes

Rapid activation of immune responses is necessary for antibacterial defense, but excessive immune activation can result in life-threatening septic shock. Understanding how these processes are balanced may provide novel therapeutic potential in treating inflammatory disease. Fc receptors are crucial for innate immune activation. However, the role of the putative Fc receptor for IgM, known as Toso/Faim3, has to this point been unclear. In this study, we generated Toso-deficient mice and used them to uncover a critical regulatory function of Toso in innate immune activation. Development of innate immune cells was intact in the absence of Toso, but Toso-deficient neutrophils exhibited more reactive oxygen species production and reduced phagocytosis of pathogens compared with controls. Cytokine production was also decreased in Toso−/− mice compared with WT animals, rendering them resistant to septic shock induced by lipopolysaccharide. However, Toso−/− mice also displayed limited cytokine production after infection with the bacterium Listeria monocytogenes that was correlated with elevated presence of Listeria throughout the body. Accordingly, Toso−/− mice succumbed to infections of L. monocytogenes, whereas WT mice successfully eliminated the infection. Taken together, our data reveal Toso to be a unique regulator of innate immune responses during bacterial infection and septic shock.

CD169+ macrophages regulate PD-L1 expression via type I interferon and thereby prevent severe immunopathology after LCMV infection

Upon infection with persistence-prone virus, type I interferon (IFN-I) mediates antiviral activity and also upregulates the expression of programmed death ligand 1 (PD-L1), and this upregulation can lead to CD8+ T-cell exhaustion. How these very diverse functions are regulated remains unknown. This study, using the lymphocytic choriomeningitis virus, showed that a subset of CD169+ macrophages in murine spleen and lymph nodes produced high amounts of IFN-I upon infection. Absence of CD169+ macrophages led to insufficient production of IFN-I, lower antiviral activity and persistence of virus. Lack of CD169+ macrophages also limited the IFN-I-dependent expression of PD-L1. Enhanced viral replication in the absence of PD-L1 led to persistence of virus and prevented CD8+ T-cell exhaustion. As a consequence, mice exhibited severe immunopathology and died quickly after infection. Therefore, CD169+ macrophages are important contributors to the IFN-I response and thereby influence antiviral activity, CD8+ T-cell exhaustion and immunopathology.

Deficiency of the B Cell-Activating Factor Receptor Results in Limited CD169+ Macrophage Function during Viral Infection

ABSTRACT The B cell-activating factor (BAFF) is critical for B cell development and humoral immunity in mice and humans. While the role of BAFF in B cells has been widely described, its role in innate immunity remains unknown. Using BAFF receptor (BAFFR)-deficient mice, we characterized BAFFR-related innate and adaptive immune functions following infection with vesicular stomatitis virus (VSV) and lymphocytic choriomeningitis virus (LCMV). We identified a critical role for BAFFR signaling in the generation and maintenance of the CD169+ macrophage compartment. Consequently, Baffr − / − mice exhibited limited induction of innate type I interferon production after viral infection. Lack of BAFFR signaling reduced virus amplification and presentation following viral infection, resulting in highly reduced antiviral adaptive immune responses. As a consequence, BAFFR-deficient mice showed exacerbated and fatal disease after viral infection. Mechanistically, transient lack of B cells in Baffr − / − animals resulted in limited lymphotoxin expression, which is critical for maintenance of CD169+ cells. In conclusion, BAFFR signaling affects both innate and adaptive immune activation during viral infections. IMPORTANCE Viruses cause acute and chronic infections in humans resulting in millions of deaths every year. Innate immunity is critical for the outcome of a viral infection. Innate type I interferon production can limit viral replication, while adaptive immune priming by innate immune cells induces pathogen-specific immunity with long-term protection. Here, we show that BAFFR deficiency not only perturbed B cells, but also resulted in limited CD169+ macrophages. These macrophages are critical in amplifying viral particles to trigger type I interferon production and initiate adaptive immune priming. Consequently, BAFFR deficiency resulted in reduced enforced viral replication, limited type I interferon production, and reduced adaptive immunity compared to BAFFR-competent controls. As a result, BAFFR-deficient mice were predisposed to fatal viral infections. Thus, BAFFR expression is critical for innate immune activation and antiviral immunity.

Toso regulates differentiation and activation of inflammatory dendritic cells during persistence-prone virus infection.

During virus infection and autoimmune disease, inflammatory dendritic cells (iDCs) differentiate from blood monocytes and infiltrate infected tissue. Following acute infection with hepatotropic viruses, iDCs are essential for re-stimulating virus-specific CD8+ T cells and therefore contribute to virus control. Here we used the lymphocytic choriomeningitis virus (LCMV) model system to identify novel signals, which influence the recruitment and activation of iDCs in the liver. We observed that intrinsic expression of Toso (Faim3, FcμR) influenced the differentiation and activation of iDCs in vivo and DCs in vitro. Lack of iDCs in Toso-deficient (Toso–/–) mice reduced CD8+ T-cell function in the liver and resulted in virus persistence. Furthermore, Toso–/– DCs failed to induce autoimmune diabetes in the rat insulin promoter-glycoprotein (RIP-GP) autoimmune diabetes model. In conclusion, we found that Toso has an essential role in the differentiation and maturation of iDCs, a process that is required for the control of persistence-prone virus infection.

Two separate mechanisms of enforced viral replication balance innate and adaptive immune activation.

The induction of innate and adaptive immunity is essential for controlling viral infections. Limited or overwhelming innate immunity can negatively impair the adaptive immune response. Therefore, balancing innate immunity separately from activating the adaptive immune response would result in a better antiviral immune response. Recently, we demonstrated that Usp18-dependent replication of virus in secondary lymphatic organs contributes to activation of the innate and adaptive immune responses. Whether specific mechanisms can balance innate and adaptive immunity separately remains unknown. In this study, using lymphocytic choriomeningitis virus (LCMV) and replication-deficient single-cycle LCMV vectors, we found that viral replication of the initial inoculum is essential for activating virus-specific CD8(+) T cells. In contrast, extracellular distribution of virus along the splenic conduits is necessary for inducing systemic levels of type I interferon (IFN-I). Although enforced virus replication is driven primarily by Usp18, B cell-derived lymphotoxin beta contributes to the extracellular distribution of virus along the splenic conduits. Therefore, lymphotoxin beta regulates IFN-I induction independently of CD8(+) T-cell activity. We found that two separate mechanisms act together in the spleen to guarantee amplification of virus during infection, thereby balancing the activation of the innate and adaptive immune system.

Virus-specific antibodies allow viral replication in the marginal zone, thereby promoting CD8(+) T-cell priming and viral control.

Clinically used human vaccination aims to induce specific antibodies that can guarantee long-term protection against a pathogen. The reasons that other immune components often fail to induce protective immunity are still debated. Recently we found that enforced viral replication in secondary lymphoid organs is essential for immune activation. In this study we used the lymphocytic choriomeningitis virus (LCMV) to determine whether enforced virus replication occurs in the presence of virus-specific antibodies or virus-specific CD8+ T cells. We found that after systemic recall infection with LCMV-WE the presence of virus-specific antibodies allowed intracellular replication of virus in the marginal zone of spleen. In contrast, specific antibodies limited viral replication in liver, lung, and kidney. Upon recall infection with the persistent virus strain LCMV-Docile, viral replication in spleen was essential for the priming of CD8+ T cells and for viral control. In contrast to specific antibodies, memory CD8+ T cells inhibited viral replication in marginal zone but failed to protect mice from persistent viral infection. We conclude that virus-specific antibodies limit viral infection in peripheral organs but still allow replication of LCMV in the marginal zone, a mechanism that allows immune boosting during recall infection and thereby guarantees control of persistent virus.

SGLT1 Deficiency Turns Listeria Infection into a Lethal Disease in Mice

Background: Cellular glucose uptake may involve either non-concentrative glucose carriers of the GLUT family or Na+-coupled glucose-carrier SGLT1, which accumulates glucose against glucose gradients and may thus accomplish cellular glucose uptake even at dramatically decreased extracellular glucose concentrations. SGLT1 is not only expressed in epithelia but as well in tumour cells and immune cells. Immune cell functions strongly depend on their metabolism, therefore we hypothesized that deficiency of SGLT1 modulates the defence against bacterial infection. To test this hypothesis, we infected wild type mice and gene targeted mice lacking functional SGLT1 with Listeria monocytogenes. Methods: SGLT1 deficient mice and wild type littermates were infected with 1x104 CFU Listeria monocytogenes intravenously. Bacterial titers were determined by colony forming assay, SGLT1, TNF-α, IL-6 and IL-12a transcript levels were determined by qRT-PCR, as well as SGLT1 protein abundance and localization by immunohistochemistry. Results: Genetic knockout of SGLT1 (Slc5a1–/– mice) significantly compromised bacterial clearance following Listeria monocytogenes infection with significantly enhanced bacterial load in liver, spleen, kidney and lung, and significantly augmented hepatic expression of TNF-α and IL-12a. While all wild type mice survived, all SGLT1 deficient mice died from the infection. Conclusions: SGLT1 is required for bacterial clearance and host survival following murine Listeria infection.

Expression of JAK3 Sensitive Na+ Coupled Glucose Carrier SGLT1 in Activated Cytotoxic T Lymphocytes.

Background: Similar to tumor cells, activated T-lymphocytes generate ATP mainly by glycolytic degradation of glucose. Lymphocyte glucose uptake involves non-concentrative glucose carriers of the GLUT family. In contrast to GLUT isoforms, Na+-coupled glucose-carrier SGLT1 accumulates glucose against glucose gradients and is effective at low extracellular glucose concentrations. The present study explored expression and regulation of SGLT1 in activated murine splenic cytotoxic T cells (CTLs) and human Jurkat T cells. Methods: FACS analysis, immunofluorescence, confocal microscopy, chemiluminescence and Western blotting were employed to estimate SGLT1 expression, function and regulation in lymphocytes, as well as dual electrode voltage clamp in SGLT1 ± JAK3 expressing Xenopus oocytes to quantify the effect of janus kinase3 (JAK3) on SGLT1 function. Results: SGLT1 is expressed in murine CTLs and also in human Jurkat T cells. 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose uptake was significantly decreased by SGLT1-blocker phloridzin (0.2 mM) and by pharmacological inhibition of JAK3 with WHI-P131 (156 µM), WHI-P154 (11.2 µM) and JAK3 inhibitor VI (0.5 µM). Electrogenic glucose transport (Iglucose) in Xenopus oocytes expressing human SGLT1 was increased by additional expression of human wild type JAK3, active A568VJAK3 but not inactive K851AJAK3. Coexpression of JAK3 enhanced the maximal transport rate without significantly modifying affinity of the carrier. Iglucose in SGLT1+JAK3 expressing oocytes was significantly decreased by WHI-P154 (11.2 µM). JAK3 increased the SGLT1 protein abundance in the cell membrane. Inhibition of carrier insertion by brefeldin A (5 µM) in SGLT1+JAK3 expressing oocytes resulted in a decline of Iglucose, which was similar in presence and absence of JAK3. Conclusions: SGLT1 is expressed in murine cytotoxic T cells and human Jurkat T cells and significantly contributes to glucose uptake in those cells post activation. JAK3 up-regulates SGLT1 activity by increasing the carrier protein abundance in the cell membrane, an effect enforcing cellular glucose uptake into activated lymphocytes and thus contributing to the immune response.

Diminished bone regeneration after debridement of posttraumatic osteomyelitis is accompanied by altered cytokine levels, elevated B cell activity, and increased osteoclast activity

Osteomyelitis is a frequent consequence of open fractures thus representing a common bone infection with subsequent alteration of bone regeneration. Impaired bone homeostasis provokes serious variations in the bone remodeling process, thereby involving multiple inflammatory cytokines to activate bone healing. Our previously established mouse model of posttraumatic osteomyelitis provides the chance to study regulation of selected cytokines after surgical debridement of osteomyelitis thus illustrating the course of initial infectious recovery. An inflammatory cytokine array revealed specifically upregulated cytokines in debrided animals after bone infection, that were verified by Western blot analysis, identifying increased levels of CCL2, CCL3, and CXCL2. Increased osteoclastogenesis after debridement of osteomyelitis was demonstrated by Calcitonin‐receptor and RANKL detection via immunohistochemical and ‐fluorescence stainings. The substantial protein analysis was complemented by uncovering diminished osteogenesis and proliferation in debrided group, tracking Osteocalcin, RUNX2, and PCNA expression. Interestingly TNF‐α expression seemed to have no effect on altered bone regeneration after bone infection. Additional flow cytometry analysis proved elevated B cell activity, subsequently increased osteoclast activity and accelerated bone resorption. Based on the variety of severely altered cytokines, we propose a RANKL‐dependent osteoclastogenesis after debridement of osteomyelitis coinciding with elevated B cells and simultaneously decreased osteogenesis. A comprehensive understanding of these mechanisms provides new therapeutic options of osteomyelitis cure and is of great importance in prospective medical treatment.

High Frequencies of Anti-Host Reactive CD8+ T Cells Ignore Non-Hematopoietic Antigen after Bone Marrow Transplantation in a Murine Model.

Background: Graft versus host disease (GvHD) occurs in 20% of cases with patients having an MHC I matched bone marrow transplantation (BMT). Mechanisms causing this disease remain to be studied. Methods: Here we used a CD8+ T cell transgenic mouse line (P14/CD45.1+) and transgenic DEE mice bearing ubiquitously the glycoprotein 33-41 (GP33) antigen derived from the major lymphocytic choriomeningitis virus (LCMV) epitope to study mechanisms of tolerance in anti-host reactive CD8+ T cells after BMT. Results: We found that anti-host reactive CD8+ T cells (P14 T cells) were not negatively selected in the thymus and that they were present in wild type (WT) recipient mice as well as in DEE recipient mice. Anti-host reactive CD8+ T cells ignored the GP33 antigen expressed ubiquitously by host cells but they could be activated ex vivo via LCMV-infection. Lipopolysaccharides (LPS) induced transient cell damage in DEE mice bearing anti-host reactive CD8+ T cells after BMT, suggesting that induction of host inflammatory response could break antigen ignorance. Introducing the GP33 antigen into BM cells led to deletion of anti-host reactive CD8+ T cells. Conclusion: We found that after BMT anti-host reactive CD8+ T cells ignored host antigen in recipients and that they were only deleted when host antigen was present in hematopoietic cells. Moreover, LPS-induced immune activation contributed to induction of alloreactivity of anti-host reactive CD8+ T cells after BMT.