Valéa Schumacher

IL-6 Controls the Innate Immune Response against Listeria monocytogenes via Classical IL-6 Signaling

The cytokine IL-6 plays a protective role in immune responses against bacterial infections. However, the mechanisms of IL-6–mediated protection are only partially understood. IL-6 can signal via the IL-6R complex composed of membrane-bound IL-6Rα (mIL-6Rα) and gp130. Owing to the restricted expression of mIL-6Rα, classical IL-6 signaling occurs only in a limited number of cells such as hepatocytes and certain leukocyte subsets. IL-6 also interacts with soluble IL-6Rα proteins and these IL-6/soluble IL-6Rα complexes can subsequently bind to membrane-bound gp130 proteins and induce signaling. Because gp130 is ubiquitously expressed, this IL-6 trans-signaling substantially increases the spectrum of cells responding to IL-6. In this study, we analyze the role of classical IL-6 signaling and IL-6 trans-signaling in the innate immune response of mice against Listeria monocytogenes infection. We demonstrate that L. monocytogenes infection causes profound systemic IL-6 production and rapid loss of IL-6Rα surface expression on neutrophils, inflammatory monocytes, and different lymphocyte subsets. IL-6–deficient mice or mice treated with neutralizing anti–IL-6 mAb displayed impaired control of L. monocytogenes infection accompanied by alterations in the expression of inflammatory cytokines and chemokines, as well as in the recruitment of inflammatory cells. In contrast, restricted blockade of IL-6 trans-signaling by application or transgenic expression of a soluble gp130 protein did not restrain the control of infection. In summary, our results demonstrate that IL-6Rα surface expression is highly dynamic during the innate response against L. monocytogenes and that the protective IL-6 function is dependent on classical IL-6 signaling via mIL-6Rα.

The transcription factor Interferon Regulatory Factor 4 is required for the generation of protective effector CD8+ T cells

Robust cytotoxic CD8+ T-cell response is important for immunity to intracellular pathogens. Here, we show that the transcription factor IFN Regulatory Factor 4 (IRF4) is crucial for the protective CD8+ T-cell response to the intracellular bacterium Listeria monocytogenes. IRF4-deficient (Irf4−/−) mice could not clear L. monocytogenes infection and generated decreased numbers of L. monocytogenes-specific CD8+ T cells with impaired effector phenotype and function. Transfer of wild-type CD8+ T cells into Irf4−/− mice improved bacterial clearance, suggesting an intrinsic defect of CD8+ T cells in Irf4−/− mice. Following transfer into wild-type recipients, Irf4−/− CD8+ T cells became activated and showed initial proliferation upon L. monocytogenes infection. However, these cells could not sustain proliferation, produced reduced amounts of IFN-γ and TNF-α, and failed to acquire cytotoxic function. Forced IRF4 expression in Irf4−/− CD8+ T cells rescued the defect. During acute infection, Irf4−/− CD8+ T cells demonstrated diminished expression of B lymphocyte-induced maturation protein-1 (Blimp-1), inhibitor of DNA binding (Id)2, and T-box expressed in T cells (T-bet), transcription factors programming effector-cell generation. IRF4 was essential for expression of Blimp-1, suggesting that altered regulation of Blimp-1 contributes to the defects of Irf4−/− CD8+ T cells. Despite increased levels of B-cell lymphoma 6 (BCL-6), Eomesodermin, and Id3, Irf4−/− CD8+ T cells showed impaired memory-cell formation, indicating additional functions for IRF4 in this process. As IRF4 governs B-cell and CD4+ T-cell differentiation, the identification of its decisive role in peripheral CD8+ T-cell differentiation, suggests a common regulatory function for IRF4 in adaptive lymphocytes fate decision.

High Sensitivity of Intestinal CD8+ T Cells to Nucleotides Indicates P2X7 as a Regulator for Intestinal T Cell Responses

The purinoreceptor P2X7 is expressed on subsets of T cells and mediates responses of these cells to extracellular nucleotides such as ATP or NAD+. We identified P2X7 as a molecule highly up-regulated on conventional CD8αβ+ and unconventional CD8αα+ T cells of the intestinal epithelium of mice. In contrast, CD8+ T cells derived from spleen, mesenteric lymph nodes, and liver expressed only marginal levels of P2X7. However, P2X7 was highly up-regulated on CD8+ T cells from spleen and lymph nodes when T cells were activated in the presence of retinoic acid. High P2X7 expression on intestinal CD8+ T cells as well as on CD8+ T cells incubated with retinoic acid resulted in enhanced sensitivity of cells to extracellular nucleotides. Both cell populations showed a high level of apoptosis following incubation with NAD+ and the ATP derivative 2′,3′-O-(benzoyl-4-benzoyl)-ATP, and injection of NAD+ caused selective in vivo depletion of intestinal CD8+ T cells. Following oral infection with Listeria monocytogenes, P2X7-deficient mice showed similar CD8+ T cell responses in the spleen, but enhanced responses in the intestinal mucosa, when compared with similarly treated wild-type control mice. Overall, our observations define P2X7 as a new regulatory element in the control of CD8+ T cell responses in the intestinal mucosa.

CD38 Controls the Innate Immune Response against Listeria monocytogenes

ABSTRACT CD38, adenosine-5′-diphosphate-ribosyl cyclase 1, is a multifunctional enzyme, expressed on a wide variety of cell types. CD38 has been assigned diverse functions, including generation of calcium-mobilizing metabolites, cell activation, and chemotaxis. Using a murine Listeria monocytogenes infection model, we found that CD38 knockout (KO) mice were highly susceptible to infection. Enhanced susceptibility was already evident within 3 days of infection, suggesting a function of CD38 in the innate immune response. CD38 was expressed on neutrophils and inflammatory monocytes, and especially inflammatory monocytes further upregulated CD38 during infection. Absence of CD38 caused alterations of the migration pattern of both cell types to sites of infection. We observed impaired accumulation of cells in the spleen but surprisingly similar or even higher accumulation of cells in the liver. CD38 KO and wild-type mice showed similar changes in the composition of neutrophils and inflammatory monocytes in blood and bone marrow, indicating that mobilization of these cells from the bone marrow was CD38 independent. In vitro, macrophages of CD38 KO mice were less efficient in uptake of listeria but still able to kill the bacteria. Dendritic cells also displayed enhanced CD38 expression following infection. However, absence of CD38 did not impair the capacity of mice to prime CD8+ T cells against L. monocytogenes, and CD38 KO mice could efficiently control secondary listeria infection. In conclusion, our results demonstrate an essential role for CD38 in the innate immune response against L. monocytogenes.

ADAM17 controls IL-6 signaling by cleavage of the murine IL-6Rα from the cell surface of leukocytes during inflammatory responses.

The cytokine IL‐6 is part of a regulatory signaling network that controls immune responses. IL‐6 binds either to the membrane‐bound IL‐6 receptor‐α (classic signaling) or to the soluble IL‐6 receptor‐α (trans‐signaling) to initiate signal transduction via gp130 activation. Because classic and trans‐signaling of IL‐6 fulfill different tasks during immune responses, controlled shedding of the membrane‐bound IL‐6 receptor‐α from the surface of immune cells can be considered a central regulator of IL‐6 function. The results from cell culture‐based experiments have implicated both a disintegrin and metalloprotease 10 and a disintegrin and metalloprotease 17 in IL‐6 receptor‐α shedding. However, the nature of the protease mediating IL‐6 receptor‐α release in vivo is not yet known. We used hypomorphic a disintegrin and metalloprotease 17 mice and conditional a disintegrin and metalloprotease 10 knock‐out mice to identify the natural protease of the murine IL‐6 receptor‐α. Circulating homeostatic soluble IL‐6 receptor‐α levels are not dependent on a disintegrin and metalloprotease 10 or 17 activity. However, during Listeria monocytogenes infection, IL‐6 receptor‐α cleavage by the α‐secretase a disintegrin and metalloprotease 17 is rapidly induced from the surface of different leukocyte populations. In contrast, CD4‐Cre‐driven a disintegrin and metalloprotease 10 deletion in T cells did not influence IL‐6 receptor‐α shedding from these cells after L. monocytogenes infection. A disintegrin and metalloprotease 17 was also required for IL‐6 receptor‐α ectodomain cleavage and release during endotoxemia. These results demonstrate a novel physiologic role for a disintegrin and metalloprotease 17 in regulating murine IL‐6 signals during inflammatory processes.

Frontrunners of T cell activation: Initial, localized Ca2+ signals mediated by NAADP and the type 1 ryanodine receptor.

High-resolution imaging of live T cells characterizes the early Ca2+ signals required for T cell activation. Calcium signals down to the millisecond Engagement of the T cell receptor (TCR) stimulates Ca2+ signaling, which is required for T cell activation. The earliest Ca2+ signals are short-lived and localized near the sites of TCR stimulation; later events are longer-lasting and more widespread. Wolf et al. used a combination of fluorescent indicator dyes and microscopy to perform high-resolution imaging of Ca2+ signals that occurred within milliseconds of the TCR stimulation of live mouse and human T cells. Microinjection of cells with the second messenger NAADP, which is generated upon T cell activation, produced a similar spatiotemporal pattern of Ca2+ signals in the absence of TCR activation. Both TCR- and NAADP-dependent signals were markedly reduced by depletion of ryanodine receptors, which are localized in the endoplasmic reticulum, implicating this internal calcium store as a source for the early Ca2+ signals required for T cell activation. The activation of T cells is the fundamental on switch for the adaptive immune system. Ca2+ signaling is essential for T cell activation and starts as initial, short-lived, localized Ca2+ signals. The second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) forms rapidly upon T cell activation and stimulates early Ca2+ signaling. We developed a high-resolution imaging technique using multiple fluorescent Ca2+ indicator dyes to characterize these early signaling events and investigate the channels involved in NAADP-dependent Ca2+ signals. In the first seconds of activation of either primary murine T cells or human Jurkat cells with beads coated with an antibody against CD3, we detected Ca2+ signals with diameters close to the limit of detection and that were close to the activation site at the plasma membrane. In Jurkat cells in which the ryanodine receptor (RyR) was knocked down or in primary T cells from RyR1−/− mice, either these early Ca2+ signals were not detected or the number of signals was markedly reduced. Local Ca2+ signals observed within 20 ms upon microinjection of Jurkat cells with NAADP were also sensitive to RyR knockdown. In contrast, TRPM2 (transient receptor potential channel, subtype melastatin 2), a potential NAADP target channel, was not required for the formation of initial Ca2+ signals in primary T cells. Thus, through our high-resolution imaging method, we characterized early Ca2+ release events in T cells and obtained evidence for the involvement of RyR and NAADP in such signals.

Interferon Regulatory Factor 4 controls TH1 cell effector function and metabolism

The transcription factor Interferon Regulatory Factor 4 (IRF4) is essential for TH2 and TH17 cell formation and controls peripheral CD8+ T cell differentiation. We used Listeria monocytogenes infection to characterize the function of IRF4 in TH1 responses. IRF4−/− mice generated only marginal numbers of listeria-specific TH1 cells. After transfer into infected mice, IRF4−/− CD4+ T cells failed to differentiate into TH1 cells as indicated by reduced T-bet and IFN-γ expression, and showed limited proliferation. Activated IRF4−/− CD4+ T cells exhibited diminished uptake of the glucose analog 2-NBDG, limited oxidative phosphorylation and strongly reduced aerobic glycolysis. Insufficient metabolic adaptation contributed to the limited proliferation and TH1 differentiation of IRF4−/− CD4+ T cells. Our study identifies IRF4 as central regulator of TH1 responses and cellular metabolism. We propose that this function of IRF4 is fundamental for the initiation and maintenance of all TH cell responses.

TH1 and TH17 cells promote crescent formation in experimental autoimmune glomerulonephritis

Autoimmunity against the Goodpasture antigen α3IV‐NC1 results in crescentic glomerulonephritis (GN). Both antibodies and T cells directed against α3IV‐NC1 have been implicated in disease development and progression. Using the model of experimental autoimmune glomerulonephritis (EAG) in DBA/1 mice, we aimed to characterize the frequency and function of α3IV‐NC1‐specific CD4+ T cells in the kidneys. DBA/1 mice repeatedly immunized with human α3IV‐NC1 developed necrotizing/crescentic GN. Kidneys with crescentic GN contained CD4+ cells responding to α3IV‐NC1 with the production of IFN‐γ or IL‐17A, demonstrating the accumulation of both α3IV‐NC1‐specific TH1 and TH17 cells. To test the functional relevance of TH1 and TH17 cells, EAG was induced in DBA/1 mice deficient in IFN‐γR, IL‐17A or IL‐23p19. Mice of all knockout groups mounted α3IV‐NC1 IgG, developed nephrotic range proteinuria, and IgG deposition to the glomerular basement membranes at levels similar to immunized wild‐type mice. However, all knockout groups showed significantly fewer glomerular crescents and attenuated tubulointerstitial damage. Our results suggest that both α3IV‐NC1‐specific TH1 and TH17 cells accumulate in the kidneys and are crucial for the development of necrotizing/crescentic GN. Copyright

CD8‐β ADP‐ribosylation affects CD8+ T‐cell function

The CD8αβ coreceptor is crucial for effective peptide: MHC‐I recognition by the TCR of CD8+ T cells. Adenosine diphosphate ribosyl transferase 2.2 (ART2.2) utilizes extracellular NAD+ to transfer ADP‐ribose to arginine residues of extracellular domains of surface proteins. Here, we show that in the presence of extracellular NAD+, ART2.2 caused ADP‐ribosylation of CD8‐β on murine CD8+ T cells in vitro and in vivo. Treatment with NAD+ prevented binding of anti‐CD8‐β mAb YTS156.7.7 but not of mAb H35–17.2, indicating that NAD+ caused modification of certain epitopes and not a general loss of CD8‐β. Loss of antibody binding was strictly dependent on ART2.2, because it was not observed on ART2‐deficient T cells or in the presence of inhibitory anti‐ART2.2 single‐domain antibodies. ADP‐ribosylation of CD8‐β occurred during cell isolation, particularly when cells were isolated from CD38‐deficient mice. Incubation of ART2‐expressing, but not of ART2‐deficient, OVA‐specific CD8+ T cells with NAD+ interfered with binding of OVA257–264:MHC‐I tetramers. In line with this result, treatment of WT mice with NAD+ resulted in reduced CD8+ T‐cell mediated cytotoxicity in vivo. We propose that ADP‐ribosylation of CD8‐β can regulate the coreceptor function of CD8 in the presence of elevated levels of extracellular NAD+.

CD38 is expressed on inflammatory cells of the intestine and promotes intestinal inflammation.

The enzyme CD38 is expressed on a variety of hematopoietic and non-hematopoietic cells and is involved in diverse processes such as generation of calcium-mobilizing metabolites, cell activation, and chemotaxis. Here, we show that under homeostatic conditions CD38 is highly expressed on immune cells of the colon mucosa of C57BL/6 mice. Myeloid cells recruited to this tissue upon inflammation also express enhanced levels of CD38. To determine the role of CD38 in intestinal inflammation, we applied the dextran sulfate sodium (DSS) colitis model. Whereas wild-type mice developed severe colitis, CD38-/- mice had only mild disease following DSS-treatment. Histologic examination of the colon mucosa revealed pronounced inflammatory damage with dense infiltrates containing numerous granulocytes and macrophages in wild-type animals, while these findings were significantly attenuated in CD38-/- mice. Despite attenuated histological findings, the mRNA expression of inflammatory cytokines and chemokines was only marginally lower in the colons of CD38-/- mice as compared to wild-type mice. In conclusion, our results identify a function for CD38 in the control of inflammatory processes in the colon.