Feeding-induced resistance to acute lethal sepsis is dependent on hepatic BMAL1 and FXR signalling.

In mice, time of day strongly influences lethality in response to LPS, with survival greatest at the beginning compared to the end of the light cycle. Here we show that feeding, rather than light, controls time-of-day dependent LPS sensitivity. Mortality following LPS administration is independent of cytokine production and the clock regulator BMAL1 expressed in myeloid cells. In contrast, deletion of BMAL1 in hepatocytes globally disrupts the transcriptional response to the feeding cycle in the liver and results in constitutively high LPS sensitivity. Using RNAseq and functional validation studies we identify hepatic farnesoid X receptor (FXR) signalling as a BMAL1 and feeding-dependent regulator of LPS susceptibility. These results show that hepatocyte-intrinsic BMAL1 and FXR signalling integrate nutritional cues to regulate survival in response to innate immune stimuli. Understanding hepatic molecular programmes operational in response to these cues could identify novel pathways for targeting to enhance endotoxemia resistance.

MicroRNA-221 and -222 modulate intestinal inflammatory Th17 cell response as negative feedback regulators downstream of interleukin-23.

MicroRNAs are important regulators of immune responses. Here, we show miR-221 and miR-222 modulate the intestinal Th17 cell response. Expression of miR-221 and miR-222 was induced by proinflammatory cytokines and repressed by the cytokine TGF-ß. Molecular targets of miR-221 and miR-222 included Maf and Il23r, and loss of miR-221 and miR-222 expression shifted the transcriptomic spectrum of intestinal Th17 cells to a proinflammatory signature. Although the loss of miR-221 and miR-222 was tolerated for maintaining intestinal Th17 cell homeostasis in healthy mice, Th17 cells lacking miR-221 and miR-222 expanded more efficiently in response to IL-23. Both global and T cell-specific deletion of miR-221 and miR-222 rendered mice prone to mucosal barrier damage. Collectively, these findings demonstrate that miR-221 and miR-222 are an integral part of intestinal Th17 cell response that are induced after IL-23 stimulation to constrain the magnitude of proinflammatory response.

The Magnitude of IFN-γ Responses Is Fine-Tuned by DNA Architecture and the Non-coding Transcript of Ifng-as1.

Interferon gamma (IFN-γ), critical for host defense and tumor surveillance, requires tight control of its expression. Multiple cis-regulatory elements exist around Ifng along with a non-coding transcript, Ifng-as1 (also termed NeST). Here, we describe two genetic models generated to dissect the molecular functions of this locus and its RNA product. DNA deletion within the Ifng-as1 locus disrupted chromatin organization of the extended Ifng locus, impaired Ifng response, and compromised host defense. Insertion of a polyA signal ablated the Ifng-as1 full-length transcript and impaired host defense, while allowing proper chromatin structure. Transient knockdown of Ifng-as1 also reduced IFN-γ production. In humans, discordant expression of IFNG and IFNG-AS1 was evident in memory T cells, with high expression of this long non-coding RNA (lncRNA) and low expression of the cytokine. These results establish Ifng-as1 as an important regulator of Ifng expression, as a DNA element and transcribed RNA, involved in dynamic and cell state-specific responses to infection.

Retinoic Acid Receptor Alpha Represses a Th9 Transcriptional and Epigenomic Program to Reduce Allergic Pathology.

CD4+ T helper (Th) differentiation is regulated by diverse inputs, including the vitamin A metabolite retinoic acid (RA). RA acts through its receptor RARα to repress transcription of inflammatory cytokines, but is also essential for Th-mediated immunity, indicating complex effects of RA on Th specification and the outcome of the immune response. We examined the impact of RA on the genome-wide transcriptional response during Th differentiation to multiple subsets. RA effects were subset-selective and were most significant in Th9 cells. RA globally antagonized Th9-promoting transcription factors and inhibited Th9 differentiation. RA directly targeted the extended Il9 locus and broadly modified the Th9 epigenome through RARα. RA-RARα activity limited murine Th9-associated pulmonary inflammation, and human allergic inflammation was associated with reduced expression of RA target genes. Thus, repression of the Th9 program is a major function of RA-RARα signaling in Th differentiation, arguing for a role for RA in interleukin 9 (IL-9) related diseases.

IL-1ß and IL-23 Promote Extrathymic Commitment of CD27+CD122- γδ T Cells to γδT17 Cells.

γδT17 cells are a subset of γδ T cells committed to IL-17 production and are characterized by the expression of IL-23R and CCR6 and lack of CD27 expression. γδT17 cells are believed to arise within a narrow time window during prenatal thymic development. In agreement with this concept, we show in this study that adult Rag1-/- recipient mice of Il23rgfp/+ (IL-23R reporter) bone marrow selectively lack IL-23R+ γδT17 cells. Despite their absence in secondary lymphoid tissues during homeostasis, γδT17 cells emerge in bone marrow chimeric mice upon induction of skin inflammation by topical treatment with imiquimod cream (Aldara). We demonstrate that IL-1ß and IL-23 together are able to promote the development of bona fide γδT17 cells from peripheral CD122-IL-23R- γδ T cells, whereas CD122+ γδ T cells fail to convert into γδT17 cells and remain stable IFN-γ producers (γδT1 cells). IL-23 is instrumental in expanding extrathymically generated γδT17 cells. In particular, TCR-Vγ4+ chain-expressing CD122-IL-23R- γδ T cells are induced to express IL-23R and IL-17 outside the thymus during skin inflammation. In contrast, TCR-Vγ1+ γδ T cells largely resist this process because prior TCR engagement in the thymus has initiated their commitment to the γδT1 lineage. In summary, our data reveal that the peripheral pool of γδ T cells retains a considerable degree of plasticity because it harbors "naive" precursors, which can be induced to produce IL-17 and replenish peripheral niches that are usually occupied by thymus-derived γδT17 cells.

IL-10 induces a STAT3-dependent autoregulatory loop in TH2 cells that promotes Blimp-1 restriction of cell expansion via antagonism of STAT5 target genes.

Blimp-1 expression in T cells extinguishes the fate of T follicular helper cells, drives terminal differentiation, and limits autoimmunity. Although various factors have been described to control Blimp-1 expression in T cells, little is known about what regulates Blimp-1 expression in T helper 2 (TH2) cells and the molecular basis of its actions. We report that signal transducer and activator of transcription 3 (STAT3) unexpectedly played a critical role in regulating Blimp-1 in TH2 cells. Furthermore, we found that the cytokine interleukin-10 (IL-10) acted directly on TH2 cells and was necessary and sufficient to induce optimal Blimp-1 expression through STAT3. Together, Blimp-1 and STAT3 amplified IL-10 production in TH2 cells, creating a strong autoregulatory loop that enhanced Blimp-1 expression. Increased Blimp-1 in T cells antagonized STAT5-regulated cell cycle and antiapoptotic genes to limit cell expansion. These data elucidate the signals required for Blimp-1 expression in TH2 cells and reveal an unexpected mechanism of action of IL-10 in T cells, providing insights into the molecular underpinning by which Blimp-1 constrains T cell expansion to limit autoimmunity.

Uncoupling Malt1 threshold function from paracaspase activity results in destructive autoimmune inflammation.

The paracaspase Malt1 is a central regulator of antigen receptor signaling that is frequently mutated in human lymphoma. As a scaffold, it assembles protein complexes for NF-κB activation, and its proteolytic domain cleaves negative NF-κB regulators for signal enforcement. Still, the physiological functions of Malt1-protease are unknown. We demonstrate that targeted Malt1-paracaspase inactivation induces a lethal inflammatory syndrome with lymphocyte-dependent neurodegeneration in vivo. Paracaspase activity is essential for regulatory T cell (Treg) and innate-like B cell development, but it is largely dispensable for overcoming Malt1-dependent thresholds for lymphocyte activation. In addition to NF-κB inhibitors, Malt1 cleaves an entire set of mRNA stability regulators, including Roquin-1, Roquin-2, and Regnase-1, and paracaspase inactivation results in excessive interferon gamma (IFNγ) production by effector lymphocytes that drive pathology. Together, our results reveal distinct threshold and modulatory functions of Malt1 that differentially control lymphocyte differentiation and activation pathways and demonstrate that selective paracaspase blockage skews systemic immunity toward destructive autoinflammation.

IL-27 and IL-12 oppose pro-inflammatory IL-23 in CD4+ T cells by inducing Blimp1.

Central nervous system (CNS) autoimmunity is regulated by the balance of pro-inflammatory cytokines and IL-10. Here we identify the transcriptional regulator Blimp1 as crucial to induce IL-10 in inflammatory T helper cells. Pre-committed Th17 cells respond to IL-27 and IL-12 by upregulating Blimp1 and adopt a Tr-1-like phenotype characterized by IL-10 and IFN-γ production. Accordingly, Blimp1-deficient effector T cells fail to produce IL-10, and deficiency in Tr-1 cell function leads to uncontrolled Th17 cell-driven CNS pathology without the need to stabilize the Th17 phenotype with IL-23. IL-23 counteracts IL-27 and IL-12-mediated effects on Tr-1-development reinforcing the pro-inflammatory phenotype of Th17 cells. Thus, the balance of IL-23 vs IL-12/IL-27 signals into CD4(+) effector T cells determines whether tissue inflammation is perpetuated or resolves.

α4-integrins control viral meningoencephalitis through differential recruitment of T helper cell subsets.

INTRODUCTION: Natalizumab blocks α4-integrins and is a prototypic agent for a series of anti-inflammatory drugs that impair trafficking of immune cells into the CNS. However, modulation of the access of immune cells to the CNS is associated with impaired immune surveillance and detrimental viral infections of the CNS. Here, we explored the potency of cellular immune responses within the CNS to protect against viral encephalitis in mice with T cell conditional disruption of VLA-4 integrin (α4ß1) expression. RESULTS: While VLA-4 expression in virus specific Th1 cells is non-redundant for their ability to access the CNS, α4-integrin deficient Th17 cells enter the CNS compartment and generate an inflammatory milieu upon intrathecal vaccinia virus (VV) infection. However, in contrast to Th1 cells that can adopt direct cytotoxic properties, Th17 cells fail to clear the virus due to insufficient Eomes induced perforin-1 expression. CONCLUSION: The quality of the intrathecal cellular antiviral response under conditions of impaired VLA-4 function jeopardizes host protection. Our functional in vivo data extend our mechanistic understanding of anti-viral immunity in the CNS and help to estimate the risk potential of upcoming therapeutic agents that target the trafficking of immune cells into distinct anatomical compartments.

Human Mas-related G protein-coupled receptors-X1 induce chemokine receptor 2 expression in rat dorsal root ganglia neurons and release of chemokine ligand 2 from the human LAD-2 mast cell line.

Primate-specific Mas-related G protein-coupled receptors-X1 (MRGPR-X1) are highly enriched in dorsal root ganglia (DRG) neurons and induce acute pain. Herein, we analyzed effects of MRGPR-X1 on serum response factors (SRF) or nuclear factors of activated T cells (NFAT), which control expression of various markers of chronic pain. Using HEK293, DRG neuron-derived F11 cells and cultured rat DRG neurons recombinantly expressing human MRGPR-X1, we found activation of a SRF reporter gene construct and induction of the early growth response protein-1 via extracellular signal-regulated kinases-1/2 known to play a significant role in the development of inflammatory pain. Furthermore, we observed MRGPR-X1-induced up-regulation of the chemokine receptor 2 (CCR2) via NFAT, which is considered as a key event in the onset of neuropathic pain and, so far, has not yet been described for any endogenous neuropeptide. Up-regulation of CCR2 is often associated with increased release of its endogenous agonist chemokine ligand 2 (CCL2). We also found MRGPR-X1-promoted release of CCL2 in a human connective tissue mast cell line endogenously expressing MRGPR-X1. Thus, we provide first evidence to suggest that MRGPR-X1 induce expression of chronic pain markers in DRG neurons and propose a so far unidentified signaling circuit that enhances chemokine signaling by acting on two distinct yet functionally co-operating cell types. Given the important role of chemokine signaling in pain chronification, we propose that interruption of this signaling circuit might be a promising new strategy to alleviate chemokine-promoted pain.

Unlike αß T cells, γδ T cells, LTi cells and NKT cells do not require IRF4 for the production of IL-17A and IL-22.

Apart from conventional CD4(+) Th17 cells, the cytokines IL-17A and IL-22 can also be produced by γδ T cells, NK cells and lymphoid tissue inducer (LTi) cells. Th17 cells develop from precursor cells after T-cell receptor stimulation in the presence of TGF-ß, IL-6 and IL-23. In contrast, a subset of γδ T cells ("γδT17") is committed for fast IL-17 production already in the thymus; however, γδ T cells can also produce IL-17 after prolonged in vitro stimulation via their γδ T-cell receptor plus IL-23. Here, we show that γδ T-, LTi- and NKT cells differ extensively from Th17 cells in their signalling requirements for the generation of IL-17A and IL-22. While production of these cytokines by Th17 cells totally depends on the transcription factor interferon regulatory factor 4 (IRF4), IRF4 is irrelevant in the other cell types. As for γδ T cells, this finding pertains to both thymic commitment and prolonged in vitro culture. Furthermore, IL-17A-producing γδ T cells accumulate in the central nervous system of IRF4 deficient (Irf4(-/-)) mice during experimental autoimmune encephalomyelitis. IL-17A-producing WT and Irf4(-/-) γδ T cells equally express CCR6 and lack CD27. The underlying IRF4-independent pathway partially involves STAT3 during in vitro stimulation.

Development and function of interleukin 17-producing γδ T cells.

Interleukin (IL) 17 is a phylogenetically ancient cytokine that has been adopted by the adaptive immune system, and the investigation of adaptive T helper (Th) 17 cells has substantially contributed to our understanding of the molecular requirements for the induction, regulation, and function of IL-17. However, IL-17 is in fact produced by a large variety of innate immune cells and exerts its most significant biological functions at the interface of the organism with its environment, such as, for example, at epithelial surfaces, where γδ T cells are a prominent source of IL-17. In this review, we will give an overview on the concepts of commitment of γδ T cells to effector phenotypes, focusing on IL-17-producing γδ T cells (γδT17 cells). The role of γδT17 cells in animal models of autoimmunity will be discussed as well as the prerequisites for the development of human γδT17 cells and their potential importance for human disease conditions.

Th17 lymphocytes traffic to the central nervous system independently of α4 integrin expression during EAE.

The integrin α4ß1 (VLA-4) is used by encephalitogenic T cells to enter the central nervous system (CNS). However, both Th1 and Th17 cells are capable of inducing experimental autoimmune encephalomyelitis (EAE), and the molecular cues mediating the infiltration of Th1 versus Th17 cells into the CNS have not yet been defined. We investigated how blocking of α4 integrins affected trafficking of Th1 and Th17 cells into the CNS during EAE. Although antibody-mediated inhibition of α4 integrins prevented EAE when MOG(35-55)-specific Th1 cells were adoptively transferred, Th17 cells entered the brain, but not the spinal cord parenchyma, irrespective of α4 blockade. Accordingly, T cell-conditional α4-deficient mice were not resistant to actively induced EAE but showed an ataxic syndrome with predominantly supraspinal infiltrates of IL-23R(+)CCR6(+)CD4(+) T cells. The entry of α4-deficient Th17 cells into the CNS was abolished by blockade of LFA-1 (αLß2 integrin). Thus, Th1 cells preferentially infiltrate the spinal cord via an α4 integrin-mediated mechanism, whereas the entry of Th17 cells into the brain parenchyma occurs in the absence of α4 integrins but is dependent on the expression of αLß2. These observations have implications for the understanding of lesion localization, immunosurveillance, and drug design in multiple sclerosis.

Expression of miRNAs miR-133b and miR-206 in the Il17a/f locus is co-regulated with IL-17 production in αß and γδ T cells.

Differentiation of T helper 17 cells (Th17) is a multistep process that involves the cytokines IL-6, TGF-ß, and IL-23 as well as IL-1ß, IL-21, and TNF-α. Thereby, robust induction of the capacity to produce IL-17 involves epigenetic modifications of the syntenic Il17a/f locus. Using inbred mouse strains, we identified co-regulation of gene transcription at the Il17a/f locus with the nearby microRNAs miR-133b and miR-206 that are clustered approximately 45 kb upstream of Il17a/f. Expression of these microRNAs was specific for Th17 as compared to other CD4(+) T cell subsets and this was equally valid for in vitro polarized and ex vivo derived cells. From all factors analyzed, IL-23 was the most important cytokine for the in vitro induction of miR-133b and miR-206 in naive CD4(+) T cells of wild type mice. However, analysis of IL-23R deficient mice revealed that IL-23R signaling was not essential for the induction of miR-133b and miR-206. Importantly, we found a similar co-regulation in CCR6(+) and other γδ T cell subsets that are predisposed to production of IL-17. Taken together, we discovered a novel feature of T cell differentiation towards an IL-17-producing phenotype that is shared between αß and γδ T cells. Notably, the specific co-regulation of miR-133b and miR-206 with the Il17a/f locus also extended to human Th17 cells. This qualifies expression of miR-133b and miR-206 in T cells as novel biomarkers for Th17-type immune reactions.

Cytokines and effector T cell subsets causing autoimmune CNS disease.

Although experimental autoimmune encephalomyelitis (EAE) is limited in its potency to reproduce the entirety of clinical and histopathologic features of multiple sclerosis (MS), this model has been successfully used to prove that MS like autoimmunity in the CNS is orchestrated by autoantigen specific T cells. EAE was also very useful to refute the idea that IFN-γ producing T helper type 1 (Th1) cells were the sole players within the pathogenic T cell response. Rather, "new" T cell lineages such as IL-17 producing Th17 cells or IL-9 producing Th9 cells have been first discovered in the context of EAE. Here, we will summarize new concepts of early and late T cell plasticity and the cytokine network that shapes T helper cell responses and lesion development in CNS specific autoimmunity.

Functional characterization of aquaporin-4 specific T cells: towards a model for neuromyelitis optica.

BACKGROUND: Antibodies to the water channel protein aquaporin-4 (AQP4), which is expressed in astrocytic endfeet at the blood brain barrier, have been identified in the serum of Neuromyelitis optica (NMO) patients and are believed to induce damage to astrocytes. However, AQP4 specific T helper cell responses that are required for the generation of anti-AQP4 antibodies and most likely also for the formation of intraparenchymal CNS lesions have not been characterized. METHODOLOGY/PRINCIPAL FINDINGS: Using overlapping 15-meric peptides of AQP4, we identified the immunogenic T cell epitopes of AQP4 that are restricted to murine major histocompatibility complex (MHC) I-A(b). The N-terminal region of AQP4 was highly immunogenic. More precisely, the intracellular epitope AQP4(22-36) was detected as major immunogenic determinant. AQP4(82-108) (located in the second transmembrane domain), AQP4(139-153) (located in the second extracellular loop), AQP4(211-225) (located in the fifth transmembrane domain), AQP4(235-249) (located in the sixth transmembrane domain), as well as AQP4(289-306) in the intracellular C-terminal region were also immunogenic epitopes. AQP4(22-36) and AQP4(289-303) specific T cells were present in the natural T cell repertoire of wild type C57BL/6 mice and T cell lines were raised. However, active immunization with these AQP4 peptides did not induce signs of spinal cord disease. Rather, sensitization with AQP4 peptides resulted in production of IFN-γ, but also IL-5 and IL-10 by antigen-specific T cells. Consistent with this cytokine profile, the AQP4 specific antibody response upon immunization with full length AQP4 included IgG1 and IgG2, which are associated with a mixed Th2/Th1 T cell response. CONCLUSIONS AND SIGNIFICANCE: AQP4 is able to induce an autoreactive T cell response. The identification of I-A(b) restricted AQP4 specific T cell epitopes will allow us to investigate how AQP4 specific autoimmune reactions are regulated and to establish faithful mouse models of NMO that include both cellular and humoral responses against AQP4.

γδ T cells enhance autoimmunity by restraining regulatory T cell responses via an interleukin-23-dependent mechanism.

Mice that lack interleukin-23 (IL-23) are resistant to T cell-mediated autoimmunity. Although IL-23 is a maturation factor for T helper 17 (Th17) cells, a subset of γδ T cells expresses the IL-23 receptor (IL-23R) constitutively. Using IL-23R reporter mice, we showed that γδ T cells were the first cells to respond to IL-23 during experimental autoimmune encephalomyelitis (EAE). Although γδ T cells produced Th17 cell-associated cytokines in response to IL-23, their major function was to prevent the development of regulatory T (Treg) cell responses. IL-23-activated γδ T cells rendered αß effector T cells refractory to the suppressive activity of Treg cells and also prevented the conversion of conventional T cells into Foxp3(+) Treg cells in vivo. Thus, IL-23, which by itself has no direct effect on Treg cells, is able to disarm Treg cell responses and promote antigen-specific effector T cell responses via activating γδ T cells.

Semaphorin 3A and 3F: key players in myelin repair in multiple sclerosis?

The presence of demyelinated plaques in the central nervous system is the hallmark of multiple sclerosis (MS). Some plaques remyelinate but others do not, leaving permanent damage. The reasons for this failure of repair are many, but one possible reason is the lack of migration of oligodendrocyte precursor cells to the lesion. The guidance molecules Semaphorin 3A and 3F, already known to direct oligodendroglial migration in development, may also be active in controlling oligodendrocyte precursor cell migration in MS, and hence may determine the ability of plaques to remyelinate. Here, in MS tissue and an experimental model of demyelination, we demonstrate a local source of these molecules around active demyelinating lesions, but not chronic plaques. We also provide evidence for their up-regulation at a distance from the lesion, in the neuronal cell bodies corresponding to the demyelinated axons. We propose that both of these mechanisms influence remyelination.