Cutting Edge: Serpine1 Negatively Regulates Th1 Cell Responses in Experimental Autoimmune Encephalomyelitis.

Th1 cells are critical in experimental autoimmune encephalomyelitis (EAE). Serine protease inhibitor clade E1 (Serpine1) has been posited as an inhibitor of IFN-γ from T cells, although its role in autoimmunity remains unclear. In this study, we show that Serpine1 knockout (KO) mice develop EAE of enhanced severity relative to wild-type (WT) controls. Serpine1 overexpression represses Th1 cell cytokine production and pathogenicity, whereas Serpine1-KO:2D2 Th1 cells transfer EAE of increased severity in comparison with WT 2D2 Th1 cells. Notably, polarized Serpine1-KO Th1 cells display delayed expression of the Th1-specific inhibitory receptor, Tim-3 (T cell Ig and mucin-domain containing-3). Serpine1-KO:Tim-3-Tg Th1 cells, which transgenically overexpress Tim-3, showed increased expression of IFN-γ and reduced expression of the checkpoint molecules Lag-3 and PD-1 relative to WT Tim-3-Tg counterparts. Furthermore, Serpine1 deficiency restored the EAE phenotype of Tim-3-Tg mice that normally develop mild disease. Taken together, we identify Serpine1 as a negative regulator of Th1 cells.

The X-linked histone demethylases KDM5C and KDM6A as regulators of T cell-driven autoimmunity in the central nervous system.

T cell-driven autoimmune responses are subject to striking sex-dependent effects. While the contributions of sex hormones are well-understood, those of sex chromosomes are meeting with increased appreciation. Here, we outline what is known about the contribution of sex chromosome-linked factors to experimental autoimmune encephalomyelitis (EAE), a mouse model that recapitulates many of the T cell-driven mechanisms of multiple sclerosis (MS) pathology. Particular attention is paid to the KDM family of histone demethylases, several of which - KDM5C, KDM5D and KDM6A - are sex chromosome encoded. Finally, we provide evidence that functional inhibition of KDM5 molecules can suppress interferon (IFN)γ production from murine male effector T cells, and that an increased ratio of inflammatory Kdm6a to immunomodulatory Kdm5c transcript is observed in T helper 17 (Th17) cells from women with the autoimmune disorder ankylosing spondylitis (AS). Histone lysine demethlyases thus represent intriguing targets for the treatment of T cell-driven autoimmune disorders.

Nociceptor neurons affect cancer immunosurveillance.

Solid tumours are innervated by nerve fibres that arise from the autonomic and sensory peripheral nervous systems1-5. Whether the neo-innervation of tumours by pain-initiating sensory neurons affects cancer immunosurveillance remains unclear. Here we show that melanoma cells interact with nociceptor neurons, leading to increases in their neurite outgrowth, responsiveness to noxious ligands and neuropeptide release. Calcitonin gene-related peptide (CGRP)-one such nociceptor-produced neuropeptide-directly increases the exhaustion of cytotoxic CD8+ T cells, which limits their capacity to eliminate melanoma. Genetic ablation of the TRPV1 lineage, local pharmacological silencing of nociceptors and antagonism of the CGRP receptor RAMP1 all reduced the exhaustion of tumour-infiltrating leukocytes and decreased the growth of tumours, nearly tripling the survival rate of mice that were inoculated with B16F10 melanoma cells. Conversely, CD8+ T cell exhaustion was rescued in sensory-neuron-depleted mice that were treated with local recombinant CGRP. As compared with wild-type CD8+ T cells, Ramp1-/- CD8+ T cells were protected against exhaustion when co-transplanted into tumour-bearing Rag1-deficient mice. Single-cell RNA sequencing of biopsies from patients with melanoma revealed that intratumoral RAMP1-expressing CD8+ T cells were more exhausted than their RAMP1-negative counterparts, whereas overexpression of RAMP1 correlated with a poorer clinical prognosis. Overall, our results suggest that reducing the release of CGRP from tumour-innervating nociceptors could be a strategy to improve anti-tumour immunity by eliminating the immunomodulatory effects of CGRP on cytotoxic CD8+ T cells.

SARS-CoV-2 and Multiple Sclerosis: Potential for Disease Exacerbation.

While the respiratory tract is the primary route of entry for SARS-CoV-2, evidence shows that the virus also impacts the central nervous system. Intriguingly, case reports have documented SARS-CoV-2 patients presenting with demyelinating lesions in the brain, spinal cord, and optic nerve, suggesting possible implications in neuroimmune disorders such as multiple sclerosis (MS) and other related neuroimmune disorders. However, the cellular mechanisms underpinning these observations remain poorly defined. The goal of this paper was to review the literature to date regarding possible links between SARS-CoV-2 infection and neuroimmune demyelinating diseases such as MS and its related disorders, with the aim of positing a hypothesis for disease exacerbation. The literature suggests that SARS-CoV, SARS-CoV-2, and orthologous murine coronaviruses invade the CNS via the olfactory bulb, spreading to connected structures via retrograde transport. We hypothesize that a glial inflammatory response may contribute to damaged oligodendrocytes and blood brain barrier (BBB) breakdown, allowing a second route for CNS invasion and lymphocyte infiltration. Potential for molecular mimicry and the stimulation of autoreactive T cells against myelin is also described. It is imperative that further studies on SARS-CoV-2 neuroinvasion address the adverse effects of the virus on myelin and exacerbation of MS symptoms, as nearly 3 million people suffer from MS worldwide.

The interaction of secreted phospholipase A2-IIA with the microbiota alters its lipidome and promotes inflammation.

Secreted phospholipase A2-IIA (sPLA2-IIA) hydrolyzes phospholipids to liberate lysophospholipids and fatty acids. Given its poor activity toward eukaryotic cell membranes, its role in the generation of proinflammatory lipid mediators is unclear. Conversely, sPLA2-IIA efficiently hydrolyzes bacterial membranes. Here, we show that sPLA2-IIA affects the immune system by acting on the intestinal microbial flora. Using mice overexpressing transgene-driven human sPLA2-IIA, we found that the intestinal microbiota was critical for both induction of an immune phenotype and promotion of inflammatory arthritis. The expression of sPLA2-IIA led to alterations of the intestinal microbiota composition, but housing in a more stringent pathogen-free facility revealed that its expression could affect the immune system in the absence of changes to the composition of this flora. In contrast, untargeted lipidomic analysis focusing on bacteria-derived lipid mediators revealed that sPLA2-IIA could profoundly alter the fecal lipidome. The data suggest that a singular protein, sPLA2-IIA, produces systemic effects on the immune system through its activity on the microbiota and its lipidome.

Biological Sex As a Critical Variable in CD4+ Effector T Cell Function in Preclinical Models of Multiple Sclerosis.

Significance: T cells play a pivotal role in maintaining adaptive immune responses against pathogens. However, misdirected T cell responses against self-tissues may lead to autoimmune disease. Biological sex has profound effects on T cell function and is an important determinant of disease incidence and severity in autoimmune diseases such as multiple sclerosis (MS). Recent Advances: Many autoimmune diseases skew toward higher female incidence, including MS; however, it is has become increasingly more accepted that men living with MS are more prone to developing a progressive disease course and to having worsened disease outcomes. Critical Issues: In this review, we discuss what is known about the role of biological sex on T cell development and differentiation, examining evidence that male sex can augment T helper 17 (Th17) responses. Next, we outline what is known about sex differences in animal models of MS, and about the distinct roles played by sex hormones versus sex chromosomes in pathogenesis in these models. Finally, we discuss recent advances that examine the molecular basis for worsened disease outcomes in males, with a particular focus on the role played by Th17 cells in these models. Future Directions: Better understanding the role of biological sex in T cell function may pave the way to effective personalized treatment strategies in MS and other autoimmune diseases. Antioxid. Redox Signal. 37, 135-149.

Tim-3 adaptor protein Bat3 is a molecular checkpoint of T cell terminal differentiation and exhaustion.

T cell exhaustion has been associated with poor prognosis in persistent viral infection and cancer. Conversely, in the context of autoimmunity, T cell exhaustion has been favorably correlated with long-term clinical outcome. Understanding the development of exhaustion in autoimmune settings may provide underlying principles that can be exploited to quell autoreactive T cells. Here, we demonstrate that the adaptor molecule Bat3 acts as a molecular checkpoint of T cell exhaustion, with deficiency of Bat3 promoting a profound exhaustion phenotype, suppressing autoreactive T cell-mediated neuroinflammation. Mechanistically, Bat3 acts as a critical mTORC2 inhibitor to suppress Akt function. As a result, Bat3 deficiency leads to increased Akt activity and FoxO1 phosphorylation, indirectly promoting Prdm1 expression. Transcriptional analysis of Bat3 -/- T cells revealed up-regulation of dysfunction-associated genes, concomitant with down-regulation of genes associated with T cell effector function, suggesting that absence of Bat3 can trigger T cell dysfunction even under highly proinflammatory autoimmune conditions.

Male sex chromosomal complement exacerbates the pathogenicity of Th17 cells in a chronic model of central nervous system autoimmunity.

Sex differences in multiple sclerosis (MS) incidence and severity have long been recognized. However, the underlying cellular and molecular mechanisms for why male sex is associated with more aggressive disease remain poorly defined. Using a T cell adoptive transfer model of chronic experimental autoimmune encephalomyelitis (EAE), we find that male Th17 cells induce disease of increased severity relative to female Th17 cells, irrespective of whether transferred to male or female recipients. Throughout the disease course, a greater frequency of male Th17 cells produce IFNγ, a hallmark of pathogenic Th17 responses. Intriguingly, XY chromosomal complement increases the pathogenicity of male Th17 cells. An X-linked immune regulator, Jarid1c, is downregulated in pathogenic male murine Th17 cells, and functional experiments reveal that it represses the severity of Th17-mediated EAE. Furthermore, Jarid1c expression is downregulated in CD4+ T cells from MS-affected individuals. Our data indicate that male sex chromosomal complement critically regulates Th17 cell pathogenicity.

Selective Immunomodulatory and Neuroprotective Effects of a NOD2 Receptor Agonist on Mouse Models of Multiple Sclerosis.

The significance of monocytes has been demonstrated in multiple sclerosis (MS). One of the therapeutic challenges is developing medications that induce mild immunomodulation that is solely targeting specific monocyte subsets without affecting microglia. Muramyl dipeptide (MDP) activates the NOD2 receptor, and systemic MDP administrations convert Ly6Chigh into Ly6Clow monocytes. Here, we report selective immunomodulatory and therapeutic effects of MDP on cuprizone and experimental autoimmune encephalomyelitis (EAE) mouse models of MS. MDP treatment exerted various therapeutic effects in EAE, including delaying EAE onset and reducing infiltration of leukocytes into the CNS before EAE onset. Of great interest is the robust beneficial effect of the MDP treatment in mice already developing the disease several days after EAE onset. Finally, we found that the NOD2 receptor plays a critical role in MDP-mediated EAE resistance. Our results demonstrate that MDP is beneficial in both early and progressive phases of EAE. Based on these results, and upon comprehensive basic and clinical research, we anticipate developing NOD2 agonist-based medications for MS in the future.

Protein translocation and retro-translocation across the endoplasmic reticulum are crucial to inflammatory effector CD4+ T cell function.

Effector CD4+ T cells can be classified by the cytokines they secrete, with T helper 1 (Th1) cells generating interferon (IFN)γ and Th17 cells secreting interleukin (IL)-17. Both Th1 and Th17 cells are strongly implicated in the initiation and chronicity of autoimmune diseases such as multiple sclerosis. The endoplasmic reticulum (ER) has been implicated as a potentially crucial site in regulating CD4+ T cell function. Secretory and transmembrane proteins are shuttled into the ER via the Sec61 translocon, where they undergo appropriate folding; misfolded proteins are retro-translocated from the ER in a p97-dependent manner. Here, we provide evidence that both processes are crucial to the secretion of inflammatory cytokines from effector CD4+ T cells. The pan-ER inhibitor eeeyarestatin-1 (ESI), which interferes with both Sec61 translocation and p97 retro-translocation, inhibited secretion of interferon (IFN)γ, interleukin (IL)-2 and tumor necrosis factor (TNF)α from Th1 cells in a dose-dependent manner. Selective inhibition of Sec61 by Apratoxin A (ApraA) revealed that ER translocation is crucial for Th1 cytokine secretion, while inhibition of p97 by NMS-873 also inhibited Th1 function, albeit to a lesser degree. By contrast, none of ESI, ApraA or NMS-873 could significantly reduce IL-17 secretion from Th17 cells. ApraA, but not NMS-873, reduced phosphorylation of Stat1 in Th1 cells, indicating the involvement of ER translocation in Th1 differentiation pathways. ApraA had modest effects on activation of the Th17 transcription factor Stat3, while NMS-873 had no effect. Interestingly, NMS-873 was able to reduce disease severity in CD4+ T cell-driven experimental autoimmune encephalomyelitis (EAE). Together, our data indicate that CD4+ T cell function, and Th1 cell function in particular, is dependent on protein translocation and dislocation across the ER.

Tim-3: A co-receptor with diverse roles in T cell exhaustion and tolerance.

T cell inhibitory co-receptors play a crucial role in maintaining the balance between physiologic immune responses and maladaptive ones. T cell immunoglobulin and mucin domain-containing-3 (Tim-3) is a unique inhibitory co-receptor in that its expression is chiefly restricted to interferon (IFN)γ-producing CD4+ and CD8+ T cells. Early reports firmly established its importance in maintaining peripheral tolerance in transplantation and autoimmunity. However, it has become increasingly clear that Tim-3 expression on T cells, together with other check-point molecules, in chronic infections and cancers can hinder productive immune responses. In this review, we outline what is currently known about the regulation of Tim-3 expression, its ligands and signaling. We discuss both its salutary and deleterious function in immune disorders, as well as the T cell-extrinsic and -intrinsic factors that regulate its function.

Publisher Correction: The metabolite BH4 controls T cell proliferation in autoimmunity and cancer.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Peripheral adaptive immunity of the triple transgenic mouse model of Alzheimer's disease.

BACKGROUND: Immunologic abnormalities have been described in peripheral blood and central nervous system of patients suffering from Alzheimer's disease (AD), yet their role in the pathogenesis still remains poorly defined. AIM AND METHODS: We used the triple transgenic mouse model (3xTg-AD) to reproduce Aß (amyloid plaques) and tau (neurofibrillary tangles) neuropathologies. We analyzed important features of the adaptive immune system in serum, primary (bone marrow) as well as secondary (spleen) lymphoid organs of 12-month-old 3xTg-AD mice using flow cytometry and ELISPOT. We further investigated serum cytokines of 9- and 13-month-old 3xTg-AD mice using multiplex ELISA. Results were compared to age-matched non-transgenic controls (NTg). RESULTS: In the bone marrow of 12-month-old 3xTg-AD mice, we detected decreased proportions of short-term reconstituting hematopoietic stem cells (0.58-fold, P = 0.0116), while lymphocyte, granulocyte, and monocyte populations remained unchanged. Our results also point to increased activation of both B and T lymphocytes. Indeed, we report elevated levels of plasma cells in bone marrow (1.3-fold, P = 0.0405) along with a 5.4-fold rise in serum IgG concentration (P < 0.0001) in 3xTg-AD animals. Furthermore, higher levels of interleukin (IL)-2 were detected in serum of 9- and 13-month-old 3xTg-AD mice (P = 0.0018). Along with increased concentrations of IL-17 (P = 0.0115) and granulocyte-macrophage colony-stimulating factor (P = 0.0085), these data support helper T lymphocyte activation with Th17 polarization. CONCLUSION: Collectively, these results suggest that the 3xTg-AD model mimics modifications of the adaptive immunity changes previously observed in human AD patients and underscore the activation of both valuable and harmful pathways of immunity in AD.

Endogenous T Cell Receptor Rearrangement Represses Aggressive Central Nervous System Autoimmunity in a TcR-Transgenic Model on the Non-Obese Diabetic Background.

The T cell response to central nervous system (CNS) antigen in experimental autoimmune encephalomyelitis (EAE) permits one to model the immune aspects of multiple sclerosis. 1C6 transgenic mice on the non-obese diabetic (NOD) background possess a class II-restricted T cell receptor (TcR; Vα5-Vß7) specific for the encephalitogenic peptide myelin oligodendrocyte glycoprotein (MOG)[35-55]. It remains to be determined what role is played by allelic inclusion in shaping the TcR repertoire of these mice. Here, we show that 1C6 T cells display substantial promiscuity in their expression of non-transgenically derived Vα chains. Further, enforced expression of the transgenic TcR in 1C6 × Rag1-/- mice profoundly disrupted thymic negative selection and led to a sharp decrease in the number of mature peripheral T cells. 1C6 × Rag1-/- mice developed spontaneous EAE at a significant frequency and rapidly developed fatal EAE upon immunization with myelin oligodendrocyte glycoprotein (MOG)[35-55]. Passive transfer of 1C6 × Rag1+/+ CD4+ T cells, but not CD8+ T cells or B cells, partially rescued 1C6 × Rag1-/- mice from severe EAE. FoxP3+ CD4+ Treg cells were present in the CNS of immunized 1C6 mice, as well as immunized 1C6 × Rag1-/- that had been supplemented with 1C6 CD4+ T cells. However, they were not observed in 1C6 × Rag1-/- that did not receive Rag1-sufficient 1C6 CD4+. Further, in vivo blockade of Treg accelerated the onset of symptoms in 1C6 mice immunized with MOG[35-55], indicating the pertinence of Treg-mediated control of autoimmune inflammation in this model. Thus, TcR allelic inclusion is crucial to the generation of FoxP3+ CD4+ T cells necessary for the suppression of severe CNS autoimmunity.

The metabolite BH4 controls T cell proliferation in autoimmunity and cancer.

Genetic regulators and environmental stimuli modulate T cell activation in autoimmunity and cancer. The enzyme co-factor tetrahydrobiopterin (BH4) is involved in the production of monoamine neurotransmitters, the generation of nitric oxide, and pain1,2. Here we uncover a link between these processes, identifying a fundamental role for BH4 in T cell biology. We find that genetic inactivation of GTP cyclohydrolase 1 (GCH1, the rate-limiting enzyme in the synthesis of BH4) and inhibition of sepiapterin reductase (the terminal enzyme in the synthetic pathway for BH4) severely impair the proliferation of mature mouse and human T cells. BH4 production in activated T cells is linked to alterations in iron metabolism and mitochondrial bioenergetics. In vivo blockade of BH4 synthesis abrogates T-cell-mediated autoimmunity and allergic inflammation, and enhancing BH4 levels through GCH1 overexpression augments responses by CD4- and CD8-expressing T cells, increasing their antitumour activity in vivo. Administration of BH4 to mice markedly reduces tumour growth and expands the population of intratumoral effector T cells. Kynurenine-a tryptophan metabolite that blocks antitumour immunity-inhibits T cell proliferation in a manner that can be rescued by BH4. Finally, we report the development of a potent SPR antagonist for possible clinical use. Our data uncover GCH1, SPR and their downstream metabolite BH4 as critical regulators of T cell biology that can be readily manipulated to either block autoimmunity or enhance anticancer immunity.

Mechanistic Differences in Neuropathic Pain Modalities Revealed by Correlating Behavior with Global Expression Profiling.

Chronic neuropathic pain is a major morbidity of neural injury, yet its mechanisms are incompletely understood. Hypersensitivity to previously non-noxious stimuli (allodynia) is a common symptom. Here, we demonstrate that the onset of cold hypersensitivity precedes tactile allodynia in a model of partial nerve injury, and this temporal divergence was associated with major differences in global gene expression in innervating dorsal root ganglia. Transcripts whose expression change correlates with the onset of cold allodynia were nociceptor related, whereas those correlating with tactile hypersensitivity were immune cell centric. Ablation of TrpV1 lineage nociceptors resulted in mice that did not acquire cold allodynia but developed normal tactile hypersensitivity, whereas depletion of macrophages or T cells reduced neuropathic tactile allodynia but not cold hypersensitivity. We conclude that neuropathic pain incorporates reactive processes of sensory neurons and immune cells, each leading to distinct forms of hypersensitivity, potentially allowing drug development targeted to each pain type.

IL-1ß enables CNS access to CCR2hi monocytes and the generation of pathogenic cells through GM-CSF released by CNS endothelial cells.

Molecular interventions that limit pathogenic CNS inflammation are used to treat autoimmune conditions such as multiple sclerosis (MS). Remarkably, IL-1ß-knockout mice are highly resistant to experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Here, we show that interfering with the IL-1ß/IL-1R1 axis severely impairs the transmigration of myeloid cells across central nervous system (CNS) endothelial cells (ECs). Notably, we report that IL-1ß expression by inflammatory CCR2hi monocytes favors their entry into the spinal cord before EAE onset. Following activation with IL-1ß, CNS ECs release GM-CSF, which in turn converts monocytes into antigen-presenting cells (APCs). Accordingly, spinal cord-infiltrated monocyte-derived APCs are associated with dividing CD4+ T cells. Factors released from the interaction between IL-1ß-competent myeloid cells and CD4+ T cells are highly toxic to neurons. Together, our results suggest that IL-1ß signaling is an entry point for targeting both the initiation and exacerbation of neuroinflammation.