BHLHE40 Regulates Myeloid Cell Polarization through IL-10-Dependent and -Independent Mechanisms.

Better understanding of the host responses to Mycobacterium tuberculosis infections is required to prevent tuberculosis and develop new therapeutic interventions. The host transcription factor BHLHE40 is essential for controlling M. tuberculosis infection, in part by repressing Il10 expression, where excess IL-10 contributes to the early susceptibility of Bhlhe40-/- mice to M. tuberculosis infection. Deletion of Bhlhe40 in lung macrophages and dendritic cells is sufficient to increase the susceptibility of mice to M. tuberculosis infection, but how BHLHE40 impacts macrophage and dendritic cell responses to M. tuberculosis is unknown. In this study, we report that BHLHE40 is required in myeloid cells exposed to GM-CSF, an abundant cytokine in the lung, to promote the expression of genes associated with a proinflammatory state and better control of M. tuberculosis infection. Loss of Bhlhe40 expression in murine bone marrow-derived myeloid cells cultured in the presence of GM-CSF results in lower levels of proinflammatory associated signaling molecules IL-1ß, IL-6, IL-12, TNF-α, inducible NO synthase, IL-2, KC, and RANTES, as well as higher levels of the anti-inflammatory-associated molecules MCP-1 and IL-10 following exposure to heat-killed M. tuberculosis. Deletion of Il10 in Bhlhe40-/- myeloid cells restored some, but not all, proinflammatory signals, demonstrating that BHLHE40 promotes proinflammatory responses via both IL-10-dependent and -independent mechanisms. In addition, we show that macrophages and neutrophils within the lungs of M. tuberculosis-infected Bhlhe40-/- mice exhibit defects in inducible NO synthase production compared with infected wild-type mice, supporting that BHLHE40 promotes proinflammatory responses in innate immune cells, which may contribute to the essential role for BHLHE40 during M. tuberculosis infection in vivo.

BHLHE40 Mediates Cross-Talk between Pathogenic TH17 Cells and Myeloid Cells during Experimental Autoimmune Encephalomyelitis.

TH17 cells are implicated in the pathogenesis of multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). We previously reported that the transcription factor basic helix-loop-helix family member e40 (BHLHE40) marks cytokine-producing pathogenic TH cells during EAE, and that its expression in T cells is required for clinical disease. In this study, using dual reporter mice, we show BHLHE40 expression within TH1/17 and ex-TH17 cells following EAE induction. Il17a-Cre-mediated deletion of BHLHE40 in TH cells led to less severe EAE with reduced TH cell cytokine production. Characterization of the leukocytes in the CNS during EAE by single-cell RNA sequencing identified differences in the infiltrating myeloid cells when BHLHE40 was present or absent in TH17 cells. Our studies highlight the importance of BHLHE40 in promoting TH17 cell encephalitogenicity and instructing myeloid cell responses during active EAE.

The ZFP36 family of RNA binding proteins regulates homeostatic and autoreactive T cell responses.

RNA binding proteins are important regulators of T cell activation, proliferation, and cytokine production. The zinc finger protein 36 (ZFP36) family genes (Zfp36, Zfp36l1, and Zfp36l2) encode RNA binding proteins that promote the degradation of transcripts containing AU-rich elements. Numerous studies have demonstrated both individual and shared functions of the ZFP36 family in immune cells, but their collective function in T cells remains unclear. Here, we found a redundant and critical role for the ZFP36 proteins in regulating T cell quiescence. T cell-specific deletion of all three ZFP36 family members in mice resulted in early lethality, immune cell activation, and multiorgan pathology characterized by inflammation of the eyes, central nervous system, kidneys, and liver. Mice with T cell-specific deletion of any two Zfp36 genes were protected from this spontaneous syndrome. Triply deficient T cells overproduced proinflammatory cytokines, including IFN-γ, TNF, and GM-CSF, due to increased mRNA stability of these transcripts. Unexpectedly, T cell-specific deletion of both Zfp36l1 and Zfp36l2 rendered mice resistant to experimental autoimmune encephalomyelitits due to failed priming of antigen-specific CD4+ T cells. ZFP36L1 and ZFP36L2 double-deficient CD4+ T cells had poor proliferation during in vitro T helper cell polarization. Thus, the ZFP36 family redundantly regulates T cell quiescence at homeostasis, but ZFP36L1 and ZFP36L2 are specifically required for antigen-specific T cell clonal expansion.

Antigen-specific depletion of CD4+ T cells by CAR T cells reveals distinct roles of higher- and lower-affinity TCRs during autoimmunity.

Both higher- and lower-affinity self-reactive CD4+ T cells are expanded in autoimmunity; however, their individual contribution to disease remains unclear. We addressed this question using peptide-MHCII chimeric antigen receptor (pMHCII-CAR) T cells to specifically deplete peptide-reactive T cells in mice. Integration of improvements in CAR engineering with TCR repertoire analysis was critical for interrogating in vivo the role of TCR affinity in autoimmunity. Our original MOG35-55 pMHCII-CAR, which targeted only higher-affinity TCRs, could prevent the induction of experimental autoimmune encephalomyelitis (EAE). However, pMHCII-CAR enhancements to pMHCII stability, as well as increased survivability via overexpression of a dominant-negative Fas, were required to target lower-affinity MOG-specific T cells and reverse ongoing clinical EAE. Thus, these data suggest a model in which higher-affinity autoreactive T cells are required to provide the "activation energy" for initiating neuroinflammatory injury, but lower-affinity cells are sufficient to maintain ongoing disease.

Antigen-guided depletion of anti-HLA antibody-producing cells by HLA-Fc fusion proteins.

Platelet transfusion and transplantation of allogeneic stem cells and solid organs are life-saving therapies. Unwanted alloantibodies to nonself human leukocyte antigens (HLAs) on donor cells increase the immunological barrier to these therapies and are important causes of platelet transfusion refractoriness and graft rejection. Although the specificities of anti-HLA antibodies can be determined at the allelic level, traditional treatments for antibody-mediated rejection nonselectively suppress humoral immunity and are not universally successful. We designed HLA-Fc fusion proteins with a bivalent targeting module derived from extracellular domains of HLA and an Fc effector module from mouse IgG2a. We found that HLA-Fc with A2 (A2Fc) and B7 (B7Fc) antigens lowered HLA-A2- and HLA-B7-specific reactivities, respectively, in sera from HLA-sensitized patients. A2Fc and B7Fc bound to B-cell hybridomas bearing surface immunoglobulins with cognate specificities and triggered antigen-specific and Fc-dependent cytotoxicity in vitro. In immunodeficient mice carrying HLA-A2-specific hybridoma cells, A2Fc treatment lowered circulating anti-HLA-A2 levels, abolished the outgrowth of hybridoma cells, and prolonged survival compared with control groups. In an in vivo anti-HLA-A2-mediated platelet transfusion refractoriness model, A2Fc treatment mitigated refractoriness. These results support HLA-Fc being a novel strategy for antigen-specific humoral suppression to improve transfusion and transplantation outcomes. With the long-term goal of targeting HLA-specific memory B cells for desensitization, further studies of HLA-Fc's efficacy in immune-competent animal models are warranted.

BHLHE40 Promotes TH2 Cell-Mediated Antihelminth Immunity and Reveals Cooperative CSF2RB Family Cytokines.

The transcription factor BHLHE40 is an emerging regulator of the immune system. Recent studies suggest that BHLHE40 regulates type 2 immunity, but this has not been demonstrated in vivo. We found that BHLHE40 is required in T cells for a protective TH2 cell response in mice infected with the helminth Heligmosomoides polygyrus bakeri H. polygyrus elicited changes in gene and cytokine expression by lamina propria CD4+ T cells, many of which were BHLHE40 dependent, including production of the common ß (CSF2RB) chain family cytokines GM-CSF and IL-5. In contrast to deficiency in GM-CSF or IL-5 alone, loss of both GM-CSF and IL-5 signaling impaired protection against H. polygyrus Overall, we show that BHLHE40 regulates the TH2 cell transcriptional program during helminth infection to support normal expression of Csf2, Il5, and other genes required for protection and reveal unexpected redundancy of common ß chain-dependent cytokines previously thought to possess substantially divergent functions.

Bhlhe40 mediates tissue-specific control of macrophage proliferation in homeostasis and type 2 immunity.

Most tissue-resident macrophage populations develop during embryogenesis, self-renew in the steady state and expand during type 2 immunity. Whether shared mechanisms regulate the proliferation of macrophages in homeostasis and disease is unclear. Here we found that the transcription factor Bhlhe40 was required in a cell-intrinsic manner for the self-renewal and maintenance of large peritoneal macrophages (LPMs), but not that of other tissue-resident macrophages. Bhlhe40 was necessary for the proliferation, but not the polarization, of LPMs in response to the cytokine IL-4. During infection with the helminth Heligmosomoides polygyrus bakeri, Bhlhe40 was required for cell cycling of LPMs. Bhlhe40 repressed the expression of genes encoding the transcription factors c-Maf and Mafb and directly promoted expression of transcripts encoding cell cycle-related proteins to enable the proliferation of LPMs. In LPMs, Bhlhe40 bound to genomic sites co-bound by the macrophage lineage-determining factor PU.1 and to unique sites, including Maf and loci encoding cell-cycle-related proteins. Our findings demonstrate a tissue-specific control mechanism that regulates the proliferation of resident macrophages in homeostasis and type 2 immunity.

Bhlhe40 is an essential repressor of IL-10 during Mycobacterium tuberculosis infection.

The cytokine IL-10 antagonizes pathways that control Mycobacterium tuberculosis (Mtb) infection. Nevertheless, the impact of IL-10 during Mtb infection has been difficult to decipher because loss-of-function studies in animal models have yielded only mild phenotypes. We have discovered that the transcription factor basic helix-loop-helix family member e40 (Bhlhe40) is required to repress Il10 expression during Mtb infection. Loss of Bhlhe40 in mice results in higher Il10 expression, higher bacterial burden, and early susceptibility similar to that observed in mice lacking IFN-γ. Deletion of Il10 in Bhlhe40-/- mice reverses these phenotypes. Bhlhe40 deletion in T cells or CD11c+ cells is sufficient to cause susceptibility to Mtb Bhlhe40 represents the first transcription factor found to be essential during Mtb infection to specifically regulate Il10 expression, revealing the importance of strict control of IL-10 production by innate and adaptive immune cells during infection. Our findings uncover a previously elusive but significant role for IL-10 in Mtb pathogenesis.

Irg1 expression in myeloid cells prevents immunopathology during M. tuberculosis infection.

Immune-Responsive Gene 1 (Irg1) is a mitochondrial enzyme that produces itaconate under inflammatory conditions, principally in cells of myeloid lineage. Cell culture studies suggest that itaconate regulates inflammation through its inhibitory effects on cytokine and reactive oxygen species production. To evaluate the functions of Irg1 in vivo, we challenged wild-type (WT) and Irg1-/- mice with Mycobacterium tuberculosis (Mtb) and monitored disease progression. Irg1-/-, but not WT, mice succumbed rapidly to Mtb, and mortality was associated with increased infection, inflammation, and pathology. Infection of LysM-Cre Irg1fl/fl, Mrp8-Cre Irg1fl/fl, and CD11c-Cre Irg1fl/fl conditional knockout mice along with neutrophil depletion experiments revealed a role for Irg1 in LysM+ myeloid cells in preventing neutrophil-mediated immunopathology and disease. RNA sequencing analyses suggest that Irg1 and its production of itaconate temper Mtb-induced inflammatory responses in myeloid cells at the transcriptional level. Thus, an Irg1 regulatory axis modulates inflammation to curtail Mtb-induced lung disease.

OTUD4 Is a Phospho-Activated K63 Deubiquitinase that Regulates MyD88-Dependent Signaling.

Ubiquitination is a major mechanism that regulates numerous cellular processes, including autophagy, DNA damage signaling, and inflammation. While hundreds of ubiquitin ligases exist to conjugate ubiquitin onto substrates, approximately 100 deubiquitinases are encoded by the human genome. Thus, deubiquitinases are likely regulated by unidentified mechanisms to target distinct substrates and cellular functions. Here, we demonstrate that the deubiquitinase OTUD4, which nominally encodes a K48-specific deubiquitinase, is phosphorylated near its catalytic domain, activating a latent K63-specific deubiquitinase. Besides phosphorylation, this latter activity requires an adjacent ubiquitin-interacting motif, which increases the affinity of OTUD4 for K63-linked chains. We reveal the Toll-like receptor (TLR)-associated factor MyD88 as a target of this K63 deubiquitinase activity. Consequently, TLR-mediated activation of NF-κB is negatively regulated by OTUD4, and macrophages from Otud4-/- mice exhibit increased inflammatory signaling upon TLR stimulation. Our results reveal insights into how a deubiquitinase may modulate diverse processes through post-translational modification.

IL-1-induced Bhlhe40 identifies pathogenic T helper cells in a model of autoimmune neuroinflammation.

The features that define autoreactive T helper (Th) cell pathogenicity remain obscure. We have previously shown that Th cells require the transcription factor Bhlhe40 to mediate experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. Here, using Bhlhe40 reporter mice and analyzing both polyclonal and TCR transgenic Th cells, we found that Bhlhe40 expression was heterogeneous after EAE induction, with Bhlhe40-expressing cells displaying marked production of IFN-γ, IL-17A, and granulocyte-macrophage colony-stimulating factor. In adoptive transfer EAE models, Bhlhe40-deficient Th1 and Th17 cells were both nonencephalitogenic. Pertussis toxin (PTX), a classical co-adjuvant for actively induced EAE, promoted IL-1ß production by myeloid cells in the draining lymph node and served as a strong stimulus for Bhlhe40 expression in Th cells. Furthermore, PTX co-adjuvanticity was Bhlhe40 dependent. IL-1ß induced Bhlhe40 expression in polarized Th17 cells, and Bhlhe40-expressing cells exhibited an encephalitogenic transcriptional signature. In vivo, IL-1R signaling was required for full Bhlhe40 expression by Th cells after immunization. Overall, we demonstrate that Bhlhe40 expression identifies encephalitogenic Th cells and defines a PTX-IL-1-Bhlhe40 pathway active in EAE.

Bhlhe40 controls cytokine production by T cells and is essential for pathogenicity in autoimmune neuroinflammation.

TH1 and TH17 cells mediate neuroinflammation in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. Pathogenic TH cells in EAE must produce the pro-inflammatory cytokine granulocyte-macrophage colony stimulating factor (GM-CSF). TH cell pathogenicity in EAE is also regulated by cell-intrinsic production of the immunosuppressive cytokine interleukin 10 (IL-10). Here we demonstrate that mice deficient for the basic helix-loop-helix (bHLH) transcription factor Bhlhe40 (Bhlhe40(-/-)) are resistant to the induction of EAE. Bhlhe40 is required in vivo in a T cell-intrinsic manner, where it positively regulates the production of GM-CSF and negatively regulates the production of IL-10. In vitro, GM-CSF secretion is selectively abrogated in polarized Bhlhe40(-/-) TH1 and TH17 cells, and these cells show increased production of IL-10. Blockade of IL-10 receptor in Bhlhe40(-/-) mice renders them susceptible to EAE. These findings identify Bhlhe40 as a critical regulator of autoreactive T-cell pathogenicity.

Batf3-dependent CD11b(low/-) peripheral dendritic cells are GM-CSF-independent and are not required for Th cell priming after subcutaneous immunization.

Dendritic cells (DCs) subsets differ in precursor cell of origin, functional properties, requirements for growth factors, and dependence on transcription factors. Lymphoid-tissue resident CD8α(+) conventional DCs (cDCs) and CD11b(low/-)CD103(+) non-lymphoid DCs are developmentally related, each being dependent on FMS-like tyrosine kinase 3 ligand (Flt3L), and requiring the transcription factors Batf3, Irf8, and Id2 for development. It was recently suggested that granulocyte/macrophage colony stimulating factor (GM-CSF) was required for the development of dermal CD11b(low/-)Langerin(+)CD103(+) DCs, and that this dermal DC subset was required for priming autoreactive T cells in experimental autoimmune encephalitis (EAE). Here, we compared development of peripheral tissue DCs and susceptibility to EAE in GM-CSF receptor deficient (Csf2rb(-/-)) and Batf3(-/-) mice. We find that Batf3-dependent dermal CD11b(low/-)Langerin(+) DCs do develop in Csf2rb(-/-) mice, but that they express reduced, but not absent, levels of CD103. Further, Batf3(-/-) mice lacking all peripheral CD11b(low/-) DCs show robust Th cell priming after subcutaneous immunization and are susceptible to EAE. Our results suggest that defective T effector priming and resistance to EAE exhibited by Csf2rb(-/-) mice does not result from the absence of dermal CD11b(low/-)Langerin(+)CD103(+) DCs.

CD8α(+) dendritic cells are an obligate cellular entry point for productive infection by Listeria monocytogenes.

CD8α(+) dendritic cells (DCs) prime cytotoxic T lymphocytes during viral infections and produce interleukin-12 in response to pathogens. Although the loss of CD8α(+) DCs in Batf3(-/-) mice increases their susceptibility to several pathogens, we observed that Batf3(-/-) mice exhibited enhanced resistance to the intracellular bacterium Listeria monocytogenes. In wild-type mice, Listeria organisms, initially located in the splenic marginal zone, migrated to the periarteriolar lymphoid sheath (PALS) where they grew exponentially and induced widespread lymphocyte apoptosis. In Batf3(-/-) mice, however, Listeria organisms remain trapped in the marginal zone, failed to traffic into the PALS, and were rapidly cleared by phagocytes. In addition, Batf3(-/-) mice, which lacked the normal population of hepatic CD103(+) peripheral DCs, also showed protection from liver infection. These results suggest that Batf3-dependent CD8α(+) and CD103(+) DCs provide initial cellular entry points within the reticuloendothelial system by which Listeria establishes productive infection.