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Regulatory T (T reg ) cells are critical in preventing aberrant immune responses. Posttranscriptional control of gene expression by microRNA (miRNA) has recently emerged as an essential genetic element for T reg cell function. Here, we report that mice with T reg cell–specific ablation of miR-142 (hereafter Foxp3 Cre miR-142 fl/fl mice) developed a fatal systemic autoimmune disorder due to a breakdown in peripheral T-cell tolerance. Foxp3 Cre miR-142 fl/fl mice displayed a significant decrease in the abundance and suppressive capacity of T reg cells. Expression profiling of miR-142 –deficient T reg cells revealed an up-regulation of multiple genes in the interferon gamma (IFNγ) signaling network. We identified several of these IFNγ-associated genes as direct miR-142-3p targets and observed excessive IFNγ production and signaling in miR-142 –deficient T reg cells. Ifng ablation rescued the T reg cell homeostatic defect and alleviated development of autoimmunity in Foxp3 Cre miR-142 fl/fl mice. Thus, our findings implicate miR-142 as an indispensable regulator of T reg cell homeostasis that exerts its function by attenuating IFNγ responses.

Neutrophils are critical as the first-line defense against fungal pathogens. Yet, previous studies indicate that neutrophil function is complex during Cryptococcus neoformans (Cn) infection. To better understand the role of neutrophils in acute pulmonary cryptococcosis, we analyzed neutrophil heterogeneity by single-cell transcriptional analysis of immune cells in the lung of Cn -infected mice from a published dataset. We identified neutrophils by reference-based annotation and identified two distinct neutrophil subsets generated during acute Cn infection: A subset with an oxidative stress signature (Ox-PMN) and another with enhanced cytokine gene expression (Cyt-PMN). Based on gene regulatory network and ligand-receptor analysis, we hypothesize that Ox-PMNs interact with the fungus and generate ROS, while Cyt-PMNs are longer-lived neutrophils that indirectly respond to Cn -derived ligands and cytokines to modulate cell-cell communication with dendritic cells and alveolar macrophages. Based on the data, we hypothesized that, during in vivo fungal infection, there is a division of labor in which each activated neutrophil becomes either Ox-PMN or Cyt-PMN.

Myelin oligodendrocyte glycoprotein (MOG) antibody-associated disease (MOGAD) is a severe, autoantibody-mediated neuroinflammatory syndrome that disproportionately impacts children. Detection of conformation-specific anti-MOG IgG1 in the serum is central to MOGAD diagnosis, yet the pathogenic role of these antibodies remains unclear. We sought to develop a clinically informed model of pediatric MOGAD to study the immunopathogenesis that recapitulates 1) early age of disease onset, 2) anti-MOG IgG1 in serum, and 3) widespread inflammation in the CNS. We employed two approaches to model MOGAD using anti-MOG IgG in mice: 1) treating young C57BL/6 mice with the murine-derived monoclonal MOG antibody 8-18C5, and 2) using young IgH transgenic (Tg) mice, engineered to express the immunoglobulin heavy chain of 8-18C5. These mice were induced with experimental autoimmune encephalomyelitis (EAE) by immunizing with MOG to elicit inflammatory demyelination. Both exogenous and endogenous anti-MOG IgG exacerbated EAE disease in young mice. Compared to wild-type littermates (+/+), young IgH Tg mice (Tg/+) exhibited reduced peripheral immune cells and greater neutrophil-to-lymphocyte ratios. Tg/+ mice also had increased CNS leukocyte infiltration compared to wild-type littermates. Tg/+ mice developed monophasic circumferential longitudinal myelitis, bilateral optic neuritis, and multifocal brain inflammation. Innate immune cells in the CNS, including microglia, showed significant downregulation of surface FcγRII/III. This downregulation on microglia was accompanied by enhanced uptake of cells expressing MOG, suggesting Fc receptor-mediated internalization of MOG immune complexes. Introduction of anti-MOG IgG in EAE recapitulates key aspects of pediatric MOGAD and enables dissection of the mechanisms underlying anti-MOG antibody-mediated immunopathogenesis.

The ability to sense, import, and detoxify copper (Cu) has been shown to be crucial for microbial pathogens to survive within an infected host. Previous studies conducted with the opportunistic human fungal pathogen Cryptococcus neoformans (Cn) have revealed two extreme Cu environments encountered during infection: a high Cu environment within the lung, and a low Cu environment within the brain. However, how Cn senses these different host Cu microenvironments and the consequences of a blunted Cu stress adaptation for pathogenesis are not well understood. In contrast to ascomycete model fungi, the basidiomycete Cn has a single transcription factor (TF), CnCuf1, to regulate adaptive responses to both high and low Cu stress. Sequence comparison with other fungal Cu-responsive TFs identified three conserved cysteine (Cys)-rich motifs located within the CnCuf1 N-terminal domain, which were therefore predicted to play a role in Cu sensing. Mutation of these conserved Cys-rich motifs demonstrated that the 1st Cys-rich motif is functionally relevant for CnCuf1 transcriptional activity during high Cu stress, while it is dispensable for low Cu stress adaptation. An inhalation model of murine infection showed that strains with defective high Cu stress regulation present a distinct and anatomically constrained pattern of yeast distribution within the infected lungs compared to a more widespread infection observed in lungs infected with the wild-type strain. Based on these findings, we hypothesize that Cuf1-driven high Cu responses modulate not absolute fitness, but the containment of Cn cells at the initial site of infection within the lung.IMPORTANCECopper is an essential micronutrient required for survival in all kingdoms of life, as it is used as a catalytic cofactor for many essential processes in the cell. In turn, this reactivity of copper ions makes elevated levels of free copper toxic to the cell. This dual nature of copper—essential for life but toxic at elevated levels—is used by our innate immune system in a process called nutritional immunity to combat and kill invading pathogens. In this work, we explore how the fungal human pathogen Cryptococcus neoformans senses high copper stress, a copper microenvironment encountered within the host lung. We identified a specific cysteine-rich motif within the copper-responsive transcription factor Cuf1 to be essential for high copper stress sensing. Mutation of this motif led to an impaired high copper stress adaptation, which did not affect the fitness of the yeast but did impact the containment and distribution of yeast cells inside the host lung.

Intestinal immunity is coordinated by specialized mononuclear phagocyte populations, constituted by a diversity of cell subsets. Although the cell subsets constituting the mononuclear phagocyte network are thought to be similar in both small and large intestine, these organs have distinct anatomy, microbial composition, and immunological demands. Whether these distinctions demand organ-specific mononuclear phagocyte populations with dedicated organ-specific roles in immunity are unknown. Here we implement a new strategy to subset murine intestinal mononuclear phagocytes and identify two novel subsets which are colon-specific: a macrophage subset and a Th17-inducing dendritic cell (DC) subset. Colon-specific DCs and macrophages co-expressed CD24 and CD14, and surprisingly, both were dependent on the transcription factor IRF4. Novel IRF4-dependent CD14 + CD24 + macrophages were markedly distinct from conventional macrophages and failed to express classical markers including CX3CR1, CD64 and CD88, and surprisingly expressed little IL-10, which was otherwise robustly expressed by all other intestinal macrophages. We further found that colon-specific CD14 + CD24 + mononuclear phagocytes were essential for Th17 immunity in the colon, and provide definitive evidence that colon and small intestine have distinct antigen presenting cell requirements for Th17 immunity. Our findings reveal unappreciated organ-specific diversity of intestine-resident mononuclear phagocytes and organ-specific requirements for Th17 immunity.

microRNA-146a (miR-146a) has been previously implicated as an essential molecular brake, preventing immune overreaction and malignant transformation by attenuating NF-κB signaling, putatively via repression of the Traf6 and Irak1 genes. The exact contribution of miR-146a–mediated silencing of these genes to the control of immune activation is currently unknown. Therefore, we defined the role of the miR-146a–Traf6 signaling axis in the regulation of immune homeostasis using a genetic epistasis analysis in miR-146a −/− mice. We have uncovered a surprising separation of functions at the level of miR-146a targets. Lowering the Traf6 gene dose and consequent attenuation of NF-κB activation rescued several significant miR-146a −/− phenotypes, such as splenomegaly, aberrant myeloproliferation, and excessive inflammatory responses. In contrast, decreasing Traf6 expression had no effect on the development of the progressive bone marrow failure phenotype, as well as lymphomagenesis in miR-146a −/− mice, indicating that miR-146a controls these biological processes through different molecular mechanisms.

Copper is an essential micronutrient required for survival in all kingdoms of life, as it is used as a catalytic cofactor for many essential processes in the cell. In turn, this reactivity of copper ions makes elevated levels of free copper toxic to the cell. This dual nature of copper—essential for life but toxic at elevated levels—is used by our innate immune system in a process called nutritional immunity to combat and kill invading pathogens. In this work, we explore how the fungal human pathogen Cryptococcus neoformans senses high copper stress, a copper microenvironment encountered within the host lung. We identified a specific cysteine-rich motif within the copper-responsive transcription factor Cuf1 to be essential for high copper stress sensing. Mutation of this motif led to an impaired high copper stress adaptation, which did not affect the fitness of the yeast but did impact the containment and distribution of yeast cells inside the host lung.

Regulatory T (T.sub.reg) cells are critical in preventing aberrant immune responses. Posttranscriptional control of gene expression by microRNA (miRNA) has recently emerged as an essential genetic element for T.sub.reg cell function. Here, we report that mice with T.sub.reg cell-specific ablation of miR-142 (hereafter Foxp3.sup.Cre miR-142.sup.fl/fl mice) developed a fatal systemic autoimmune disorder due to a breakdown in peripheral T-cell tolerance. Foxp3.sup.Cre miR-142.sup.fl/fl mice displayed a significant decrease in the abundance and suppressive capacity of T.sub.reg cells. Expression profiling of miR-142-deficient T.sub.reg cells revealed an up-regulation of multiple genes in the interferon gamma (IFN[gamma]) signaling network. We identified several of these IFN[gamma]-associated genes as direct miR-142-3p targets and observed excessive IFN[gamma] production and signaling in miR-142-deficient T.sub.reg cells. Ifng ablation rescued the T.sub.reg cell homeostatic defect and alleviated development of autoimmunity in Foxp3.sup.Cre miR-142.sup.fl/fl mice. Thus, our findings implicate miR-142 as an indispensable regulator of T.sub.reg cell homeostasis that exerts its function by attenuating IFN[gamma] responses.

An immune engram is a recently described phenomenon in which neuronal populations encode functional aspects of an immune challenge. Here we investigate an immune engram arising from respiratory infection with influenza A virus, demonstrating a molecular mechanism with differential influence over behavioral and immunological aspects of the engram. We first define a cellular response to acute non-neurotropic influenza A/Puerto Rico/8/1934 (PR8) infection by mapping cFos+ cells and microglia morphology across brain regions. In the posterior insula, this response has an early peak at 3 days post infection. Using a cre-dependent excitatory chemogenetic system in TRAP2 mice, we capture an engram at this same region and infection timepoint. Activation of this PR8 engram results in anxiety behavior and increased transcriptional expression of cytokines in lung tissue but not spleen tissue. We further explore how pulmonary signals contribute to this PR8 engram. Using tissue-specific, cre-dependent expression of diphtheria toxin fragment in mice, we ablate expressing cells including pulmonary neuroendocrine cells in respiratory tissue. Loss of -expressing cells prevents changes in synaptic engulfment by microglia in the insula during PR8 infection without altering the cellular response to infection in pulmonary tissue. Signaling of calcitonin gene related peptide (CGRP), a peptide encoded by , can be blocked with the small molecule CGRP receptor antagonist rimegepant. Using rimegepant during acute PR8 infection we again demonstrate that loss of signaling prevents the cellular response to PR8 infection in the insula. Finally, applying rimegepant alongside the chemogenetic system in TRAP2 mice we show that CGRP receptor antagonism during engram formation prevents anxiety behavior but not peripheral gene expression changes resulting from PR8 engram activation.

The ability to sense, import and detoxify copper (Cu) has been shown to be crucial for microbial pathogens to survive within an infected host. Previous studies conducted with the opportunistic human fungal pathogen Cryptococcus neoformans (Cn) have revealed two extreme Cu environments encountered during infection: a high Cu environment within the lung and a low Cu environment within the brain. However, how Cn senses these different host Cu microenvironments, and the consequences of a blunted Cu stress adaptation for pathogenesis are not well understood. In contrast to ascomycete model fungi, the basidiomycete Cn has a single transcription factor (TF), CnCuf1, to regulate adaptive responses to both high- and low- Cu stress. Sequence comparison with other fungal Cu-responsive TFs identified three conserved cysteine (Cys)-rich motifs located within the CnCuf1 N-terminal domain, which were therefore predicted to play a role in Cu sensing. Mutation of these conserved Cys-rich motifs demonstrated that the 1st Cys-rich motif is functionally relevant for CnCuf1 transcriptional activity during high Cu stress, while it is dispensable for low Cu stress adaptation. An inhalation model of murine infection showed that strains with defective high Cu stress regulation present a distinct and anatomically constrained pattern of yeast distribution within the infected lungs compared to a more widespread infection observed in lungs infected with the wild-type strain. Based on these findings, we hypothesize that Cuf1-driven high Cu responses modulate not absolute fitness but containment of Cn cells at the initial site of infection within the lung. Copper is an essential micronutrient required for survival in all kingdoms of life as it is used as a catalytic cofactor for many essential processes in the cell. In turn, this reactivity of copper ions makes elevated levels of free copper toxic for the cell. This dual nature of copper – essential for life but toxic at elevated levels – is used by our innate immune system in a process called nutritional immunity to combat and kill invading pathogens. In this work we explore how the fungal human pathogen Cryptococcus neoformans senses high copper stress, a copper microenvironment encountered within the host lung. We identified a specific cysteine-rich motif within the copper responsive transcription factor Cuf1 to be essential for high copper stress sensing. Mutation of this motif led to an impaired high copper stress adaptation, which did not affect fitness of the yeast but did impact containment and distribution of yeast cells inside the host lung.