Molecular control of steady-state dendritic cell maturation and immune homeostasis.

Dendritic cells (DCs) are specialized sentinels responsible for coordinating adaptive immunity. This function is dependent upon coupled sensitivity to environmental signs of inflammation and infection to cellular maturation-the programmed alteration of DC phenotype and function to enhance immune cell activation. Although DCs are thus well equipped to respond to pathogens, maturation triggers are not unique to infection. Given that immune cells are exquisitely sensitive to the biological functions of DCs, we now appreciate that multiple layers of suppression are required to restrict the environmental sensitivity, cellular maturation, and even life span of DCs to prevent aberrant immune activation during the steady state. At the same time, steady-state DCs are not quiescent but rather perform key functions that support homeostasis of numerous cell types. Here we review these functions and molecular mechanisms of suppression that control steady-state DC maturation. Corruption of these steady-state operatives has diverse immunological consequences and pinpoints DCs as potent drivers of autoimmune and inflammatory disease.

Expression of A20 by dendritic cells preserves immune homeostasis and prevents colitis and spondyloarthritis.

Dendritic cells (DCs), which are known to support immune activation during infection, may also regulate immune homeostasis in resting animals. Here we show that mice lacking the ubiquitin-editing molecule A20 specifically in DCs spontaneously showed DC activation and population expansion of activated T cells. Analysis of DC-specific epistasis in compound mice lacking both A20 and the signaling adaptor MyD88 specifically in DCs showed that A20 restricted both MyD88-independent signals, which drive activation of DCs and T cells, and MyD88-dependent signals, which drive population expansion of T cells. In addition, mice lacking A20 specifically in DCs spontaneously developed lymphocyte-dependent colitis, seronegative ankylosing arthritis and enthesitis, conditions stereotypical of human inflammatory bowel disease (IBD). Our findings indicate that DCs need A20 to preserve immune quiescence and suggest that A20-dependent DC functions may underlie IBD and IBD-associated arthritides.

Irgm1-deficiency leads to myeloid dysfunction in colon lamina propria and susceptibility to the intestinal pathogen Citrobacter rodentium.

IRGM and its mouse orthologue Irgm1 are dynamin-like proteins that regulate vesicular remodeling, intracellular microbial killing, and pathogen immunity. IRGM dysfunction is linked to inflammatory bowel disease (IBD), and while it is thought that defective intracellular killing of microbes underscores IBD susceptibility, studies have yet to address how IRGM/Irgm1 regulates immunity to microbes relevant to intestinal inflammation. Here we find that loss of Irgm1 confers marked susceptibility to Citrobacter rodentium, a noninvasive intestinal pathogen that models inflammatory responses to intestinal bacteria. Irgm1-deficient mice fail to control C. rodentium outgrowth in the intestine, leading to systemic pathogen spread and host mortality. Surprisingly, susceptibility due to loss of Irgm1 function was not linked to defective intracellular killing of C. rodentium or exaggerated inflammation, but was instead linked to failure to remodel specific colon lamina propria (C-LP) myeloid cells that expand in response to C. rodentium infection and are essential for C. rodentium immunity. Defective immune remodeling was most striking in C-LP monocytes, which were successfully recruited to the infected C-LP, but subsequently underwent apoptosis. Apoptotic susceptibility was induced by C. rodentium infection and was specific to this setting of pathogen infection, and was not apparent in other settings of intestinal inflammation. These studies reveal a novel role for Irgm1 in host defense and suggest that deficiencies in survival and remodeling of C-LP myeloid cells that control inflammatory intestinal bacteria may underpin IBD pathogenesis linked to IRGM dysfunction.

Dimerization and ubiquitin mediated recruitment of A20, a complex deubiquitinating enzyme.

A20 is an anti-inflammatory protein linked to multiple human autoimmune diseases and lymphomas. A20 possesses a deubiquitinating motif and a zinc finger, ZF4, that binds ubiquitin and supports its E3 ubiquitin ligase activity. To understand how these activities mediate A20's physiological functions, we generated two lines of gene-targeted mice, abrogating either A20's deubiquitinating activity (Tnfaip3(OTU) mice) or A20's ZF4 (Tnfaip3(ZF4) mice). Both Tnfaip3(OTU) and Tnfaip3(ZF4) mice exhibited increased responses to TNF and sensitivity to colitis. A20's C103 deubiquitinating motif restricted both K48- and K63-linked ubiquitination of receptor interacting protein 1 (RIP1). A20's ZF4 was required for recruiting A20 to ubiquitinated RIP1. A20(OTU) proteins and A20(ZF4) proteins complemented each other to regulate RIP1 ubiquitination and NFκB signaling normally in compound mutant Tnfaip3(OTU/ZF4) cells. This complementation involved homodimerization of A20 proteins, and we have defined an extensive dimerization interface in A20. These studies reveal how A20 proteins collaborate to restrict TNF signaling.

A20 in Myeloid Cells Protects Against Hypertension by Inhibiting Dendritic Cell-Mediated T-Cell Activation.

RATIONALE: The ubiquitin-editing protein A20 in dendritic cells (DCs) suppresses NF-κB (nuclear factor-κB) signaling and constrains DC-mediated T-cell stimulation, but the role of A20 in modulating the hypertensive response requires elucidation. OBJECTIVE: Here, we tested the hypothesis that A20 in CD11c-expressing myeloid cells mitigates Ang II (angiotensin II)-induced hypertension by limiting renal T-cell activation. METHODS AND RESULTS: Mice with heterozygous deletion of A20 in CD11c-expressing myeloid cells (DC ACT[Cd11c-Cre+A20flox/wt]) have spontaneous DC activation but have normal baseline blood pressures. In response to low-dose chronic Ang II infusion, DC ACT mice compared with WT (wild type) controls had an exaggerated hypertensive response and augmented proportions of CD62LloCD44hi effector memory T lymphocytes in the kidney lymph node. After 10 days of Ang II, DC ACT kidneys had increased numbers of memory effector CD8+, but not CD4+ T cells, compared with WTs. Moreover, the expressions of TNF-α (tumor necrosis factor-α) and IFN-γ (interferon-γ) were upregulated in the DC ACT renal CD8+ T cells but not CD4+ T cells. Saline challenge testing revealed enhanced renal fluid retention in the DC ACT mice. DC ACT kidneys showed augmented protein expression of γ-epithelial sodium channel and NHE3 (sodium-hydrogen antiporter 3). DC ACT mice also had greater reductions in renal blood flow following acute injections with Ang II and enhanced oxidant stress in the vasculature as evidenced by higher circulating levels of malondialdehyde compared with WT controls. To directly test whether enhanced T-cell activation in the DC ACT cohort was responsible for their exaggerated hypertensive response, we chronically infused Ang II into lymphocyte-deficient DC ACT Rag1 (recombination activating protein 1)-deficient (Rag1-/-) mice and WT (Cd11c-Cre-A20flox/wt) Rag1-/- controls. The difference in blood pressure elevation accruing from DC activation was abrogated on the Rag1-/- strain. CONCLUSIONS: Following stimulation of the renin-angiotensin system, A20 suppresses DC activation and thereby mitigates T-cell-dependent blood pressure elevation.

The Ubiquitin-Modifying Enzyme A20 Terminates C-Type Lectin Receptor Signals and Is a Suppressor of Host Defense against Systemic Fungal Infection.

C-type lectin receptors (CLRs) play key roles in antifungal defense. CLR-induced NF-κB is central to CLR functions in immunity, and thus, molecules that control the amplitude of CLR-induced NF-κB could profoundly influence host defense against fungal pathogens. However, little is known about the mechanisms that negatively regulate CLR-induced NF-κB, and molecules which act on the CLR family broadly and which directly regulate acute CLR-signaling cascades remain unidentified. Here, we identify the ubiquitin-editing enzyme A20 as a negative regulator of acute NF-κB activation downstream of multiple CLR pathways. Absence of A20 suppression results in exaggerated CLR responses in cells which are A20 deficient and also cells which are A20 haplosufficient, including multiple primary immune cells. Loss of a single allele of A20 results in enhanced defense against systemic Candida albicans infection and prolonged host survival. Thus, A20 restricts CLR-induced innate immune responses in vivo and is a suppressor of host defense against systemic fungal infection.

Cutting edge: ABIN-1 protects against psoriasis by restricting MyD88 signals in dendritic cells.

Psoriasis is a chronic, inflammatory skin disease caused by a combination of environmental and genetic factors. The Tnip1 gene encodes A20 binding and inhibitor of NF-κB-1 (ABIN-1) protein and is strongly associated with susceptibility to psoriasis in humans. ABIN-1, a widely expressed ubiquitin-binding protein, restricts TNF- and TLR-induced signals. In this study, we report that mice lacking ABIN-1 specifically in dendritic cells (DCs), ABIN-1(fl) CD11c-Cre mice, exhibit perturbed immune homeostasis. ABIN-1-deficient DCs display exaggerated NF-κB and MAPK signaling and produce more IL-23 than do normal cells in response to TLR ligands. Challenge of ABIN-1(fl) CD11c-Cre mice with topical TLR7 ligand leads to greater numbers of Th17 and TCRγδ T cells and exacerbated development of psoriaform lesions. These phenotypes are reversed by DC-specific deletion of the TLR adaptor MyD88. These studies link ABIN-1 with IL-23 and IL-17, and they provide cellular and molecular mechanisms by which ABIN-1 regulates susceptibility to psoriasis.

A binary module for microbiota-mediated regulation of γδ17 cells, hallmarked by microbiota-driven expression of programmed cell death protein 1.

Little is known about how microbiota regulate innate-like γδ T cells or how these restrict their effector functions within mucosal barriers, where microbiota provide chronic stimulation. Here, we show that microbiota-mediated regulation of γδ17 cells is binary, where microbiota instruct in situ interleukin-17 (IL-17) production and concomitant expression of the inhibitory receptor programmed cell death protein 1 (PD-1). Microbiota-driven expression of PD-1 and IL-17 and preferential adoption of a PD-1high phenotype are conserved for γδ17 cells across multiple mucosal barriers. Importantly, microbiota-driven PD-1 inhibits in situ IL-17 production by mucosa-resident γδ17 effectors, linking microbiota to their simultaneous activation and suppression. We further show the dynamic nature of this microbiota-driven module and define an inflammation-associated activation state for γδ17 cells marked by augmented PD-1, IL-17, and lipid uptake, thus linking the microbiota to dynamic subset-specific activation and metabolic remodeling to support γδ17 effector functions in a microbiota-dense tissue environment.

Corrigendum: A Requirement of Protein Geranylgeranylation for Chemokine Receptor Signaling and Th17 Cell Function in an Animal Model of Multiple Sclerosis.

[This corrects the article DOI: 10.3389/fimmu.2021.641188.].

A Requirement of Protein Geranylgeranylation for Chemokine Receptor Signaling and Th17 Cell Function in an Animal Model of Multiple Sclerosis.

Precisely controlled lymphocyte migration is critically required for immune surveillance and successful immune responses. Lymphocyte migration is strictly regulated by chemokines and chemokine receptors. Here we show that protein geranylgeranylation, a form of post-translational protein lipid modification, is required for chemokine receptor-proximal signaling. Mature thymocytes deficient for protein geranylgeranylation are impaired for thymus egress. Circulating mature T cells lacking protein geranylgeranylation fail to home to secondary lymphoid organs or to transmigrate in response to chemokines in vitro. Mechanistically, protein geranylgeranylation modifies the γ-subunits of the heterotrimeric small GTPases that are essential for chemokine receptor signaling. In addition, protein geranylgeranylation also promotes the differentiation of IL-17-producing T helper cells while inhibiting the differentiation of Foxp3+ regulatory T cells. Finally, mice with T cell lineage-specific deficiency of protein geranylgeranylation are resistant to experimental autoimmune encephalomyelitis induction. This study elucidated a critical role of protein geranylgeranylation in regulating T lymphocyte migration and function.

Th17 Immunity in the Colon Is Controlled by Two Novel Subsets of Colon-Specific Mononuclear Phagocytes.

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.

C-C Motif Chemokine Receptor 7 Exacerbates Hypertension Through Effects on T Lymphocyte Trafficking.

Activated T lymphocytes that infiltrate blood pressure control organs make a critical contribution to the pathogenesis of hypertension. Dendritic cells act as potent antigen-presenting cells to stimulate prohypertensive T cells. However, the mechanisms that facilitate the recruitment of prohypertensive T cells and dendritic cells into the kidney's draining lymph node during hypertension require elucidation. As CCR7 (C-C motif chemokine receptor type 7) directs the homing of lymphocytes and dendritic cells into lymph nodes, we posited that dendritic cell-mediated T lymphocyte stimulation in the renal lymph node is CCR7 dependent and required for a full hypertensive response. We found that CCR7-deficient (CCR7 KO) mice had a blunted hypertensive response in our model of chronic Ang II (angiotensin II) infusion. Ang II-infused CCR7 KO animals had exaggerated accumulation of CD8+ T cells in the kidney but reduced numbers of CD4+ and CD8+ T cells in the kidney's draining lymph node. To understand whether CCR7-dependent homing of T lymphocytes or dendritic cells into the lymph node regulates the hypertensive response, we injected CCR7 KO or wild-type T cells or dendritic cells into CCR7 KO recipients, neither of which restored the full hypertensive response to Ang II infusion. However, adoptive transfer of wild-type but not CCR7 KO T lymphocytes into RAG1 (recombination-activating gene 1)-deficient mice that lack a lymphocyte niche restored full blood pressure elevation during Ang II infusion. Thus, CCR7-dependent interactions between T lymphocytes and dendritic cells are essential for T lymphocyte stimulation and hypertension accruing from inappropriate activation of the renin-angiotensin system.

Control of antiviral innate immune response by protein geranylgeranylation.

The mitochondrial antiviral signaling protein (MAVS) orchestrates host antiviral innate immune response to RNA virus infection. However, how MAVS signaling is controlled to eradicate virus while preventing self-destructive inflammation remains obscure. Here, we show that protein geranylgeranylation, a posttranslational lipid modification of proteins, limits MAVS-mediated immune signaling by targeting Rho family small guanosine triphosphatase Rac1 into the mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) at the mitochondria-ER junction. Protein geranylgeranylation and subsequent palmitoylation promote Rac1 translocation into MAMs upon viral infection. MAM-localized Rac1 limits MAVS' interaction with E3 ligase Trim31 and hence inhibits MAVS ubiquitination, aggregation, and activation. Rac1 also facilitates the recruitment of caspase-8 and cFLIPL to the MAVS signalosome and the subsequent cleavage of Ripk1 that terminates MAVS signaling. Consistently, mice with myeloid deficiency of protein geranylgeranylation showed improved survival upon influenza A virus infection. Our work revealed a critical role of protein geranylgeranylation in regulating antiviral innate immune response.

Opportunities and Challenges for Single-Unit Recordings from Enteric Neurons in Awake Animals.

Advanced electrode designs have made single-unit neural recordings commonplace in modern neuroscience research. However, single-unit resolution remains out of reach for the intrinsic neurons of the gastrointestinal system. Single-unit recordings of the enteric (gut) nervous system have been conducted in anesthetized animal models and excised tissue, but there is a large physiological gap between awake and anesthetized animals, particularly for the enteric nervous system. Here, we describe the opportunity for advancing enteric neuroscience offered by single-unit recording capabilities in awake animals. We highlight the primary challenges to microelectrodes in the gastrointestinal system including structural, physiological, and signal quality challenges, and we provide design criteria recommendations for enteric microelectrodes.

Salmonella Activation of STAT3 Signaling by SarA Effector Promotes Intracellular Replication and Production of IL-10.

Salmonella enterica is an important foodborne pathogen that uses secreted effector proteins to manipulate host pathways to facilitate survival and dissemination. Different S. enterica serovars cause disease syndromes ranging from gastroenteritis to typhoid fever and vary in their effector repertoire. We leveraged this natural diversity to identify stm2585, here designated sarA (Salmonella anti-inflammatory response activator), as a Salmonella effector that induces production of the anti-inflammatory cytokine IL-10. RNA-seq of cells infected with either ΔsarA or wild-type S. Typhimurium revealed that SarA activates STAT3 transcriptional targets. Consistent with this, SarA is necessary and sufficient for STAT3 phosphorylation, STAT3 inhibition blocks IL-10 production, and SarA and STAT3 interact by co-immunoprecipitation. These effects of SarA contribute to intracellular replication in vitro and bacterial load at systemic sites in mice. Our results demonstrate the power of using comparative genomics for identifying effectors and that Salmonella has evolved mechanisms for activating an important anti-inflammatory pathway.

Inflammatory Th1 and Th17 in the Intestine Are Each Driven by Functionally Specialized Dendritic Cells with Distinct Requirements for MyD88.

Normal dynamics between microbiota and dendritic cells (DCs) support modest numbers of T cells, yet these do not cause inflammation. The DCs that induce inflammatory T cells and the signals that drive this process remain unclear. Here, we demonstrate that small intestine DCs lacking the signaling attenuator A20 induce inflammatory T cells and that the signals perceived and antigen-presenting cell (APC) functions are unique for different DC subsets. Thus, although CD103+CD11b- DCs exclusively instruct IFNγ+ T cells, CD103+CD11b+ DCs exclusively instruct IL-17+ T cells. Surprisingly, APC functions of both DC subsets are upregulated in a MyD88-independent fashion. In contrast, CD103-CD11b+ DCs instruct both IFNγ+ and IL-17+ T cells, and only the IL-17-inducing APC functions require MyD88. In disease pathogenesis, both CD103-CD11b+ and CD103+CD11b+ DCs expand pathologic Th17 cells. Thus, in disease pathogenesis, specific DCs instruct specific inflammatory T cells.

Autophagy enhances NFκB activity in specific tissue macrophages by sequestering A20 to boost antifungal immunity.

Immune responses must be well restrained in a steady state to avoid excessive inflammation. However, such restraints are quickly removed to exert antimicrobial responses. Here we report a role of autophagy in an early host antifungal response by enhancing NFκB activity through A20 sequestration. Enhancement of NFκB activation is achieved by autophagic depletion of A20, an NFκB inhibitor, in F4/80(hi) macrophages in the spleen, peritoneum and kidney. We show that p62, an autophagic adaptor protein, captures A20 to sequester it in the autophagosome. This allows the macrophages to release chemokines to recruit neutrophils. Indeed, mice lacking autophagy in myeloid cells show higher susceptibility to Candida albicans infection due to impairment in neutrophil recruitment. Thus, at least in the specific aforementioned tissues, autophagy appears to break A20-dependent suppression in F4/80(hi) macrophages, which express abundant A20 and contribute to the initiation of efficient innate immune responses.

The final touches make perfect the peptide-MHC class I repertoire.

Major histocompatibility complex (MHC) class I molecules present short, perfectly cleaved peptides on the cell surface for immune surveillance by CD8(+) T cells. The pathway for generating these peptides begins in the cytoplasm, and the peptide-MHC I (pMHC I) repertoire is finalized in the endoplasmic reticulum. Recent studies show that the peptides for MHC I are customized by the ER aminopeptidase associated with antigen processing and by dynamic interactions within the MHC peptide-loading complex. Failure to customize the pMHC I repertoire has profound immunological consequences.