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Interleukin (IL)-37, an antiinflammatory IL-1 family cytokine, is a key suppressor of innate immunity. IL-37 signaling requires the heterodimeric IL-18R1 and IL-1R8 receptor, which is abundantly expressed in the gastrointestinal tract. Here we report a 4-mo-old male from a consanguineous family with a homozygous loss-of-function IL37 mutation. The patient presented with persistent diarrhea and was found to have infantile inflammatory bowel disease (I-IBD). Patient cells showed increased intracellular IL-37 expression and increased proinflammatory cytokine production. In cell lines, mutant IL-37 was not stably expressed or properly secreted and was thus unable to functionally suppress proinflammatory cytokine expression. Furthermore, induced pluripotent stem cell–derived macrophages from the patient revealed an activated macrophage phenotype, which is more prone to lipopolysaccharide and IL-1β stimulation, resulting in hyperinflammatory tumor necrosis factor production. Insights from this patient will not only shed light on monogenic contributions of I-IBD but may also reveal the significance of the IL-18 and IL-37 axis in colonic homeostasis.

Apoptosis is a genetically regulated program of cell death that plays a key role in immune disease processes. We identified EBF4, a little-studied member of the early B cell factor (EBF) family of transcription factors, in a whole-genome CRISPR screen for regulators of Fas/APO-1/CD95-mediated T cell death. Loss of EBF4 increases the half-life of the c-FLIP protein, and its presence in the Fas signaling complex impairs caspase-8 cleavage and apoptosis. Transcriptome analysis revealed that EBF4 regulates molecules such as TBX21, EOMES, granzyme, and perforin that are important for human natural killer (NK) and CD8+T cell functions. Proximity-dependent biotin identification (Bio-ID) mass spectrometry analyses showed EBF4 binding to STAT3, STAT5, and MAP kinase 3 and a strong pathway relationship to interleukin-2 regulated genes, which are known to govern cytotoxicity pathways. Chromatin immunoprecipitation and DNA sequencing analysis defined a canonical EBF4 binding motif, 5′-CCCNNGG/AG-3′, closely related to the EBF1 binding site; using a luciferase-based reporter, we found a dose-dependent transcriptional response of this motif to EBF4. We also conducted assay for transposase-accessible chromatin sequencing in EBF4-overexpressing cells and found increased chromatin accessibility upstream of granzyme and perforin and in topologically associated domains in human lymphocytes. Finally, we discovered that the EBF4 has basal expression in human but not mouse NK cells and CD8+T cells and vanishes following activating stimulation. Together, our data reveal key features of a previously unknown transcriptional regulator of human cytotoxic immune function.

Clustered regularly interspaced short palindromic repeats (CRISPR)–based methods have revolutionized genome engineering and the study of gene-phenotype relationships. However, modifying cells of the innate immune system, especially macrophages, has been challenging because of cell pathology and low targeting efficiency resulting from nucleic acid activation of intracellular sensors. Likewise, lymphocytes of the adaptive immune system are difficult to modify using CRISPR-enhanced homology-directed repair because of inefficient or toxic delivery of donor templates using transient transfection methods. To overcome these challenges and limitations, we modified existing tools and developed three alternative methods for CRISPR-based genome editing using a hit-and-run transient expression strategy, together with a convenient system for promoting gene expression using CRISPRa. Overall, our CRISPR tools and strategies designed to tackle both murine and human immune cell genome engineering provide efficient alternatives to existing methods and have wide application not only in terms of hematopoietic cells but also other mammalian cell types of interest.

Inborn errors of immunity (IEIs) unveil regulatory pathways of human immunity. We describe a new IEI caused by mutations in the GTPase of the immune-associated protein 6 (GIMAP6) gene in patients with infections, lymphoproliferation, autoimmunity, and multiorgan vasculitis. Patients and Gimap6−/− mice show defects in autophagy, redox regulation, and polyunsaturated fatty acid (PUFA)–containing lipids. We find that GIMAP6 complexes with GABARAPL2 and GIMAP7 to regulate GTPase activity. Also, GIMAP6 is induced by IFN-γ and plays a critical role in antibacterial immunity. Finally, we observed that Gimap6−/− mice died prematurely from microangiopathic glomerulosclerosis most likely due to GIMAP6 deficiency in kidney endothelial cells.

Intestinal mucus forms the first line of defense against bacterial invasion while providing nutrition to support microbial symbiosis. How the host controls mucus barrier integrity and commensalism is unclear. We show that terminal sialylation of glycans on intestinal mucus by ST6GALNAC1 (ST6), the dominant sialyltransferase specifically expressed in goblet cells and induced by microbial pathogen-associated molecular patterns, is essential for mucus integrity and protecting against excessive bacterial proteolytic degradation. Glycoproteomic profiling and biochemical analysis of ST6 mutations identified in patients show that decreased sialylation causes defective mucus proteins and congenital inflammatory bowel disease (IBD). Mice harboring a patient ST6 mutation have compromised mucus barriers, dysbiosis, and susceptibility to intestinal inflammation. Based on our understanding of the ST6 regulatory network, we show that treatment with sialylated mucin or a Foxo3 inhibitor can ameliorate IBD.

The balance between gut microbiota and host is critical for maintaining host health. Although dysregulation of the gut microbiota triggers the development of various inflammatory diseases, including colitis, the molecular mechanism of microbiota-driven colitis development is largely unknown. Here, we found that gasdermin D (GSDMD) was activated during acute colitis. In the dextran sulfate sodium (DSS)-induced colitis model, compared to wild-type mice, Gsdmd-deficient mice had less colitis severity. Mechanistically, GSDMD expression in intestinal epithelial cells (IECs), but not infiltrating immune cells, was critical for GSDMD-mediated colitis progression. Moreover, commensal Escherichia coli (E. coli) largely overgrew during colitis, and then the dysregulated commensal E. coli mediated GSDMD activation. Furthermore, the activated GSDMD promoted the release of interleukin-18 (IL-18), but not the transcript or maturation level of IL-18, which in turn mediated goblet cell loss to induce colitis development. Thus, GSDMD promotes colitis development by mediating IL-18 release, and the microbiota can mediate colitis pathogenesis through regulation of GSDMD activation. Our results provide a potential molecular mechanism by which the microbiota-driven GSDMD activation contributes to colitis pathogenesis.

Necroptosis is a recently defined type of programmed cell death with the specific signaling cascade of receptor-interacting protein 1 (RIPK1) and RIPK3 complex to activate the executor MLKL. However, the pathophysiological roles of necroptosis are largely unexplored. Here, we report that fungus triggers myeloid cell necroptosis and this type of cell death contributes to host defense against the pathogen infection. Candida albicans as well as its sensor Dectin-1 activation strongly induced necroptosis in myeloid cells through the RIPK1-RIPK3-MLKL cascade. CARD9, a key adaptor in Dectin-1 signaling, was identified to bridge the RIPK1 and RIPK3 complex-mediated necroptosis pathway. RIPK1 and RIPK3 also potentiated Dectin-1-induced MLKL-independent inflammatory response. Both the MLKL-dependent and MLKL-independent pathways were required for host defense against C. albicans infection. Thus, our study demonstrates a new type of host defense system against fungal infection.

We report a pleiotropic disease due to loss-of-function mutations in RHBDF2, the gene encoding iRHOM2, in two kindreds with recurrent infections in different organs. One patient had recurrent pneumonia but no colon involvement, another had recurrent infectious hemorrhagic colitis but no lung involvement and the other two experienced recurrent respiratory infections. Loss of iRHOM2, a rhomboid superfamily member that regulates the ADAM17 metalloproteinase, caused defective ADAM17-dependent cleavage and release of cytokines, including tumor-necrosis factor and amphiregulin. To understand the diverse clinical phenotypes, we challenged Rhbdf2

Background TRAF3 interacting protein 2 (TRAF3IP2) (Act1) is an adapter protein that interacts with IL-17R via its similar expression to fibroblast growth factor genes and IL-17R domain and coordinates 2 separate proinflammatory pathways following IL-17 cytokine stimulation. Objective We sought to elucidate the immunologic consequences of TRAF3IP2 homozygous mutations to improve treatments for immunodeficiency patients with chronic mucocutaneous candidiasis. Methods We describe 2 patients presenting with chronic mucocutaneous candidiasis who harbor biallelic nonsense mutations in TRAF3IP2. The cellular and molecular features of this genetic defect were assessed using in vitro cytokine assays and protein analysis. Results We show that the homozygous mutation causes complete loss of protein expression. We also show that the absence of TRAF3IP2 was associated with a defective response to combined IL-2/IL-25 (IL-17E) stimulation. Conclusions Failure to initiate normal signaling downstream of IL-17R engagement likely contributes to the patients’ recurrent fungal infections. These findings add to our molecular understanding of genetic defects affecting this critical pathway of antifungal immunity.

The connection between innate and adaptive immunity is best exemplified by antigen presentation. Although antigen-presenting cells (APCs) are required for antigen receptor-mediated T-cell activation, how T-cells feedback to APCs to sustain an antigen-specific immune response is not completely clear. Here we show that CD8+ T-cell (also called cytotoxic T lymphocytes, CTL) feedback activates the NLRP3 inflammasome in APCs in an antigen-dependent manner to promote IL-1β maturation. Perforin from antigen-specific CTLs is required for NLRP3 inflammasome activation in APCs. Furthermore, such activation of NLRP3 inflammasome contributes to the induction of antigen-specific antitumour immunity and pathogenesis of graft-versus-host diseases. Our study reveals a positive feedback loop between antigen-specific CTLs and APC to amplify adaptive immunity., Perforin is part of the cytotoxic effector mechanism of CD8+ T cells. Here the authors show that antigen-induced perforin release from CD8 T cells into antigen-presenting cells can activate NLRP3 inflammasome to constitute a positive feedback loop to promote anti-tumour immunity and allo-responses.

Immune cells and mesenchymal stem/stromal cells are the major cellular components in tumor microenvironment that actively migrate to tumor sites by sensing "signals" released from tumor cells. Together with other stromal cells, they form the soil for malignant cell progression. In the crosstalk between tumor cells and its surrounded microenvironment, exosomes exert multiple functions in shaping tumor immune responses. In tumor cells, their exosomes can lead to pro-tumor immune responses, whereas in immune cells, their derived exosomes can operate on tumor cells and regulate their ability to growth, metastasis, even reaction to chemotherapy. Employing exosomes as vehicles for the delivery products to initiate anti-tumor immune responses has striking therapeutic effects on tumor progression. Thus, exosomes are potential therapeutic targets in tumor-related clinical conditions. Here we discuss the role of exosomes in regulating tumor immune microenvironment and future indications for the clinical application of exosomes.

Cells use mitophagy to remove damaged or unwanted mitochondria to maintain homeostasis. Here we report that the intracellular bacterial pathogen Listeria monocytogenes exploits host mitophagy to evade killing. We found that L. monocytogenes induced mitophagy in macrophages through the virulence factor listeriolysin O (LLO). We discovered that NLRX1, the only Nod-like receptor (NLR) family member with a mitochondrial targeting sequence, contains an LC3-interacting region (LIR) and directly associated with LC3 through the LIR. NLRX1 and its LIR motif were essential for L. monocytogenes-induced mitophagy. NLRX1 deficiency and use of a mitophagy inhibitor both increased mitochondrial production of reactive oxygen species and thereby suppressed the survival of L. monocytogenes. Mechanistically, L. monocytogenes and LLO induced oligomerization of NLRX1 to promote binding of its LIR motif to LC3 for induction of mitophagy. Our study identifies NLRX1 as a novel mitophagy receptor and discovers a previously unappreciated strategy used by pathogens to hijack a host cell homeostasis system for their survival.

Melanoma is the most aggressive type of skin cancer. Melanoma has an extremely poor prognosis because of its high potential for vascular invasion, metastasis and recurrence. The mechanism of melanoma metastasis is not well understood. ATP-binding cassette sub-family B member 5 (ABCB5) plays a key role in melanoma growth. However, it is uncertain what function ABCB5 may exert in melanoma metastasis. In this report, we for the first time demonstrate ABCB5 as a crucial factor that promotes melanoma metastasis. ABCB5 positive (ABCB5+) malignant melanoma initiating cells (MMICs) display a higher metastatic potential compared with ABCB5 negative (ABCB5-) melanoma subpopulation. Knockdown of ABCB5 expression reduces melanoma cell migration and invasion in vitro and melanoma pulmonary metastasis in tumor xenograft mice. ABCB5 and NF-κB p65 expression levels are positively correlated in both melanoma tissues and cell lines. Consequently, ABCB5 activates the NF-κB pathway by inhibiting p65 ubiquitination to enhance p65 protein stability. Our finding highlights ABCB5 as a novel pro-metastasis factor and provides a potential therapeutic target for melanoma.

IL-17 is a pro-inflammatory cytokine implicated a variety of autoimmune diseases. We have recently reported that FGF2 cooperates with IL-17 to protect intestinal epithelium during dextran sodium sulfate (DSS)-induced colitis. Here, we report a pathogenic role of the FGF2-IL-17 cooperation in the pathogenesis of autoimmune arthritis. Combined treatment with FGF2 and IL-17 synergistically induced ERK activation as well as the production of cytokines and chemokines in human synovial intimal resident fibroblast-like synoviocytes (FLS). Furthermore, ectopic expression of FGF2 in mouse joints potentiated IL-17-induced inflammatory cytokine and chemokine production in the tissue. In the collagen-induced arthritis (CIA) model, while ectopic expression of FGF2 in vivo exacerbated tissue inflammation and disease symptom in the wild-type controls, the effect was largely blunted in Il17a−/− mice. Taken together, our study suggests that FGF2 cooperates with IL-17 to promote the pathogenesis of autoimmune arthritis by cooperating with IL-17 to induce inflammatory response.

SummaryAlthough the microbiota has been shown to drive production of interleukin-17A (IL-17A) from T helper 17 cells to promote cell proliferation and tumor growth in colorectal cancer, the molecular mechanisms for microbiota-mediated regulation of tumorigenesis are largely unknown. Here, we found that the innate-like cytokine IL-17C was upregulated in human colorectal cancers and in mouse intestinal tumor models. Alterations in the microbiota drove IL-17C upregulation specifically in intestinal epithelial cells (IECs) through Toll-like receptor (TLR)-MyD88-dependent signaling during intestinal tumorigenesis. Microbiota-driven IL-17C induced Bcl-2 and Bcl-xL expression in IECs in an autocrine manner to promote cell survival and tumorigenesis in both chemically induced and spontaneous intestinal tumor models. Thus, IL-17C promotes cancer development by increasing IEC survival, and the microbiota can mediate cancer pathogenesis through regulation of IL-17C.

The NOD like receptors (NLRs), a class of intracellular receptors that respond to pathogen attack or cellular stress, have gained increasing attention. NLRC5, the largest member of the NLR protein family, has recently been identified as a critical regulator of immune responses. While NLRC5 is constitutively and widely expressed, it can be dramatically induced by interferons during pathogen infections. Both in vitro and in vivo studies have demonstrated that NLRC5 is a specific and master regulator of major mistocompatibility complex (MHC) class I genes as well as related genes involved in MHC class I antigen presentation. The expression of MHC class I genes is regulated by NLRC5 in coordination with the RFX components through an enhanceosome-dependent manner. And the involvement of NLRC5 in MHC class I mediated CD8+ T cell activation, proliferation and cytotoxicity is proved to be critical for host defense against intracellular bacterial infections. Nevertheless, the role of NLRC5 in innate immunity remains to be further explored. Here, we review the research advances on the structure, expression regulation and function of NLRC5.

Summary Iron deposition is frequently observed in human autoinflammatory diseases, but its functional significance is largely unknown. Here we showed that iron promoted proinflammatory cytokine expression in T cells, including GM-CSF and IL-2, via regulating the stability of an RNA-binding protein PCBP1. Iron depletion or Pcbp1 deficiency in T cells inhibited GM-CSF production by attenuating Csf2 3′ untranslated region (UTR) activity and messenger RNA stability. Pcbp1 deficiency or iron uptake blockade in autoreactive T cells abolished their capacity to induce experimental autoimmune encephalomyelitis, an animal model for multiple sclerosis. Mechanistically, intracellular iron protected PCBP1 protein from caspase-mediated proteolysis, and PCBP1 promoted messenger RNA stability of Csf2 and Il2 by recognizing UC-rich elements in the 3′ UTRs. Our study suggests that iron accumulation can precipitate autoimmune diseases by promoting proinflammatory cytokine production. RNA-binding protein-mediated iron sensing may represent a simple yet effective means to adjust the inflammatory response to tissue homeostatic alterations.

SummaryThe intestinal epithelial barrier plays a critical role in the mucosal immunity. However, it remains largely unknown how the epithelial barrier is maintained after damage. Here we show that growth factor FGF2 synergized with interleukin-17 (IL-17) to induce genes for repairing of damaged epithelium. FGF2 or IL-17 deficiency resulted in impaired epithelial proliferation, increased pro-inflammatory microbiota outgrowth, and consequently worse pathology in a DSS-induced colitis model. The dysregulated microbiota in the model induced transforming growth factor beta 1 (TGFβ1) expression, which in turn induced FGF2 expression mainly in regulatory T cells. Act1, an essential adaptor in IL-17 signaling, suppressed FGF2-induced ERK activation through binding to adaptor molecule GRB2 to interfere with its association with guanine nucleotide exchange factor SOS1. Act1 preferentially bound to IL-17 receptor complex, releasing its suppressive effect on FGF2 signaling. Thus, microbiota-driven FGF2 and IL-17 cooperate to repair the damaged intestinal epithelium through Act1-mediated direct signaling cross-talk.