Group 3 innate lymphoid cells continuously require the transcription factor GATA-3 after commitment.

The transcription factor GATA-3 is indispensable for the development of all innate lymphoid cells (ILCs) that express the interleukin 7 receptor α-chain (IL-7Rα). However, the function of low GATA-3 expression in committed group 3 ILCs (ILC3 cells) has not been identified. We found that GATA-3 regulated the homeostasis of ILC3 cells by controlling IL-7Rα expression. In addition, GATA-3 served a critical function in the development of the NKp46(+) ILC3 subset by regulating the balance between the transcription factors T-bet and RORγt. Among NKp46(+) ILC3 cells, although GATA-3 positively regulated genes specific to the NKp46(+) ILC3 subset, it negatively regulated genes specific to lymphoid tissue-inducer (LTi) or LTi-like ILC3 cells. Furthermore, GATA-3 was required for IL-22 production in both ILC3 subsets. Thus, despite its low expression, GATA-3 was critical for the homeostasis, development and function of ILC3 subsets.

Innate and adaptive lymphocytes sequentially shape the gut microbiota and lipid metabolism.

The mammalian gut is colonized by numerous microorganisms collectively termed the microbiota, which have a mutually beneficial relationship with their host. Normally, the gut microbiota matures during ontogeny to a state of balanced commensalism marked by the absence of adverse inflammation. Subsets of innate lymphoid cells (ILCs) and conventional T cells are considered to have redundant functions in containment and clearance of microbial pathogens, but how these two major lymphoid-cell populations each contribute to shaping the mature commensal microbiome and help to maintain tissue homeostasis has not been determined. Here we identify, using advanced multiplex quantitative imaging methods, an extensive and persistent phosphorylated-STAT3 signature in group 3 ILCs and intestinal epithelial cells that is induced by interleukin (IL)-23 and IL-22 in mice that lack CD4+ T cells. By contrast, in immune-competent mice, phosphorylated-STAT3 activation is induced only transiently by microbial colonization at weaning. This early signature is extinguished as CD4+ T cell immunity develops in response to the expanding commensal burden. Physiologically, the persistent IL-22 production from group 3 ILCs that occurs in the absence of adaptive CD4+ T-cell activity results in impaired host lipid metabolism by decreasing lipid transporter expression in the small bowel. These findings provide new insights into how innate and adaptive lymphocytes operate sequentially and in distinct ways during normal development to establish steady-state commensalism and tissue metabolic homeostasis.

B cell residency but not T cell-independent IgA switching in the gut requires innate lymphoid cells.

Immunoglobulin A (IgA)-producing plasma cells derived from conventional B cells in the gut play an important role in maintaining the homeostasis of gut flora. Both T cell-dependent and T cell-independent IgA class switching occurs in the lymphoid structures in the gut, whose formation depends on lymphoid tissue inducers (LTis), a subset of innate lymphoid cells (ILCs). However, our knowledge on the functions of non-LTi helper-like ILCs, the innate counter parts of CD4 T helper cells, in promoting IgA production is still limited. By cell adoptive transfer and utilizing a unique mouse strain, we demonstrated that the generation of IgA-producing plasma cells from B cells in the gut occurred efficiently in the absence of both T cells and helper-like ILCs and without engaging TGF-ß signaling. Nevertheless, B cell recruitment and/or retention in the gut required functional NKp46-CCR6+ LTis. Therefore, while CCR6+ LTis contribute to the accumulation of B cells in the gut through inducing lymphoid structure formation, helper-like ILCs are not essential for the T cell-independent generation of IgA-producing plasma cells.

ECM1 controls T(H)2 cell egress from lymph nodes through re-expression of S1P(1).

Type 2 helper T cells (T(H)2) are critically involved in allergies and asthma. Here we demonstrate that extracellular matrix protein-1 (ECM1) is highly and selectively expressed in T(H)2 cells. ECM1 deficiency caused impaired T(H)2 responses and reduced allergic airway inflammation in vivo. Functional analysis demonstrated that although the T(H)2 polarization of ECM1-deficient cells was unimpaired, these cells had a defect in migration and were retained in peripheral lymphoid organs. This was associated with reduced expression of KLF2 and S1P(1). We also found that ECM1 could directly bind the interleukin-2 (IL-2) receptor to inhibit IL-2 signaling and activate S1P(1) expression. Our data identify a previously unknown function of ECM1 in regulating T(H)2 cell migration through control of KLF2 and S1P(1) expression.

Thinking differently about ILCs-Not just tissue resident and not just the same as CD4+ T-cell effectors.

Innate lymphoid cells (ILCs) resemble adaptive T lymphocytes based on transcription factor expression, cytokine production, and their presumptive roles in immunity, but are activated for effector function through cytokine signaling and not antigen-specific receptors. The prevailing view is that ILCs adapt to specific microenvironments during development and operate as tissue-resident cells in co-operation with antigen-specific T cells to provide host protection and contribute to tissue maintenance. In particular, conventional models equate the activity of different ILC subsets with CD4+ effector T-cell types based on corresponding transcription factor expression and a potential for comparable cytokine production. Based on recent data from our laboratory, we suggest that these views on tissue residence and parallel functioning to CD4+ T cells are too restrictive. Our findings show that ILC2s can be mobilized from the gut under inflammatory conditions and contribute to distal immunity in the lungs during infection, whereas gut-resident ILC3s operate in a quite distinct manner from Th17 CD4+ effector cells in responding to commensal microbes, with important implications for control of metabolic homeostasis. In this review, we discuss the recent advances leading to these revised views of ILC inter-organ trafficking and the distinct and complementary function of ILCs with respect to adaptive T cells in establishing and maintaining a physiologic host environment.

TRIM30 alpha negatively regulates TLR-mediated NF-kappa B activation by targeting TAB2 and TAB3 for degradation.

Toll-like receptor (TLR) signaling is pivotal to innate and adaptive immune responses and must be tightly controlled. The mechanisms of TLR signaling have been the focus of extensive studies. Here we report that the tripartite-motif protein TRIM30alpha, a RING protein, was induced by TLR agonists and interacted with the TAB2-TAB3-TAK1 adaptor-kinase complex involved in the activation of transcription factor NF-kappaB. TRIM30alpha promoted the degradation of TAB2 and TAB3 and inhibited NF-kappaB activation induced by TLR signaling. In vivo studies showed that transfected or transgenic mice overexpressing TRIM30alpha were more resistant to endotoxic shock. Consistent with that, in vivo 'knockdown' of TRIM30alpha mRNA by small interfering RNA impaired lipopolysaccharide-induced tolerance. Finally, expression of TRIM30alpha depended on NF-kappaB activation. Our results collectively indicate that TRIM30alpha negatively regulates TLR-mediated NF-kappaB activation by targeting degradation of TAB2 and TAB3 by a 'feedback' mechanism.

ß-arrestin1 is critical for the full activation of NLRP3 and NLRC4 inflammasomes.

Inflammasomes are multiprotein complexes that trigger the activation of caspase-1 and the maturation of IL-1ß, which are critical for inflammation and control of pathogen infection. Although the function of inflammasomes in immune response and disease development is well studied, the molecular mechanism by which inflammasomes are activated and assembled remains largely unknown. In this study, we found that ß-arrestin1, a key regulator of the G protein-coupled receptor signaling pathway, was required for nucleotide-binding domain and leucine-rich repeat containing (NLR) family pyrin domain-containing 3 (NLRP3) and NLR family CARD domain-containing protein 4 (NLRC4) inflammasome-mediated IL-1ß production and caspase-1 activation, but it had no effect on absent in melanoma 2 (AIM2) inflammasome activation. Moreover, apoptosis-associated speck-like protein containing a CARD (ASC) pyroptosome, which is ASC aggregation mediating caspase-1 activation, was also impaired in ß-arrestin1-deficient macrophages upon NLRP3 or NLRC4, but not AIM2 inflammasome activation. Mechanistic study revealed that ß-arrestin1 specifically interacted with NLRP3 and NLRC4 and promoted their self-oligomerization. In vivo, in a monosodium urate crystal (MSU)-induced NLRP3-dependent peritonitis model, MSU-induced IL-1ß production and neutrophil flux were significantly reduced in ß-arrestin1 knockout mice. Additionally, ß-arrestin1 deficiency rescued the weight loss of mice upon log-phase Salmonella typhimurium infection, with less IL-1ß production. Taken together, our results indicate that ß-arrestin1 plays a critical role in the assembly and activation of two major canonical inflammasomes, and it may provide a new therapeutic target for inflammatory diseases.

Negative regulation of Nmi on virus-triggered type I IFN production by targeting IRF7.

Viral infection causes host cells to produce type I IFNs, which play a critical role in viral clearance. IFN regulatory factor (IRF) 7 is the master regulator of type I IFN-dependent immune responses. In this article, we report that N-Myc and STATs interactor (Nmi), a Sendai virus-inducible protein, interacted with IRF7 and inhibited virus-triggered type I IFN production. The overexpression of Nmi inhibited the Sendai virus-triggered induction of type I IFNs, whereas the knockdown of Nmi promoted IFN production. Furthermore, the enhanced production of IFNs resulting from Nmi knockdown was sufficient to protect cells from infection by vesicular stomatitis virus. In addition, Nmi was found to promote the K48-linked ubiquitination of IRF7 and the proteasome-dependent degradation of this protein. Finally, an impairment of antiviral responses is also detectable in Nmi-transgenic mice. These findings suggest that Nmi is a negative regulator of the virus-triggered induction of type I IFNs that targets IRF7.

Transcription factor TCF-1 regulates the functions, but not the development, of lymphoid tissue inducer subsets in different tissues.

Lymphoid tissue inducer (LTi) cells, a subset of innate lymphoid cells (ILCs), play an essential role in the formation of secondary lymphoid tissues. However, the regulation of the development and functions of this ILC subset is still elusive. In this study, we report that the transcription factor T cell factor 1 (TCF-1), just as GATA3, is indispensable for the development of non-LTi ILC subsets. While LTi cells are still present in TCF-1-deficient mice, the organogenesis of Peyer's patches (PPs), but not of lymph nodes, is impaired in these mice. LTi cells from different tissues have distinct gene expression patterns, and TCF-1 regulates the expression of lymphotoxin specifically in PP LTi cells. Mechanistically, TCF-1 may directly and/or indirectly regulate Lta, including through promoting the expression of GATA3. Thus, the TCF-1-GATA3 axis, which plays an important role during T cell development, also critically regulates the development of non-LTi cells and tissue-specific functions of LTi cells.

The glycolysis/HIF-1α axis defines the inflammatory role of IL-4-primed macrophages.

T helper type 2 (Th2) cytokine-activated M2 macrophages contribute to inflammation resolution and wound healing. This study shows that IL-4-primed macrophages exhibit a stronger response to lipopolysaccharide stimulation while maintaining M2 signature gene expression. Metabolic divergence between canonical M2 and non-canonical proinflammatory-prone M2 (M2INF) macrophages occurs after the IL-4Rα/Stat6 axis. Glycolysis supports Hif-1α stabilization and proinflammatory phenotype of M2INF macrophages. Inhibiting glycolysis blunts Hif-1α accumulation and M2INF phenotype. Wdr5-dependent H3K4me3 mediates the long-lasting effect of IL-4, with Wdr5 knockdown inhibiting M2INF macrophages. Our results also show that the induction of M2INF macrophages by IL-4 intraperitoneal injection and transferring of M2INF macrophages confer a survival advantage against bacterial infection in vivo. In conclusion, our findings highlight the previously neglected non-canonical role of M2INF macrophages and broaden our understanding of IL-4-mediated physiological changes. These results have immediate implications for how Th2-skewed infections could redirect disease progression in response to pathogen infection.

The transcription factor Zeb1 controls homeostasis and function of type 1 conventional dendritic cells.

Type 1 conventional dendritic cells (cDC1) are the most efficient cross-presenting cells that induce protective cytotoxic T cell response. However, the regulation of their homeostasis and function is incompletely understood. Here we observe a selective reduction of splenic cDC1 accompanied by excessive cell death in mice with Zeb1 deficiency in dendritic cells, rendering the mice more resistant to Listeria infection. Additionally, cDC1 from other sources of Zeb1-deficient mice display impaired cross-presentation of exogenous antigens, compromising antitumor CD8+ T cell responses. Mechanistically, Zeb1 represses the expression of microRNA-96/182 that target Cybb mRNA of NADPH oxidase Nox2, and consequently facilitates reactive-oxygen-species-dependent rupture of phagosomal membrane to allow antigen export to the cytosol. Cybb re-expression in Zeb1-deficient cDC1 fully restores the defective cross-presentation while microRNA-96/182 overexpression in Zeb1-sufficient cDC1 inhibits cross-presentation. Therefore, our results identify a Zeb1-microRNA-96/182-Cybb pathway that controls cross-presentation in cDC1 and uncover an essential role of Zeb1 in cDC1 homeostasis.

Tripartite-motif protein 30 negatively regulates NLRP3 inflammasome activation by modulating reactive oxygen species production.

The NLR family, pyrin domain-containing 3 (NLRP3) inflammasome is critical for caspase-1 activation and the proteolytic processing of pro-IL-1ß. However, the mechanism that regulates NLRP3 inflammasome activation remains unclear. In this paper, we demonstrate that tripartite-motif protein 30 (TRIM30) negatively regulates NLRP3 inflammasome activation. After stimulation with ATP, an agonist of the NLRP3 inflammasome, knockdown of TRIM30 enhanced caspase-1 activation and increased production of IL-1ß in both J774 cells and bone marrow-derived macrophages. Similarly with ATP, knockdown of TRIM30 increased caspase-1 activation and IL-1ß production triggered by other NLRP3 inflammasome agonists, including nigericin, monosodium urate, and silica. Production of reactive oxygen species was increased in TRIM30 knockdown cells, and its increase was required for enhanced NLRP3 inflammasome activation, because antioxidant treatment blocked excess IL-1ß production. Conversely, overexpression of TRIM30 attenuated reactive oxygen species production and NLRP3 inflammasome activation. Finally, in a crystal-induced NLRP3 inflammasome-dependent peritonitis model, monosodium urate-induced neutrophil flux and IL-1ß production was reduced significantly in TRIM30 transgenic mice as compared with that in their nontransgenic littermates. Taken together, our results indicate that TRIM30 is a negative regulator of NLRP3 inflammasome activation and provide insights into the role of TRIM30 in maintaining inflammatory responses.

Dec2 promotes Th2 cell differentiation by enhancing IL-2R signaling.

Th cell differentiation is precisely regulated by thousands of genes at different stages. In the present study, we demonstrate that Dec2, a transcription factor belonging to the bHLH (basic helix-loop-helix) superfamily, is progressively induced during the course of Th2 differentiation, especially at the late stage. The up-regulated Dec2 can strongly promote Th2 development under Th2-inducing conditions, as evidenced by retrovirus-mediated gene transfer or transgenic manipulation. In addition, an enhancement of Th2 responses is also detectable in Dec2 transgenic mice in vivo. Conversely, RNA interference-mediated suppression of endogenous Dec2 could attenuate Th2 differentiation. Finally, we show that the enhanced Th2 development is at least in part due to substantial up-regulation of CD25 expression elicited by Dec2, thereby resulting in hyperresponsiveness to IL-2 stimulation.

Transient T-bet expression functionally specifies a distinct T follicular helper subset.

T follicular helper (Tfh) cells express transcription factor BCL-6 and cytokine IL-21. Mature Tfh cells are also capable of producing IFN-γ without expressing the Th1 transcription factor T-bet. Whether this IFN-γ-producing Tfh population represents a unique Tfh subset with a distinct differentiation pathway is poorly understood. By using T-bet fate-mapping mouse strains, we discovered that almost all the IFN-γ-producing Tfh cells have previously expressed T-bet and express high levels of NKG2D. DNase I hypersensitivity analysis indicated that the Ifng gene locus is partially accessible in this "ex-T-bet" population with a history of T-bet expression. Furthermore, multicolor tissue imaging revealed that the ex-T-bet Tfh cells found in germinal centers express IFN-γ in situ. Finally, we found that IFN-γ-expressing Tfh cells are absent in T-bet-deficient mice, but fully present in mice with T-bet deletion at late stages of T cell differentiation. Together, our findings demonstrate that transient expression of T-bet epigenetically imprints the Ifng locus for cytokine production in this Th1-like Tfh cell subset.

S1P-dependent interorgan trafficking of group 2 innate lymphoid cells supports host defense.

Innate lymphoid cells (ILCs) are innate counterparts of adaptive T lymphocytes, contributing to host defense, tissue repair, metabolic homeostasis, and inflammatory diseases. ILCs have been considered to be tissue-resident cells, but whether ILCs move between tissue sites during infection has been unclear. We show here that interleukin-25- or helminth-induced inflammatory ILC2s are circulating cells that arise from resting ILC2s residing in intestinal lamina propria. They migrate to diverse tissues based on sphingosine 1-phosphate (S1P)-mediated chemotaxis that promotes lymphatic entry, blood circulation, and accumulation in peripheral sites, including the lung, where they contribute to anti-helminth defense and tissue repair. This ILC2 expansion and migration is a behavioral parallel to the antigen-driven proliferation and migration of adaptive lymphocytes to effector sites and indicates that ILCs complement adaptive immunity by providing both local and distant tissue protection during infection.

Cellular localization of NLRP3 inflammasome.

Inflammasome is a large protein complex activated upon cellular stress or microbial infection, which triggers maturation of pro-inflammatory cytokines interleukin-1ß and interleukin-18 through caspase-1 activation. Nod-like receptor family protein 3 (NLRP3) is the most characterized inflammasome activated by various stimuli. However, the mechanism of its activation is unclear and its exact cellular localization is still unknown. We examined the potential co-localization of NLRP3 inflammasome with mitochondria and seven other organelles under adenosine triphosphate, nigericin or monosodium urate stimulation in mouse peritoneal macrophages using confocal microscopy approach. Our results revealed that the activated endogenous apoptosis-associated speck-like protein containing a CARD (ASC) pyroptosome forms in the cytoplasm and co-localizes with NLRP3 and caspase-1, but not with any of the organelles screened. This study indicates that the ASC pyroptosome universally localizes within the cytoplasm rather than with any specific organelles.

Nitric oxide suppresses NLRP3 inflammasome activation and protects against LPS-induced septic shock.

Inflammasomes are multi-protein complexes that trigger the activation of caspase-1 and the maturation of interleukin-1ß (IL-1ß), yet the regulation of these complexes remains poorly characterized. Here we show that nitric oxide (NO) inhibited the NLRP3-mediated ASC pyroptosome formation, caspase-1 activation and IL-1ß secretion in myeloid cells from both mice and humans. Meanwhile, endogenous NO derived from iNOS (inducible form of NO synthase) also negatively regulated NLRP3 inflammasome activation. Depletion of iNOS resulted in increased accumulation of dysfunctional mitochondria in response to LPS and ATP, which was responsible for the increased IL-1ß production and caspase-1 activation. iNOS deficiency or pharmacological inhibition of NO production enhanced NLRP3-dependent cytokine production in vivo, thus increasing mortality from LPS-induced sepsis in mice, which was prevented by NLRP3 deficiency. Our results thus identify NO as a critical negative regulator of the NLRP3 inflammasome via the stabilization of mitochondria. This study has important implications for the design of new strategies to control NLRP3-related diseases.