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We assessed the contribution of IL1 signaling molecules to malignant tumor growth using IL1β−/−, IL1α−/−, and IL1R1−/− mice. Tumors grew progressively in IL1R−/− and IL1α−/− mice but were often absent in IL1β−/− mice. This was observed whether tumors were implanted intradermally or injected intravenously and was true across multiple distinct tumor lineages. Antibodies to IL1β prevented tumor growth in wild-type (WT) mice but not in IL1R1−/− or IL1α−/− mice. Antibodies to IL1α promoted tumor growth in IL1β−/− mice and reversed the tumor-suppressive effect of anti-IL1β in WT mice. Depletion of CD8+ T cells and blockade of lymphocyte mobilization abrogated the IL1β−/− tumor suppressive effect, as did crossing IL1β−/− mice to SCID or Rag1−/− mice. Finally, blockade of IL1β synergized with blockade of PD-1 to inhibit tumor growth in WT mice. These results suggest that IL1β promotes tumor growth, whereas IL1α inhibits tumor growth by enhancing T-cell–mediated antitumor immunity.

Gasdermins, a family of five pore-forming proteins (GSDMA–GSDME) in humans expressed predominantly in the skin, mucosa and immune sentinel cells, are key executioners of inflammatory cell death (pyroptosis), which recruits immune cells to infection sites and promotes protective immunity(1,2). Pore formation is triggered by gasdermin cleavage(1,2). Although the proteases that activate GSDMB, C, D and E have been identified, how GSDMA—the dominant gasdermin in the skin—is activated, remains unknown. Streptococcus pyogenes, also known as group A Streptococcus (GAS), is a major skin pathogen that causes substantial morbidity and mortality worldwide(3). Here we show that the GAS cysteine protease SpeB virulence factor triggers keratinocyte pyroptosis by cleaving GSDMA after Gln246, unleashing an active N-terminal fragment that triggers pyroptosis. Gsdma1 genetic deficiency blunts mouse immune responses to GAS, resulting in uncontrolled bacterial dissemination and death. GSDMA acts as both a sensor and substrate of GAS SpeB and as an effector to trigger pyroptosis, adding a simple one-molecule mechanism for host recognition and control of virulence of a dangerous microbial pathogen.

Background Natural killer T (NKT) cells are unconventional T cells that bridge innate and adaptive immunity. NKT cells have been implicated in the development of atopic dermatitis (AD). Objective We aimed to investigate the role of NKT cells in AD development, especially in skin. Methods Global proteomic and transcriptomic analyses were performed by using skin and blood from human healthy–controls and patients with AD. Levels of CXCR4 and CXCL12 expression in skin NKT cells were analyzed in human AD and mouse AD models. By using parabiosis and intravital imaging, the role of skin CXCR4+ NKT cells was further evaluated in models of mice with AD by using CXCR4–conditionally deficient or CXCL12 transgenic mice. Results CXCR4 and its cognate ligand CXCL12 were significantly upregulated in the skin of humans with AD by global transcriptomic and proteomic analyses. CXCR4+ NKT cells were enriched in AD skin, and their levels were consistently elevated in our models of mice with AD. Allergen-induced NKT cells participate in cutaneous allergic inflammation. Similar to tissue-resident memory T cells, the predominant skin NKT cells were CXCR4+ and CD69+. Skin-resident NKT cells uniquely expressed CXCR4, unlike NKT cells in the liver, spleen, and lymph nodes. Skin fibroblasts were the main source of CXCL12. CXCR4+ NKT cells preferentially trafficked to CXCL12-rich areas, forming an enriched CXCR4+ tissue-resident NKT cells/CXCL12+ cell cluster that developed in acute and chronic allergic inflammation in our models of mice with AD. Conclusions CXCR4+ tissue-resident NKT cells may form a niche that contributes to AD, in which CXCL12 is highly expressed.

We assessed the contribution of IL1 signaling molecules to malignant tumor growth using IL1β−/−, IL1α−/−, and IL1R1−/− mice. Tumors grew progressively in IL1R−/− and IL1α−/− mice but were often absent in IL1β−/− mice. This was observed whether tumors were implanted intradermally or injected intravenously and was true across multiple distinct tumor lineages. Antibodies to IL1β prevented tumor growth in wild-type (WT) mice but not in IL1R1−/− or IL1α−/− mice. Antibodies to IL1α promoted tumor growth in IL1β−/− mice and reversed the tumor-suppressive effect of anti-IL1β in WT mice. Depletion of CD8+ T cells and blockade of lymphocyte mobilization abrogated the IL1β−/− tumor suppressive effect, as did crossing IL1β−/− mice to SCID or Rag1−/− mice. Finally, blockade of IL1β synergized with blockade of PD-1 to inhibit tumor growth in WT mice. These results suggest that IL1β promotes tumor growth, whereas IL1α inhibits tumor growth by enhancing T-cell–mediated antitumor immunity.

INTRODUCTION An inflammatory innate immune response is a first line of host defense against invading pathogens. Inflammation recruits immune cells to the infection site and activates protective adaptive immune responses. Invasive bacteria have evolved multiple ways to interfere with host innate immune signaling to facilitate infection. The Yersinia effector protein YopJ suppresses proinflammatory cytokine production by inhibiting transforming growth factor–β–activated kinase 1 (TAK1) and nuclear factor κB (NF-κB) activation. To counteract this virulence factor, host cells initiate receptor-interacting serine-threonine protein kinase 1 (RIPK1)–dependent caspase-8–directed gasdermin D (GSDMD) cleavage and inflammatory cell death (pyroptosis) when TAK1 is inhibited. However, how the RIPK1–caspase-8–GSDMD axis is instructed during Yersinia infection is unclear. RATIONALE The best-studied mechanism by which pathogens stimulate inflammatory cell death involves triggering cytosolic sensors, called inflammasomes, which activate inflammatory caspases (1/4/5/11) to process proinflammatory cytokines and cause pyroptosis. Inflammatory caspase cleavage of GSDMD causes cell membrane pores that mediate both pyroptosis and proinflammatory cytokine secretion. An alternate pyroptotic pathway, mediated by activation of RIPK1 and caspase-8, is triggered when the YopJ virulence factor secreted during pathogenic Yersinia infection blocks TAK1 activation. To determine the molecular mechanisms underlying Yersinia activation of RIPK1–caspase-8–dependent pyroptosis, we performed a genome-wide CRISPR screen using Cas9-expressing immortalized mouse bone marrow–derived macrophages infected with a genome-wide library of single-guide RNA–encoding lentiviruses. The genomes of cells resistant to caspase-8– or caspase-11–dependent pyroptosis were sequenced to identify the knocked-out genes required for pyroptosis. RESULTS The screen identified multiple genes in the lysosomal membrane–anchored Folliculin (Flcn)–Folliculin-interacting protein 2 (Fnip2)–Rag-Ragulator complex as necessary for caspase-8– but not caspase-11–mediated pyroptosis. Deficiency of Rag-Ragulator complex genes rendered cells highly resistant to TAK1 inhibition–triggered pyroptosis, indicating a critical and unexpected role of the lysosomal membrane–tethered Rag-Ragulator supercomplex in RIPK1-dependent caspase-8–directed pyroptosis. In response to pathogenic Yersinia or its mimic [lipopolysaccharide (LPS) plus TAK1 inhibitor], a Fas-associated death domain (FADD)–RIPK1–caspase-8–containing complex was recruited to lysosomal membrane–tethered Rag-Ragulator. Activation of RIPK1 phosphorylation, caspase-8 activation, and pyroptosis depended on Rag guanosine triphosphatase (GTPase) activity and Rag-Ragulator lysosomal binding but was independent of the mechanistic target of rapamycin complex 1 (mTORC1), a well-known Rag-Ragulator–regulated complex. By contrast, Rag-Ragulator did not regulate canonical or noncanonical inflammasome-triggered pyroptosis. CONCLUSION Our study revealed an instructive role of metabolic signaling in directing TAK1 inhibition–induced pyroptosis during a pathogenic bacterial infection. Rag-Ragulator is a well-known critical regulator of cellular responses to changes in nutrient availability and metabolism. Here, Rag-Ragulator served as a platform for activating a FADD–RIPK1–caspase-8 complex formed in response to Toll-like receptor (TLR) or tumor necrosis factor receptor (TNFR) ligation. Rag GTPase activity was critical for triggering the pathway. The role found here for Rag-Ragulator in pyroptosis expands its known roles in metabolic regulation to include regulation of the response to pathogenic infection. Rag-Ragulator monitors both metabolism and infection to serve as a central hub for helping to decide whether available nutrients are adequate for cell proliferation and if an infected cell should die and send out inflammatory danger signals. Future studies can further explore the conditions that stimulate caspase-8–mediated pyroptosis and provide more mechanistic details of how it is regulated, as well as investigate whether manipulating this pathway could have therapeutic benefit.

Background While the incidence of patients with atopic dermatitis (AD) with atopic march (AM) showing respiratory allergy is steadily rising, the pathomechanism is still unknown. There are currently no biomarkers to predict progression of AM. Methods To explore the mechanism of AM, patients with AD and AM and healthy controls were recruited and RNA microarray, flow cytometry, quantitative real-time polymerase chain reaction, and immunofluorescence staining were performed. We also co-cultured dendritic cells and CD4+ T cells with various Dermatophagoides farinae allergen fractions. Cytokine levels were evaluated using enzyme-linked immunosorbent assay. Findings Both fatty-acid-binding protein 5 (FABP5) and Th17-related genes were more highly expressed in AM. FABP5 knockdown significantly decreased Th17-inducing cytokines in keratinocytes and IL-17A in T cells from AM patients. Further confirmation was obtained using an AM mice model compared to mice without AM. Der f 1, a major D. farinae allergen, increased FABP5 and IL-17A expression in T cells from AM patients. Higher serum FABP5 levels from AM patients were positively correlated with serum IL-17A levels. Interpretation FABP5 expression, possibly enhanced by higher epicutaneous and respiratory sensitization to Der f 1, may directly promote Th17 responses in AD patients with AM. Thus, AM progression can be explained by Th17 reaction induced by FABP5. FABP5 was identified as a potential biomarker in AM. Funding This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (Ministry of Science and ICT; No. NRF-2017R1A2B4009568), grants of the Korean Health Technology R&D Project, Ministry for Health, Welfare & Family Affairs, and the Republic of Korea (HI13C0010, HI14C1324, HI14C1799).

Background β7 integrins are responsible for the efficient recruitment of lymphocytes from the blood and their retention in gut-associated lymphoid tissues. Integrin α4β7 binds MAdCAM-1, mediating rolling adhesion of lymphocytes on blood vessel walls when inactive and firm adhesion when activated, thereby controlling two critical steps of lymphocyte homing to the gut. By contrast, integrin αEβ7 mediates the adhesion of lymphocytes to gut epithelial cells by interacting with E-cadherin. Integrin β7 blocking antibodies have shown efficacy in clinical management of inflammatory bowel disease (IBD); however, fully blocking β7 function leads to the depletion of colonic regulatory T (Treg) cells and exacerbates dextran sulfate sodium (DSS)-induced colitis by evoking aberrant innate immunity, implying its potential adverse effect for IBD management. Thus, a better therapeutic strategy targeting integrin β7 is required to avoid this adverse effect. Results Herein, we inhibited integrin α4β7 activation in vivo by creating mice that carry in their integrin β7 gene a mutation (F185A) which from structural studies is known to lock α4β7 in its resting state. Lymphocytes from β7-F185A knock-in (KI) mice expressed α4β7 integrins that could not be activated by chemokines and showed significantly impaired homing to the gut. The β7-F185A mutation did not inhibit αEβ7 activation, but led to the depletion of αEβ7+ lymphocytes in the spleen and a significantly reduced population of αEβ7+ lymphocytes in the gut of KI mice. β7-F185A KI mice were resistant to T cell transfer-induced chronic colitis, but did not show an increased susceptibility to DSS-induced innate colitis, the adverse effect of fully blocking β7 function. Conclusions Our findings demonstrate that specific inhibition of integrin α4β7 activation is a potentially better strategy than fully blocking α4β7 function for IBD treatment.

Modified Vaccinia Ankara (MVA) was recently approved as a Smallpox vaccine. Transmission of Variola is by respiratory droplets, and MVA delivered by skin scarification (s.s.) protected mice far more effectively against lethal respiratory challenge with VACV than any other route of delivery, and at much lower doses. Comparisons of s.s. with intradermal, subcutaneous or intramuscular routes showed that MVAOVAs.s.-generated T cells were both more abundant and transcriptionally distinct. MVAOVAs.s. produced greater numbers of lung Ova-specific CD8+TRMand was superior in protecting mice against lethal VACVOVArespiratory challenge. Nearly as many lung TRMwere generated with MVAOVAs.s. compared to direct pulmonary immunization with MVAOVA, and both routes vaccination protected mice against lethal pulmonary challenge with VACVOVA. Strikingly, MVAOVAs.s.-generated effector T cells exhibited overlapping gene transcriptional profiles to those generated via direct pulmonary immunization. Overall, our data suggest that heterologous MVA vectors delivered via s.s. are uniquely well-suited as vaccine vectors for respiratory pathogens like COVID-19. In addition, MVA delivered via s.s. could represent a more effective dose-sparing smallpox vaccine.

Additional file 1: Figure S1. The secondary lymphoid tissues other than GALT appear normal in β7-F185A KI and β7-KO mice. Representative histological sections of the peripheral lymph node (PLN), mesenteric lymph node (MLN) and spleen (SP) of WT, β7-F185A KI (KI) and β7-KO (KO) mice were analyzed by hematoxylin and eosin staining. Scale bars, 500μm. Figure S2. Expression of integrins β7 in splenic lymphocytes of WT and β7-F185A mice. (A) Quantitative PCR analysis of integrin β7 expression in splenic lymphocytes from WT and KI mice. Results are normalized to GAPDH. (B) Total (cell surface plus intracellular) protein expression of integrin β7 in splenic lymphocytes was determined by flow cytometry using permeabilized cells. Data are mean ± s.d. of at least 3 independent experiments (A-B). Figure S3. Impaired adhesion and transmigration of Jurkat T-β7 F185A cells. (A) Expession of β7 and CCR9 in Jurkat T-β7 WT and Jurkat T-β7 F185A cell lines were determined by flow cytometry. (B) Adhesion of Jurkat T-β7 WT, Jurkat T-β7 F185A and Jurkat T cells to MAdCAM-1 substrates at 1 dyn/cm2 or 2 dyn/cm2 before and after chemokine stimulation. (C) Transmigration of Jurkat T-β7 WT, Jurkat T-F185A and Jurkat T cells through MAdCAM-1-coated insert. Data are mean ± s.d. of at least 3 independent experiments (B C). *** P < 0.001; ns, not significant (Student’s t-test). Asterisk in B indicates the changes of total adherent cells. Figure S4. β7-F185A mutation does not affect αEβ7-mediated cell adhesion to E-cadherin substrates. (A) Expession of β7 and αE in Jurkat T-αEβ7 WT and Jurkat T-αEβ7 F185A cell lines were determined by flow cytometry. The numbers within the panels show the specific mean fluorescence intensities of FIB504 (anti-β7) and M290 (anti-αE) mAbs. (B) Adhesion of Jurkat T-αEβ7 WT, Jurkat T-αEβ7 F185A and Jurkat T cells to the immobilized E-cadherin substrates (40 μg/ml) at 1dyn/cm2 before and after chemokine stimulation. αEβ7-E-cadherin binding was inhibited by pre-treatment of cells with 10 μg/ml αE blocking antibody M290. Data are mean ± s.d. of at least 3 independent experiments (A-B).*** P < 0.001; ns, not significant (Student’s t-test). Figure S5. Integrin αE+ lymphocytes in spleen, SI and colon. (A) Expression of αE in WT, +/-, KI and KO splenic lymphocytes was determined by flow cytometry. (B-C) Expression of αE in intestinal IEL and LPL was detected by flow cytometry. The numbers within the panels (A-B) show the percentage of αE+ lymphocytes. The numbers within the table (C) show the specific mean fluorescence intensities of M290 (anti-αE) mAb in αE+ lymphocytes. Data are mean ± s.d. of at least 3 independent experiments (A-C). AAP < 0.05; AAAP < 0.001 (Student's t-test in C). SP, spleen; SI, small intestine; IEL, intraepithelial lymphocyte; LPL, lamina propria lymphocyte.

Additional file 2: Table S1. The analysis of integrin β7-high splenic lymphocytes subsets in WT mice. Table S2. The list of primers used for genotyping. Table S3. The list of primers used for real-time quantitative PCR analyses

Background: While incidence of patients with atopic dermatitis (AD) with atopic march (AM) showing respiratory allergy is steadily rising, the pathomachanism is still unclear. Methods: To explore the mechanism of AM, RNA microarray, flow cytometry, quantitative real-time polymerase chain reaction, and immunofluorescence staining were performed to compare the skin, lungs, and T cells from two groups; patients and mice with AD and AM versus AD without AM. We also co-cultured dendritic cells and CD4+ T cells with various Dermatophagoides farinae allergen fractions. Cytokine levels were evaluated using enzyme-linked immunosorbent assay. Findings: Fatty-acid-binding protein 5 (FABP5) was more highly expressed in AM patients and Th17-related genes were also strongly associated with AM patients. Correspondingly, FABP5 knockdown significantly decreased Th17-inducing cytokines in keratinocytes and IL-17A in T cells from AM patients. Further confirmation was done by using AM mice models compared to those without AM. Der f 1, a major D. farinae allergen, increased FABP5 and IL-17A expression in T cells from AM patients. Higher serum FABP5 levels from AM patients were positively correlated with serum IL-17A levels. Interpretation: FABP5 expression, possibly enhanced by higher epicutaneous and respiratory sensitization to Der f 1, may directly promote Th17 responses in AD patients with AM. Funding Statement: This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (Ministry of Science and ICT; No. NRF-2017R1A2B4009568), grants of the Korean Health Technology R&D Project, Ministry for Health, Welfare & Family Affairs, and the Republic of Korea (HI13C0010, HI14C1324, HI14C1799). Declaration of Interests: The authors state no conflict of interest. Ethics Approval Statement: Informed consent was obtained from all subjects, and the protocols were approved by the Institutional Review Board (no. 4-2013-0624). All animal procedures were approved by the Institutional Animal Care and Use Committee of the Department of Laboratory Animal Resources, Yonsei Biomedical Research Institute, Yonsei University College of Medicine (IACUC No. 2016-0007).

Tissue-resident memory T (TRM) cells persist indefinitely in epithelial barrier tissues and protect the host against pathogens. However, the biological pathways that enable the long-term survival of TRM cells are obscure. Here we show that mouse CD8+ TRM cells generated by viral infection of the skin differentially express high levels of several molecules that mediate lipid uptake and intracellular transport, including fatty-acid-binding proteins 4 and 5 (FABP4 and FABP5). We further show that T-cell-specific deficiency of Fabp4 and Fabp5 (Fabp4/Fabp5) impairs exogenous free fatty acid (FFA) uptake by CD8+ TRM cells and greatly reduces their long-term survival in vivo, while having no effect on the survival of central memory T (TCM) cells in lymph nodes. In vitro, CD8+ TRM cells, but not CD8+ TCM cells, demonstrated increased mitochondrial oxidative metabolism in the presence of exogenous FFAs; this increase was not seen in Fabp4/Fabp5 double-knockout CD8+ TRM cells. The persistence of CD8+ TRM cells in the skin was strongly diminished by inhibition of mitochondrial FFA β-oxidation in vivo. Moreover, skin CD8+ TRM cells that lacked Fabp4/Fabp5 were less effective at protecting mice from cutaneous viral infection, and lung Fabp4/Fabp5 double-knockout CD8+ TRM cells generated by skin vaccinia virus (VACV) infection were less effective at protecting mice from a lethal pulmonary challenge with VACV. Consistent with the mouse data, increased FABP4 and FABP5 expression and enhanced extracellular FFA uptake were also demonstrated in human CD8+ TRM cells in normal and psoriatic skin. These results suggest that FABP4 and FABP5 have a critical role in the maintenance, longevity and function of CD8+ TRM cells, and suggest that CD8+ TRM cells use exogenous FFAs and their oxidative metabolism to persist in tissue and to mediate protective immunity.

Cytosolic DNA triggers innate immune responses through the activation of cyclic GMP–AMP synthase (cGAS) and production of the cyclic dinucleotide second messenger 2′,3′-cyclic GMP–AMP (cGAMP)1–4. 2′,3′-cGAMP is a potent inducer of immune signalling; however, no intracellular nucleases are known to cleave 2′,3′-cGAMP and prevent the activation of the receptor stimulator of interferon genes (STING)5–7. Here we develop a biochemical screen to analyse 24 mammalian viruses, and identify poxvirus immune nucleases (poxins) as a family of 2′,3′-cGAMP-degrading enzymes. Poxins cleave 2′,3′-cGAMP to restrict STING-dependent signalling and deletion of the poxin gene (B2R) attenuates vaccinia virus replication in vivo. Crystal structures of vaccinia virus poxin in pre- and post-reactive states define the mechanism of selective 2′,3′-cGAMP degradation through metal-independent cleavage of the 3′–5′ bond, converting 2′,3′-cGAMP into linear Gp[2′–5′]Ap[3′]. Poxins are conserved in mammalian poxviruses. In addition, we identify functional poxin homologues in the genomes of moths and butterflies and the baculoviruses that infect these insects. Baculovirus and insect host poxin homologues retain selective 2′,3′-cGAMP degradation activity, suggesting an ancient role for poxins in cGAS–STING regulation. Our results define poxins as a family of 2′,3′-cGAMP-specific nucleases and demonstrate a mechanism for how viruses evade innate immunity. Poxvirus immune nucleases (poxins) degrade 2′,3′-cyclic GMP–AMP that is produced by cyclic GMP–AMP synthase (cGAS) to evade the innate immune system of the host.

Ubiquitin is a versatile molecular signature that modulates diverse cellular processes via proteasome-dependent and proteasome-independent mechanisms. The covalent and/or non-covalent binding of mono-ubiquitin and/or poly-ubiquitin chains to a target protein broadens the dynamic and functional spectra for signal integration. Different linkages of poly-ubiquitin chains determine specific physiological or pathological functions of target proteins. Accumulating evidences has revealed the essential roles of ubiquitination in orchestrating the host defenses against cytosolic RNA or DNA from viral infections. In this review, we summarize the current progress regarding the understanding of ubiquitin-mediated regulation of the RIG-I and STING antiviral signaling pathways and discuss certain critical issues that remain to be resolved in future studies.

BACKGROUND: Candida albicans is a dimorphic fungus to which humans are exposed early in life and by adulthood it is part of the mycobiome of skin and other tissues. Neonatal skin lacks resident memory T (T(RM)) cells, but in adults the C. albicans skin test is a surrogate for immunocompetence. Young adult mice raised under SPF conditions are naive to C. albicans, and have recently been shown to have an immune system resembling that of neonatal humans. OBJECTIVE: We studied the evolution of the adaptive cutaneous immune response to Candida. METHODS: We examined both human skin T cells and the de novo and memory immune responses in a mouse model of C. albicans skin infection. RESULTS: In mice, the initial IL-17 producing cells after C. albicans infection were dermal γδ T cells, but by day 7 αβ Th17 T effector cells were predominant. By day 30, the majority of C. albicans reactive IL-17 producing T cells were CD4 T(RM) cells. Intravital microscopy showed that CD4 effector T cells were recruited to the site of primary infection and were highly motile 10 days post infection. Between 30–90 days post infection, these CD4 T cells became increasingly sessile, acquired expression of CD69 and CD103, and localized to the papillary dermis. These established T(RM) produced IL-17 upon challenge, while motile migratory memory T (T(MM)) cells did not. T(RM) rapidly clear an infectious challenge with C. albicans more effectively than re-circulating T cells, though both populations participate. We found that in normal human skin, IL-17 producing CD4+ T(RM) that responded to C. albicans in an MHC Class II restricted fashion could be readily identified. CONCLUSIONS: These studies demonstrate that C. albicans infection of skin preferentially generates CD4+ IL-17 producing T(RM), which mediate durable protective immunity.

SummaryImmune surveillance and host defense depend on the precisely regulated trafficking of lymphocytes. Integrin α4β7 mediates lymphocyte homing to the gut through its interaction with mucosal vascular address in cell adhesion molecule-1 (MAdCAM-1). α4β7 also binds vascular cell adhesion molecule-1 (VCAM-1), which is expressed in other tissues. To maintain the tissue specificity of lymphocyte homing, α4β7 must distinguish one ligand from the other. Here, we demonstrate that α4β7 is activated by different chemokines in a ligand-specific manner. CCL25 stimulation promotes α4β7-mediated lymphocyte adhesion to MAdCAM-1 but suppresses adhesion to VCAM-1, whereas CXCL10 stimulation has the opposite effect. Using separate pathways, CCL25 and CXCL10 stimulate differential phosphorylation states of the β7 tail and distinct talin and kindlin-3 binding patterns, resulting in different binding affinities of MAdCAM-1 and VCAM-1 to α4β7. Thus, our findings provide a mechanism for lymphocyte traffic control through the unique ligand-specific regulation of integrin adhesion by different chemokines.

Aim To investigate tumor necrosis factor (TNF)-alpha expression and its relationship with serum bile acids in placental trophoblasts from patients with intrahepatic cholestasis of pregnancy (ICP). Methods Human placenta, including normal pregnancies (n = 10) and patients with ICP (n = 10), were collected at term and subject to TNF-alpha measurements. Bile acid-induced TNF-alpha expression and cell apoptosis were evaluated in cultured syncytiotrophoblasts in vitro. Results ICP placental trophoblasts displayed apoptotic histological abnormalities. TNF-alpha levels in ICP tissue were significantly greater than those of controls as measured by quantitative polymerase chain reaction and western blot. Levels of placental TNF-alpha mRNA were positively correlated with serum bile acid concentration in ICP patients. In vitro, lithocholic acid (LCA) significantly enhanced TNF-alpha mRNA at both doses, by 2.07-fold at 15 mu m and by 3.41-fold at 30 mu m, whereas deoxycholic acid mildly increased TNF-alpha mRNA by 1.41-fold at 100 mu m only. LCA treatment produced significantly higher percentage of caspase-3 positive cells than vehicle treatment, rescuable by the addition of a TNF-alpha inhibitor, indicative of apoptosis induced by LCA-TNF-alpha pathway. Conclusion This study shows that the increase of TNF-alpha expression in placental trophoblasts is strongly associated with ICP pathology and is inducible by LCA in vitro, suggesting its potential value in the clinical prevention, diagnosis and treatment of ICP.

STUDY DESIGN A three-level rat tail caudal intervertebral disc (IVD) degeneration (IVDD) model was established to study effects of static compression on extracellular matrix (ECM) remodeling and integrin signaling in IVDs during IVDD. OBJECTIVE The aim of this study was to investigate the effect of compression force on ECM remodeling and integrin signaling in IVDs during IVDD. SUMMARY OF BACKGROUND DATA Integrins sense mechanical environment alteration via binding to ECM ligands and trigger intracellular signaling for pathological ECM remodeling during IVDD. However, the role of compression force in ECM remodeling and integrin signaling during IVDD remains elusive. METHODS Compared with the classical one-level rat tail IVDD model that exerts axial stress on the 8th to 9th caudal vertebral bodies, a three-level model was established by using an Ilizarov-type apparatus to exert stress on the 7th to 10th caudal vertebral bodies in rat tails for four weeks. To exclude side effects from surgical stab injury on manipulated discs, intact coccygeal (Co) disc Co8-9 was analyzed. RESULTS In three-level IVDD model, significant degeneration of the Co8-9 disc was observed. Quantitative real-time polymerase chain reaction (qRT-PCR) showed elevated mRNA expression of collagen types I, III, and V; matrix metalloproteinases (MMPs) 2, 3, 9, 13, 14; and decreased mRNA expression of collagen type II in Co8-9 disc. Compression loading altered the expression of integrin α2β1 (upregulated) and α10β1 (downregulated) in NP cells, and activated integrin downstream signaling. By contrast, one-level model showed more severe disc degeneration and ECM remodeling. Integrin α1, α2, α11, and β1 were upregulated, whereas α10 was downregulated. Similar activation of integrin signaling was observed. CONCLUSION Static compression altered collagen and MMP expression, and promoted β1 integrin expression and signaling in IVD. Compared with one-level rat tail IVDD model, three-level model showed milder effects on disc degeneration, ECM remodeling, and integrin expression, suggesting one-level model might involve other causes that induce IVDD via mechanisms independent of compression force. LEVEL OF EVIDENCE N/A.

Inflammatory caspases (caspases 1, 4, 5 and 11) are activated in response to microbial infection and danger signals. When activated, they cleave mouse and human gasdermin D (GSDMD) after Asp276 and Asp275, respectively, to generate an N-terminal cleavage product (GSDMD-NT) that triggers inflammatory death (pyroptosis) and release of inflammatory cytokines such as interleukin-1β1,2. Cleavage removes the C-terminal fragment (GSDMD-CT), which is thought to fold back on GSDMD-NT to inhibit its activation. However, how GSDMD-NT causes cell death is unknown. Here we show that GSDMD-NT oligomerizes in membranes to form pores that are visible by electron microscopy. GSDMD-NT binds to phosphatidylinositol phosphates and phosphatidylserine (restricted to the cell membrane inner leaflet) and cardiolipin (present in the inner and outer leaflets of bacterial membranes). Mutation of four evolutionarily conserved basic residues blocks GSDMD-NT oligomerization, membrane binding, pore formation and pyroptosis. Because of its lipid-binding preferences, GSDMD-NT kills from within the cell, but does not harm neighbouring mammalian cells when it is released during pyroptosis. GSDMD-NT also kills cell-free bacteria in vitro and may have a direct bactericidal effect within the cytosol of host cells, but the importance of direct bacterial killing in controlling in vivo infection remains to be determined.

Lymphocyte homing is regulated by the dynamic interaction between integrins and their ligands. Integrin α4β7 mediates both rolling and firm adhesion of lymphocytes by modulating its affinity to the ligand, mucosal addressin cell adhesion molecule-1 (MAdCAM-1). Although previous studies have revealed some mechanisms of α4β7-MAdCAM-1 binding, little is known about the different molecular bases of the low- and high-affinity α4β7-MAdCAM-1 interactions, which mediate rolling and firm adhesion of lymphocytes, respectively. Here, we found that two loops in immunoglobulin domains 1 and 2 (D1 and D2) of MAdCAM-1 played different roles in MAdCAM-1 binding to low-affinity (inactive) and high-affinity (activated) α4β7. The Asp-42 in the CC′ loop of D1 was indispensable for MAdCAM-1 binding to both low-affinity and high-affinity α4β7. The other CC′ loop residues except for Arg-39 and Ser-44 were essential for MAdCAM-1 binding to both inactive α4β7 and α4β7 activated by SDF-1α or talin, but not required for MAdCAM-1 binding to Mn2+-activated α4β7. Single amino acid substitution of the DE loop residues mildly decreased MAdCAM-1 binding to both inactive and activated α4β7. Notably, removal of the DE loop greatly impaired MAdCAM-1 binding to inactive and SDF-1α- or talin-activated α4β7, but only decreased 60% of MAdCAM-1 binding to Mn2+-activated α4β7. Moreover, DE loop residues were important for stabilizing the low-affinity α4β7-MAdCAM-1 interaction. Thus, our findings demonstrate the distinct roles of the CC′ and DE loops in the recognition of MAdCAM-1 by low- and high-affinity α4β7 and suggest that the inactive α4β7 and α4β7 activated by different stimuli have distinct conformations with different structural requirements for MAdCAM-1 binding.

Integrin α 4 β 7 mediates rolling and firm adhesion of leucocytes, two of the critical steps in leukocyte migration and tissue specific homing. Affinity of α 4 β 7 for ligand is dynamically regulated by three interlinked metal ion-binding sites in β 7 -subunit I domain. In this study, we found that Phe185 (F185), a highly conserved aromatic residue in β 7 -subunit, links the specificity-determining loop and the synergistic metal ion-binding site (SyMBS) through cation-π interaction. Mutations of F185 that disrupted the SyMBS cation-F185 interaction led to deficient firm cell adhesion mediated by high affinity α 4 β 7 , and only slightly affected rolling adhesion mediated by low affinity α 4 β 7 . Disruption of SyMBS cation-F185 interaction induced partial extension of integrin ectodomain and separation of cytoplasmic tails, and impaired α 4 β 7 -mediated bidirectional signaling. In addition, loss of SyMBS cation-F185 interaction increased paxillin expression and promoted paxillin-integrin binding, leading to deficient cell spreading. Furthermore, integrin α 4 β 7 -mediated cell migration was decreased by the abolishment of SyMBS cation-F185 interaction. Thus, these findings reveal a cation-π interaction playing vital roles in the regulation of integrin affinity, signaling, and biological functions.

Summary Widespread mRNA decay, an unappreciated feature of apoptosis, enhances cell death and depends on mitochondrial outer membrane permeabilization (MOMP), TUTases, and DIS3L2. Which RNAs are decayed and the decay-initiating event are unknown. Here, we show extensive decay of mRNAs and poly(A) noncoding (nc)RNAs at the 3′ end, triggered by the mitochondrial intermembrane space 3′-to-5′ exoribonuclease PNPT1, released during MOMP. PNPT1 knockdown inhibits apoptotic RNA decay and reduces apoptosis, while ectopic expression of PNPT1, but not an RNase-deficient mutant, increases RNA decay and cell death. The 3′ end of PNPT1 substrates thread through a narrow channel. Many non-poly(A) ncRNAs contain 3′-secondary structures or bind proteins that may block PNPT1 activity. Indeed, mutations that disrupt the 3′-stem-loop of a decay-resistant ncRNA render the transcript susceptible, while adding a 3′-stem-loop to an mRNA prevents its decay. Thus, PNPT1 release from mitochondria during MOMP initiates apoptotic decay of RNAs lacking 3′-structures.

Intrahepatic cholestasis of pregnancy (ICP) is a cholestatic disorder with potentially deleterious consequences for fetuses. Although a clear correlation between the elevated levels of maternal serum bile acids and deficient fetal outcome has been established in clinical practice, the underlying mechanisms remain elusive. Herein, we report that bile acids induce NF-κB pathway activation via G protein-coupled bile acid receptor 1 (Gpbar1), with consequent upregulation of inflammatory genes in trophoblasts, leading to aberrant leukocyte infiltration and inflammation in placenta. Ursodeoxycholic acid (UDCA), a drug used clinically to treat ICP, competes with other bile acids for binding with Gpbar1 and thus inhibits bile acid-induced inflammatory response in trophoblasts and improves fetal survival in pregnant rats with obstructive cholestasis. Notably, inhibition of NF-κB by andrographolide is more effective than UDCA in benefiting placentas and fetuses. Thus, anti-inflammation therapy targeting Gpbar1/NF-κB pathway could be effective in suppressing bile acid-induced inflammation and alleviating ICP-associated fetal disorders.

Integrin α4β7 controls lymphocyte trafficking into the gut and has essential roles in inflammatory bowel disease (IBD). The α4β7-blocking antibody vedolizumab is approved for IBD treatment; however, high dose of vedolizumab aggravates colitis in a small percentage of patients. Herein, we show that integrin β7 deficiency results in colonic regulatory T (Treg) cell depletion and exacerbates dextran sulfate sodium (DSS) colitis by evoking aberrant innate immunity. In DSS-treated β7-deficient mice, the loss of colonic Treg cells induces excessive macrophage infiltration in the colon via upregulation of colonic epithelial intercellular adhesion molecule 1 and increases proinflammatory cytokine expression, thereby exacerbating DSS-induced colitis. Moreover, reconstitution of the colonic Treg cell population in β7-deficient mice suppresses aberrant innate immune response in the colon and attenuates DSS colitis. Thus, integrin α4β7 is essential for suppression of DSS colitis as it regulates the colonic Treg cell population and innate immunity.

SummaryStimulator of interferon genes (STING, also known as MITA, ERIS, or MPYS) is essential for host immune responses triggered by microbial DNAs. However, the regulatory mechanisms underlying STING-mediated signaling are not fully understood. We report here that, upon cytoplasmic DNA stimulation, the endoplasmic reticulum (ER) protein AMFR was recruited to and interacted with STING in an insulin-induced gene 1 (INSIG1)-dependent manner. AMFR and INSIG1, an E3 ubiquitin ligase complex, then catalyzed the K27-linked polyubiquitination of STING. This modification served as an anchoring platform for recruiting TANK-binding kinase 1 (TBK1) and facilitating its translocation to the perinuclear microsomes. Depletion of AMFR or INSIG1 impaired STING-mediated antiviral gene induction. Consistently, myeloid-cell-specific Insig1−/− mice were more susceptible to herpes simplex virus 1 (HSV-1) infection than wild-type mice. This study uncovers an essential role of the ER proteins AMFR and INSIG1 in innate immunity, revealing an important missing link in the STING signaling pathway.

Control of integrin activation and signaling plays crucial roles in cell adhesion, spreading and migration. Here, we report that selective breakage of two conserved disulfide bonds located at the knees of integrin α4C589-C594 and β7C494-C526 activated α4β7. This activated integrin had a unique structure that was different from the typical extended conformation of active integrin. In addition, these activated α4β7 integrins spontaneously clustered on the cell membrane and triggered integrin downstream signaling independent of ligand binding. Although these disulfide bonds were not broken during α4β7 activation by inside-out signaling or Mn(2+), they could be specifically reduced by 0.1 mM dithiothreitol, a reducing strength that could be produced in vivo under certain conditions. Our findings reveal a novel mechanism of integrin activation under specific reducing conditions by which integrin can signal and promote cell spreading in the absence of ligand.

Integrin α4β7 mediates rolling and firm adhesion of lymphocytes pre- and post-activation, which is distinct from most integrins only mediating firm cell adhesion upon activation. This two-phase cell adhesion suggests a unique molecular basis for the dynamic interaction of α4β7 with its ligand, mucosal addressin cell adhesion molecule 1 (MAdCAM-1). Here we report that a disulfide bond-stabilized W1 β4-β1 loop in α4 β-propeller domain plays critical roles in regulating integrin α4β7 affinity and signaling. Either breaking the disulfide bond or deleting the disulfide bond-occluded segment in the W1 β4-β1 loop inhibited rolling cell adhesion supported by the low-affinity interaction between MAdCAM-1 and inactive α4β7 but negligibly affected firm cell adhesion supported by the high-affinity interaction between MAdCAM-1 and Mn(2+)-activated α4β7. Additionally, disrupting the disulfide bond or deleting the disulfide bond-occluded segment not only blocked the conformational change and activation of α4β7 triggered by talin or phorbol-12-myristate-13-acetate via inside-out signaling but also disrupted integrin-mediated outside-in signaling and impaired phosphorylation of focal adhesion kinase and paxillin. Thus, these findings reveal a particular molecular basis for α4β7-mediated rolling cell adhesion and a novel regulatory element of integrin affinity and signaling.

To investigate tumor necrosis factor (TNF)-α expression and its relationship with serum bile acids in placental trophoblasts from patients with intrahepatic cholestasis of pregnancy (ICP).Human placenta, including normal pregnancies (n = 10) and patients with ICP (n = 10), were collected at term and subject to TNF-α measurements. Bile acid-induced TNF-α expression and cell apoptosis were evaluated in cultured syncytiotrophoblasts in vitro.ICP placental trophoblasts displayed apoptotic histological abnormalities. TNF-α levels in ICP tissue were significantly greater than those of controls as measured by quantitative polymerase chain reaction and western blot. Levels of placental TNF-α mRNA were positively correlated with serum bile acid concentration in ICP patients. In vitro, lithocholic acid (LCA) significantly enhanced TNF-α mRNA at both doses, by 2.07-fold at 15 μm and by 3.41-fold at 30 μm, whereas deoxycholic acid mildly increased TNF-α mRNA by 1.41-fold at 100 μm only. LCA treatment produced significantly higher percentage of caspase-3 positive cells than vehicle treatment, rescuable by the addition of a TNF-α inhibitor, indicative of apoptosis induced by LCA-TNF-α pathway.This study shows that the increase of TNF-α expression in placental trophoblasts is strongly associated with ICP pathology and is inducible by LCA in vitro, suggesting its potential value in the clinical prevention, diagnosis and treatment of ICP.