Bacterial Peptidoglycan Fragments Differentially Regulate Innate Immune Signaling.

The human innate immune system responds to both pathogen and commensal bacteria at the molecular level using bacterial peptidoglycan (PG) recognition elements. Traditionally, synthetic and commercially accessible PG monosaccharide units known as muramyl dipeptide (MDP) and N-glycolyl MDP (ng-MDP) have been used to probe the mechanism of innate immune activation of pattern recognition receptors, such as NOD-like receptors. However, bacterial PG is a dynamic and complex structure, with various chemical modifications and trimming mechanisms that result in the production of disaccharide-containing elements. These molecules pose as attractive targets for immunostimulatory screening; however, studies are limited because of their synthetic accessibility. Inspired by disaccharide-containing compounds produced from the gut microbe Lactobacillus acidophilus, a robust and scalable chemical synthesis of PG-based disaccharide ligands was implemented. Together with a monosaccharide PG library, compounds were screened for their ability to stimulate proinflammatory genes in bone-marrow-derived macrophages. The data reveal distinct gene induction patterns for monosaccharide and disaccharide PG units, suggesting that PG innate immune signaling is more complex than a one activator-one pathway program, as biologically relevant fragments induce transcriptional programs to different degrees. These disaccharide molecules will serve as critical immunostimulatory tools to more precisely define specialized innate immune regulatory mechanisms that distinguish between commensal and pathogenic bacteria residing in the microbiome.

Vascular Patterning as Integrative Readout of Complex Molecular and Physiological Signaling by VESsel GENeration Analysis.

The molecular signaling cascades that regulate angiogenesis and microvascular remodeling are fundamental to normal development, healthy physiology, and pathologies such as inflammation and cancer. Yet quantifying such complex, fractally branching vascular patterns remains difficult. We review application of NASA's globally available, freely downloadable VESsel GENeration (VESGEN) Analysis software to numerous examples of 2D vascular trees, networks, and tree-network composites. Upon input of a binary vascular image, automated output includes informative vascular maps and quantification of parameters such as tortuosity, fractal dimension, vessel diameter, area, length, number, and branch point. Previous research has demonstrated that cytokines and therapeutics such as vascular endothelial growth factor, basic fibroblast growth factor (fibroblast growth factor-2), transforming growth factor-beta-1, and steroid triamcinolone acetonide specify unique "fingerprint" or "biomarker" vascular patterns that integrate dominant signaling with physiological response. In vivo experimental examples described here include vascular response to keratinocyte growth factor, a novel vessel tortuosity factor; angiogenic inhibition in humanized tumor xenografts by the anti-angiogenesis drug leronlimab; intestinal vascular inflammation with probiotic protection by Saccharomyces boulardii, and a workflow programming of vascular architecture for 3D bioprinting of regenerative tissues from 2D images. Microvascular remodeling in the human retina is described for astronaut risks in microgravity, vessel tortuosity in diabetic retinopathy, and venous occlusive disease.

The persistence of interleukin-6 is regulated by a blood buffer system derived from dendritic cells.

The interleukin-6 (IL-6) membrane receptor and its circulating soluble form, sIL-6R, can be targeted by antibody therapy to reduce deleterious immune signaling caused by chronic overexpression of the pro-inflammatory cytokine IL-6. This strategy may also hold promise for treating acute hyperinflammation, such as observed in coronavirus disease 2019 (COVID-19), highlighting a need to define regulators of IL-6 homeostasis. We found that conventional dendritic cells (cDCs), defined in mice via expression of the transcription factor Zbtb46, were a major source of circulating sIL-6R and, thus, systemically regulated IL-6 signaling. This was uncovered through identification of a cDC-dependent but T cell-independent modality that naturally adjuvants plasma cell differentiation and antibody responses to protein antigens. This pathway was then revealed as part of a broader biological buffer system in which cDC-derived sIL-6R set the in-solution persistence of IL-6. This control axis may further inform the development of therapeutic agents to modulate pro-inflammatory immune reactions.

Hepatic gap junctions amplify alcohol liver injury by propagating cGAS-mediated IRF3 activation.

Alcohol-related liver disease (ALD) accounts for the majority of cirrhosis and liver-related deaths worldwide. Activation of IFN-regulatory factor (IRF3) initiates alcohol-induced hepatocyte apoptosis, which fuels a robust secondary inflammatory response that drives ALD. The dominant molecular mechanism by which alcohol activates IRF3 and the pathways that amplify inflammatory signals in ALD remains unknown. Here we show that cytoplasmic sensor cyclic guanosine monophosphate-adenosine monophosphate (AMP) synthase (cGAS) drives IRF3 activation in both alcohol-injured hepatocytes and the neighboring parenchyma via a gap junction intercellular communication pathway. Hepatic RNA-seq analysis of patients with a wide spectrum of ALD revealed that expression of the cGAS-IRF3 pathway correlated positively with disease severity. Alcohol-fed mice demonstrated increased hepatic expression of the cGAS-IRF3 pathway. Mice genetically deficient in cGAS and IRF3 were protected against ALD. Ablation of cGAS in hepatocytes only phenocopied this hepatoprotection, highlighting the critical role of hepatocytes in fueling the cGAS-IRF3 response to alcohol. We identified connexin 32 (Cx32), the predominant hepatic gap junction, as a critical regulator of spreading cGAS-driven IRF3 activation through the liver parenchyma. Disruption of Cx32 in ALD impaired IRF3-stimulated gene expression, resulting in decreased hepatic injury despite an increase in hepatic steatosis. Taken together, these results identify cGAS and Cx32 as key factors in ALD pathogenesis and as potential therapeutic targets for hepatoprotection.

T cell fate following Salmonella infection is determined by a STING-IRF1 signaling axis in mice.

The innate immune response following infection with entero-invasive bacterial species is triggered upon release of cyclic di-guanylate monophosphate (c-di-GMP) into the host cell cytosol. Bacterial c-di-GMP activates the intracellular Sensor Stimulator of Interferon Genes (STING), encoded by Tmem173 in mice. Here we identify Interferon Regulatory Factor (IRF) 1 as a critical effector of STING-mediated microbial DNA sensing that is responsible for TH17 cell generation in the mucosal immune system. We find that STING activation induces IRF1-dependent transcriptional programs in dendritic cells (DCs) that define T cell fate determination, including induction of Gasdermin D, IL-1 family member cytokines, and enzymes for eicosanoid synthesis. Our results show that IRF1-dependent transcriptional programs in DCs are a prerequisite for antigen-specific TH17 subspecification in response to microbial c-di-GMP and Salmonella typhimurium infection. Our identification of a STING-IRF1 signaling axis for adaptive host defense control will aid further understanding of infectious disease mechanisms.

GEF-H1 Signaling upon Microtubule Destabilization Is Required for Dendritic Cell Activation and Specific Anti-tumor Responses.

Dendritic cell (DC) activation is a critical step for anti-tumor T cell responses. Certain chemotherapeutics can influence DC function. Here we demonstrate that chemotherapy capable of microtubule destabilization has direct effects on DC function; namely, it induces potent DC maturation and elicits anti-tumor immunity. Guanine nucleotide exchange factor-H1 (GEF-H1) is specifically released upon microtubule destabilization and is required for DC activation. In response to chemotherapy, GEF-H1 drives a distinct cell signaling program in DCs dominated by the c-Jun N-terminal kinase (JNK) pathway and AP-1/ATF transcriptional response for control of innate and adaptive immune responses. Microtubule destabilization, and subsequent GEF-H1 signaling, enhances cross-presentation of tumor antigens to CD8 T cells. In absence of GEF-H1, anti-tumor immunity is hampered. In cancer patients, high expression of the GEF-H1 immune gene signature is associated with prolonged survival. Our study identifies an alternate intracellular axis in DCs induced upon microtubule destabilization in which GEF-H1 promotes protective anti-tumor immunity.

Microbial recognition by GEF-H1 controls IKKε mediated activation of IRF5.

During infection, transcription factor interferon regulatory factor 5 (IRF5) is essential for the control of host defense. Here we show that the microtubule-associated guanine nucleotide exchange factor (GEF)-H1, is required for the phosphorylation of IRF5 by microbial muramyl-dipeptides (MDP), the minimal structural motif of peptidoglycan of both Gram-positive and Gram-negative bacteria. Specifically, GEF-H1 functions in a microtubule based recognition system for microbial peptidoglycans that mediates the activation of IKKε which we identify as a new upstream IKKα/ß and IRF5 kinase. The deletion of GEF-H1 or dominant-negative variants of GEF-H1 prevent activation of IKKε and phosphorylation of IRF5. The GEF-H1-IKKε-IRF5 signaling axis functions independent of NOD-like receptors and is critically required for the recognition of intracellular peptidoglycans and host defenses against Listeria monocytogenes.

CX3CR1+ Macrophages and CD8+ T Cells Control Intestinal IgA Production.

Secretory IgA is a key host defense mechanism that controls the intestinal microbiota. We investigated the role of CD11c+CX3CR1+CD64+ macrophages in IgA production in the intestine. Intestinal CX3CR1+ macrophages directly induced IgA secretion by B cells. Ag delivery to lamina propria (LP) CX3CR1+ macrophages specifically induced intestinal IgA production. The induction of IgA by CX3CR1+ macrophages required BAFF, a proliferation-inducing ligand, and TNF-α, but was surprisingly independent of TLR-mediated microbial recognition and retinoic acid signaling. IgA secretion by CX3CR1+ macrophages was enhanced by LP CD8+ T cells through the secretion of IL-9 and IL-13. CX3CR1+ macrophages and CD8+ T cells induced IgA production by B cells independently of mesenteric lymph nodes and Peyer patches. Our data reveal a previously unrecognized cellular circuitry in which LP CX3CR1+ macrophages, B cells, and CD8+ T cells coordinate the protective Ig secretion in the small intestine upon peripheral Ag delivery.

C1orf106 is a colitis risk gene that regulates stability of epithelial adherens junctions.

Polymorphisms in C1orf106 are associated with increased risk of inflammatory bowel disease (IBD). However, the function of C1orf106 and the consequences of disease-associated polymorphisms are unknown. Here we demonstrate that C1orf106 regulates adherens junction stability by regulating the degradation of cytohesin-1, a guanine nucleotide exchange factor that controls activation of ARF6. By limiting cytohesin-1-dependent ARF6 activation, C1orf106 stabilizes adherens junctions. Consistent with this model, C1orf106-/- mice exhibit defects in the intestinal epithelial cell barrier, a phenotype observed in IBD patients that confers increased susceptibility to intestinal pathogens. Furthermore, the IBD risk variant increases C1orf106 ubiquitination and turnover with consequent functional impairments. These findings delineate a mechanism by which a genetic polymorphism fine-tunes intestinal epithelial barrier integrity and elucidate a fundamental mechanism of cellular junctional control.

Shigella depends on SepA to destabilize the intestinal epithelial integrity via cofilin activation.

Shigella is unique among enteric pathogens, as it invades colonic epithelia through the basolateral pole. Therefore, it has evolved the ability to breach the intestinal epithelial barrier to deploy an arsenal of effector proteins, which permits bacterial invasion and leads to a severe inflammatory response. However, the mechanisms used by Shigella to regulate epithelial barrier permeability remain unknown. To address this question, we used both an intestinal polarized model and a human ex-vivo model to further characterize the early events of host-bacteria interactions. Our results showed that secreted Serine Protease A (SepA), which belongs to the serine protease autotransporter of Enterobacteriaceae family, is responsible for critically disrupting the intestinal epithelial barrier. Such disruption facilitates bacterial transit to the basolateral pole of the epithelium, ultimately fostering the hallmarks of the disease pathology. SepA was found to cause a decrease in active LIM Kinase 1 (LIMK1) levels, a negative inhibitor of actin-remodeling proteins, namely cofilin. Correspondingly, we observed increased activation of cofilin, a major actin-polymerization factor known to control opening of tight junctions at the epithelial barrier. Furthermore, we resolved the crystal structure of SepA to elucidate its role on actin-dynamics and barrier disruption. The serine protease activity of SepA was found to be required for the regulatory effects on LIMK1 and cofilin, resulting in the disruption of the epithelial barrier during infection. Altogether, we demonstrate that SepA is indispensable for barrier disruption, ultimately facilitating Shigella transit to the basolateral pole where it effectively invades the epithelium.

Reovirus infection triggers inflammatory responses to dietary antigens and development of celiac disease.

Viral infections have been proposed to elicit pathological processes leading to the initiation of T helper 1 (TH1) immunity against dietary gluten and celiac disease (CeD). To test this hypothesis and gain insights into mechanisms underlying virus-induced loss of tolerance to dietary antigens, we developed a viral infection model that makes use of two reovirus strains that infect the intestine but differ in their immunopathological outcomes. Reovirus is an avirulent pathogen that elicits protective immunity, but we discovered that it can nonetheless disrupt intestinal immune homeostasis at inductive and effector sites of oral tolerance by suppressing peripheral regulatory T cell (pTreg) conversion and promoting TH1 immunity to dietary antigen. Initiation of TH1 immunity to dietary antigen was dependent on interferon regulatory factor 1 and dissociated from suppression of pTreg conversion, which was mediated by type-1 interferon. Last, our study in humans supports a role for infection with reovirus, a seemingly innocuous virus, in triggering the development of CeD.

Homozygous ARHGEF2 mutation causes intellectual disability and midbrain-hindbrain malformation.

Mid-hindbrain malformations can occur during embryogenesis through a disturbance of transient and localized gene expression patterns within these distinct brain structures. Rho guanine nucleotide exchange factor (ARHGEF) family members are key for controlling the spatiotemporal activation of Rho GTPase, to modulate cytoskeleton dynamics, cell division, and cell migration. We identified, by means of whole exome sequencing, a homozygous frameshift mutation in the ARHGEF2 as a cause of intellectual disability, a midbrain-hindbrain malformation, and mild microcephaly in a consanguineous pedigree of Kurdish-Turkish descent. We show that loss of ARHGEF2 perturbs progenitor cell differentiation and that this is associated with a shift of mitotic spindle plane orientation, putatively favoring more symmetric divisions. The ARHGEF2 mutation leads to reduction in the activation of the RhoA/ROCK/MLC pathway crucial for cell migration. We demonstrate that the human brain malformation is recapitulated in Arhgef2 mutant mice and identify an aberrant migration of distinct components of the precerebellar system as a pathomechanism underlying the midbrain-hindbrain phenotype. Our results highlight the crucial function of ARHGEF2 in human brain development and identify a mutation in ARHGEF2 as novel cause of a neurodevelopmental disorder.

Induction and suppression of antiviral RNA interference by influenza A virus in mammalian cells.

Influenza A virus (IAV) causes annual epidemics and occasional pandemics, and is one of the best-characterized human RNA viral pathogens1. However, a physiologically relevant role for the RNA interference (RNAi) suppressor activity of the IAV non-structural protein 1 (NS1), reported over a decade ago2, remains unknown3. Plant and insect viruses have evolved diverse virulence proteins to suppress RNAi as their hosts produce virus-derived small interfering RNAs (siRNAs) that direct specific antiviral defence4-7 by an RNAi mechanism dependent on the slicing activity of Argonaute proteins (AGOs)8,9. Recent studies have documented induction and suppression of antiviral RNAi in mouse embryonic stem cells and suckling mice10,11. However, it is still under debate whether infection by IAV or any other RNA virus that infects humans induces and/or suppresses antiviral RNAi in mature mammalian somatic cells12-21. Here, we demonstrate that mature human somatic cells produce abundant virus-derived siRNAs co-immunoprecipitated with AGOs in response to IAV infection. We show that the biogenesis of viral siRNAs from IAV double-stranded RNA (dsRNA) precursors in infected cells is mediated by wild-type human Dicer and potently suppressed by both NS1 of IAV as well as virion protein 35 (VP35) of Ebola and Marburg filoviruses. We further demonstrate that the slicing catalytic activity of AGO2 inhibits IAV and other RNA viruses in mature mammalian cells, in an interferon-independent fashion. Altogether, our work shows that IAV infection induces and suppresses antiviral RNAi in differentiated mammalian somatic cells.

Gliadin Induces Neutrophil Migration via Engagement of the Formyl Peptide Receptor, FPR1.

BACKGROUND: Gliadin, the immunogenic component within gluten and trigger of celiac disease, is known to induce the production of Interleukin-8, a potent neutrophil-activating and chemoattractant chemokine. We sought to study the involvement of neutrophils in the early immunological changes following gliadin exposure. METHODS: Utilizing immunofluorescence microscopy and flow cytometry, the redistribution of major tight junction protein, Zonula occludens (ZO)-1, and neutrophil recruitment were assessed in duodenal tissues of gliadin-gavaged C57BL/6 wild-type and Lys-GFP reporter mice, respectively. Intravital microscopy with Lys-GFP mice allowed monitoring of neutrophil recruitment in response to luminal gliadin exposure in real time. In vitro chemotaxis assays were used to study murine and human neutrophil chemotaxis to gliadin, synthetic alpha-gliadin peptides and the neutrophil chemoattractant, fMet-Leu-Phe, in the presence or absence of a specific inhibitor of the fMet-Leu-Phe receptor-1 (FPR1), cyclosporine H. An irrelevant protein, zein, served as a control. RESULTS: Redistribution of ZO-1 and an influx of CD11b+Lys6G+ cells in the lamina propria of the small intestine were observed upon oral gavage of gliadin. In vivo intravital microscopy revealed a slowing down of GFP+ cells within the vessels and influx in the mucosal tissue within 2 hours after challenge. In vitro chemotaxis assays showed that gliadin strongly induced neutrophil migration, similar to fMet-Leu-Phe. We identified thirteen synthetic gliadin peptide motifs that induced cell migration. Blocking of FPR1 completely abrogated the fMet-Leu-Phe-, gliadin- and synthetic peptide-induced migration. CONCLUSIONS: Gliadin possesses neutrophil chemoattractant properties similar to the classical neutrophil chemoattractant, fMet-Leu-Phe, and likewise uses FPR1 in the process.

SLAMF4 Is a Negative Regulator of Expansion of Cytotoxic Intraepithelial CD8+ T Cells That Maintains Homeostasis in the Small Intestine.

BACKGROUND & AIMS: Intraepithelial T lymphocyte cells (IEL) are the first immune cells to respond to pathogens; they help maintain the integrity of the epithelial barrier. We studied the function of the mouse glycoprotein Signaling Lymphocyte Activation Molecule Family receptor (SLAMF) 4 (encoded by Slamf4) on the surface of CD8αß αß T-cell receptor (TCR)(+) IELs, and the roles of these cells in homeostasis of the small intestine in mice. METHODS: SLAMF4(-) CD8(+) αßTCR(+) cells isolated from spleens of OT-I Rag1(-/-) mice were induced to express gut-homing receptors and transferred to C57BL/6J mice; levels of SLAMF4(+) cells were measured in small intestine tissues. After administration of anti-CD3 or antigen, with or without anti-SLAM4, to C57BL/6J and Slamf4(-/-) mice, CD8αß αßTCR(+) IELs were collected; cytokine production and cytotoxicity were measured. Depletion of CX3CR1(+) phagocytes was assessed in mice by live-cell confocal imaging or by cytofluorometry; small intestine tissues were analyzed by histology and inflammation was quantified. RESULTS: Splenic CD8(+) αßTCR(+) cells began to express SLAMF4 only after migrating to the small intestine. Injection of C57BL/6J mice with anti-SLAMF4 and anti-CD3 increased levels of interleukin 10 and interferon gamma secretion by IEL, compared with injection of anti-CD3 only. Similarly, the number of granzyme B(+) cytotoxic CD8(+) αßTCR(+) IELs increased in Slamf4(-/-) mice after injection of anti-CD3 and anti-SLAMF4, administration of antigen, or injection of anti-CD3. Surprisingly, in vivo activation of CD8αß(+) IELs with anti-CD3 or antigen caused transient depletion of CX3CR1(+) phagocytes, which was prolonged by co-injection with anti-SLAMF4 or in Slamf4(-/-) mice. Anti-CD3 aggravated inflammation in the small intestines of Slamf4(-/-) mice and Eat2a(-/-)Eat2b(-/-) mice, indicated by flattened villi and crypt hyperplasia. CONCLUSIONS: In mice, the intestinal environment induces SLAMF4 expression and localization to the surface of CD8(+) αßTCR(+) IELs. Signaling via SLAMF4 controls expansion of cytotoxic CD8αß(+) IELs, which regulate the reversible depletion of lamina propria phagocytes and inflammation in the small intestine.

Small-molecule screening identifies inhibition of salt-inducible kinases as a therapeutic strategy to enhance immunoregulatory functions of dendritic cells.

Genetic alterations that reduce the function of the immunoregulatory cytokine IL-10 contribute to colitis in mouse and man. Myeloid cells such as macrophages (MΦs) and dendritic cells (DCs) play an essential role in determining the relative abundance of IL-10 versus inflammatory cytokines in the gut. As such, using small molecules to boost IL-10 production by DCs-MΦs represents a promising approach to increase levels of this cytokine specifically in gut tissues. Toward this end, we screened a library of well-annotated kinase inhibitors for compounds that enhance production of IL-10 by murine bone-marrow-derived DCs stimulated with the yeast cell wall preparation zymosan. This approach identified a number of kinase inhibitors that robustly up-regulate IL-10 production including the Food and Drug Administration (FDA)-approved drugs dasatinib, bosutinib, and saracatinib that target ABL, SRC-family, and numerous other kinases. Correlating the kinase selectivity profiles of the active compounds with their effect on IL-10 production suggests that inhibition of salt-inducible kinases (SIKs) mediates the observed IL-10 increase. This was confirmed using the SIK-targeting inhibitor HG-9-91-01 and a series of structural analogs. The stimulatory effect of SIK inhibition on IL-10 is also associated with decreased production of the proinflammatory cytokines IL-1ß, IL-6, IL-12, and TNF-α, and these coordinated effects are observed in human DCs-MΦs and anti-inflammatory CD11c(+) CX3CR1(hi) cells isolated from murine gut tissue. Collectively, these studies demonstrate that SIK inhibition promotes an anti-inflammatory phenotype in activated myeloid cells marked by robust IL-10 production and establish these effects as a previously unidentified activity associated with several FDA-approved multikinase inhibitors.

Plasmacytoid dendritic cells mediate anti-inflammatory responses to a gut commensal molecule via both innate and adaptive mechanisms.

Polysaccharide A (PSA), the archetypical immunomodulatory molecule of the gut commensal Bacteroides fragilis, induces regulatory T cells to secrete the anti-inflammatory cytokine interleukin-10 (IL-10). The cellular mediators of PSA's immunomodulatory properties are incompletely understood. In a mouse model of colitis, we find that PSA requires both innate and adaptive immune mechanisms to generate protection. Plasmacytoid DCs (PDCs) exposed to PSA do not produce proinflammatory cytokines, but instead they specifically stimulate IL-10 secretion by CD4+ T cells and efficiently mediate PSA-afforded immunoprotection. PSA induces and preferentially ligates Toll-like receptor 2 on PDCs but not on conventional DCs. Compared with other TLR2 ligands, PSA is better at enhancing PDC expression of costimulatory molecules required for protection against colitis. PDCs can thus orchestrate the beneficial immunoregulatory interaction of commensal microbial molecules, such as PSA, through both innate and adaptive immune mechanisms.

Glucocorticoid-Induced TNF Receptor Family-Related Protein Ligand is Requisite for Optimal Functioning of Regulatory CD4(+) T Cells.

Glucocorticoid-induced tumor necrosis factor receptor family-related protein (TNFRSF18, CD357) is constitutively expressed on regulatory T cells (Tregs) and is inducible on effector T cells. In this report, we examine the role of glucocorticoid-induced TNF receptor family-related protein ligand (GITR-L), which is expressed by antigen presenting cells, on the development and expansion of Tregs. We found that GITR-L is dispensable for the development of naturally occurring FoxP3(+) Treg cells in the thymus. However, the expansion of Treg in GITR-L (-/-) mice is impaired after injection of the dendritic cells (DCs) inducing factor Flt3 ligand. Furthermore, DCs from the liver of GITR-L (-/-) mice were less efficient in inducing proliferation of antigen-specific Treg cells in vitro than the same cells from WT littermates. Upon gene transfer of ovalbumin into hepatocytes of GITR-L (-/-)FoxP3(GFP) reporter mice using adeno-associated virus (AAV8-OVA) the number of antigen-specific Treg in liver and spleen is reduced. The reduced number of Tregs resulted in an increase in the number of ovalbumin specific CD8(+) T effector cells. This is highly significant because proliferation of antigen-specific CD8(+) cells itself is dependent on the presence of GITR-L, as shown by in vitro experiments and by adoptive transfers into GITR-L (-/-) Rag (-/-) and Rag (-/-) mice that had received AAV8-OVA. Surprisingly, administering αCD3 significantly reduced the numbers of FoxP3(+) Treg cells in the liver and spleen of GITR-L (-/-) but not WT mice. Because soluble Fc-GITR-L partially rescues αCD3 induced in vitro depletion of the CD103(+) subset of FoxP3(+)CD4(+) Treg cells, we conclude that expression of GITR-L by antigen presenting cells is requisite for optimal Treg-mediated regulation of immune responses including those in response during gene transfer.

GEF-H1 controls microtubule-dependent sensing of nucleic acids for antiviral host defenses.

Detailed understanding of the signaling intermediates that confer the sensing of intracellular viral nucleic acids for induction of type I interferons is critical for strategies to curtail viral mechanisms that impede innate immune defenses. Here we show that the activation of the microtubule-associated guanine nucleotide exchange factor GEF-H1, encoded by Arhgef2, is essential for sensing of foreign RNA by RIG-I-like receptors. Activation of GEF-H1 controls RIG-I-dependent and Mda5-dependent phosphorylation of IRF3 and induction of IFN-ß expression in macrophages. Generation of Arhgef2(-/-) mice revealed a pronounced signaling defect that prevented antiviral host responses to encephalomyocarditis virus and influenza A virus. Microtubule networks sequester GEF-H1 that upon activation is released to enable antiviral signaling by intracellular nucleic acid detection pathways.

Atg16l1 is required for autophagy in intestinal epithelial cells and protection of mice from Salmonella infection.

BACKGROUND & AIMS: Intestinal epithelial cells aid in mucosal defense by providing a physical barrier against entry of pathogenic bacteria and secreting antimicrobial peptides (AMPs). Autophagy is an important component of immune homeostasis. However, little is known about its role in specific cell types during bacterial infection in vivo. We investigated the role of autophagy in the response of intestinal epithelial and antigen-presenting cells to Salmonella infection in mice. METHODS: We generated mice deficient in Atg16l1 in epithelial cells (Atg16l1(f/f) × Villin-cre) or CD11c(+) cells (Atg16l1(f/f) × CD11c-cre); these mice were used to assess cell type-specific antibacterial autophagy. All responses were compared with Atg16l1(f/f) mice (controls). Mice were infected with Salmonella enterica serovar typhimurium; cecum and small-intestine tissues were collected for immunofluorescence, histology, and quantitative reverse-transcription polymerase chain reaction analyses of cytokines and AMPs. Modulators of autophagy were screened to evaluate their effects on antibacterial responses in human epithelial cells. RESULTS: Autophagy was induced in small intestine and cecum after infection with S typhimurium, and required Atg16l1. S typhimurium colocalized with microtubule-associated protein 1 light chain 3ß (Map1lc3b or LC3) in the intestinal epithelium of control mice but not in Atg16l1(f/f) × Villin-cre mice. Atg16l1(f/f) × Villin-cre mice also had fewer Paneth cells and abnormal granule morphology, leading to reduced expression of AMPs. Consistent with these defective immune responses, Atg16l1(f/f) × Villin-cre mice had increased inflammation and systemic translocation of bacteria compared with control mice. In contrast, we observed few differences between Atg16l1(f/f) × CD11c-cre and control mice. Trifluoperazine promoted autophagy and bacterial clearance in HeLa cells; these effects were reduced upon knockdown of ATG16L1. CONCLUSIONS: Atg16l1 regulates autophagy in intestinal epithelial cells and is required for bacterial clearance. It also is required to prevent systemic infection of mice with enteric bacteria.