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Chronic autoimmune diseases often lead to long-term sequelae and require lifelong immunosuppression because of an incomplete understanding of the triggers and drivers in genetically predisposed patients. Gut bacteria that escape the gut barrier, known as translocating gut pathobionts, have been implicated as instigators and perpetuators of extraintestinal autoimmune diseases in mice. The gut microbial contributions to autoimmunity in humans remain largely unclear, including whether specific pathological human adaptive immune responses are triggered by such pathobionts. Here, we show that the translocating pathobiont Enterococcus gallinarum can induce both human and mouse interferon-γ + T helper 17 (T H 17) differentiation and immunoglobulin G3 (IgG3) subclass switch of anti- E. gallinarum RNA antibodies, which correlated with anti-human RNA autoantibody responses in patients with systemic lupus erythematosus (SLE) and autoimmune hepatitis, two extraintestinal autoimmune diseases. E. gallinarum RNA, but not human RNA, triggered Toll-like receptor 8 (TLR8), and TLR8-mediated human monocyte activation promoted human T H 17 induction by E. gallinarum . Translocation of the pathobiont triggered increased anti-RNA autoantibody titers that correlated with renal autoimmune pathophysiology in murine gnotobiotic lupus models and with disease activity in patients with SLE. These studies elucidate cellular mechanisms of how a translocating gut pathobiont induces systemic human T cell- and B cell-dependent autoimmune responses and provide a framework for developing host- and microbiota-derived biomarkers and targeted therapies in autoimmune diseases.

Tertiary lymphoid structures play important roles in autoimmune and non-autoimmune conditions. While many of the molecular mechanisms involved in tertiary lymphoid structure formation have been identified, the cellular sources and temporal and spatial relationship remain unknown. Here we use combine single-cell RNA-sequencing, spatial transcriptomics and proteomics of minor salivary glands of patients with Sjogren's disease and Sicca Syndrome, with ex-vivo functional studies to construct a cellular and spatial map of key components involved in the formation and function of tertiary lymphoid structures. We confirm the presence of a fibroblast cell state and identify a pericyte/mural cell state with potential immunological functions. The identification of cellular properties associated with these structures and the molecular and functional interactions identified by this analysis may provide key therapeutic cues for tertiary lymphoid structures associated conditions in autoimmunity and cancer., Competing Interests: Competing interests: F.B. is an employee of Candel Therapeutics. C.D.B is the founder of Mestag Therapeutics. B.A.F. has undertaken consultancy for: Novartis, BMS, Servier, Galapagos, Roche, UCB, Sanofi, Janssen, A.Z., Otsuka, Amgen, Kiniksa and received research funding from Janssen, Servier, Galapagos, and Celgene. A.F. has consulted for Janssen and Sonoma, and has received research funding from: BMS, Roche, UCB, Nascient, Mestag, GSK, and Janssen. S.J.B. has undertaken consultancy for: Abbvie, Amgen, Argenx, Aurinia, Bain, BMS, EcoR1, Iqvia, J&J/Janssen, Kiniksa & Novartis. K.G.L. and A.P.F. are employees of F. Hoffmann-La Roche Ltd. The remaining authors declare no competing interests., (© 2024. The Author(s).)

Omnivorous animals, including mice and humans, tend to prefer energy-dense nutrients rich in fat over plant-based diets, especially for short periods of time, but the health consequences of this short-term consumption of energy-dense nutrients are unclear. Here, we show that short-term reiterative switching to 'feast diets', mimicking our social eating behavior, breaches the potential buffering effect of the intestinal microbiota and reorganizes the immunological architecture of mucosa-associated lymphoid tissues. The first dietary switch was sufficient to induce transient mucosal immune depression and suppress systemic immunity, leading to higher susceptibility to Salmonella enterica serovar Typhimurium and Listeria monocytogenes infections. The ability to respond to antigenic challenges with a model antigen was also impaired. These observations could be explained by a reduction of CD4 + T cell metabolic fitness and cytokine production due to impaired mTOR activity in response to reduced microbial provision of fiber metabolites. Reintroducing dietary fiber rewired T cell metabolism and restored mucosal and systemic CD4 + T cell functions and immunity. Finally, dietary intervention with human volunteers confirmed the effect of short-term dietary switches on human CD4 + T cell functionality. Therefore, short-term nutritional changes cause a transient depression of mucosal and systemic immunity, creating a window of opportunity for pathogenic infection., (© 2023. The Author(s).)

Extraintestinal autoimmune diseases are multifactorial with translocating gut pathobionts implicated as instigators and perpetuators in mice. However, the microbial contributions to autoimmunity in humans remain largely unclear, including whether specific pathological human adaptive immune responses are triggered by such pathobionts. We show here that the translocating pathobiont Enterococcus gallinarum induces human IFNγ + Th17 differentiation and IgG3 subclass switch of anti- E. gallinarum RNA and correlating anti-human RNA autoantibody responses in patients with systemic lupus erythematosus and autoimmune hepatitis. Human Th17 induction by E. gallinarum is cell-contact dependent and involves TLR8-mediated human monocyte activation. In murine gnotobiotic lupus models, E. gallinarum translocation triggers IgG3 anti-RNA autoantibody titers that correlate with renal autoimmune pathophysiology and with disease activity in patients. Overall, we define cellular mechanisms of how a translocating pathobiont induces human T- and B-cell-dependent autoimmune responses, providing a framework for developing host- and microbiota-derived biomarkers and targeted therapies in extraintestinal autoimmune diseases., One Sentence Summary: Translocating pathobiont Enterococcus gallinarum promotes human Th17 and IgG3 autoantibody responses linked to disease activity in autoimmune patients.

Transcription factor EB (TFEB) activates lysosomal biogenesis genes in response to environmental cues. Given implications of impaired TFEB signaling and lysosomal dysfunction in metabolic, neurological, and infectious diseases, we aim to systematically identify TFEB-directed circuits by examining transcriptional responses to TFEB subcellular localization and stimulation. We reveal that steady-state nuclear TFEB is sufficient to activate transcription of lysosomal, autophagy, and innate immunity genes, whereas other targets require higher thresholds of stimulation. Furthermore, we identify shared and distinct transcriptional signatures between mTOR inhibition and bacterial autophagy. Using a genome-wide CRISPR library, we find TFEB targets that protect cells from or sensitize cells to lysosomal cell death. BHLHE40 and BHLHE41, genes responsive to high, sustained levels of nuclear TFEB, act in opposition to TFEB upon lysosomal cell death induction. Further investigation identifies genes counter-regulated by TFEB and BHLHE40/41, adding this negative feedback to the current understanding of TFEB regulatory mechanisms., Competing Interests: Declaration of Interests P.B., N.R., and B.N. are employees of Novartis. D.R.B. and H.W.V. are employees of Vir Biotechnology. R.J.X. is a consultant to Novartis and a cofounder of Jnana Therapeutics and Celsius Therapeutics. H.W.V. is a founder of Casma Therapeutics and PierianDx. These organizations did not participate in funding this work., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

The clear role of autophagy in human inflammatory diseases such as Crohn disease was first identified by genome-wide association studies and subsequently dissected in multiple mechanistic studies. ATG16L1 has been particularly well studied in knockout and hypomorph settings as well as models recapitulating the Crohn disease-associated T300A polymorphism. Interestingly, ATG16L1 has a single homolog, ATG16L2, which is independently implicated in diseases, including Crohn disease and systemic lupus erythematosus. However, the contribution of ATG16L2 to canonical autophagy pathways and other cellular functions is poorly understood. To better understand its role, we generated and analyzed the first, to our knowledge, ATG16L2 knockout mouse. Our results show that ATG16L1 and ATG16L2 contribute very distinctly to autophagy and cellular ontogeny in myeloid, lymphoid, and epithelial lineages. Dysregulation of any of these lineages could contribute to complex diseases like Crohn disease and systemic lupus erythematosus, highlighting the value of examining cell-specific effects. We also identify a novel genetic interaction between ATG16L2 and epithelial ATG16L1. These findings are discussed in the context of how these genes may contribute distinctly to human disease., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Tetraspanins are a family of proteins possessing four transmembrane domains that help in lateral organization of plasma membrane proteins. These proteins interact with each other as well as other receptors and signaling proteins, resulting in functional complexes called "tetraspanin microdomains." Tetraspanins, including CD82, play an essential role in the pathogenesis of fungal infections. Dectin-1, a receptor for the fungal cell wall carbohydrate β-1,3-glucan, is vital to host defense against fungal infections. The current study identifies a novel association between tetraspanin CD82 and Dectin-1 on the plasma membrane of Candida albicans -containing phagosomes independent of phagocytic ability. Deletion of CD82 in mice resulted in diminished fungicidal activity, increased C. albicans viability within macrophages, and decreased cytokine production (TNF-α, IL-1β) at both mRNA and protein level in macrophages. Additionally, CD82 organized Dectin-1 clustering in the phagocytic cup. Deletion of CD82 modulates Dectin-1 signaling, resulting in a reduction of Src and Syk phosphorylation and reactive oxygen species production. CD82 knockout mice were more susceptible to C. albicans as compared with wild-type mice. Furthermore, patient C. albicans -induced cytokine production was influenced by two human CD82 single nucleotide polymorphisms, whereas an additional CD82 single nucleotide polymorphism increased the risk for candidemia independent of cytokine production. Together, these data demonstrate that CD82 organizes the proper assembly of Dectin-1 signaling machinery in response to C. albicans ., (Copyright © 2019 by The American Association of Immunologists, Inc.)

The transition from mitotic to meiotic cell cycles is essential for haploid gamete formation and fertility. Stimulated by retinoic acid gene 8 (Stra8) is an essential gatekeeper of meiotic initiation in vertebrates; yet, the molecular role of STRA8 remains principally unknown. Here we demonstrate that STRA8 functions as a suppressor of autophagy during spermatogenesis in mice. Stra8-deficient germ cells fail to enter meiosis and present aberrant upregulation of autophagy-lysosome genes, commensurate with autophagy activation. Biochemical assays show that ectopic expression of STRA8 alone is sufficient to inhibit both autophagy induction and maturation. Studies also revealed that, Nr1d1, a nuclear hormone receptor gene, is upregulated in Stra8-deficient testes and that STRA8 binds to the Nr1d1 promoter, indicating that Nr1d1 is a direct target of STRA8 transcriptional repression. In addition, it was found that NR1D1 binds to the promoter of Ulk1, a gene essential for autophagy initiation, and that Nr1d1 is required for the upregulated Ulk1 expression in Stra8-deficient testes. Furthermore, both genetic deletion of Nr1d1 and pharmacologic inhibition of NR1D1 by its synthetic antagonist SR8278 exhibit rescuing effects on the meiotic initiation defects observed in Stra8-deficient male germ cells. Together, the data suggest a novel link between STRA8-mediated autophagy suppression and meiotic initiation., Competing Interests: NO authors have competing interests

Inflammatory bowel disease (IBD) is driven by dysfunction between host genetics, the microbiota, and immune system. Knowledge gaps remain regarding how IBD genetic risk loci drive gut microbiota changes. The Crohn's disease risk allele ATG16L1 T300A results in abnormal Paneth cells due to decreased selective autophagy, increased cytokine release, and decreased intracellular bacterial clearance. To unravel the effects of ATG16L1 T300A on the microbiota and immune system, we employed a gnotobiotic model using human fecal transfers into ATG16L1 T300A knock-in mice. We observed increases in Bacteroides ovatus and Th1 and Th17 cells in ATG16L1 T300A mice. Association of altered Schaedler flora mice with B. ovatus specifically increased Th17 cells selectively in ATG16L1 T300A knock-in mice. Changes occur before disease onset, suggesting that ATG16L1 T300A contributes to dysbiosis and immune infiltration prior to disease symptoms. Our work provides insight for future studies on IBD subtypes, IBD patient treatment and diagnostics., Competing Interests: SL, KC, KL, HJ, HP, EC, MM, JL, CG, JD, JG, HV, AA, AK, RX No competing interests declared, WG Senior editor, eLife, (© 2019, Lavoie et al.)

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., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The discovery of numerous genetic variants in the human genome that are associated with inflammatory bowel disease (IBD) has revealed critical pathways that play important roles in intestinal homeostasis. These genetic studies have identified a critical role for macroautophagy/autophagy and more recently, lysosomal function, in maintaining the intestinal barrier and mucosal homeostasis. This review highlights recent work on the functional characterization of IBD-associated human genetic variants in cell type-specific functions for autophagy.

Understanding the transcription factors that modulate epithelial resistance to injury is necessary for understanding intestinal homeostasis and injury repair processes. Recently, transcription factor EB (TFEB) was implicated in expression of autophagy and host defense genes in nematodes and mammalian cells. However, the in vivo roles of TFEB in the mammalian intestinal epithelium were not known. Here, we used mice with a conditional deletion of Tfeb in the intestinal epithelium (Tfeb ΔIEC ) to examine its importance in defense against injury. Unperturbed Tfeb ΔIEC mice exhibited grossly normal intestinal epithelia, except for a defect in Paneth cell granules. Tfeb ΔIEC mice exhibited lower levels of lipoprotein ApoA1 expression, which is downregulated in Crohn's disease patients and causally linked to colitis susceptibility. Upon environmental epithelial injury using dextran sodium sulfate (DSS), Tfeb ΔIEC mice exhibited exaggerated colitis. Thus, our study reveals that TFEB is critical for resistance to intestinal epithelial cell injury, potentially mediated by APOA1.

Autophagy contributes to cellular homeostasis in the face of nutrient deprivation and other cellular stresses. Cell type-specific functions for autophagy are critical in maintaining homeostasis at both the tissue level and at the whole-organism level. Recent work has highlighted the ways in which human genetic variants modulate autophagy to alter epithelial and immune responses in inflammatory bowel disease.

Xenophagy is a form of selective autophagy that involves the targeting and elimination of intracellular pathogens through several recognition, recruitment, and ubiquitination events. E3 ubiquitin ligases control substrate selectivity in the ubiquitination cascade; however, systematic approaches to map the role of E3 ligases in antibacterial autophagy have been lacking. We screened more than 600 putative human E3 ligases, identifying E3 ligases that are required for adaptor protein recruitment and LC3-bacteria colocalization, critical steps in antibacterial autophagy. An unbiased informatics approach pinpointed RNF166 as a key gene that interacts with the autophagy network and controls the recruitment of ubiquitin as well as the autophagy adaptors p62 and NDP52 to bacteria. Mechanistic studies demonstrated that RNF166 catalyzes K29- and K33-linked polyubiquitination of p62 at residues K91 and K189. Thus, our study expands the catalog of E3 ligases that mediate antibacterial autophagy and identifies a critical role for RNF166 in this process., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Although numerous polymorphisms have been associated with inflammatory bowel disease (IBD), identifying the function of these genetic factors has proved challenging. Here we identified a role for nine genes in IBD susceptibility loci in antibacterial autophagy and characterized a role for one of these genes, GPR65, in maintaining lysosome function. Mice lacking Gpr65, a proton-sensing G protein-coupled receptor, showed increased susceptibly to bacteria-induced colitis. Epithelial cells and macrophages lacking GPR65 exhibited impaired clearance of intracellular bacteria and accumulation of aberrant lysosomes. Similarly, IBD patient cells and epithelial cells expressing an IBD-associated missense variant, GPR65 I231L, displayed aberrant lysosomal pH resulting in lysosomal dysfunction, impaired bacterial restriction, and altered lipid droplet formation. The GPR65 I231L polymorphism was sufficient to confer decreased GPR65 signaling. Collectively, these data establish a role for GPR65 in IBD susceptibility and identify lysosomal dysfunction as a potentially causative element in IBD pathogenesis with effects on cellular homeostasis and defense., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Unlabelled: A core set of autophagy proteins is required for gamma interferon (IFN-γ)-mediated clearance of Toxoplasma gondii in the mouse because of their control of several downstream effectors, including immunity-related GTPases (IRGs) and guanylate-binding proteins (GBPs). However, these effectors are absent (i.e., IRGs) from or nonessential (i.e., GBPs) in IFN-γ-activated human cells, raising the question of how these cells control parasite replication. Here, we define a novel role for ubiquitination and recruitment of autophagy adaptors in the strain-specific control of T. gondii replication in IFN-γ-activated human cells. Vacuoles containing susceptible strains of T. gondii became ubiquitinated, recruited the adaptors p62 and NDP52, and were decorated with LC3. Parasites within LC3-positive vacuoles became enclosed in multiple layers of host membranes, resulting in stunting of parasite replication. However, LC3-positive T. gondii-containing vacuoles did not fuse with endosomes and lysosomes, indicating that this process is fundamentally different from xenophagy, a form of autophagy involved in the control of intracellular bacterial pathogens. Genetic knockout of ATG16L or ATG7 reverted the membrane encapsulation and restored parasite replication, indicating that core autophagy proteins involved in LC3 conjugation are important in the control of parasite growth. Despite a role for the core autophagy machinery in this process, upstream activation through Beclin 1 was not sufficient to enhance the ubiquitination of T. gondii-containing vacuoles, suggesting a lack of reliance on canonical autophagy. These findings demonstrate a new mechanism for IFN-γ-dependent control of T. gondii in human cells that depends on ubiquitination and core autophagy proteins that mediate membrane engulfment and restricted growth., Importance: Autophagy is a process of cellular remodeling that allows the cell to recycle senescent organelles and recapture nutrients. During innate immune responses in the mouse, autophagy is recruited to help target intracellular pathogens and thus eliminate them. However, the antimicrobial mediators that depend on autophagy in the mouse are not conserved in humans, raising the issue of how human cells control intracellular pathogens. Our study defines a new pathway for the control of the ubiquitous intracellular parasite T. gondii in human cells activated by IFN-γ. Recruitment of autophagy adaptors resulted in engulfment of the parasite in multiple membranes and growth impairment. Although susceptible type 2 and 3 stains of T. gondii were captured by this autophagy-dependent pathway, type 1 strains were able to avoid entrapment., (Copyright © 2015 Selleck et al.)

Studies of human genetics and pathophysiology have implicated the regulation of autophagy in inflammation, neurodegeneration, infection, and autoimmunity. These findings have motivated the use of small-molecule probes to study how modulation of autophagy affects disease-associated phenotypes. Here, we describe the discovery of the small-molecule probe BRD5631 that is derived from diversity-oriented synthesis and enhances autophagy through an mTOR-independent pathway. We demonstrate that BRD5631 affects several cellular disease phenotypes previously linked to autophagy, including protein aggregation, cell survival, bacterial replication, and inflammatory cytokine production. BRD5631 can serve as a valuable tool for studying the role of autophagy in the context of cellular homeostasis and disease.

The polymorphism ATG16L1 T300A, associated with increased risk of Crohn's disease, impairs pathogen defense mechanisms including selective autophagy, but specific pathway interactions altered by the risk allele remain unknown. Here, we use perturbational profiling of human peripheral blood cells to reveal that CLEC12A is regulated in an ATG16L1-T300A-dependent manner. Antibacterial autophagy is impaired in CLEC12A-deficient cells, and this effect is exacerbated in the presence of the ATG16L1(∗)300A risk allele. Clec12a(-/-) mice are more susceptible to Salmonella infection, supporting a role for CLEC12A in antibacterial defense pathways in vivo. CLEC12A is recruited to sites of bacterial entry, bacteria-autophagosome complexes, and sites of sterile membrane damage. Integrated genomics identified a functional interaction between CLEC12A and an E3-ubiquitin ligase complex that functions in antibacterial autophagy. These data identify CLEC12A as early adaptor molecule for antibacterial autophagy and highlight perturbational profiling as a method to elucidate defense pathways in complex genetic disease., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Individuals who harbor a common coding polymorphism (Thr300Ala) within a structurally unclassified region of ATG16L1 are at increased risk for the development of Crohn disease. Recently, we reported on the generation and characterization of knockin mice carrying the ATG16L1 T300A variant. We demonstrate that multiple cell types from T300A knock-in mice exhibit reduced selective autophagy, and we mechanistically link this phenotype with an increased susceptibility of ATG16L1 T300A to CASP3- and CASP7-mediated cleavage. These findings demonstrate how a single polymorphism can result in cell type- and pathway-specific disruptions of selective autophagy and alterations in the inflammatory milieu that can contribute to disease.

The pathogenesis of complex diseases, such as type 1 diabetes (T1D), derives from interactions between host genetics and environmental factors. Previous studies have suggested that viral infection plays a significant role in initiation of T1D in genetically predisposed individuals. T1D susceptibility loci may therefore be enriched in previously uncharacterized genes functioning in antiviral defense pathways. To identify genes involved in antiviral immunity, we performed an image-based high-throughput genetic screen using short hairpin RNAs (shRNAs) against 161 genes within T1D susceptibility loci. RAW 264.7 cells transduced with shRNAs were infected with GFP-expressing herpes simplex virus type 1 (HSV-1) and fluorescent microscopy was performed to assess the viral infectivity by fluorescence reporter activity. Of the 14 candidates identified with high confidence, two candidates were selected for further investigation, Il27 and Tagap. Administration of recombinant IL-27 during viral infection was found to act synergistically with interferon gamma (IFN-γ) to activate expression of type I IFNs and proinflammatory cytokines, and to enhance the activities of interferon regulatory factor 3 (IRF3). Consistent with a role in antiviral immunity, Tagap-deficient macrophages demonstrated increased viral replication, reduced expression of proinflammatory chemokines and cytokines, and decreased production of IFN-β. Taken together, our unbiased loss-of-function genetic screen identifies genes that play a role in host antiviral immunity and delineates roles for IL-27 and Tagap in the production of antiviral cytokines.

A coding polymorphism (Thr300Ala) in the essential autophagy gene, autophagy related 16-like 1 (ATG16L1), confers increased risk for the development of Crohn disease, although the mechanisms by which single disease-associated polymorphisms contribute to pathogenesis have been difficult to dissect given that environmental factors likely influence disease initiation in these patients. Here we introduce a knock-in mouse model expressing the Atg16L1 T300A variant. Consistent with the human polymorphism, T300A knock-in mice do not develop spontaneous intestinal inflammation, but exhibit morphological defects in Paneth and goblet cells. Selective autophagy is reduced in multiple cell types from T300A knock-in mice compared with WT mice. The T300A polymorphism significantly increases caspase 3- and caspase 7-mediated cleavage of Atg16L1, resulting in lower levels of full-length Atg16Ll T300A protein. Moreover, Atg16L1 T300A is associated with decreased antibacterial autophagy and increased IL-1β production in primary cells and in vivo. Quantitative proteomics for protein interactors of ATG16L1 identified previously unknown nonoverlapping sets of proteins involved in ATG16L1-dependent antibacterial autophagy or IL-1β production. These findings demonstrate how the T300A polymorphism leads to cell type- and pathway-specific disruptions of selective autophagy and suggest a mechanism by which this polymorphism contributes to disease.

Autophagy is an evolutionarily conserved catabolic process that directs cytoplasmic proteins, organelles and microbes to lysosomes for degradation. Autophagy acts at the intersection of pathways involved in cellular stress, host defense, and modulation of inflammatory and immune responses; however, the details of how the autophagy network intersects with these processes remain largely undefined. Given the role of autophagy in several human diseases, it is important to determine the extent to which modulators of autophagy also modify inflammatory or immune pathways and whether it is possible to modulate a subset of these pathways selectively. Here, we identify small-molecule inducers of basal autophagy (including several FDA-approved drugs) and characterize their effects on IL-1β production, autophagic engulfment and killing of intracellular bacteria, and development of Treg, TH17, and TH1 subsets from naïve T cells. Autophagy inducers with distinct, selective activity profiles were identified that reveal the functional architecture of connections between autophagy, and innate and adaptive immunity. In macrophages from mice bearing a conditional deletion of the essential autophagy gene Atg16L1, the small molecules inhibit IL-1β production to varying degrees suggesting that individual compounds may possess both autophagy-dependent and autophagy-independent activity on immune pathways. The small molecule autophagy inducers constitute useful probes to test the contributions of autophagy-related pathways in diseases marked by impaired autophagy or elevated IL-1β and to test novel therapeutic hypotheses.

Attempts to eradicate HIV have been thwarted by the persistence of a small pool of quiescent memory CD4 T cells that harbor a transcriptionally silent, integrated form of the virus that can produce infectious virions following an anamnestic immune response. Transcription factors downstream of T-cell receptor activation, such as NF-κB/Rel and nuclear factor of activated T cells (NFAT) transcription members, are considered important regulators of HIV transcription during acute HIV infection. We now report studies exploring their precise role as antagonists of HIV latency using cell and primary CD4 T cell models of HIV-1 latency. Surprisingly, RNA interference studies performed in J-Lat CD4 T cells suggested that none of the NFATs, including NFATc1, NFATc2, NFATc3, and NFAT5, played a key role in the reactivation of latent HIV. However, cyclosporin A markedly inhibited the reactivation response. These results were reconciled when calcium signaling through calcineurin was shown to potentiate prostratin induced activation of NF-κB that in turn stimulated the latent HIV long terminal repeat (LTR). Similar effects of calcineurin were confirmed in a primary CD4 T cell model of HIV latency. These findings highlight an important role for calcineurin in NF-κB-dependent induction of latent HIV transcription. Innovative approaches exploiting the synergistic actions of calcineurin and prostratin in the absence of generalized T-cell activation merit exploration as a means to attack the latent viral reservoir.

Studies of the genetics underlying inflammatory bowel diseases have increased our understanding of the pathways involved in both ulcerative colitis and Crohn's disease and focused attention on the role of the microbiome in these diseases. Full understanding of pathogenesis will require a comprehensive grasp of the delicate homeostasis between gut bacteria and the human host. In this review, we present current evidence of microbiome-gene interactions in the context of other known risk factors and mechanisms, and describe the next steps necessary to pair genetic variant and microbiome sequencing data from patient cohorts. We discuss the concept of dysbiosis, proposing that the functional composition of the gut microbiome may provide a more consistent definition of dysbiosis and may more readily provide evidence of genome-microbiome interactions in future exploratory studies.

Latently infected cells form the major obstacle to HIV eradication. Studies of HIV latency have been generally hindered by the lack of a robust and rapidly deployable cell model that involves primary human CD4 T lymphocytes. Latently infected cell lines have proven useful, but it is unclear how closely these proliferating cells recapitulate the conditions of viral latency in non-dividing CD4 T lymphocytes in vivo. Current primary lymphocyte models more closely reflect the in vivo state of HIV latency, but they are limited by protracted culture periods and often low cell yields. Additionally, these models are always established in a single latently infected cell type that may not reflect the heterogeneous nature of the latent reservoir. Here we describe a rapid, sensitive, and quantitative primary cell model of HIV-1 latency with replication competent proviruses and multiple reporters to enhance the flexibility of the system. In this model, post-integration HIV-1 latency can be established in all populations of CD4 T cells, and reactivation of latent provirus assessed within 7 days. The kinetics and magnitude of reactivation were evaluated after stimulation with various cytokines, small molecules, and T-cell receptor agonists. Reactivation of latent HIV proviruses was readily detected in the presence of strong activators of NF-κB. Latently infected transitional memory CD4 T cells proved more responsive to these T-cell activators than latently infected central memory cells. These findings reveal potentially important biological differences within the latently infected pool of memory CD4 T cells and describe a flexible primary CD4 T-cell system to evaluate novel antagonists of HIV latency.

The essential transactivator function of the HIV Tat protein is regulated by multiple posttranslational modifications. Although individual modifications are well characterized, their crosstalk and dynamics of occurrence during the HIV transcription cycle remain unclear.We examine interactions between two critical modifications within the RNA-binding domain of Tat: monomethylation of lysine 51 (K51) mediated by Set7/9/KMT7, an early event in the Tat transactivation cycle that strengthens the interaction of Tat with TAR RNA, and acetylation of lysine 50 (K50) mediated by p300/KAT3B, a later process that dissociates the complex formed by Tat, TAR RNA and the cyclin T1 subunit of the positive transcription elongation factor b (P-TEFb). We find K51 monomethylation inhibited in synthetic Tat peptides carrying an acetyl group at K50 while acetylation can occur in methylated peptides, albeit at a reduced rate. To examine whether Tat is subject to sequential monomethylation and acetylation in cells, we performed mass spectrometry on immunoprecipitated Tat proteins and generated new modification-specific Tat antibodies against monomethylated/acetylated Tat. No bimodified Tat protein was detected in cells pointing to a demethylation step during the Tat transactivation cycle. We identify lysine-specific demethylase 1 (LSD1/KDM1) as a Tat K51-specific demethylase, which is required for the activation of HIV transcription in latently infected T cells. LSD1/KDM1 and its cofactor CoREST associates with the HIV promoter in vivo and activate Tat transcriptional activity in a K51-dependent manner. In addition, small hairpin RNAs directed against LSD1/KDM1 or inhibition of its activity with the monoamine oxidase inhibitor phenelzine suppresses the activation of HIV transcription in latently infected T cells.Our data support the model that a LSD1/KDM1/CoREST complex, normally known as a transcriptional suppressor, acts as a novel activator of HIV transcription through demethylation of K51 in Tat. Small molecule inhibitors of LSD1/KDM1 show therapeutic promise by enforcing HIV latency in infected T cells.

Purpose of Review: Differential rates of disease progression are obviously multifactorial, but the virulence of the actual infecting virus is most frequently ignored as potential source of slow or rapid disease progression. In this review, the argument will be made that nearly all elite suppressors are infected by weak HIV-1 strain (in terms of replicative capacity). Whether this poor virus replication is the cause of elite suppression or the consequence of a strong immune response remains a leading question in the field., Recent Findings: Although numerous research studies have related HIV-1 replicative capacity/fitness in tissue culture to virulence within patients, this review will focus on several recent and key discoveries on the important role of HIV-1 fitness in elite suppression. First, elite suppressors appear to harbor HIV-1 variants that encode Gag, Pol, and Env proteins that are less efficient than their counterparts of HIV-1 in typical/chronic progressors. Second, the actual HIV-1 clone(s) that establish acute infection may be less fit in patients who become elite controllers as compared with typical progressors. Finally, the fitness costs of cytotoxic T lymphocyte escape in HIV-1 may be easily compensated by secondary mutations if the infecting strain is capable of high replication kinetics and rapid evolution. A strain with weak replicative capacity might not compensate for fitness loss or even generate the initial escape mutations., Summary: A combination of good, anti-HIV-1 host genetics (e.g. HLA-B*57) along with infection by a 'whimpy' HIV-1 strain may be necessary for elite suppression, whereas only one of these may lead to slow progression and viremia.

The mechanism by which CD4 T cells are depleted in HIV-infected hosts remains poorly understood. In ex vivo cultures of human tonsil tissue, CD4 T cells undergo a pronounced cytopathic response following HIV infection. Strikingly, >95% of these dying cells are not productively infected but instead correspond to bystander cells. We now show that the death of these "bystander" cells involves abortive HIV infection. Inhibitors blocking HIV entry or early steps of reverse transcription prevent CD4 T cell death while inhibition of later events in the viral life cycle does not. We demonstrate that the nonpermissive state exhibited by the majority of resting CD4 tonsil T cells leads to accumulation of incomplete reverse transcripts. These cytoplasmic nucleic acids activate a host defense program that elicits a coordinated proapoptotic and proinflammatory response involving caspase-3 and caspase-1 activation. While this response likely evolved to protect the host, it centrally contributes to the immunopathogenic effects of HIV., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Approximately half of all human genes undergo alternative mRNA splicing. This process often yields homologous gene products exhibiting diverse functions. Alternative splicing of APOBEC3G (A3G) and APOBEC3F (A3F), the major host resistance factors targeted by the HIV-1 protein Vif, has not been explored. We investigated the effects of alternative splicing on A3G/A3F gene expression and antiviral activity. Three alternatively spliced A3G mRNAs and two alternatively spliced A3F mRNAs were detected in peripheral blood mononuclear cells in each of 10 uninfected, healthy donors. Expression of these splice variants was altered in different cell subsets and in response to cellular stimulation. Alternatively spliced A3G variants were insensitive to degradation by Vif but displayed no antiviral activity against HIV-1. Conversely, alternative splicing of A3F produced a 37-kDa variant lacking exon 2 (A3FΔ2) that was prominently expressed in macrophages and monocytes and was resistant to Vif-mediated degradation. Alternative splicing also produced a 24-kDa variant of A3F lacking exons 2-4 (A3FΔ2-4) that was highly sensitive to Vif. Both A3FΔ2 and A3FΔ2-4 displayed reduced cytidine deaminase activity and moderate antiviral activity. These alternatively spliced A3F gene products, particularly A3FΔ2, were incorporated into HIV virions, albeit at levels less than wild-type A3F. Thus, alternative splicing of A3F mRNA generates truncated antiviral proteins that differ sharply in their sensitivity to Vif.

Elite suppressors (ES) are a rare subset of HIV-1-infected individuals who are able to maintain HIV-1 viral loads below the limit of detection by ultra-sensitive clinical assays in the absence of antiretroviral therapy. Mechanism(s) responsible for this elite control are poorly understood but likely involve both host and viral factors. This study assesses ES plasma-derived envelope glycoprotein (env) fitness as a function of entry efficiency as a possible contributor to viral suppression. Fitness of virus entry was first evaluated using a novel inducible cell line with controlled surface expression levels of CD4 (receptor) and CCR5 (co-receptor). In the context of physiologic CCR5 and CD4 surface densities, ES envs exhibited significantly decreased entry efficiency relative to chronically infected viremic progressors. ES envs also demonstrated slow entry kinetics indicating the presence of virus with reduced entry fitness. Overall, ES env clones were less efficient at mediating entry than chronic progressor envs. Interestingly, acute infection envs exhibited an intermediate phenotypic pattern not distinctly different from ES or chronic progressor envs. These results imply that lower env fitness may be established early and may directly contribute to viral suppression in ES individuals.

HIV-1 latency in resting CD4+ T cells represents a major barrier to virus eradication in patients on highly active antiretroviral therapy (HAART). We describe here a novel post-transcriptional block in HIV-1 gene expression in resting CD4+ T cells from patients on HAART. This block involves the aberrant localization of multiply spliced (MS) HIV-1 RNAs encoding the critical positive regulators Tat and Rev. Although these RNAs had no previously described export defect, we show that they exhibit strict nuclear localization in resting CD4+ T cells from patients on HAART. Overexpression of the transcriptional activator Tat from non-HIV vectors allowed virus production in these cells. Thus, the nuclear retention of MS HIV-1 RNA interrupts a positive feedback loop and contributes to the non-productive nature of infection of resting CD4+ T cells. To define the mechanism of nuclear retention, proteomic analysis was used to identify proteins that bind MS HIV-1 RNA. Polypyrimidine tract binding protein (PTB) was identified as an HIV-1 RNA-binding protein differentially expressed in resting and activated CD4+ T cells. Overexpression of PTB in resting CD4+ T cells from patients on HAART allowed cytoplasmic accumulation of HIV-1 RNAs. PTB overexpression also induced virus production by resting CD4+ T cells. Virus culture experiments showed that overexpression of PTB in resting CD4+ T cells from patients on HAART allowed release of replication-competent virus, while preserving a resting cellular phenotype. Whether through effects on RNA export or another mechanism, the ability of PTB to reverse latency without inducing cellular activation is a result with therapeutic implications.

Neutralizing antibodies (NAb) against autologous virus can reach high titers in human immunodeficiency virus type 1 (HIV-1)-infected patients with progressive disease. Less is known about the role of NAb in HIV-1-infected patients with viral loads of <50 copies/ml of plasma, including patients on effective highly active antiretroviral therapy (HAART) and elite suppressors, who control HIV-1 replication without antiretroviral therapy. In this study, we analyzed full-length env sequences from plasma viruses and proviruses in resting CD4(+) T cells of HAART-treated patients, elite suppressors, and untreated HIV-1-infected patients with progressive disease. For each patient group, we assessed plasma virus neutralization by autologous, contemporaneous plasma. The degree of env diversity, the number of N-linked glycosylation sites, and the lengths of variable loops were all lower in elite suppressors than in HAART-treated and untreated viremic patients. Both elite suppressors and HAART-treated patients had lower titers of NAb against HIV-1 lab strains than those of untreated viremic patients. Surprisingly, titers of NAb against autologous, contemporaneous plasma viruses were similarly low in chronic progressors, elite suppressors, and HAART-treated patients. In elite suppressors and HAART-treated patients, titers of NAb against autologous plasma viruses also did not differ significantly from titers against autologous proviruses from resting CD4(+) T cells. These results suggest that high-titer NAb are not required for maintenance of viral suppression in elite suppressors and that NAb do not select plasma virus variants in most HAART-treated patients. Both drug-mediated and natural suppression of HIV-1 replication to levels below 50 copies/ml may limit the stimulation and maintenance of effective NAb responses.

A stable latent reservoir for human immunodeficiency virus type 1 (HIV-1) in resting memory CD4+ T cells presents a barrier to eradication of the infection even in patients on highly active antiretroviral therapy. Potential mechanisms for latency include inaccessibility of the integrated viral genome, absence of key host transcription factors, premature termination of HIV-1 RNAs, and abnormal splicing patterns. To differentiate among these mechanisms, we isolated extremely pure populations of resting CD4+ T cells from patients on highly active antiretroviral therapy. These cells did not produce virus but retained the capacity to do so if appropriately stimulated. Products of HIV-1 transcription were examined in purified resting CD4+ T cells. Although short, prematurely terminated HIV-1 transcripts have been suggested as a marker for latently infected cells, the production of short transcripts had not been previously demonstrated in purified populations of resting CD4+ T cells. By separating RNA into polyadenylated and nonpolyadenylated fractions, we showed that resting CD4+ T cells from patients on highly active antiretroviral therapy produce abortive transcripts that lack a poly(A) tail and that terminate prior to nucleotide 181. Short transcripts dominated the pool of total HIV-1 transcripts in resting CD4+ T cells. Processive, polyadenylated HIV-1 mRNAs were also present at a low level. Both unspliced and multiply spliced forms were found. Taken together, these results show that the nonproductive nature of the infection in resting CD4+ T cells from patients on highly active antiretroviral therapy is not due to absolute blocks at the level of either transcriptional initiation or elongation but rather relative inefficiencies at multiple steps.