Host-microbiome co-evolution drives homeostasis and disease susceptibility, yet regulatory principles governing the integrated intestinal host-commensal microenvironment remain obscure. While ...inflammasome signaling participates in these interactions, its activators and microbiome-modulating mechanisms are unknown. Here, we demonstrate that the microbiota-associated metabolites taurine, histamine, and spermine shape the host-microbiome interface by co-modulating NLRP6 inflammasome signaling, epithelial IL-18 secretion, and downstream anti-microbial peptide (AMP) profiles. Distortion of this balanced AMP landscape by inflammasome deficiency drives dysbiosis development. Upon fecal transfer, colitis-inducing microbiota hijacks this microenvironment-orchestrating machinery through metabolite-mediated inflammasome suppression, leading to distorted AMP balance favoring its preferential colonization. Restoration of the metabolite-inflammasome-AMP axis reinstates a normal microbiota and ameliorates colitis. Together, we identify microbial modulators of the NLRP6 inflammasome and highlight mechanisms by which microbiome-host interactions cooperatively drive microbial community stability through metabolite-mediated innate immune modulation. Therefore, targeted “postbiotic” metabolomic intervention may restore a normal microenvironment as treatment or prevention of dysbiosis-driven diseases.
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•Microbiota-modulated metabolites regulate NLRP6 inflammasome and intestinal IL-18•Inflammasome-derived IL-18 orchestrates colonic anti-microbial peptide expression•Inflammasome modulation by metabolites enables dysbiotic community transfer•Integrated metabolite signaling determines the severity of intestinal inflammation
Microbiota-associated metabolites shape the host-microbiome interface by modulating NLRP6 inflammasome signaling, epithelial IL-18 secretion, and the generation of downstream anti-microbial peptides. This axis, therefore, determines both host indigenous microbiome profiles and the susceptibility to intestinal inflammation.
Abstract
The transcriptional regulator Rbpj is involved in T-helper (T
H
) subset polarization, but its function in T
reg
cells remains unclear. Here we show that T
reg
-specific Rbpj deletion leads ...to splenomegaly and lymphadenopathy despite increased numbers of T
reg
cells with a polyclonal TCR repertoire. A specific defect of Rbpj-deficient T
reg
cells in controlling T
H
2 polarization and B cell responses is observed, leading to the spontaneous formation of germinal centers and a T
H
2-associated immunoglobulin class switch. The observed phenotype is environment-dependent and can be induced by infection with parasitic nematodes. Rbpj-deficient T
reg
cells adopt open chromatin landscapes and gene expression profiles reminiscent of tissue-derived T
H
2-polarized T
reg
cells, with a prevailing signature of the transcription factor Gata-3. Taken together, our study suggests that T
reg
cells require Rbpj to specifically restrain T
H
2 responses, including their own excessive T
H
2-like differentiation potential.
Tail-anchored (TA) proteins have a single C-terminal transmembrane domain, making their biogenesis dependent on posttranslational translocation. Despite their importance, no dedicated insertion ...machinery has been uncovered for mitochondrial outer membrane (MOM) TA proteins. To decipher the molecular mechanisms guiding MOM TA protein insertion, we performed two independent systematic microscopic screens in which we visualized the localization of model MOM TA proteins on the background of mutants in all yeast genes. We could find no mutant in which insertion was completely blocked. However, both screens demonstrated that MOM TA proteins were partially localized to the endoplasmic reticulum (ER) in spf1 cells. Spf1, an ER ATPase with unknown function, is the first protein shown to affect MOM TA protein insertion. We found that ER membranes in spf1 cells become similar in their ergosterol content to mitochondrial membranes. Indeed, when we visualized MOM TA protein distribution in yeast strains with reduced ergosterol content, they phenocopied the loss of Spf1. We therefore suggest that the inherent differences in membrane composition between organelle membranes are sufficient to determine membrane integration specificity in a eukaryotic cell.
The endoplasmic reticulum (ER) is the site of synthesis of secreted and membrane proteins. To exit the ER, proteins are packaged into COPII vesicles through direct interaction with the COPII coat or ...aided by specific cargo receptors. Despite the fundamental role of such cargo receptors in protein traffic, only a few have been identified; their cargo spectrum is unknown and the signals they recognize remain poorly understood. We present here an approach we term "PAIRS" (pairing analysis of cargo receptors), which combines systematic genetic manipulations of yeast with automated microscopy screening, to map the spectrum of cargo for a known receptor or to uncover a novel receptor for a particular cargo. Using PAIRS we followed the fate of ∼150 cargos on the background of mutations in nine putative cargo receptors and identified novel cargo for most of these receptors. Deletion of the Erv14 cargo receptor affected the widest range of cargo. Erv14 substrates have a wide array of functions and structures; however, they are all membrane-spanning proteins of the late secretory pathway or plasma membrane. Proteins residing in these organelles have longer transmembrane domains (TMDs). Detailed examination of one cargo supported the hypothesis that Erv14 dependency reflects the length rather than the sequence of the TMD. The PAIRS approach allowed us to uncover new cargo for known cargo receptors and to obtain an unbiased look at specificity in cargo selection. Obtaining the spectrum of cargo for a cargo receptor allows a novel perspective on its mode of action. The rules that appear to guide Erv14 substrate recognition suggest that sorting of membrane proteins at multiple points in the secretory pathway could depend on the physical properties of TMDs. Such a mechanism would allow diverse proteins to utilize a few receptors without the constraints of evolving location-specific sorting motifs.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Aire is a transcriptional regulator that induces the promiscuous expression of thousands of tissue-restricted antigens (TRAs) in medullary thymic epithelial cells (mTECs), a step critical for the ...induction of immunological self-tolerance. Studies have offered molecular insights into how Aire operates, but more comprehensive understanding of this process still remains elusive. Here we found abundant expression of the protein deacetylase Sirtuin-1 (Sirt1) in mature Aire(+) mTECs, wherein it was required for the expression of Aire-dependent TRA-encoding genes and the subsequent induction of immunological self-tolerance. Our study elucidates a previously unknown molecular mechanism for Aire-mediated transcriptional regulation and identifies a unique function for Sirt1 in preventing organ-specific autoimmunity.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
The thymus provides a unique microenvironment enabling development and selection of T lymphocytes. Medullary thymic epithelial cells (mTECs) play a pivotal role in this process by facilitating ...negative selection of self-reactive thymocytes and the generation of Foxp3+ regulatory T cells. Although studies have highlighted the non-canonical nuclear factor κB (NF-κB) pathway as the key regulator of mTEC development, comprehensive understanding of the molecular pathways regulating this process still remains incomplete. Here, we demonstrate that the development of functionally competent mTECs is regulated by the histone deacetylase 3 (Hdac3). Although histone deacetylases are global transcriptional regulators, this effect is highly specific only to Hdac3, as neither Hdac1 nor Hdac2 inactivation caused mTEC ablation. Interestingly, Hdac3 induces an mTEC-specific transcriptional program independently of the previously recognized RANK-NFκB signaling pathway. Thus, our findings uncover yet another layer of complexity of TEC lineage divergence and highlight Hdac3 as a major and specific molecular switch crucial for mTEC differentiation.
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•Hdac3 uniquely regulates mTEC development independently of NF-κB or the NCoR complex•Hdac3 induces the mTEC transcriptional program while repressing that of cTECs•Repression of Notch signaling by Hdac3 is crucial for mTEC development
Goldfarb et al. show that Hdac3 is essential for normal development and function of medullary thymic epithelial cells (mTECs) independently of non-canonical NFκB signaling. Their findings highlight Hdac3 as a master switch inducing the mTEC transcriptional program in immature TECs.
Regulatory T cells are important regulators of the immune system and have versatile functions for the homeostasis and repair of tissues. They express the forkhead box transcription factor Foxp3 as a ...lineage-defining protein. Negative regulators of Foxp3 expression are not well understood. Here, we generated double-stranded DNA probes complementary to the Foxp3 promoter sequence and performed a pull-down with nuclear protein in vitro, followed by elution of bound proteins and quantitative mass spectrometry. Of the Foxp3-promoter-binding transcription factors identified with this approach, one was T cell factor 1 (TCF1). Using viral over-expression, we identified TCF1 as a repressor of Foxp3 expression. In TCF1-deficient animals, increased levels of Foxp3intermediateCD25negative T cells were identified. CRISPR-Cas9 knockout studies in primary human and mouse conventional CD4 T (Tconv) cells revealed that TCF1 protects Tconv cells from inadvertent Foxp3 expression. Our data implicate a role of TCF1 in suppressing Foxp3 expression in activated T cells.
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•Quantitative proteomics identifies proteins bound to the Foxp3 gene promoter•Promoter-binding proteins are suppressing Foxp3 expression•TCF1-deficient animals have more Foxp3-expressing CTLA4−CD25−CD4+ T cells•TCF1 suppresses Foxp3 expression in activated non-Treg cells
Molecular Biology; Molecular Mechanism of Gene Regulation; Immunology; Proteomics
Aire is a transcriptional regulator that induces promiscuous expression of thousands of genes encoding tissue-restricted antigens (TRAs) in medullary thymic epithelial cells (mTECs). While the target ...genes of Aire are well characterized, the transcriptional programs that regulate its own expression have remained elusive. Here we comprehensively analyzed both cis-acting and trans-acting regulatory mechanisms and found that the Aire locus was insulated by the global chromatin organizer CTCF and was hypermethylated in cells and tissues that did not express Aire. In mTECs, however, Aire expression was facilitated by concurrent eviction of CTCF, specific demethylation of exon 2 and the proximal promoter, and the coordinated action of several transcription activators, including Irf4, Irf8, Tbx21, Tcf7 and Ctcfl, which acted on mTEC-specific accessible regions in the Aire locus.
Ameloblasts are specialized epithelial cells in the jaw that have an indispensable role in tooth enamel formation-amelogenesis
. Amelogenesis depends on multiple ameloblast-derived proteins that ...function as a scaffold for hydroxyapatite crystals. The loss of function of ameloblast-derived proteins results in a group of rare congenital disorders called amelogenesis imperfecta
. Defects in enamel formation are also found in patients with autoimmune polyglandular syndrome type-1 (APS-1), caused by AIRE deficiency
, and in patients diagnosed with coeliac disease
. However, the underlying mechanisms remain unclear. Here we show that the vast majority of patients with APS-1 and coeliac disease develop autoantibodies (mostly of the IgA isotype) against ameloblast-specific proteins, the expression of which is induced by AIRE in the thymus. This in turn results in a breakdown of central tolerance, and subsequent generation of corresponding autoantibodies that interfere with enamel formation. However, in coeliac disease, the generation of such autoantibodies seems to be driven by a breakdown of peripheral tolerance to intestinal antigens that are also expressed in enamel tissue. Both conditions are examples of a previously unidentified type of IgA-dependent autoimmune disorder that we collectively name autoimmune amelogenesis imperfecta.
Promiscuous expression of tissue-restricted self-antigens (TRAs) in medullar thymic epithelial cells (mTECs) is critical for induction of T cell tolerance to self. This process is largely mediated by ...the Autoimmune regulator (Aire) gene. Although there has been a dramatic progress in our understanding of Aire’s physiological function and significance, several key questions remain unanswered. Specifically, our understanding of the transcriptional programs regulating the expression of Aire itself and the mechanisms that define its own activity and gene recognition remain elusive. Moreover, the identity of the self-antigens targeted upon Aire-deficiency remains largely obscure. The work presented herein focused on addressing the above open questions. Specifically, in order to understand what regulates Aire expression, we combined thorough DNA methylation analysis and gene expression profiling of Aire expressing cells. These revealed demethylated CpG residues and transcriptional regulator patterns that are unique only to those cells. Next, by utilizing several independent reporter-based screening systems we were able to focus on 15 promising candidate Aire-regulators. Importantly, subsequent functional and biochemical analyses highlighted a unique multimolecular complex (Irf4, Irf8, Tbx21 and Tcf7) that binds to the proximal Aire promoter and is critical for effective Aire expression in-vitro and in-vivo. Moreover, subsequent molecular dissection highlighted the transcription modulator CTCF as an insulator of Aire expression. Moreover, in a parallel project we sought to gain a better understanding into the transcriptional and epigenetic mechanisms defining Aire-dependent gene activation. We demonstrate that Aire preferentially targets genes that are marked with various histone modifications linked to transcriptional silencing. Furthermore, follow-up siRNA screens targeting a large proportion of all histone modifying enzymes and chromatin remodelers highlighted several specific factors (p300, Brg1, Baz1b, Kmt2d and Kmt3a) to be essential for Aire-dependent gene activation in HEK 293FT cells. Finally, in a different set of experiments we sought to identify tooth specific antigens that are targeted by auto-antibodies in Aire-deficient individuals. Indeed, our experiments identified Ameloblastin (Ambn) as a putative enamel-specific antigen target. Collectively, these data provide significant new insights into the layered molecular architectures controlling the very tight and specific expression of the Aire gene in mTECs and the unique epigenetic landscape governing its gene recognition and activity.
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