IgG exacerbates genital chlamydial pathology in females by enhancing pathogenic CD8+ T cell responses.

Chlamydia trachomatis infections are an important sexually transmitted infection that can lead to inflammation, scarring and hydrosalpinx/infertility. However, infections are commonly clinically asymptomatic and do not receive treatment. The underlying cause of asymptomatic immunopathology remains unknown. Here, we demonstrate that IgG produced during male infection enhanced the incidence of immunopathology and infertility in females. Human endocervical cells expressing the neonatal Fc Receptor (FcRn) increased translocation of human IgG-opsonized C. trachomatis. Using total IgG purified from infected male mice, we opsonized C. muridarum and then infected female mice, mimicking sexual transmission. Following infection, IgG-opsonized Chlamydia was found to transcytose the epithelial barrier in the uterus, where it was phagocytosed by antigen-presenting cells (APCs) and trafficked to the draining lymph nodes. APCs then expanded both CD4+ and CD8+ T cell populations and caused significantly more infertility in female mice infected with non-opsonized Chlamydia. Enhanced phagocytosis of IgG-opsonized Chlamydia significantly increased pro-inflammatory signalling and T cell proliferation. As IgG is transcytosed by FcRn, we utilized FcRn-/- mice and observed that shedding kinetics of Chlamydia were only affected in FcRn-/- mice infected with IgG-opsonized Chlamydia. Depletion of CD8+ T cells in FcRn-/- mice lead to a significant reduction in the incidence of infertility. Taken together, these data demonstrate that IgG seroconversion during male infection can amplify female immunopathology, dependent on FcRn transcytosis, APC differentiation and enhanced CD8 T cell responses.

Interplay of gut microbiota and host epithelial mitochondrial dysfunction is necessary for the development of spontaneous intestinal inflammation in mice.

BACKGROUND: Intestinal epithelial cell (IEC) mitochondrial dysfunction involvement in inflammatory bowel diseases (IBD), including Crohn's disease affecting the small intestine, is emerging in recent studies. As the interface between the self and the gut microbiota, IECs serve as hubs of bidirectional cross-talk between host and luminal microbiota. However, the role of mitochondrial-microbiota interaction in the ileum is largely unexplored. Prohibitin 1 (PHB1), a chaperone protein of the inner mitochondrial membrane required for optimal electron transport chain function, is decreased during IBD. We previously demonstrated that mice deficient in PHB1 specifically in IECs (Phb1i∆IEC) exhibited mitochondrial impairment, Paneth cell defects, gut microbiota dysbiosis, and spontaneous inflammation in the ileum (ileitis). Mice deficient in PHB1 in Paneth cells (epithelial secretory cells of the small intestine; Phb1∆PC) also exhibited mitochondrial impairment, Paneth cell defects, and spontaneous ileitis. Here, we determined whether this phenotype is driven by Phb1 deficiency-associated ileal microbiota alterations or direct effects of loss of PHB1 in host IECs. RESULTS: Depletion of gut microbiota by broad-spectrum antibiotic treatment in Phb1∆PC or Phb1i∆IEC mice revealed a necessary role of microbiota to cause ileitis. Using germ-free mice colonized with ileal microbiota from Phb1-deficient mice, we show that this microbiota could not independently induce ileitis without host mitochondrial dysfunction. The luminal microbiota phenotype of Phb1i∆IEC mice included a loss of the short-chain fatty acid butyrate. Supplementation of butyrate in Phb1-deficient mice ameliorated Paneth cell abnormalities and ileitis. Phb1-deficient ileal enteroid models suggest deleterious epithelial-intrinsic responses to ileal microbiota that were protected by butyrate. CONCLUSIONS: These results suggest a mutual and essential reinforcing interplay of gut microbiota and host IEC, including Paneth cell, mitochondrial health in influencing ileitis. Restoration of butyrate is a potential therapeutic option in Crohn's disease patients harboring epithelial cell mitochondrial dysfunction. Video Abstract.

IL-22: Immunity's bittersweet symphony.

Epithelial cells play a crucial role in barrier defense. Here, Moniruzzaman et al. (2023. J. Exp. Med.https://doi.org/10.1084/jem.20230106) discovered that interleukin-22 (IL-22) represses MHC class II expression by epithelial cells with an opposite impact on chronic inflammatory disease and viral infection.

Characterizing CD4 T cell differentiation in mouse small intestine using T cell transfer, lamina propria preparation, and flow cytometry.

Studying gene function in T cells is crucial for understanding physiology and disease pathogenesis. Here, we provide a protocol to examine the role of specific genes in CD4+ T cell differentiation in the intestine. We describe steps for isolating naïve CD4+ T cells from mouse spleens and transferring them to recipient mice. We detail procedures to isolate lamina propria cells and analyze CD4+ T subsets using flow cytometry. This protocol is useful in the study of mucosal immune functions. For complete details on the use and execution of this protocol, please refer to Duan et al.1.

Expression of HMGCS2 in intestinal epithelial cells is downregulated in inflammatory bowel disease associated with endoplasmic reticulum stress.

Introduction: The Unfolded Protein Response, a mechanism triggered by the cell in response to Endoplasmic reticulum stress, is linked to inflammatory responses. Our aim was to identify novel Unfolded Protein Response-mechanisms that might be involved in triggering or perpetuating the inflammatory response carried out by the Intestinal Epithelial Cells in the context of Inflammatory Bowel Disease. Methods: We analyzed the transcriptional profile of human Intestinal Epithelial Cell lines treated with an Endoplasmic Reticulum stress inducer (thapsigargin) and/or proinflammatory stimuli. Several genes were further analyzed in colonic biopsies from Ulcerative Colitis patients and healthy controls. Lastly, we generated Caco-2 cells lacking HMGCS2 by CRISPR Cas-9 and analyzed the functional implications of its absence in Intestinal Epithelial Cells. Results: Exposure to a TLR ligand after thapsigargin treatment resulted in a powerful synergistic modulation of gene expression, which led us to identify new genes and pathways that could be involved in inflammatory responses linked to the Unfolded Protein Response. Key differentially expressed genes in the array also exhibited transcriptional alterations in colonic biopsies from active Ulcerative Colitis patients, including NKG2D ligands and the enzyme HMGCS2. Moreover, functional studies showed altered metabolic responses and epithelial barrier integrity in HMGCS2 deficient cell lines. Conclusion: We have identified new genes and pathways that are regulated by the Unfolded Protein Response in the context of Inflammatory Bowel Disease including HMGCS2, a gene involved in the metabolism of Short Chain Fatty Acids that may have an important role in intestinal inflammation linked to Endoplasmic Reticulum stress and the resolution of the epithelial damage.

T Cell CEACAM1-TIM-3 Crosstalk Alleviates Liver Transplant Injury in Mice and Humans.

BACKGROUND & AIMS: Carcinoembryonic antigen-related cell adhesion molecule 1 (CC1) acts through homophilic and heterophilic interactions with T cell immunoglobulin domain and mucin domain-containing protein 3 (TIM-3), which regulates innate immune activation in orthotopic liver transplantation (OLT). We investigated whether cluster of differentiation (CD) 4+ T cell-dependent CC1-TIM-3 crosstalk may affect OLT outcomes in mice and humans. METHODS: Wild-type (WT) and CC1-deficient (CC1 knock-out [KO]) mouse livers were transplanted into WT, CC1KO, or T-cell TIM-3 transgenic (TIM-3Tg)/CC1KO double-mutant recipients. CD4+ T cells were adoptively transferred into T/B cell-deficient recombination activating gene 2 protein (Rag2) KO recipients, followed by OLT. The perioperative liver-associated CC1 increase was analyzed in 50 OLT patients. RESULTS: OLT injury in WT livers deteriorated in CC1KO compared with CC1-proficient (WT) recipients. The frequency of TIM-3+CD4+ T cells was higher in WT than CC1KO hosts. Reconstitution of Rag2KO mice with CC1KO-T cells increased nuclear factor (NF)-κB phosphorylation and OLT damage compared with recipients repopulated with WT T cells. T-cell TIM-3 enhancement in CC1KO recipients (WT â†’ TIM3Tg/CC1KO) suppressed NF-κB phosphorylation in Kupffer cells and mitigated OLT injury. However, TIM-3-mediated protection was lost by pharmacologic TIM-3 blockade or an absence of CC1 in the donor liver (CC1KO â†’ TIM-3Tg/CC1KO). The perioperative CC1 increase in human OLT reduced hepatocellular injury, early allograft dysfunction, and the cumulative rejection rate. CONCLUSIONS: This translational study identifies T cell-specific CC1 signaling as a therapeutic means to alleviate OLT injury by promoting T cell-intrinsic TIM-3, which in turn interacts with liver-associated CC1 to suppress NF-κB in Kupffer cells. By suppressing peritransplant liver damage, promoting T-cell homeostasis, and improving OLT outcomes, recipient CC1 signaling serves as a novel cytoprotective sentinel.

Endoplasmic reticulum stress in the intestinal epithelium initiates purine metabolite synthesis and promotes Th17 cell differentiation in the gut.

Intestinal IL-17-producing T helper (Th17) cells are dependent on adherent microbes in the gut for their development. However, how microbial adherence to intestinal epithelial cells (IECs) promotes Th17 cell differentiation remains enigmatic. Here, we found that Th17 cell-inducing gut bacteria generated an unfolded protein response (UPR) in IECs. Furthermore, subtilase cytotoxin expression or genetic removal of X-box binding protein 1 (Xbp1) in IECs caused a UPR and increased Th17 cells, even in antibiotic-treated or germ-free conditions. Mechanistically, UPR activation in IECs enhanced their production of both reactive oxygen species (ROS) and purine metabolites. Treating mice with N-acetyl-cysteine or allopurinol to reduce ROS production and xanthine, respectively, decreased Th17 cells that were associated with an elevated UPR. Th17-related genes also correlated with ER stress and the UPR in humans with inflammatory bowel disease. Overall, we identify a mechanism of intestinal Th17 cell differentiation that emerges from an IEC-associated UPR.

The therapeutic age of the neonatal Fc receptor.

IgGs are essential soluble components of the adaptive immune response that evolved to protect the body from infection. Compared with other immunoglobulins, the role of IgGs is distinguished and enhanced by their high circulating levels, long half-life and ability to transfer from mother to offspring, properties that are conferred by interactions with neonatal Fc receptor (FcRn). FcRn binds to the Fc portion of IgGs in a pH-dependent manner and protects them from intracellular degradation. It also allows their transport across polarized cells that separate tissue compartments, such as the endothelium and epithelium. Further, it is becoming apparent that FcRn functions to potentiate cellular immune responses when IgGs, bound to their antigens, form IgG immune complexes. Besides the protective role of IgG, IgG autoantibodies are associated with numerous pathological conditions. As such, FcRn blockade is a novel and effective strategy to reduce circulating levels of pathogenic IgG autoantibodies and curtail IgG-mediated diseases, with several FcRn-blocking strategies on the path to therapeutic use. Here, we describe the current state of knowledge of FcRn-IgG immunobiology, with an emphasis on the functional and pathological aspects, and an overview of FcRn-targeted therapy development.

Intelectin-1 binds and alters the localization of the mucus barrier-modifying bacterium Akkermansia muciniphila.

Intelectin-1 (ITLN1) is a lectin secreted by intestinal epithelial cells (IECs) and upregulated in human ulcerative colitis (UC). We investigated how ITLN1 production is regulated in IECs and the biological effects of ITLN1 at the host-microbiota interface using mouse models. Our data show that ITLN1 upregulation in IECs from UC patients is a consequence of activating the unfolded protein response. Analysis of microbes coated by ITLN1 in vivo revealed a restricted subset of microorganisms, including the mucolytic bacterium Akkermansia muciniphila. Mice overexpressing intestinal ITLN1 exhibited decreased inner colonic mucus layer thickness and closer apposition of A. muciniphila to the epithelial cell surface, similar to alterations reported in UC. The changes in the inner mucus layer were microbiota and A. muciniphila dependent and associated with enhanced sensitivity to chemically induced and T cell-mediated colitis. We conclude that by determining the localization of a select group of bacteria to the mucus layer, ITLN1 modifies this critical barrier. Together, these findings may explain the impact of ITLN1 dysregulation on UC pathogenesis.

The γδ IEL effector API5 masks genetic susceptibility to Paneth cell death.

Loss of Paneth cells and their antimicrobial granules compromises the intestinal epithelial barrier and is associated with Crohn's disease, a major type of inflammatory bowel disease1-7. Non-classical lymphoid cells, broadly referred to as intraepithelial lymphocytes (IELs), intercalate the intestinal epithelium8,9. This anatomical position has implicated them as first-line defenders in resistance to infections, but their role in inflammatory disease pathogenesis requires clarification. The identification of mediators that coordinate crosstalk between specific IEL and epithelial subsets could provide insight into intestinal barrier mechanisms in health and disease. Here we show that the subset of IELs that express γ and δ T cell receptor subunits (γδ IELs) promotes the viability of Paneth cells deficient in the Crohn's disease susceptibility gene ATG16L1. Using an ex vivo lymphocyte-epithelium co-culture system, we identified apoptosis inhibitor 5 (API5) as a Paneth cell-protective factor secreted by γδ IELs. In the Atg16l1-mutant mouse model, viral infection induced a loss of Paneth cells and enhanced susceptibility to intestinal injury by inhibiting the secretion of API5 from γδ IELs. Therapeutic administration of recombinant API5 protected Paneth cells in vivo in mice and ex vivo in human organoids with the ATG16L1 risk allele. Thus, we identify API5 as a protective γδ IEL effector that masks genetic susceptibility to Paneth cell death.

Structural analysis of human CEACAM1 oligomerization.

The human (h) CEACAM1 GFCC' face serves as a binding site for homophilic and heterophilic interactions with various microbial and host ligands. hCEACAM1 has also been observed to form oligomers and micro-clusters on the cell surface which are thought to regulate hCEACAM1-mediated signaling. However, the structural basis for hCEACAM1 higher-order oligomerization is currently unknown. To understand this, we report a hCEACAM1 IgV oligomer crystal structure which shows how GFCC' face-mediated homodimerization enables highly flexible ABED face interactions to arise. Structural modeling and nuclear magnetic resonance (NMR) studies predict that such oligomerization is not impeded by the presence of carbohydrate side-chain modifications. In addition, using UV spectroscopy and NMR studies, we show that oligomerization is further facilitated by the presence of a conserved metal ion (Zn++ or Ni++) binding site on the G strand of the FG loop. Together these studies provide biophysical insights on how GFCC' and ABED face interactions together with metal ion binding may facilitate hCEACAM1 oligomerization beyond dimerization.

Screening for modulators of the cellular composition of gut epithelia via organoid models of intestinal stem cell differentiation.

The cellular composition of barrier epithelia is essential to organismal homoeostasis. In particular, within the small intestine, adult stem cells establish tissue cellularity, and may provide a means to control the abundance and quality of specialized epithelial cells. Yet, methods for the identification of biological targets regulating epithelial composition and function, and of small molecules modulating them, are lacking. Here we show that druggable biological targets and small-molecule regulators of intestinal stem cell differentiation can be identified via multiplexed phenotypic screening using thousands of miniaturized organoid models of intestinal stem cell differentiation into Paneth cells, and validated via longitudinal single-cell RNA-sequencing. We found that inhibitors of the nuclear exporter Exportin 1 modulate the fate of intestinal stem cells, independently of known differentiation cues, significantly increasing the abundance of Paneth cells in the organoids and in wild-type mice. Physiological organoid models of the differentiation of intestinal stem cells could find broader utility for the screening of biological targets and small molecules that can modulate the composition and function of other barrier epithelia.

Genetic and pharmacological inhibition of XBP1 protects against APAP hepatotoxicity through the activation of autophagy.

Acetaminophen (APAP) hepatotoxicity induces endoplasmic reticulum (ER) stress which triggers the unfolded protein response (UPR) in hepatocytes. However, the mechanisms underlying ER stress remain poorly understood, thus reducing the options for exploring new pharmacological therapies for patients with hyperacute liver injury. Eight-to-twelve-week-old C57BL/6J Xbp1-floxed (Xbp1f/f) and hepatocyte-specific knockout Xbp1 mice (Xbp1∆hepa) were challenged with either high dose APAP [500 mg/kg] and sacrificed at early (1-2 h) and late (24 h) stages of hepatotoxicity. Histopathological examination of livers, immunofluorescence and immunohistochemistry, Western blot, real time (RT)-qPCR studies and transmission electron microscopy (TEM) were performed. Pharmacological inhibition of XBP1 using pre-treatment with STF-083010 [STF, 75 mg/kg] and autophagy induction with Rapamycin [RAPA, 8 mg/kg] or blockade with Chloroquine [CQ, 60 mg/kg] was also undertaken in vivo. Cytoplasmic expression of XBP1 coincided with severity of human and murine hyperacute liver injury. Transcriptional and translational activation of the UPR and sustained activation of JNK1/2 were major events in APAP hepatotoxicity, both in a human hepatocytic cell line and in a preclinical model. Xbp1∆hepa livers showed decreased UPR and JNK1/2 activation but enhanced autophagy in response to high dose APAP. Additionally, blockade of XBP1 splicing by STF, mitigated APAP-induced liver injury and without non-specific off-target effects (e.g., CYP2E1 activity). Furthermore, enhanced autophagy might be responsible for modulating CYP2E1 activity in Xbp1∆hepa animals. Genetic and pharmacological inhibition of Xbp1 specifically in hepatocytes ameliorated APAP-induced liver injury by enhancing autophagy and decreasing CYP2E1 expression. These findings provide the basis for the therapeutic restoration of ER stress and/or induction of autophagy in patients with hyperacute liver injury.

A purine metabolic checkpoint that prevents autoimmunity and autoinflammation.

Still's disease, the paradigm of autoinflammation-cum-autoimmunity, predisposes for a cytokine storm with excessive T lymphocyte activation upon viral infection. Loss of function of the purine nucleoside enzyme FAMIN is the sole known cause for monogenic Still's disease. Here we discovered that a FAMIN-enabled purine metabolon in dendritic cells (DCs) restrains CD4+ and CD8+ T cell priming. DCs with absent FAMIN activity prime for enhanced antigen-specific cytotoxicity, IFNγ secretion, and T cell expansion, resulting in excessive influenza A virus-specific responses. Enhanced priming is already manifest with hypomorphic FAMIN-I254V, for which ∼6% of mankind is homozygous. FAMIN controls membrane trafficking and restrains antigen presentation in an NADH/NAD+-dependent manner by balancing flux through adenine-guanine nucleotide interconversion cycles. FAMIN additionally converts hypoxanthine into inosine, which DCs release to dampen T cell activation. Compromised FAMIN consequently enhances immunosurveillance of syngeneic tumors. FAMIN is a biochemical checkpoint that protects against excessive antiviral T cell responses, autoimmunity, and autoinflammation.

A model of preferential pairing between epithelial and dendritic cells in thymic antigen transfer.

Medullary thymic epithelial cells (mTECs), which produce and present self-antigens, are essential for the establishment of central tolerance. Since mTEC numbers are limited, their function is complemented by thymic dendritic cells (DCs), which transfer mTEC-produced self-antigens via cooperative antigen transfer (CAT). While CAT is required for effective T cell selection, many aspects remain enigmatic. Given the recently described heterogeneity of mTECs and DCs, it is unclear whether the antigen acquisition from a particular TEC subset is mediated by preferential pairing with a specific subset of DCs. Using several relevant Cre-based mouse models that control for the expression of fluorescent proteins, we have found that, in regards to CAT, each subset of thymic DCs preferentially targets a distinct mTEC subset(s). Importantly, XCR1+-activated DC subset represented the most potent subset in CAT. Interestingly, thymic DCs can also acquire antigens from more than one mTEC, and of these, monocyte-derived dendritic cells (moDCs) were determined to be the most efficient. moDCs also represented the most potent DC subset in the acquisition of antigen from other DCs. These findings suggest a preferential pairing model for the distribution of mTEC-derived antigens among distinct populations of thymic DCs.

PUFA-Induced Metabolic Enteritis as a Fuel for Crohn's Disease.

BACKGROUND & AIMS: Crohn's disease (CD) globally emerges with Westernization of lifestyle and nutritional habits. However, a specific dietary constituent that comprehensively evokes gut inflammation in human inflammatory bowel diseases remains elusive. We aimed to delineate how increased intake of polyunsaturated fatty acids (PUFAs) in a Western diet, known to impart risk for developing CD, affects gut inflammation and disease course. We hypothesized that the unfolded protein response and antioxidative activity of glutathione peroxidase 4 (GPX4), which are compromised in human CD epithelium, compensates for metabolic perturbation evoked by dietary PUFAs. METHODS: We phenotyped and mechanistically dissected enteritis evoked by a PUFA-enriched Western diet in 2 mouse models exhibiting endoplasmic reticulum (ER) stress consequent to intestinal epithelial cell (IEC)-specific deletion of X-box binding protein 1 (Xbp1) or Gpx4. We translated the findings to human CD epithelial organoids and correlated PUFA intake, as estimated by a dietary questionnaire or stool metabolomics, with clinical disease course in 2 independent CD cohorts. RESULTS: PUFA excess in a Western diet potently induced ER stress, driving enteritis in Xbp1-/-IEC and Gpx4+/-IEC mice. ω-3 and ω-6 PUFAs activated the epithelial endoplasmic reticulum sensor inositol-requiring enzyme 1α (IRE1α) by toll-like receptor 2 (TLR2) sensing of oxidation-specific epitopes. TLR2-controlled IRE1α activity governed PUFA-induced chemokine production and enteritis. In active human CD, ω-3 and ω-6 PUFAs instigated epithelial chemokine expression, and patients displayed a compatible inflammatory stress signature in the serum. Estimated PUFA intake correlated with clinical and biochemical disease activity in a cohort of 160 CD patients, which was similarly demonstrable in an independent metabolomic stool analysis from 199 CD patients. CONCLUSIONS: We provide evidence for the concept of PUFA-induced metabolic gut inflammation which may worsen the course of human CD. Our findings provide a basis for targeted nutritional therapy.

Immunoglobulin A-specific deficiency induces spontaneous inflammation specifically in the ileum.

OBJECTIVE: Although immunoglobulin A (IgA) is abundantly expressed in the gut and known to be an important component of mucosal barriers against luminal pathogens, its precise function remains unclear. Therefore, we tried to elucidate the effect of IgA on gut homeostasis maintenance and its mechanism. DESIGN: We generated various IgA mutant mouse lines using the CRISPR/Cas9 genome editing system. Then, we evaluated the effect on the small intestinal homeostasis, pathology, intestinal microbiota, cytokine production, and immune cell activation using intravital imaging. RESULTS: We obtained two lines, with one that contained a <50 base pair deletion in the cytoplasmic region of the IgA allele (IgA tail-mutant; IgAtm/tm) and the other that lacked the most constant region of the IgH α chain, which resulted in the deficiency of IgA production (IgA-/-). IgA-/- exhibited spontaneous inflammation in the ileum but not the other parts of the gastrointestinal tract. Associated with this, there were significantly increased lamina propria CD4+ T cells, elevated productions of IFN-γ and IL-17, increased ileal segmented filamentous bacteria and skewed intestinal microflora composition. Intravital imaging using Ca2+ biosensor showed that IgA-/- had elevated Ca2+ signalling in Peyer's patch B cells. On the other hand, IgAtm/tm seemed to be normal, suggesting that the IgA cytoplasmic tail is dispensable for the prevention of the intestinal disorder. CONCLUSION: IgA plays an important role in the mucosal homeostasis associated with the regulation of intestinal microbiota and protection against mucosal inflammation especially in the ileum.

Host immunomodulatory lipids created by symbionts from dietary amino acids.

Small molecules derived from symbiotic microbiota critically contribute to intestinal immune maturation and regulation1. However, little is known about the molecular mechanisms that control immune development in the host-microbiota environment. Here, using a targeted lipidomic analysis and synthetic approach, we carried out a multifaceted investigation of immunomodulatory α-galactosylceramides from the human symbiont Bacteroides fragilis (BfaGCs). The characteristic terminal branching of BfaGCs is the result of incorporation of branched-chain amino acids taken up in the host gut by B. fragilis. A B. fragilis knockout strain that cannot metabolize branched-chain amino acids showed reduced branching in BfaGCs, and mice monocolonized with this mutant strain had impaired colonic natural killer T (NKT) cell regulation, implying structure-specific immunomodulatory activity. The sphinganine chain branching of BfaGCs is a critical determinant of NKT cell activation, which induces specific immunomodulatory gene expression signatures and effector functions. Co-crystal structure and affinity analyses of CD1d-BfaGC-NKT cell receptor complexes confirmed the interaction of BfaGCs as CD1d-restricted ligands. We present a structural and molecular-level paradigm of immunomodulatory control by interactions of endobiotic metabolites with diet, microbiota and the immune system.