Serine metabolism is crucial for cGAS-STING signaling and viral defense control in the gut.

Inflammatory bowel diseases are characterized by the chronic relapsing inflammation of the gastrointestinal tract. While the molecular causality between endoplasmic reticulum (ER) stress and intestinal inflammation is widely accepted, the metabolic consequences of chronic ER stress on the pathophysiology of IBD remain unclear. By using in vitro, in vivo models, and patient datasets, we identified a distinct polarization of the mitochondrial one-carbon metabolism and a fine-tuning of the amino acid uptake in intestinal epithelial cells tailored to support GSH and NADPH metabolism upon ER stress. This metabolic phenotype strongly correlates with IBD severity and therapy response. Mechanistically, we uncover that both chronic ER stress and serine limitation disrupt cGAS-STING signaling, impairing the epithelial response against viral and bacterial infection and fueling experimental enteritis. Consequently, the antioxidant treatment restores STING function and virus control. Collectively, our data highlight the importance of serine metabolism to allow proper cGAS-STING signaling and innate immune responses upon gut inflammation.

Modeling microbiota-associated human diseases: from minimal models to complex systems.

Alterations in the intestinal microbiota are associated with various human diseases of the digestive system, including obesity and its associated metabolic diseases, inflammatory bowel diseases (IBD), and colorectal cancer (CRC). All three diseases are characterized by modifications of the richness, composition, and metabolic functions of the human intestinal microbiota. Despite being multi-factorial diseases, studies in germ-free animal models have unarguably identified the intestinal microbiota as a causal driver of disease pathogenesis. However, for an increased mechanistic understanding of microbial signatures in human diseases, models require detailed refinement to closely mimic the human microbiota and reflect the complexity and range of dysbiosis observed in patients. The transplantation of human fecal microbiota into animal models represents a powerful tool for studying the causal and functional role of the dysbiotic human microbiome in a pathological context. While human microbiota-associated models were initially employed to study obesity, an increasing number of studies have applied this approach in the context of IBD and CRC over the past decade. In this review, we discuss different approaches that allow the functional validation of the bacterial contribution to human diseases, with emphasis on obesity and its associated metabolic diseases, IBD, and CRC. We discuss the utility of simple models, such as in vitro fermentation systems of the human microbiota and ex vivo intestinal organoids, as well as more complex whole organism models. Our focus here lies on human microbiota-associated mouse models in the context of all three diseases, as well as highlighting the advantages and limitations of this approach.

Microbe-Mucus Interface in the Pathogenesis of Colorectal Cancer.

Overlying gastrointestinal epithelial cells is the transparent mucus layer that separates the lumen from the host. The dynamic mucus layer serves to lubricate the mucosal surface, to protect underlying epithelial cells, and as a transport medium between luminal contents and epithelial cells. Furthermore, it provides a habitat for commensal bacteria and signals to the underlying immune system. Mucins are highly glycosylated proteins, and their glycocode is tissue-specific and closely linked to the resident microbiota. Aberrant mucin expression and glycosylation are linked to chronic inflammation and gastrointestinal cancers, including colorectal cancer (CRC). Aberrant mucus production compromises the mucus layer and allows bacteria to come into close contact with the intestinal epithelium, potentially triggering unfavorable host responses and the subsequent development of tumors. Here, we review our current understanding of the interaction between the intestinal microbiota and mucus in healthy and CRC subjects. Deep knowledge of the intricate mechanisms of microbe-mucus interactions may contribute to the development of novel treatment strategies for CRC, in which a dysfunctional mucus layer is observed.

Activating Transcription Factor 6 Mediates Inflammatory Signals in Intestinal Epithelial Cells Upon Endoplasmic Reticulum Stress.

BACKGROUND & AIMS: Excess and unresolved endoplasmic reticulum (ER) stress in intestinal epithelial cells (IECs) promotes intestinal inflammation. Activating transcription factor 6 (ATF6) is one of the signaling mediators of ER stress. We studied the pathways that regulate ATF6 and its role for inflammation in IECs. METHODS: We performed an RNA interference screen, using 23,349 unique small interfering RNAs targeting 7783 genes and a luciferase reporter controlled by an ATF6-dependent ERSE (ER stress-response element) promoter, to identify proteins that activate or inhibit the ATF6 signaling pathway in HEK293 cells. To validate the screening results, intestinal epithelial cell lines (Caco-2 cells) were transfected with small interfering RNAs or with a plasmid overexpressing a constitutively active form of ATF6. Caco-2 cells with a CRISPR-mediated disruption of autophagy related 16 like 1 gene (ATG16L1) were used to study the effect of ATF6 on ER stress in autophagy-deficient cells. We also studied intestinal organoids derived from mice that overexpress constitutively active ATF6, from mice with deletion of the autophagy related 16 like 1 or X-Box binding protein 1 gene in IECs (Atg16l1ΔIEC or Xbp1ΔIEC, which both develop spontaneous ileitis), from patients with Crohn's disease (CD) and healthy individuals (controls). Cells and organoids were incubated with tunicamycin to induce ER stress and/or chemical inhibitors of newly identified activator proteins of ATF6 signaling, and analyzed by real-time polymerase chain reaction and immunoblots. Atg16l1ΔIEC and control (Atg16l1fl/fl) mice were given intraperitoneal injections of tunicamycin and were treated with chemical inhibitors of ATF6 activating proteins. RESULTS: We identified and validated 15 suppressors and 7 activators of the ATF6 signaling pathway; activators included the regulatory subunit of casein kinase 2 (CSNK2B) and acyl-CoA synthetase long chain family member 1 (ACSL1). Knockdown or chemical inhibition of CSNK2B and ACSL1 in Caco-2 cells reduced activity of the ATF6-dependent ERSE reporter gene, diminished transcription of the ATF6 target genes HSP90B1 and HSPA5 and reduced NF-κB reporter gene activation on tunicamycin stimulation. Atg16l1ΔIEC and or Xbp1ΔIEC organoids showed increased expression of ATF6 and its target genes. Inhibitors of ACSL1 or CSNK2B prevented activation of ATF6 and reduced CXCL1 and tumor necrosis factor (TNF) expression in these organoids on induction of ER stress with tunicamycin. Injection of mice with inhibitors of ACSL1 or CSNK2B significantly reduced tunicamycin-mediated intestinal inflammation and IEC death and expression of CXCL1 and TNF in Atg16l1ΔIEC mice. Purified ileal IECs from patients with CD had higher levels of ATF6, CSNK2B, and HSPA5 messenger RNAs than controls; early-passage organoids from patients with active CD show increased levels of activated ATF6 protein, incubation of these organoids with inhibitors of ACSL1 or CSNK2B reduced transcription of ATF6 target genes, including TNF. CONCLUSIONS: Ileal IECs from patients with CD have higher levels of activated ATF6, which is regulated by CSNK2B and HSPA5. ATF6 increases expression of TNF and other inflammatory cytokines in response to ER stress in these cells and in organoids from Atg16l1ΔIEC and Xbp1ΔIEC mice. Strategies to inhibit the ATF6 signaling pathway might be developed for treatment of inflammatory bowel diseases.

ER Stress and the UPR in Shaping Intestinal Tissue Homeostasis and Immunity.

An imbalance in the correct protein folding milieu of the endoplasmic reticulum (ER) can cause ER stress, which leads to the activation of the unfolded protein response (UPR). The UPR constitutes a highly conserved and intricately regulated group of pathways that serve to restore ER homeostasis through adaptation or apoptosis. Numerous studies over the last decade have shown that the UPR plays a critical role in shaping immunity and inflammation, resulting in the recognition of the UPR as a key player in pathological processes including complex inflammatory, autoimmune and neoplastic diseases. The intestinal epithelium, with its many highly secretory cells, forms an important barrier and messenger between the luminal environment and the host immune system. It is not surprising, that numerous studies have associated ER stress and the UPR with intestinal diseases such as inflammatory bowel disease (IBD) and colorectal cancer (CRC). In this review, we discuss our current understanding of the roles of ER stress and the UPR in shaping immune responses and maintaining tissue homeostasis. Furthermore, the role played by the UPR in disease, with emphasis on IBD and CRC, is described here. As a key player in immunity and inflammation, the UPR has been increasingly recognized as an important pharmacological target in the development of therapeutic strategies for immune-mediated pathologies. We summarize available strategies targeting the UPR and their therapeutic implications. Understanding the balance between homeostasis and pathophysiology, as well as means of manipulating this balance, provides an important avenue for future research.

Activated ATF6 Induces Intestinal Dysbiosis and Innate Immune Response to Promote Colorectal Tumorigenesis.

BACKGROUND & AIMS: Activating transcription factor 6 (ATF6) regulates endoplasmic reticulum stress. We studied whether ATF6 contributes to the development of colorectal cancer (CRC) using tissue from patients and transgenic mice. METHODS: We analyzed data from 541 patients with CRC in The Cancer Genome Atlas database for genetic variants and aberrant expression levels of unfolded protein response genes. Findings were validated in a cohort of 83 patients with CRC in Germany. We generated mice with intestinal epithelial cell-specific expression of the active form of Atf6 (nATF6IEC) from 2 alleles (homozygous), mice with expression of nATF6IEC from 1 allele (heterozygous), and nATF6IECfl/fl mice (controls). All nATF6IEC mice were housed under either specific-pathogen-free or germ-free conditions. Cecal microbiota from homozygous nATF6IEC mice or control mice was transferred into homozygous nATF6IEC mice or control mice. nATF6IEC mice were crossed with mice with disruptions in the myeloid differentiation primary response gene 88 and toll-like receptor adaptor molecule 1 gene (Myd88/Trif-knockout mice). Intestinal tissues were collected from mice and analyzed by histology, immunohistochemistry, immunoblots, gene expression profiling of unfolded protein response and inflammatory genes, array-based comparative genome hybridization, and 16S ribosomal RNA gene sequencing. RESULTS: Increased expression of ATF6 was associated with reduced disease-free survival times of patients with CRC. Homozygous nATF6IEC mice developed spontaneous colon adenomas at 12 weeks of age. Compared with controls, homozygous nATF6IEC mice had changes in the profile of their cecal microbiota, increased proliferation of intestinal epithelial cells, and loss of the mucus barrier-all preceding tumor formation. These mice had increased penetration of bacteria into the inner mucus layer and activation of signal transducer and activator of transcription 3, yet inflammation was not observed at the pretumor or tumor stages. Administration of antibiotics to homozygous nATF6IEC mice greatly reduced tumor incidence, and germ-free housing completely prevented tumorigenesis. Analysis of nATF6IEC MyD88/TRIF-knockout mice showed that tumor initiation and growth required MyD88/TRIF-dependent activation of signal transducer and activator of transcription 3. Transplantation of cecal microbiota from nATF6IEC mice and control mice, collected before tumor formation, caused tumor formation in ex-germ-free nATF6IEC mice. CONCLUSIONS: In patients with CRC, ATF6 was associated with reduced time of disease-free survival. In studies of nATF6IEC mice, we found sustained intestinal activation of ATF6 in the colon to promote dysbiosis and microbiota-dependent tumorigenesis.

Bacterial Signaling at the Intestinal Epithelial Interface in Inflammation and Cancer.

The gastrointestinal (GI) tract provides a compartmentalized interface with an enormous repertoire of immune and metabolic activities, where the multicellular structure of the mucosa has acquired mechanisms to sense luminal factors, such as nutrients, microbes, and a variety of host-derived and microbial metabolites. The GI tract is colonized by a complex ecosystem of microorganisms, which have developed a highly coevolved relationship with the host's cellular and immune system. Intestinal epithelial pattern recognition receptors (PRRs) substantially contribute to tissue homeostasis and immune surveillance. The role of bacteria-derived signals in intestinal epithelial homeostasis and repair has been addressed in mouse models deficient in PRRs and signaling adaptors. While critical for host physiology and the fortification of barrier function, the intestinal microbiota poses a considerable health challenge. Accumulating evidence indicates that dysbiosis is associated with the pathogenesis of numerous GI tract diseases, including inflammatory bowel diseases (IBD) and colorectal cancer (CRC). Aberrant signal integration at the epithelial cell level contributes to such diseases. An increased understanding of bacterial-specific structure recognition and signaling mechanisms at the intestinal epithelial interface is of great importance in the translation to future treatment strategies. In this review, we summarize the growing understanding of the regulation and function of the intestinal epithelial barrier, and discuss microbial signaling in the dynamic host-microbe mutualism in both health and disease.

Role of the Microbiota in Colorectal Cancer: Updates on Microbial Associations and Therapeutic Implications.

Genetic, environmental, and dietary factors have been found to influence the development and progression of colorectal cancer (CRC). More recently, accumulating evidence associates the intestinal microbiota with the initiation and progression of this disease. While studies have shown that individuals with CRC display alterations in gut bacterial composition, it remains somewhat unclear whether such differences drive cancer development or whether they are a response to tumorigenesis. In this review, the authors assess new evidence linking the community structure or specific bacterial factors of the intestinal microbiota to CRC development and progression, with insights into therapeutic implications.

Mitochondrial function controls intestinal epithelial stemness and proliferation.

Control of intestinal epithelial stemness is crucial for tissue homeostasis. Disturbances in epithelial function are implicated in inflammatory and neoplastic diseases of the gastrointestinal tract. Here we report that mitochondrial function plays a critical role in maintaining intestinal stemness and homeostasis. Using intestinal epithelial cell (IEC)-specific mouse models, we show that loss of HSP60, a mitochondrial chaperone, activates the mitochondrial unfolded protein response (MT-UPR) and results in mitochondrial dysfunction. HSP60-deficient crypts display loss of stemness and cell proliferation, accompanied by epithelial release of WNT10A and RSPO1. Sporadic failure of Cre-mediated Hsp60 deletion gives rise to hyperproliferative crypt foci originating from OLFM4+ stem cells. These effects are independent of the MT-UPR-associated transcription factor CHOP. In conclusion, compensatory hyperproliferation of HSP60+ escaper stem cells suggests paracrine release of WNT-related factors from HSP60-deficient, functionally impaired IEC to be pivotal in the control of the proliferative capacity of the stem cell niche.