Highly multiplexed spatial analysis identifies tissue-resident memory T cells as drivers of ulcerative and immune checkpoint inhibitor colitis.

Colitis is a prevalent adverse event associated with immune checkpoint inhibitor (ICI) therapy with similarities to inflammatory bowel disease. Incomplete mechanistic understanding of ICI colitis curtails evidence-based treatment. Given the often-overlooked connection between tissue architecture and mucosal immune cell function, we here applied imaging mass cytometry (IMC) to gain spatial proteomic insight in ICI colitis in comparison to ulcerative colitis (UC). Using a cell segmentation pipeline that simultaneously utilizes high-resolution nuclear imaging and high-multiplexity IMC, we show that intra-epithelial CD8+ T cells are significantly more abundant (and numerically dominant) in anti-PD-1 ± anti-CTLA-4-induced colitis compared to anti-CTLA-4-induced colitis and UC. We identified activated, cycling CD8+ tissue-resident memory T(RM) cells at the lamina propria-epithelial interface as drivers of cytotoxicity in ICI colitis and UC. Moreover, we found that combined ICI-induced colitis featured highest granzyme B levels both in tissue and serum. Together, these data reinforce CD8+ TRM cells as potentially targetable drivers of ICI colitis.

Compartment-driven imprinting of intestinal CD4 T cells in inflammatory bowel disease and homeostasis.

The mucosal immune system is implicated in the etiology and progression of inflammatory bowel diseases. The lamina propria and epithelium of the gut mucosa constitute two separate compartments, containing distinct T-cell populations. Human CD4 T-cell programming and regulation of lamina propria and epithelium CD4 T cells, especially during inflammation, remain incompletely understood. We performed flow cytometry, bulk, and single-cell RNA-sequencing to profile ileal lamina propria and intraepithelial CD4 T cells (CD4CD8αα, regulatory T cells (Tregs), CD69- and CD69high Trm T cells) in controls and Crohn's disease (CD) patients (paired non-inflamed and inflamed). Inflammation results in alterations of the CD4 T-cell population with a pronounced increase in Tregs and migrating/infiltrating cells. On a transcriptional level, inflammation within the epithelium induced T-cell activation, increased IFNγ responses, and an effector Treg profile. Conversely, few transcriptional changes within the lamina propria were observed. Key regulators including the chromatin remodelers ARID4B and SATB1 were found to drive compartment-specific transcriptional programming of CD4 T(reg) cells. In summary, inflammation in CD patients primarily induces changes within the epithelium and not the lamina propria. Additionally, there is compartment-specific CD4 T-cell imprinting, driven by shared regulators, between the lamina propria and the epithelium. The main consequence of intraepithelial adaptation, irrespective of inflammation, seems to be an overall dampening of broad (pro-inflammatory) responses and tight regulation of lifespan. These data suggest differential regulation of the lamina propria and epithelium, with a specific regulatory role in the inflamed epithelium.

Functional dyspepsia patients have lower mucosal cholecystokinin concentrations in response to duodenal lipid.

BACKGROUND: Dyspeptic symptoms are frequently induced, or exacerbated, by fatty food ingestion. Excessive release of, and/or hypersensitivity to, cholecystokinin (CCK) may explain the exaggerated response to lipid in patients with functional dyspepsia (FD). Thus far, plasma CCK response has been evaluated. However, stimulation of CCK1 receptors on duodenal vagal afferents occurs in a paracrine manner, suggesting that mucosal CCK concentrations are relevant to quantify. Apolipoprotein A-IV stimulates mucosal CCK release. AIM: To investigate the hypothesis that fat-induced release of CCK and apolipoprotein A-IV (apoA-IV) is enhanced in the duodenum of FD patients. PATIENTS AND METHODS: Sixteen symptomatic FD patients and 10 healthy volunteers (HV) underwent duodenal perfusion with intralipid 20%, 2 kcal/min, for 60 min. Symptoms were scored and blood samples were collected every 15 min during lipid perfusion and 15 min after discontinuation when duodenal biopsies were taken. Plasma and mucosal concentrations of CCK and apoA-IV were quantified. RESULTS: Abdominal discomfort (P=0.001), nausea (P=0.05), and fullness (P=0.005) in response to duodenal lipid increased significantly only in FD patients. Following lipid infusion, the mean mucosal CCK concentration was lower in FD patients compared with HV (P<0.0001). Fasting concentrations and plasma response of CCK were comparable in FD patients and HV. Plasma apoA-IV response appeared to differ between patients and HV, whereas mucosal apoA-IV concentrations were similar. CONCLUSION: Our results suggest excessive local release of CCK in response to duodenal lipid in FD. This likely causes exaggerated stimulation of duodenal vagal afferents, explaining dyspeptic symptom generation. The mechanisms underlying elevated mucosal CCK release warrant further investigation.

In gastroesophageal reflux disease, differential gene expression in the duodenum points towards enhanced chylomicron production and secretion.

BACKGROUND: Duodenal signaling affects esophageal motility and perception, both pathophysiological factors in gastroesophageal reflux disease (GERD). Duodenal gene expression abnormalities, contributing to altered esophageal sensorimotor function, have not been reported to date. AIM: To identify differentially expressed genes in GERD patients' duodenum. METHODS: Twenty GERD patients (total 24-h acid exposure 6-12%, SAP ≥95%) and ten healthy controls (HC) were included. Two weeks prior to duodenal biopsy collection, ten patients discontinued proton pump inhibitor (PPI) treatment and ten took maximum dose PPI. RNA was profiled on an Affymetrix Human Genome U133 Plus 2.0 array (Affymetrix, Santa Clara, CA, USA). Genes exhibiting a fold change ≥ 1.4 (t test p value <1E-4) were considered differentially expressed. A subset of 21 differentially expressed genes was selected for confirmatory TaqMan low-density array RT-PCR. Mucosal apolipoprotein A-IV (apoA-IV) and cholecystokinin (CCK) concentrations were determined by ELISA and RIA, respectively. RESULTS: In GERD patients off PPI, 23 up- and 23 down-regulated genes relative to HC were found. In GERD patients on PPI, 33 and five genes were higher, respectively, lower expressed. The majority of up-regulated genes were associated with lipid absorption, particularly triglyceride resynthesis and intracellular vesicular transport, rate-limiting processes for chylomicron production and secretion. Differential expression of 11 genes was confirmed by RT-PCR. Mucosal apoA-IV and CCK concentrations (signaling proteins released upon chylomicron secretion) were similar in GERD patients and HC. CONCLUSIONS: The identified mRNA expression differences suggest that in GERD patients' duodenum, the chylomicron production and secretion potential is elevated, and may underlie a mechanism by which postprandial duodenal signaling contributes to GERD symptom generation.

SERT and TPH-1 mRNA expression are reduced in irritable bowel syndrome patients regardless of visceral sensitivity state in large intestine.

Colorectal visceral hypersensitivity has been demonstrated in a subset of irritable bowel syndrome (IBS) patients. Serine protease and serotonergic signaling modulate gastrointestinal visceral sensitivity. We evaluated whether altered mucosal serine protease and serotonergic pathway components are related to rectal visceral hypersensitivity in IBS patients. Colorectal mucosal biopsies of 23 IBS patients and 15 controls were collected. Gene transcripts of protease-activated receptor (PAR)-2, trypsinogen IV, tryptophan hydroxylase (TPH)-1, and serotonin reuptake transporter (SERT) were quantified using real-time polymerase chain reaction. Substance P and 5-HT contents were measured by ELISA. The number of enterochromaffin cells, mast cells, and intraepithelial lymphocytes was determined using immunohistochemistry. Rectal visceral sensitivity was determined in IBS patients using barostat programmed for phasic ascending distension. Rectal hypersensitivity (+) and (-) IBS patients showed lower TPH-1 and SERT mRNA levels in the rectum compared with controls (P ≤ 0.05). Rectal hypersensitivity (+) IBS patients (n = 12) showed lower TPH-1 mRNA level in the sigmoid compared with controls (P = 0.015). No significant differences were observed in PAR-2 and trypsinogen IV expression between controls and IBS patients. Rectal substance P content was increased in IBS patients compared with controls (P = 0.045). No significant differences were found in transcript levels, cell counts, and substance P and 5-HT contents between rectal hypersensitivity (+) and (-) IBS patients. In conclusion, regardless of visceral hypersensitivity state, several serotonergic signaling components are altered in IBS patients.

Serotonin synthesis and uptake in symptomatic patients with Crohn's disease in remission.

BACKGROUND & AIMS: Symptoms resembling irritable bowel syndrome (IBS) are reported frequently in Crohn's disease (CD) patients in remission. Studies of the mucosal content of serotonin, which is a pivotal neurotransmitter in the gut, suggest that serotonin availability is altered in IBS patients. We aimed to study the role of serotonin in the generation of IBS-like symptoms in CD patients in remission. METHODS: Ileal and colonic biopsy specimens were obtained from 20 CD patients in remission, 10 with and 10 without IBS-like symptoms, and 11 healthy controls. Enterochromaffin cells were counted, and messenger RNA expression levels of tryptophan hydroxylase (TpH)-1 and serotonin reuptake transporter were determined. RESULTS: The levels of mucosal serotonin reuptake transporter expression were significantly higher in the ileum than in the colon, in all groups studied (P < .02). When the ileum and colon were analyzed separately, TpH-1 expression in the colon of CD patients with IBS-like symptoms was found to be significantly higher compared with the 2 other studied groups (controls, P < .005; CD patients without IBS-like symptoms, P < .01). The number of enterochromaffin cells per gland was comparable for the patient groups in the ileum and colon. CONCLUSIONS: CD patients in remission who experience IBS-like symptoms have increased mucosal TpH-1 levels in the colon, suggesting that increased serotonin biosynthesis in the colon plays a role in the generation of the symptoms.

A microarray-assisted screen for potential Hap1 and Rox1 target genes in Saccharomyces cerevisiae.

Saccharomyces cerevisiae adapts to altered oxygen availability by differentially expressing a number of genes. Under aerobic conditions oxygen control of gene expression is exerted through the activator Hap1 and the repressor Rox1. The Hap1 transcription factor senses cellular heme status and increases expression of aerobic genes in response to oxygen. The repression of hypoxic genes under normoxic conditions results from Hap1-mediated activation of ROX1 transcription. To allow the identification of additional Hap1 and Rox1 target genes, genome-wide expression was analysed in aerobically, chemostat-cultivated hap1 and rox1 null mutants. The microarray results show that deletion of HAP1 causes a lower transcript level of 51 genes. Transcription of 40 genes was increased in rox1 mutant cells compared to wild-type cells. Combining these results with our previously described transcriptome data of aerobically and anaerobically grown cells and with computational analysis of the promoters identified 24 genes that are potentially regulated by Hap1, and 38 genes satisfied the criteria of being direct targets of Rox1. In addition, this work provides further evidence that Rox1 controls transcription of anaerobic genes through repression under normoxic conditions.