Etrolizumab-s fails to control E-Cadherin-dependent co-stimulation of highly activated cytotoxic T cells.

Despite promising preclinical and earlier clinical data, a recent phase III trial on the anti-ß7 integrin antibody etrolizumab in Crohn's disease (CD) did not reach its primary endpoint. The mechanisms leading to this outcome are not well understood. Here we characterize the ß7+ T cell compartment from patients with CD in comparison to cells from individuals without inflammatory bowel disease. By flow cytometric, transcriptomic and functional profiling of circulating T cells, we find that triple-integrin-expressing (α4+ß7+ß1hi) T cells have the potential to home to the gut despite α4ß7 blockade and have a specific cytotoxic signature. A subset of triple-integrin-expressing cells readily acquires αE expression and could be co-stimulated via E-Cadherin-αEß7 interactions in vitro. Etrolizumab-s fails to block such αEß7 signalling at high levels of T cell stimulation. Consistently, in CD patients treated with etrolizumab, T cell activation correlates with cytotoxic signatures. Collectively, our findings might add one important piece to the puzzle to explain phase III trial results with etrolizumab, while they also highlight that αEß7 remains an interesting target for future therapeutic approaches in inflammatory bowel disease.

ATP citrate lyase (ACLY)-dependent immunometabolism in mucosal T cells drives experimental colitis in vivo.

OBJECTIVE: Mucosal T cells play a major role in inflammatory bowel disease (IBD). However, their immunometabolism during intestinal inflammation is poorly understood. Due to its impact on cellular metabolism and proinflammatory immune cell function, we here focus on the enzyme ATP citrate lyase (ACLY) in mucosal T cell immunometabolism and its relevance for IBD. DESIGN: ACLY expression and its immunometabolic impact on colitogenic T cell function were analysed in mucosal T cells from patients with IBD and in two experimental colitis models. RESULTS: ACLY was markedly expressed in colon tissue under steady-state conditions but was significantly downregulated in lamina propria mononuclear cells in experimental dextran sodium sulfate-induced colitis and in CD4+ and to a lesser extent in CD8+ T cells infiltrating the inflamed gut in patients with IBD. ACLY-deficient CD4+ T cells showed an impaired capacity to induce intestinal inflammation in a transfer colitis model as compared with wild-type T cells. Assessment of T cell immunometabolism revealed that ACLY deficiency dampened the production of IBD-relevant cytokines and impaired glycolytic ATP production but enriched metabolites involved in the biosynthesis of phospholipids and phosphatidylcholine. Interestingly, the short-chain fatty acid butyrate was identified as a potent suppressor of ACLY expression in T cells, while IL-36α and resolvin E1 induced ACLY levels. In a translational approach, in vivo administration of the butyrate prodrug tributyrin downregulated mucosal infiltration of ACLYhigh CD4+ T cells and ameliorated chronic colitis. CONCLUSION: ACLY controls mucosal T cell immunometabolism and experimental colitis. Therapeutic modulation of ACLY expression in T cells emerges as a novel strategy to promote the resolution of intestinal inflammation.

Blocking GPR15 Counteracts Integrin-dependent T Cell Gut Homing in Vivo.

BACKGROUND AND AIMS: The G protein coupled receptor GPR15 is expressed on and functionally important for T cells homing to the large intestine. However, the precise mechanisms by which GPR15 controls gut homing have been unclear. Thus, we aimed to elucidate these mechanisms as well as to explore the potential of targeting GPR15 for interfering with T cell recruitment to the colon in inflammatory bowel disease [IBD]. METHODS: We used dynamic adhesion and transmigration assays, as well as a humanised in vivo model of intestinal cell trafficking, to study GPR15-dependent effects on gut homing. Moreover, we analysed GPR15 and integrin expression in patients with and without IBD, cross-sectionally and longitudinally. RESULTS: GPR15 controlled T cell adhesion to MAdCAM-1 and VCAM-1 upstream of α4ß7 and α4ß1 integrin, respectively. Consistently, high co-expression of these integrins with GPR15 was found on T cells from patients with IBD, and GPR15 also promoted T cell recruitment to the colon in humanised mice. Anti-GPR15 antibodies effectively blocked T cell gut homing in vitro and in vivo. In vitro data, as well as observations in a cohort of patients treated with vedolizumab, suggest that this might be more effective than inhibiting α4ß7. CONCLUSIONS: GPR15 seems to have a broad, but organ-selective, impact on T cell trafficking and is therefore a promising target for future therapy of IBD. Further studies are needed.

Adhesion GPCR Gpr126 (Adgrg6) Expression Profiling in Zebrafish, Mouse, and Human Kidney.

Adhesion G protein-coupled receptors (aGPCRs) comprise the second-largest class of GPCRs, the most common target for approved pharmacological therapies. aGPCRs play an important role in development and disease and have recently been associated with the kidney. Several aGPCRs are expressed in the kidney and some aGPCRs are either required for kidney development or their expression level is altered in diseased kidneys. Yet, general aGPCR function and their physiological role in the kidney are poorly understood. Here, we characterize in detail Gpr126 (Adgrg6) expression based on RNAscope® technology in zebrafish, mice, and humans during kidney development in adults. Gpr126 expression is enriched in the epithelial linage during nephrogenesis and persists in the adult kidney in parietal epithelial cells, collecting ducts, and urothelium. Single-cell RNAseq analysis shows that gpr126 expression is detected in zebrafish in a distinct ionocyte sub-population. It is co-detected selectively with slc9a3.2, slc4a4a, and trpv6, known to be involved in apical acid secretion, buffering blood or intracellular pH, and to maintain high cytoplasmic Ca2+ concentration, respectively. Furthermore, gpr126-expressing cells were enriched in the expression of potassium transporter kcnj1a.1 and gcm2, which regulate the expression of a calcium sensor receptor. Notably, the expression patterns of Trpv6, Kcnj1a.1, and Gpr126 in mouse kidneys are highly similar. Collectively, our approach permits a detailed insight into the spatio-temporal expression of Gpr126 and provides a basis to elucidate a possible role of Gpr126 in kidney physiology.

IL-3 receptor signalling suppresses chronic intestinal inflammation by controlling mechanobiology and tissue egress of regulatory T cells.

IL-3 has been reported to be involved in various inflammatory disorders, but its role in inflammatory bowel disease (IBD) has not been addressed so far. Here, we determined IL-3 expression in samples from patients with IBD and studied the impact of Il3 or Il3r deficiency on T cell-dependent experimental colitis. We explored the mechanical, cytoskeletal and migratory properties of Il3r -/- and Il3r +/+ T cells using real-time deformability cytometry, atomic force microscopy, scanning electron microscopy, fluorescence recovery after photobleaching and in vitro and in vivo cell trafficking assays. We observed that, in patients with IBD, the levels of IL-3 in the inflamed mucosa were increased. In vivo, experimental chronic colitis on T cell transfer was exacerbated in the absence of Il-3 or Il-3r signalling. This was attributable to Il-3r signalling-induced changes in kinase phosphorylation and actin cytoskeleton structure, resulting in increased mechanical deformability and enhanced egress of Tregs from the inflamed colon mucosa. Similarly, IL-3 controlled mechanobiology in human Tregs and was associated with increased mucosal Treg abundance in patients with IBD. Collectively, our data reveal that IL-3 signaling exerts an important regulatory role at the interface of biophysical and migratory T cell features in intestinal inflammation and suggest that this might be an interesting target for future intervention.

Manual cell selection in single cell transcriptomics using scSELpy supports the analysis of immune cell subsets.

Introduction: Single cell RNA sequencing plays an increasing and indispensable role in immunological research such as in the field of inflammatory bowel diseases (IBD). Professional pipelines are complex, but tools for the manual selection and further downstream analysis of single cell populations are missing so far. Methods: We developed a tool called scSELpy, which can easily be integrated into Scanpy-based pipelines, allowing the manual selection of cells on single cell transcriptomic datasets by drawing polygons on various data representations. The tool further supports the downstream analysis of the selected cells and the plotting of results. Results: Taking advantage of two previously published single cell RNA sequencing datasets we show that this tool is useful for the positive and negative selection of T cell subsets implicated in IBD beyond standard clustering. We further demonstrate the feasibility for subphenotyping T cell subsets and use scSELpy to corroborate earlier conclusions drawn from the dataset. Moreover, we also show its usefulness in the context of T cell receptor sequencing. Discussion: Collectively, scSELpy is a promising additive tool fulfilling a so far unmet need in the field of single cell transcriptomic analysis that might support future immunological research.

Differential Effects of Ontamalimab Versus Vedolizumab on Immune Cell Trafficking in Intestinal Inflammation and Inflammatory Bowel Disease.

BACKGROUND AND AIMS: The anti-MAdCAM-1 antibody ontamalimab demonstrated efficacy in a phase II trial in ulcerative colitis and results of early terminated phase III trials are pending, but its precise mechanisms of action are still unclear. Thus, we explored the mechanisms of action of ontamalimab and compared it to the anti-α4ß7 antibody vedolizumab. METHODS: We studied MAdCAM-1 expression with RNA sequencing and immunohistochemistry. The mechanisms of action of ontamalimab were assessed with fluorescence microscopy, dynamic adhesion and rolling assays. We performed in vivo cell trafficking studies in mice and compared ontamalimab and vedolizumab surrogate [-s] antibodies in experimental models of colitis and wound healing. We analysed immune cell infiltration under anti-MAdCAM-1 and anti-α4ß7 treatment by single-cell transcriptomics and studied compensatory trafficking pathways. RESULTS: MAdCAM-1 expression was increased in active inflammatory bowel disease. Binding of ontamalimab to MAdCAM-1 induced the internalization of the complex. Functionally, ontamalimab blocked T cell adhesion similar to vedolizumab, but also inhibited L-selectin-dependent rolling of innate and adaptive immune cells. Despite conserved mechanisms in mice, the impact of ontamalimab-s and vedolizumab-s on experimental colitis and wound healing was similar. Single-cell RNA sequencing demonstrated enrichment of ontamalimab-s-treated lamina propria cells in specific clusters, and in vitro experiments indicated that redundant adhesion pathways are active in these cells. CONCLUSIONS: Ontamalimab has unique and broader mechanisms of action compared to vedolizumab. However, this seems to be compensated for by redundant cell trafficking circuits and leads to similar preclinical efficacy of anti-α4ß7 and anti-MAdCAM-1 treatment. These results will be important for the interpretation of pending phase III data.

Anti-ß7 integrin treatment impedes the recruitment on non-classical monocytes to the gut and delays macrophage-mediated intestinal wound healing.

BACKGROUND: Closing mucosal defects to reach mucosal healing is an important goal of therapy in inflammatory bowel disease (IBD). Among other cells, monocyte-derived macrophages are centrally involved in such intestinal wound healing. We had previously demonstrated that the anti-α4ß7 integrin antibody vedolizumab blocks the recruitment of non-classical monocytes as biased progenitors of wound healing macrophages to the gut and delays wound healing. However, although important for the interpretation of disappointing results in recent phase III trials in IBD, the effects of the anti-ß7 antibody etrolizumab on wound healing are unclear so far. METHODS: We analyzed the expression of etrolizumab targets on human and mouse monocyte subsets by flow cytometry and assessed their function in adhesion and homing assays. We explored wound-associated monocyte recruitment dynamics with multi-photon microscopy and compared the effects of etrolizumab and vedolizumab surrogate (-s) antibodies on experimental wound healing and wound-associated macrophage abundance. Finally, we investigated wound healing macrophage signatures in the large intestinal transcriptome of patients with Crohn's disease treated with etrolizumab. RESULTS: Human and mouse non-classical monocytes expressed more αEß7 integrin than classical monocytes and were a target of etrolizumab-s, which blocked non-classical monocyte adhesion to MAdCAM-1 and E-Cadherin as well as gut homing in vivo. Intestinal wound healing was delayed on treatment with etrolizumab-s along with a reduction of peri-lesional wound healing macrophages. Wound healing macrophage signatures in the colon of patients with Crohn's disease were substantially down-regulated on treatment with etrolizumab, but not with placebo. CONCLUSIONS: Combined blockade of αEß7 and α4ß7 with etrolizumab seems to exceed the effect of anti-α4ß7 treatment on intestinal wound healing, which might help to inform further investigations to understand the recent observations in the etrolizumab phase III trial program.

Residual homing of α4ß7-expressing ß1+PI16+ regulatory T cells with potent suppressive activity correlates with exposure-efficacy of vedolizumab.

OBJECTIVE: The anti-α4ß7 integrin antibody vedolizumab is administered at a fixed dose for the treatment of IBDs. This leads to a wide range of serum concentrations in patients and previous studies had suggested that highest exposure levels are associated with suboptimal clinical response. We aimed to determine the mechanisms underlying these non-linear exposure-efficacy characteristics of vedolizumab. DESIGN: We characterised over 500 samples from more than 300 subjects. We studied the binding of vedolizumab to T cells and investigated the functional consequences for dynamic adhesion, transmigration, gut homing and free binding sites in vivo. Employing single-cell RNA sequencing, we characterised α4ß7 integrin-expressing T cell populations 'resistant' to vedolizumab and validated our findings in vitro and in samples from vedolizumab-treated patients with IBD. We also correlated our findings with a post-hoc analysis of the Gemini II and III studies. RESULTS: Regulatory T (TReg) cells exhibited a right-shifted vedolizumab binding profile compared with effector T (TEff) cells. Consistently, in a certain concentration range, the residual adhesion, transmigration, homing of and availability of functional α4ß7 on TReg cells in vivo was higher than that of/on TEff cells. We identified a vedolizumab-'resistant' α4ß7-expressing ß1+PI16+ TReg cell subset with pronounced regulatory properties as the substrate for this effect. Our observations correlated with exposure-efficacy data from Gemini II and III trials. CONCLUSION: Completely blocking TEff cell trafficking with vedolizumab, while simultaneously permitting residual homing of powerful TReg cells in an optimal 'therapeutic window' based on target exposure levels might be a strategy to optimise treatment outcomes in patients with IBD.

Implementation of high-resolution melting analysis of the porcupine (PORCN) gene for molecular diagnosis of focal dermal hypoplasia: Identification of a novel mutation.

BACKGROUND: Focal dermal hypoplasia (FDH) is rare X-linked dominant disease characterized by atrophy and linear pigmentation of the skin, split hand/foot deformities and ocular anomalies. FDH is caused by mutations of the Porcupine (PORCN) gene, which encodes an enzyme that catalyzes the palmitoylation of Wnt ligands required for their secretion. High resolution melting analysis (HRM) is a technique that allows rapid, labor-efficient, low-cost detection of genomic variants. In the present study, we report the successful implementation of HRM in the molecular diagnosis of FDH. METHODS: Polymerase chain reaction and HRM assays were designed and optimized for each of the coding exons of the PORCN gene, processing genomic DNA samples form a non-affected control and a patient complying with the FDH diagnostic criteria. The causal mutation was characterized by Sanger sequencing from an amplicon showing a HRM trace suggesting heterozygous variation and was validated using an amplification-refractory mutation system (ARMS) assay. RESULTS: The melting profiles suggested the presence of a variant in the patient within exon 1. Sanger sequencing revealed a previously unknown C to T transition replacing a glutamine codon for a premature stop codon at position 28, which was validated using ARMS. CONCLUSIONS: Next-generation sequencing facilitates the molecular diagnosis of monogenic disorders; however, its cost-benefit ratio is not optimal when a single, small or medium size causal gene is already identified and the clinical diagnostic presumption is strong. Under those conditions, as it is the case for FDH, HRM represents a cost- and labor-effective approach.

Identification of human miR-1839-5p by small RNA-seq, a miRNA enriched in neoplastic tissues.

BACKGROUND: MicroRNAs (miRNAs) modulate gene expression through destabilization or translational inhibition of cytoplasmic transcripts or by transcriptional regulation through binding to genomic DNA. Although miRNAs are globally down-regulated in cancer, some are overexpressed in neoplastic tissues, playing key roles in tumorigenesis (oncomiRs), sometimes behaving as effective cancer markers. METHODS: Using total RNA from human uterus adenocarcinoma and non-neoplastic uterus, we conducted a small RNA-sequencing experiment followed by prediction of novel miRNAs using MirDeep* software. Synteny analysis and whole genome alignments were performed using BLAST. We also evaluated expression by a reverse transcriptase-polymerase chain reaction (RT-PCR) in normal tissues of the FSD2 gene, which spans the human miR-1839-5p gene in the opposite direction. RESULTS: MirDeep* analysis predicted a miRNA not previously annotated in databases, identical to and likely the orthologue of mouse miR-1839-5p. Whole-genome local alignments of this miRNA revealed a single perfect hit that is indeed syntenic to mouse miR-1839-5p. Alignments with other mammalian orthologues showed considerable conservation. We validated the prediction via a stem-loop RT-PCR assay, also employed to screen RNA samples from several additional normal and cancer tissues, showing increased expression in neoplastic tissues compared to their respective non neoplastic counterparts. Human heart tissue expresses both miR-1839-5p and FSD2. CONCLUSIONS: Human tissues express an orthologue of mouse miR-1839-5p and, given its expression pattern, we suggest that this miRNA could be explored as a potential oncomiR or cancer marker. Also, according to the genomic organization of miR-1839-5p and FSD2, perfect complementarity exists between the two elements, making possible miRNA-directed cleavage in human cardiac tissue.

CRISPR-Cas9-mediated genome editing in vancomycin-resistant Enterococcus faecium.

The Gram-positive bacterium Enterococcus faecium is becoming increasingly prevalent as a cause of hospital-acquired, antibiotic-resistant infections. A fundamental part of research into E. faecium biology relies on the ability to generate targeted mutants but this process is currently labour-intensive and time-consuming, taking 4 to 5 weeks per mutant. In this report, we describe a method relying on the high recombination rates of E. faecium and the application of the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas9 genome editing tool to more efficiently generate targeted mutants in the E. faecium chromosome. Using this tool and the multi-drug resistant clinical E. faecium strain E745, we generated a deletion mutant in the lacL gene, which encodes the large subunit of the E. faeciumß-galactosidase. Blue/white screening using 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal) could be used to distinguish between the wild-type and lacL deletion mutant. We also inserted two copies of gfp into the intrinsic E. faecium macrolide resistance gene msrC to generate stable green fluorescent cells. We conclude that CRISPR-Cas9 can be used to generate targeted genome modifications in E. faecium in 3 weeks, with limited hands-on time. This method can potentially be implemented in other Gram-positive bacteria with high intrinsic recombination rates.