Tracking in situ checkpoint inhibitor-bound target T cells in patients with checkpoint-induced colitis.

The success of checkpoint inhibitors (CPIs) for cancer has been tempered by immune-related adverse effects including colitis. CPI-induced colitis is hallmarked by expansion of resident mucosal IFNγ cytotoxic CD8+ T cells, but how these arise is unclear. Here, we track CPI-bound T cells in intestinal tissue using multimodal single-cell and subcellular spatial transcriptomics (ST). Target occupancy was increased in inflamed tissue, with drug-bound T cells located in distinct microdomains distinguished by specific intercellular signaling and transcriptional gradients. CPI-bound cells were largely CD4+ T cells, including enrichment in CPI-bound peripheral helper, follicular helper, and regulatory T cells. IFNγ CD8+ T cells emerged from both tissue-resident memory (TRM) and peripheral populations, displayed more restricted target occupancy profiles, and co-localized with damaged epithelial microdomains lacking effective regulatory cues. Our multimodal analysis identifies causal pathways and constitutes a resource to inform novel preventive strategies.

Dual RNA sequencing reveals dendritic cell reprogramming in response to typhoidal Salmonella invasion.

Salmonella enterica represent a major disease burden worldwide. S. enterica serovar Typhi (S. Typhi) is responsible for potentially life-threatening Typhoid fever affecting 10.9 million people annually. While non-typhoidal Salmonella (NTS) serovars usually trigger self-limiting diarrhoea, invasive NTS bacteraemia is a growing public health challenge. Dendritic cells (DCs) are key professional antigen presenting cells of the human immune system. The ability of pathogenic bacteria to subvert DC functions and prevent T cell recognition contributes to their survival and dissemination within the host. Here, we adapted dual RNA-sequencing to define how different Salmonella pathovariants remodel their gene expression in tandem with that of infected DCs. We find DCs harness iron handling pathways to defend against invading Salmonellas, which S. Typhi is able to circumvent by mounting a robust response to nitrosative stress. In parallel, we uncover the alternative strategies invasive NTS employ to impair DC functions.

IL-6 effector function of group 2 innate lymphoid cells (ILC2) is NOD2 dependent.

Cutaneous group 2 innate lymphoid cells (ILC2) are spatially and epigenetically poised to respond to barrier compromise and associated immunological threats. ILC2, lacking rearranged antigen-specific receptors, are primarily activated by damage-associated cytokines and respond with type 2 cytokine production. To investigate ILC2 potential for direct sensing of skin pathogens and allergens, we performed RNA sequencing of ILC2 derived from in vivo challenged human skin or blood. We detected expression of NOD2 and TLR2 by skin and blood ILC2. Stimulation of ILC2 with TLR2 agonist alone not only induced interleukin-5 (IL-5) and IL-13 expression but also elicited IL-6 expression in combination with Staphylococcus aureus muramyl dipeptide (MDP). Heat-killed skin-resident bacteria provoked an IL-6 profile in ILC2 in vitro that was notably impaired in ILC2 derived from patients with nucleotide-binding oligomerization domain-containing protein 2 (NOD2) mutations. In addition, we show that NOD2 signaling can stimulate autophagy in ILC2, which was also impaired in patients with NOD2 mutations. Here, we have identified a role for ILC2 NOD2 signaling in the differential regulation of ILC2-derived IL-6 and have reported a previously unrecognized pathway of direct ILC2 bacterial sensing.

Spatiotemporal analysis of human intestinal development at single-cell resolution.

Development of the human intestine is not well understood. Here, we link single-cell RNA sequencing and spatial transcriptomics to characterize intestinal morphogenesis through time. We identify 101 cell states including epithelial and mesenchymal progenitor populations and programs linked to key morphogenetic milestones. We describe principles of crypt-villus axis formation; neural, vascular, mesenchymal morphogenesis, and immune population of the developing gut. We identify the differentiation hierarchies of developing fibroblast and myofibroblast subtypes and describe diverse functions for these including as vascular niche cells. We pinpoint the origins of Peyer's patches and gut-associated lymphoid tissue (GALT) and describe location-specific immune programs. We use our resource to present an unbiased analysis of morphogen gradients that direct sequential waves of cellular differentiation and define cells and locations linked to rare developmental intestinal disorders. We compile a publicly available online resource, spatio-temporal analysis resource of fetal intestinal development (STAR-FINDer), to facilitate further work.

Unexpectedly High Levels of Inverted Re-Insertions Using Paired sgRNAs for Genomic Deletions.

Use of dual sgRNAs is a common CRISPR/Cas9-based strategy for the creation of genetic deletions. The ease of screening combined with a rather high rate of success makes this approach a reliable genome engineering procedure. Recently, a number of studies using CRISPR/Cas9 have revealed unwanted large-scale rearrangements, duplications, inversions or larger-than-expected deletions. Strict quality control measures are required to validate the model system, and this crucially depends on knowing which potential experimental outcomes to expect. Using the dual sgRNA deletion approach, our team discovered high levels of excision, inversion and re-insertion at the site of targeting. We detected those at a variety of genomic loci and in several immortalized cell lines, demonstrating that inverted re-insertions are a common by-product with an overall frequency between 3% and 20%. Our findings imply an inherent danger in the misinterpretation of screening data when using only a single PCR screening. While amplification of the region of interest might classify clones as wild type (WT) based on amplicon size, secondary analyses can discover heterozygous (HET) clones among presumptive WTs, and events deemed as HET clones could potentially be full KO. As such, screening for inverted re-insertions helps in decreasing the number of clones required to obtain a full KO. With this technical note, we want to raise awareness of this phenomenon and suggest implementing a standard secondary PCR while screening for deletions.

Single-cell atlas of colonic CD8+ T cells in ulcerative colitis.

Colonic antigen-experienced lymphocytes such as tissue-resident memory CD8+ T cells can respond rapidly to repeated antigen exposure. However, their cellular phenotypes and the mechanisms by which they drive immune regulation and inflammation remain unclear. Here we compiled an unbiased atlas of human colonic CD8+ T cells in health and ulcerative colitis (UC) using single-cell transcriptomics with T-cell receptor repertoire analysis and mass cytometry. We reveal extensive heterogeneity in CD8+ T-cell composition, including expanded effector and post-effector terminally differentiated CD8+ T cells. While UC-associated CD8+ effector T cells can trigger tissue destruction and produce tumor necrosis factor (TNF)-α, post-effector cells acquire innate signatures to adopt regulatory functions that may mitigate excessive inflammation. Thus, we identify colonic CD8+ T-cell phenotypes in health and UC, define their clonal relationships and characterize terminally differentiated dysfunctional UC CD8+ T cells expressing IL-26, which attenuate acute colitis in a humanized IL-26 transgenic mouse model.

Colonic epithelial cell diversity in health and inflammatory bowel disease.

The colonic epithelium facilitates host-microorganism interactions to control mucosal immunity, coordinate nutrient recycling and form a mucus barrier. Breakdown of the epithelial barrier underpins inflammatory bowel disease (IBD). However, the specific contributions of each epithelial-cell subtype to this process are unknown. Here we profile single colonic epithelial cells from patients with IBD and unaffected controls. We identify previously unknown cellular subtypes, including gradients of progenitor cells, colonocytes and goblet cells within intestinal crypts. At the top of the crypts, we find a previously unknown absorptive cell, expressing the proton channel OTOP2 and the satiety peptide uroguanylin, that senses pH and is dysregulated in inflammation and cancer. In IBD, we observe a positional remodelling of goblet cells that coincides with downregulation of WFDC2-an antiprotease molecule that we find to be expressed by goblet cells and that inhibits bacterial growth. In vivo, WFDC2 preserves the integrity of tight junctions between epithelial cells and prevents invasion by commensal bacteria and mucosal inflammation. We delineate markers and transcriptional states, identify a colonic epithelial cell and uncover fundamental determinants of barrier breakdown in IBD.

Structural Remodeling of the Human Colonic Mesenchyme in Inflammatory Bowel Disease.

Intestinal mesenchymal cells play essential roles in epithelial homeostasis, matrix remodeling, immunity, and inflammation. But the extent of heterogeneity within the colonic mesenchyme in these processes remains unknown. Using unbiased single-cell profiling of over 16,500 colonic mesenchymal cells, we reveal four subsets of fibroblasts expressing divergent transcriptional regulators and functional pathways, in addition to pericytes and myofibroblasts. We identified a niche population located in proximity to epithelial crypts expressing SOX6, F3 (CD142), and WNT genes essential for colonic epithelial stem cell function. In colitis, we observed dysregulation of this niche and emergence of an activated mesenchymal population. This subset expressed TNF superfamily member 14 (TNFSF14), fibroblastic reticular cell-associated genes, IL-33, and Lysyl oxidases. Further, it induced factors that impaired epithelial proliferation and maturation and contributed to oxidative stress and disease severity in vivo. Our work defines how the colonic mesenchyme remodels to fuel inflammation and barrier dysfunction in IBD.

Plasma Nuclear Magnetic Resonance Metabolomics Discriminates Between High and Low Endoscopic Activity and Predicts Progression in a Prospective Cohort of Patients With Ulcerative Colitis.

BACKGROUND AND AIMS: Endoscopic assessment of ulcerative colitis [UC] is one of the most accurate measures of disease activity, but frequent endoscopic investigations are disliked by patients and expensive for the healthcare system. A minimally invasive test that provides a surrogate measure of endoscopic activity is required. METHODS: Plasma nuclear magnetic resonance [NMR] spectra from 40 patients with UC followed prospectively over 6 months were analysed with multivariate statistics. NMR metabolite profiles were compared with endoscopic [Ulcerative Colitis Endoscopic Index of Severity: UCEIS], histological [Nancy Index] and clinical [Simple Clinical Colitis Activity Index: SCCAI] severity indices, along with routine blood measurements. RESULTS: A blinded principal component analysis spontaneously separated metabolite profiles of patients with low [≤3] and high [>3] UCEIS. Orthogonal partial least squares discrimination analysis identified low and high UCEIS metabolite profiles with an accuracy of 77 ± 5%. Plasma metabolites driving discrimination included decreases in lipoproteins and increases in isoleucine, valine, glucose and myo-inositol in high compared to low UCEIS. This same metabolite profile distinguished between low [Nancy 0-1] and high histological activity [Nancy 3-4] with a modest although significant accuracy [65 ± 6%] but was independent of SCCAI and all blood parameters measured. A different metabolite profile, dominated by changes in lysine, histidine, phenylalanine and tyrosine, distinguished between improvement in UCEIS [decrease ≥1] and worsening [increase ≥1] over 6 months with an accuracy of 74 ± 4%. CONCLUSION: Plasma NMR metabolite analysis has the potential to provide a low-cost, minimally invasive technique that may be a surrogate for endoscopic assessment, with predictive capacity.

ICG-001 affects DRP1 activity and ER stress correlative with its anti-proliferative effect.

Mitochondria form a highly dynamic network driven by opposing scission and fusion events. DRP1 is an essential modulator of mitochondrial fission and dynamics within mammalian cells. Its fission activity is regulated by posttranslational modifications such as activating phosphorylation at serine 616. DRP1 activity has recently been implicated as being dysregulated in numerous human disorders such as cancer and neurodegenerative diseases. Here we describe the development of a cell-based screening assay to detect DRP1 activation. We utilized this to undertake focused compound library screening and identified potent modulators that affected DRP1 activity including ICG-001, which is described as WNT/ß-catenin signaling inhibitor. Our findings elucidate novel details about ICG-001's mechanism of action (MOA) in mediating anti-proliferative activity. We show ICG-001 both inhibits mitochondrial fission and activates an early endoplasmic reticulum (ER) stress response to induce cell death in susceptible colorectal cancer cell lines.