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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. Profiling of single epithelial cells in healthy and inflamed colons identifies specialized cellular subpopulations, including a type of goblet cell that secretes the antibacterial protein WFDC2, which preserves the integrity of the epithelial barrier layer.

Non-typhoidal Salmonella (NTS) are highly prevalent food-borne pathogens. Recently, a highly invasive, multi-drug resistant S . Typhimurium, ST313, emerged as a major cause of bacteraemia in children and immunosuppressed adults, however the pathogenic mechanisms remain unclear. Here, we utilize invasive and non-invasive Salmonella strains combined with single-cell RNA-sequencing to study the transcriptome of individual infected and bystander monocyte-derived dendritic cells (MoDCs) implicated in disseminating invasive ST313. Compared with non-invasive Salmonella , ST313 directs a highly heterogeneous innate immune response. Bystander MoDCs exhibit a hyper-activated profile potentially diverting adaptive immunity away from infected cells. MoDCs harbouring invasive Salmonella display higher expression of IL10 and MARCH1 concomitant with lower expression of CD83 to evade adaptive immune detection. Finally, we demonstrate how these mechanisms conjointly restrain MoDC-mediated activation of Salmonella -specific CD4 + T cell clones. Here, we show how invasive ST313 exploits discrete evasion strategies within infected and bystander MoDCs to mediate its dissemination in vivo. Salmonella employ a range of strategies to counter host defences during infection. Here, Aulicino et al. use single-cell RNA-sequencing to examine the effects of invasive and non-invasive strains of Salmonella, revealing discrete and divergent immune evasion strategies in infected and bystander dendritic cells.

The recently described ESX-5 secretion system of Mycobacterium tuberculosis is one of the most important modulators of host-pathogen interactions due to its crucial impact on PPE protein secretion, cell wall stability and virulence. Although various components of the ESX-5 secretion machinery have been defined, other ESX-5 core components still remain to be characterized. In this study, we focused on EccB(5) and EccC(5), a transmembrane protein (EccB(5)) and a membrane-bound ATPase (EccC(5)), both predicted to be building blocks of the M. tuberculosis ESX-5 membrane-associated complex. In vitro expression studies demonstrated that EccB(5) and EccC(5) encoding genes constitute an operon. The expression of this operon is essential for M. tuberculosis, since the deletion of the eccB(5)-eccC(5) genomic segment at the ESX-5 locus is possible only after the integration of a second functional copy of eccB(5)-eccC(5) genes into the M. tuberculosis chromosome. The characterization of two M. tuberculosis conditional mutant strains (Mtb(Pptr)eccB(5) and Mtb(Pptr)eccC(5)), in which the eccB(5)-eccC(5) operon or the eccC(5) gene, respectively, were expressed under the control of an anhydrotetracycline-repressible promoter, confirmed that the repression of eccB(5)-eccC(5) genes is detrimental for growth of M. tuberculosis both in vitro and in THP-1 human macrophage cell line. Moreover, analysis of the secretome of Mtb(Pptr)eccB(5)-eccC(5) and Mtb(Pptr)eccC(5) strains revealed that both EccB(5) and EccC(5) are required for secretion of ESX-5 specific substrates, thus confirming that they are indeed components of the ESX-5 secretion machinery. Taken together these findings demonstrate the importance of an intact and functional ESX-5 system for viability of M. tuberculosis, thus opening new interesting options for alternative antimycobacterial control strategies.

The interplay between NOD2 and TLR2 following recognition of components of the bacterial cell wall peptidoglycan is well-established, however their role in redirecting metabolic pathways in myeloid cells to degrade pathogens and mount antigen presentation remains unclear. We show NOD2 and TLR2 mediate phosphorylation of the deubiquitinase ataxin-3 via RIPK2 and TBK1. In myeloid cells ataxin-3 associates with the mitochondrial cristae protein MIC60, and is required for oxidative phosphorylation. Depletion of ataxin-3 leads to impaired induction of mitochondrial reactive oxygen species (mROS) and defective bacterial killing. A mass spectrometry analysis of NOD2/TLR2 triggered ataxin-3 deubiquitination targets revealed immunometabolic regulators, including HIF-1α and LAMTOR1 that may contribute to these effects. Thus, we define how ataxin-3 plays an essential role in NOD2 and TLR2 sensing and effector functions in myeloid cells.

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. Aulicino, Antanaviciute et al investigate the transcriptional response to invasive Salmonella strains in dendritic cells (DCs). They show that S. Typhi mount a response against nitrosative stress pathways and propose a role of iron uptake and transport in preventing infection, which the pathogen can bypass. In parallel, they find that invasive Salmonella employs several mechanisms targeting more classic aspects of immunity to impair DC function.

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. Multimodal single-cell profiling reveals heterogeneity of colonic CD8 + T cells in patients with ulcerative colitis, including expansion of a chronically activated IL-26-expressing subpopulation with innate-like features.

Colonic antigen-experienced lymphocytes such as tissue-resident memory CD8.sup.+ 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.sup.+ 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.sup.+ T-cell composition, including expanded effector and post-effector terminally differentiated CD8.sup.+ T cells. While UC-associated CD8.sup.+ effector T cells can trigger tissue destruction and produce tumor necrosis factor (TNF)-[alpha], post-effector cells acquire innate signatures to adopt regulatory functions that may mitigate excessive inflammation. Thus, we identify colonic CD8.sup.+ T-cell phenotypes in health and UC, define their clonal relationships and characterize terminally differentiated dysfunctional UC CD8.sup.+ T cells expressing IL-26, which attenuate acute colitis in a humanized IL-26 transgenic mouse model.

Mucosal-associated invariant T (MAIT) cells are innate T lymphocytes activated by bacteria that produce vitamin B2 metabolites. Mouse models of infection have demonstrated a role for MAIT cells in antimicrobial defense. However, proposed protective roles of MAIT cells in human infections remain unproven and clinical conditions associated with selective absence of MAIT cells have not been identified. We report that typhoidal and nontyphoidal Salmonella enterica strains activate MAIT cells. However, S. Typhimurium sequence type 313 (ST313) lineage 2 strains, which are responsible for the burden of multidrug-resistant nontyphoidal invasive disease in Africa, escape MAIT cell recognition through overexpression of ribB. This bacterial gene encodes the 4-dihydroxy-2-butanone-4-phosphate synthase enzyme of the riboflavin biosynthetic pathway. The MAIT cell-specific phenotype did not extend to other innate lymphocytes. We propose that ribB overexpression is an evolved trait that facilitates evasion from immune recognition by MAIT cells and contributes to the invasive pathogenesis of S. Typhimurium ST313 lineage 2.

The recently described ESX-5 secretion system of Mycobacterium tuberculosis is one of the most important modulators of host-pathogen interactions due to its crucial impact on PPE protein secretion, cell wall stability and virulence. Although various components of the ESX-5 secretion machinery have been defined, other ESX-5 core components still remain to be characterized. In this study, we focused on EccB5 and EccC5, a transmembrane protein (EccB5) and a membrane-bound ATPase (EccC5), both predicted to be building blocks of the M. tuberculosis ESX-5 membrane-associated complex. In vitro expression studies demonstrated that EccB5 and EccC5 encoding genes constitute an operon. The expression of this operon is essential for M. tuberculosis, since the deletion of the eccB5-eccC5 genomic segment at the ESX-5 locus is possible only after the integration of a second functional copy of eccB5-eccC5 genes into the M. tuberculosis chromosome. The characterization of two M. tuberculosis conditional mutant strains (MtbPptreccB5 and MtbPptreccC5), in which the eccB5-eccC5 operon or the eccC5 gene, respectively, were expressed under the control of an anhydrotetracycline-repressible promoter, confirmed that the repression of eccB5-eccC5 genes is detrimental for growth of M. tuberculosis both in vitro and in THP-1 human macrophage cell line. Moreover, analysis of the secretome of MtbPptreccB5-eccC5 and MtbPptreccC5 strains revealed that both EccB5 and EccC5 are required for secretion of ESX-5 specific substrates, thus confirming that they are indeed components of the ESX-5 secretion machinery. Taken together these findings demonstrate the importance of an intact and functional ESX-5 system for viability of M. tuberculosis, thus opening new interesting options for alternative antimycobacterial control strategies.

To tackle the complexity of cross-reactive and pathogen-specific T cell responses against related Salmonella serovars, we used mass cytometry, unbiased single-cell cloning, live fluorescence barcoding, and T cell–receptor sequencing to reconstruct the Salmonella -specific repertoire of circulating effector CD4 + T cells, isolated from volunteers challenged with Salmonella enterica serovar Typhi ( S . Typhi) or Salmonella Paratyphi A ( S . Paratyphi). We describe the expansion of cross-reactive responses against distantly related Salmonella serovars and of clonotypes recognizing immunodominant antigens uniquely expressed by S . Typhi or S . Paratyphi A. In addition, single–amino acid variations in two immunodominant proteins, CdtB and PhoN, lead to the accumulation of T cells that do not cross-react against the different serovars, thus demonstrating how minor sequence variations in a complex microorganism shape the pathogen-specific T cell repertoire. Our results identify immune-dominant, serovar-specific, and cross-reactive T cell antigens, which should aid in the design of T cell–vaccination strategies against Salmonella . Typhoidal Salmonella is a major pathogen, but there is still a lack of knowledge about suitable vaccine antigens. Cerundolo and colleagues deep-phenotype bacteria-specific CD4 + T cells of Salmonella -infected volunteers to define cross-reactive and serovar-specific responses.