Highlights • MD-2 and CD14 regulate the transport and signaling functions of TLR4. • LPS-induced CD14 aggregation modulates PIP5K activity to promote myddosome signaling downstream of TLR4. • ...Pathogenic and commensal bacteria modulate LPS structures to evade CD14 and MD-2 functions.
Crohn's disease (CD) is a chronic transmural inflammation of intestinal segments caused by dysregulated interaction between microbiome and gut immune system. Here, we profile, via multiple ...single-cell technologies, T cells purified from the intestinal epithelium and lamina propria (LP) from terminal ileum resections of adult severe CD cases. We find that intraepithelial lymphocytes (IEL) contain several unique T cell subsets, including NKp30
γδT cells expressing RORγt and producing IL-26 upon NKp30 engagement. Further analyses comparing tissues from non-inflamed and inflamed regions of patients with CD versus healthy controls show increased activated T
17 but decreased CD8
T, γδT, T
and Treg cells in inflamed tissues. Similar analyses of LP find increased CD8
, as well as reduced CD4
T cells with an elevated T
17 over Treg/T
ratio. Our analyses of CD tissues thus suggest a potential link, pending additional validations, between transmural inflammation, reduced IEL γδT cells and altered spatial distribution of IEL and LP T cell subsets.
The Gram-negative bacteria Yersinia pestis , causative agent of plague, is extremely virulent. One mechanism contributing to Y. pestis virulence is the presence of a type-three secretion system, ...which injects effector proteins, Yops, directly into immune cells of the infected host. One of these Yop proteins, YopJ, is proapoptotic and inhibits mammalian NF-κB and MAP-kinase signal transduction pathways. Although the molecular mechanism remained elusive for some time, recent work has shown that YopJ acts as a serine/threonine acetyl-transferase targeting MAP2 kinases. Using Drosophila as a model system, we find that YopJ inhibits one innate immune NF-κB signaling pathway (IMD) but not the other (Toll). In fact, we show YopJ mediated serine/threonine acetylation and inhibition of dTAK1, the critical MAP3 kinase in the IMD pathway. Acetylation of critical serine/threonine residues in the activation loop of Drosophila TAK1 blocks phosphorylation of the protein and subsequent kinase activation. In addition, studies in mammalian cells show similar modification and inhibition of hTAK1. These data present evidence that TAK1 is a target for YopJ-mediated inhibition.
Deficiency in mevalonate kinase (MVK) causes systemic inflammation. However, the molecular mechanisms linking the mevalonate pathway to inflammation remain obscure. Geranylgeranyl pyrophosphate, a ...non-sterol intermediate of the mevalonate pathway, is the substrate for protein geranylgeranylation, a protein post-translational modification that is catalyzed by protein geranylgeranyl transferase I (GGTase I). Pyrin is an innate immune sensor that forms an active inflammasome in response to bacterial toxins. Mutations in MEFV (encoding human PYRIN) result in autoinflammatory familial Mediterranean fever syndrome. We found that protein geranylgeranylation enabled Toll-like receptor (TLR)-induced activation of phosphatidylinositol-3-OH kinase (PI(3)K) by promoting the interaction between the small GTPase Kras and the PI(3)K catalytic subunit p110δ. Macrophages that were deficient in GGTase I or p110δ exhibited constitutive release of interleukin 1β that was dependent on MEFV but independent of the NLRP3, AIM2 and NLRC4 inflammasomes. In the absence of protein geranylgeranylation, compromised PI(3)K activity allows an unchecked TLR-induced inflammatory responses and constitutive activation of the Pyrin inflammasome.
Highlights • We assemble a collection of bacterial and viral mechanisms for blocking TLR signaling networks directly. • We analyze recent findings of novel microbial strategies that inhibit TLR ...signaling and identify where future research is likely to be most productive. • We discuss the limited knowledge of microbial systems for antagonizing the very earliest module of TLR signaling: the receptor module.
Abstract
Background
Patient-derived organoid (PDO) models offer potential to transform drug discovery for inflammatory bowel disease (IBD) but are limited by inconsistencies with differentiation and ...functional characterization. We profiled molecular and cellular features across a range of intestinal organoid models and examined differentiation and establishment of a functional epithelial barrier.
Methods
Patient-derived organoids or monolayers were generated from control or IBD patient–derived colon or ileum and were molecularly or functionally profiled. Biological or technical replicates were examined for transcriptional responses under conditions of expansion or differentiation. Cell-type composition was determined by deconvolution of cell-associated gene signatures and histological features. Differentiated control or IBD-derived monolayers were examined for establishment of transepithelial electrical resistance (TEER), loss of barrier integrity in response to a cocktail of interferon (IFN)-γ and tumor necrosis factor (TNF)-α, and prevention of cytokine-induced barrier disruption by the JAK inhibitor, tofacitinib.
Results
In response to differentiation media, intestinal organoids and monolayers displayed gene expression patterns consistent with maturation of epithelial cell types found in the human gut. Upon differentiation, both colon- and ileum-derived monolayers formed functional barriers, with sustained TEER. Barrier integrity was compromised by inflammatory cytokines IFN-γ and TNF-α, and damage was inhibited in a dose-dependent manner by tofacitinib.
Conclusions
We describe the generation and characterization of human colonic or ileal organoid models capable of functional differentiation to mature epithelial cell types. In monolayer culture, these cells formed a robust epithelial barrier with sustained TEER and responses to pharmacological modulation. Our findings demonstrate that control and IBD patient-derived organoids possess consistent transcriptional and functional profiles that can enable development of epithelial-targeted therapies.
Graphical Abstract
Graphical Abstract
During bacterial infections, Toll-like receptor 4 (TLR4) signals through the MyD88- and TRIF-dependent pathways to promote pro-inflammatory and interferon (IFN) responses, respectively. Bacteria can ...inhibit the MyD88 pathway, but if the TRIF pathway is also targeted is unclear. We demonstrate that, in addition to MyD88, Yersinia pseudotuberculosis inhibits TRIF signaling through the type III secretion system effector YopJ. Suppression of TRIF signaling occurs during dendritic cell (DC) and macrophage infection and prevents expression of type I IFN and pro-inflammatory cytokines. YopJ-mediated inhibition of TRIF prevents DCs from inducing natural killer (NK) cell production of antibacterial IFNγ. During infection of DCs, YopJ potently inhibits MAPK pathways but does not prevent activation of IKK- or TBK1-dependent pathways. This singular YopJ activity efficiently inhibits TLR4 transcription-inducing activities, thus illustrating a simple means by which pathogens impede innate immunity.
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•The Yersinia effector YopJ blocks TRIF signaling and IFNβ production in DCs•YopJ does not block myddosome formation, NF-κB, or IRF3 activation induced by TLR4•Blocking MAPKs is sufficient to prevent MyD88- and TRIF-dependent gene expression•YopJ interference with TRIF signaling prevents DC-mediated activation of NK cells
The mechanisms by which pathogens block TLR4 signaling are unclear. Rosadini et al. demonstrate that Yersinia pseudotuberculosis utilizes the effector YopJ to interfere with MAPK pathways downstream of TRIF and MyD88 in dendritic cells. This singular target of YopJ is sufficient to dismantle the entire TLR4-dependent transcriptional response.
Haemophilus influenzae efficiently colonizes and persists at the human nasopharyngeal mucosa, causing disease when it spreads to other sites. Nitric oxide (NO) represents a major antimicrobial ...defense deployed by host cells in locations colonized by H. influenzae during pathogenesis that are likely to vary in oxygen levels. Formate-dependent nitrite reductase regulator (FNR) is an oxygen-sensitive regulator in several bacterial pathogens. We report that fnr of H. influenzae is required for anaerobic defense against exposure to NO donors and to resist NO-dependent effects of gamma interferon (IFN-γ)-activated murine bone marrow-derived macrophages. To understand the mechanism of resistance, we investigated the role of FNR-regulated genes in defense against NO sources. Expression analysis revealed FNR-dependent activation of nrfA, dmsA, napA, and ytfE. Nonpolar deletion mutants of nrfA and ytfE exhibited sensitivity to NO donors, and the ytfE gene was more critical for survival. Compared to the wild-type strain, the ytfE mutant exhibited decreased survival when exposed to macrophages, a defect that was more pronounced after prior stimulation of macrophages with IFN-γ or lipopolysaccharide. Complementation restored survival of the mutant to the level in the parental strain. Increased sensitivity of the ytfE mutant relative to that of the parent was abrogated by treatment of macrophages with a NO synthase inhibitor, implicating YtfE in resistance to a NO-dependent pathway. These results identify a requirement for FNR in positive control of ytfE and indicate a critical role for ytfE in resistance of H. influenzae to reactive nitrogen species and the antibacterial effects of macrophages.
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