Cx3cr1 controls kidney resident macrophage heterogeneity.

Kidney macrophages are comprised of both monocyte-derived and tissue resident populations; however, the heterogeneity of kidney macrophages and factors that regulate their heterogeneity are poorly understood. Herein, we performed single cell RNA sequencing (scRNAseq), fate mapping, and parabiosis to define the cellular heterogeneity of kidney macrophages in healthy mice. Our data indicate that healthy mouse kidneys contain four major subsets of monocytes and two major subsets of kidney resident macrophages (KRM) including a population with enriched Ccr2 expression, suggesting monocyte origin. Surprisingly, fate mapping data using the newly developed Ms4a3Cre Rosa Stopf/f TdT model indicate that less than 50% of Ccr2+ KRM are derived from Ly6chi monocytes. Instead, we find that Ccr2 expression in KRM reflects their spatial distribution as this cell population is almost exclusively found in the kidney cortex. We also identified Cx3cr1 as a gene that governs cortex specific accumulation of Ccr2+ KRM and show that loss of Ccr2+ KRM reduces the severity of cystic kidney disease in a mouse model where cysts are mainly localized to the kidney cortex. Collectively, our data indicate that Cx3cr1 regulates KRM heterogeneity and niche-specific disease progression.

Discovery of Hippo signaling as a regulator of CSPG4 expression and as a therapeutic target for Clostridioides difficile disease.

The signaling pathways and networks regulating expression of chondroitin sulfate proteoglycan 4 (CSPG4), a cancer-related protein that serves as a receptor for Clostridiodes difficile TcdB, are poorly defined. In this study, TcdB-resistant/CSPG4-negative HeLa cells were generated by exposure to increasing concentrations of the toxin. The cells that emerged (HeLa R5) lost expression of CSPG4 mRNA and were resistant to binding by TcdB. mRNA expression profiles paired with integrated pathway analysis correlated changes in the Hippo and estrogen signaling pathways with a CSPG4 decrease in HeLa R5 cells. Both signaling pathways altered CSPG4 expression when modulated chemically or through CRISPR-mediated deletion of key transcriptional regulators in the Hippo pathway. Based on the in vitro findings, we predicted and experimentally confirmed that a Hippo pathway inactivating drug (XMU-MP-1) provides protection from C. difficile disease in a mouse model. These results provide insights into key regulators of CSPG4 expression and identify a therapeutic for C. difficile disease.

A Comprehensive Immune Cell Atlas of Cystic Kidney Disease Reveals the Involvement of Adaptive Immune Cells in Injury-Mediated Cyst Progression in Mice.

BACKGROUND: Inducible disruption of cilia-related genes in adult mice results in slowly progressive cystic disease, which can be greatly accelerated by renal injury. METHODS: To identify in an unbiased manner modifier cells that may be influencing the differential rate of cyst growth in injured versus non-injured cilia mutant kidneys at a time of similar cyst severity, we generated a single-cell atlas of cystic kidney disease. We conducted RNA-seq on 79,355 cells from control mice and adult-induced conditional Ift88 mice (hereafter referred to as cilia mutant mice) that were harvested approximately 7 months post-induction or 8 weeks post 30-minute unilateral ischemia reperfusion injury. RESULTS: Analyses of single-cell RNA-seq data of CD45+ immune cells revealed that adaptive immune cells differed more in cluster composition, cell proportion, and gene expression than cells of myeloid origin when comparing cystic models with one another and with non-cystic controls. Surprisingly, genetic deletion of adaptive immune cells significantly reduced injury-accelerated cystic disease but had no effect on cyst growth in non-injured cilia mutant mice, independent of the rate of cyst growth or underlying genetic mutation. Using NicheNet, we identified a list of candidate cell types and ligands that were enriched in injured cilia mutant mice compared with aged cilia mutant mice and non-cystic controls that may be responsible for the observed dependence on adaptive immune cells during injury-accelerated cystic disease. CONCLUSIONS: Collectively, these data highlight the diversity of immune cell involvement in cystic kidney disease.

Autoinducing peptide-based quorum signaling systems in Clostridioides difficile.

The autoinducing peptide-based Agr system in Clostridioides difficile is involved in virulence factor expression, motility, and sporulation. This review highlights several of the recent discoveries regarding C. difficile Agr. Typical Agr systems rely on the combined activities of four proteins involved in peptide expression, peptide processing, peptide sensing, and transcriptional regulation. As emphasized in this review, at least two C. difficile Agr systems (Agr1 and Agr3) lack the set of proteins associated with this regulatory network. In line with this, recent finding indicate Agr1 can function in ways that may not depend on accumulation of extracellular peptide. Also, described are the similarities and differences in Agr systems within the pathogenic Clostridia.

Combined and Distinct Roles of Agr Proteins in Clostridioides difficile 630 Sporulation, Motility, and Toxin Production.

The Clostridioides difficile accessory gene regulator 1 (agr1) locus consists of two genes, agrB1 and agrD1, that presumably constitute an autoinducing peptide (AIP) system. Typically, AIP systems function through the AgrB-mediated processing of AgrD to generate a processed form of the AIP that provides a concentration-dependent extracellular signal. Here, we show that the C. difficile 630 Agr1 system has multiple functions, not all of which depend on AgrB1. CRISPR-Cas9n deletion of agrB1, agrD1, or the entire locus resulted in changes in transcription of sporulation-related factors and an overall loss in spore formation. Sporulation was recovered in the mutants by providing supernatant from stationary-phase cultures of the parental strain. In contrast, C. difficile motility was reduced only when both AgrB1 and AgrD1 were disrupted. Finally, in the absence of AgrB1, the AgrD1 peptide accumulated within the cytoplasm and this correlated with increased expression of tcdR (15-fold), as well as tcdA (20-fold) and tcdB (5-fold), which encode the two major C. difficile toxins. The combined deletion of agrB1/agrD1 or deletion of only agrD1 did not significantly alter expression of tcdR or tcdB but did show a minor effect on tcdA expression. Overall, these data indicate that the Agr1-based system in C. difficile 630 carries out multiple functions, some of which are associated with prototypical AIP signaling and others of which involve previously undescribed mechanisms of action.IMPORTANCEC. difficile is a spore-forming, toxigenic, anaerobic bacterium that causes severe gastrointestinal illness. Understanding the ways in which C. difficile senses growth conditions to regulate toxin expression and sporulation is essential to advancing our understanding of this pathogen. The Agr1 system in C. difficile has been thought to function by generating an extracellular autoinducing peptide that accumulates and exogenously activates two-component signaling. The absence of the peptide or protease should, in theory, result in similar phenotypes. However, in contrast to longstanding assumptions about Agr, we found that mutants of individual agr1 genes exhibit distinct phenotypes in C. difficile These findings suggest that the Agr1 system may have other regulatory mechanisms independent of the typical Agr quorum sensing system. These data not only challenge models for Agr's mechanism of action in C. difficile but also may expand our conceptions of how this system works in other Gram-positive pathogens.