Polymorphisms in the gene encoding the transcription factor IRF5 that lead to higher mRNA expression are associated with many autoimmune diseases. Here we show that IRF5 expression in macrophages was reversibly induced by inflammatory stimuli and contributed to the plasticity of macrophage polarization. High expression of IRF5 was characteristic of M1 macrophages, in which it directly activated transcription of the genes encoding interleukin 12 subunit p40 (IL-12p40), IL-12p35 and IL-23p19 and repressed the gene encoding IL-10. Consequently, those macrophages set up the environment for a potent T helper type 1 (T(H)1)-T(H)17 response. Global gene expression analysis demonstrated that exogenous IRF5 upregulated or downregulated expression of established phenotypic markers of M1 or M2 macrophages, respectively. Our data suggest a critical role for IRF5 in M1 macrophage polarization and define a previously unknown function for IRF5 as a transcriptional repressor.

Foxp3+ regulatory T cells are abundant in the intestine where they prevent dysregulated inflammatory responses to self and environmental stimuli. It is now appreciated that Treg cells acquire tissue-specific adaptations that facilitate their survival and function1; however, key host factors controlling the Treg response in the intestine are poorly understood. IL-1 family member IL-33 is constitutively expressed in epithelial cells at barrier sites2 where it functions as an endogenous danger signal or alarmin following tissue damage3. Recent studies in humans have described high levels of IL-33 in inflamed lesions of inflammatory bowel disease (IBD) patients4-7 suggesting a role for this cytokine in the pathogenesis of IBD. In the intestine, both protective and pathologic roles for IL-33 have been described in murine models of acute colitis8-11 but its contribution to chronic inflammation remains ill defined. Here we show that the IL-33 receptor ST2 is preferentially expressed on colonic Treg (cTreg) cells, where it promotes Treg function and adaptation to the inflammatory environment. IL-33 signaling into T cells stimulates Treg responses in several ways. Firstly, it enhances transforming growth factor-β1 (TGF-β1) mediated differentiation of Treg cells and secondly, it provides a necessary signal for Treg accumulation and maintenance in inflamed tissues. Strikingly, IL-23, a key pro-inflammatory cytokine in the pathogenesis of IBD, restrained Treg responses through inhibition of IL-33 responsiveness. These results demonstrate a hitherto unrecognized link between an endogenous mediator of tissue damage and a major anti-inflammatory pathway, and suggest that the balance between IL-33 and IL-23 may be a key controller of intestinal immune responses.

CRISPR-based genetic screens are accelerating biological discovery, but current methods have inherent limitations. Widely used pooled screens are restricted to simple readouts including cell proliferation and sortable marker proteins. Arrayed screens allow for comprehensive molecular readouts such as transcriptome profiling, but at much lower throughput. Here we combine pooled CRISPR screening with single-cell RNA sequencing into a broadly applicable workflow, directly linking guide-RNA expression to transcriptome responses in thousands of individual cells. Our method for CRISPR droplet sequencing (CROP-seq) enables pooled CRISPR screens with single-cell transcriptome resolution, which will facilitate high-throughput functional dissection of complex regulatory mechanisms and heterogeneous cell populations.

The mammalian immune system implements a remarkably effective set of mechanisms for fighting pathogens 1 . Its main components are hematopoietic immune cells, including myeloid cells that control innate immunity and lymphoid cells that constitute adaptive immunity 2 . However, immune functions are not unique to hematopoietic cells, and many other cell types display basic mechanisms of pathogen defence 3 – 5 . To advance our understanding of immunology outside the haematopoietic system, here we systematically investigate the regulation of immune genes in the three major types of structural cells: epithelium, endothelium, and fibroblasts. We characterize these cell types across twelve organs in mice, using cellular phenotyping, transcriptome sequencing, chromatin-accessibility profiling, and epigenome mapping. This comprehensive dataset revealed complex immune gene activity and regulation in structural cells. The observed patterns were highly organ-specific and seem to modulate the extensive interactions between structural cells and haematopoietic immune cells. Moreover, we identified an epigenetically encoded immune potential in structural cells under tissue homeostasis, which was triggered in response to systemic viral infection. This study highlights the prevalence and organ-specific complexity of immune gene activity in non-haematopoietic structural cells, and it provides a high-resolution, multi-omics atlas of the epigenetic and transcriptional networks that regulate structural cells in the mouse.

Summary Non-lymphoid tissues (NLTs) harbor a pool of adaptive immune cells with largely unexplored phenotype and development. We used single-cell RNA-seq to characterize 35,000 CD4 + regulatory (Treg) and memory (Tmem) T cells in mouse skin and colon, their respective draining lymph nodes (LNs) and spleen. In these tissues, we identified Treg cell subpopulations with distinct degrees of NLT phenotype. Subpopulation pseudotime ordering and gene kinetics were consistent in recruitment to skin and colon, yet the initial NLT-priming in LNs and the final stages of NLT functional adaptation reflected tissue-specific differences. Predicted kinetics were recapitulated using an in vivo melanoma-induction model, validating key regulators and receptors. Finally, we profiled human blood and NLT Treg and Tmem cells, and identified cross-mammalian conserved tissue signatures. In summary, we describe the relationship between Treg cell heterogeneity and recruitment to NLTs through the combined use of computational prediction and in vivo validation.

Highlights d Stool butyrate levels are reduced in patients with RA compared to healthy controls d Supplementation with butyrate suppresses arthritis severity in a mouse model d Suppression of arthritis by butyrate supplementation depends upon AhR + Bregs d Butyrate increases serotonin-derived 5-HIAA, which directly activates AhR + Bregs

SummaryNon-lymphoid tissues (NLTs) harbour a pool of adaptive immune cells, the development and phenotype of which remains largely unexplored. Here, we used single-cell RNA-seq to characterise CD4 + regulatory (Treg) and memory (Tmem) T cells in mouse skin and colon, the respective draining lymph nodes and spleen. From this data, we modelled a continuous lymphoid-to-NLT trajectory for Treg, and reconstructed the mechanisms of cell migration and NLT adaption. This revealed a shared transcriptional programme of NLT priming in both skin and colon-associated lymph nodes, followed by tissue-specific adaptation. Predicted migration kinetics were validated using a melanoma-induction model, emphasizing the relevance of key regulators and receptors, including Batf, Rora, Ccr8, Samsn1 . Finally, we profiled human blood and NLT Treg and Tmem cells, identifying cross-mammalian conserved tissue signatures. In summary, we have identified molecular signals mediating 1 . CC-BY 4.0 International license not peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/217489 doi: bioRxiv preprint first posted online Nov. 22, 2017; NLT Treg recruitment and tissue adaptation through the combined use of computational prediction and in vivo validation.

SummaryThe role of intestinal eosinophils in immune homeostasis is enigmatic and the molecular signals that drive them from protective to tissue damaging are unknown. Most commonly associated with Th2 cell-mediated diseases, we describe a role for eosinophils as crucial effectors of the interleukin-23 (IL-23)-granulocyte macrophage colony-stimulating factor (GM-CSF) axis in colitis. Chronic intestinal inflammation was characterized by increased bone marrow eosinopoiesis and accumulation of activated intestinal eosinophils. IL-5 blockade or eosinophil depletion ameliorated colitis, implicating eosinophils in disease pathogenesis. GM-CSF was a potent activator of eosinophil effector functions and intestinal accumulation, and GM-CSF blockade inhibited chronic colitis. By contrast neutrophil accumulation was GM-CSF independent and dispensable for colitis. In addition to TNF secretion, release of eosinophil peroxidase promoted colitis identifying direct tissue-toxic mechanisms. Thus, eosinophils are key perpetrators of chronic inflammation and tissue damage in IL-23-mediated immune diseases and it suggests the GM-CSF-eosinophil axis as an attractive therapeutic target.