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Rationale: Workers' exposure to metalworking fluid (MWF) has been associated with respiratory disease.Objectives: As part of a public health investigation of a manufacturing facility, we performed a cross-sectional study using paired environmental and human sampling to evaluate the cross-pollination of microbes between the environment and the host and possible effects on lung pathology present among workers.Methods: Workplace environmental microbiota were evaluated in air and MWF samples. Human microbiota were evaluated in lung tissue samples from workers with respiratory symptoms found to have lymphocytic bronchiolitis and alveolar ductitis with B-cell follicles and emphysema, in lung tissue samples from control subjects, and in skin, nasal, and oral samples from 302 workers from different areas of the facility. In vitro effects of MWF exposure on murine B cells were assessed.Measurements and Main Results: An increased similarity of microbial composition was found between MWF samples and lung tissue samples of case workers compared with control subjects. Among workers in different locations within the facility, those that worked in the machine shop area had skin, nasal, and oral microbiota more closely related to the microbiota present in the MWF samples. Lung samples from four index cases and skin and nasal samples from workers in the machine shop area were enriched with Pseudomonas, the dominant taxa in MWF. Exposure to used MWF stimulated murine B-cell proliferation in vitro, a hallmark cell subtype found in the pathology of index cases.Conclusions: Evaluation of a manufacturing facility with a cluster of workers with respiratory disease supports cross-pollination of microbes from MWF to humans and suggests the potential for exposure to these microbes to be a health hazard.

SUMMARY The immunoglobulin heavy chain (Igh) locus features a dynamic chromatin landscape to promote class switch recombination (CSR), yet the mechanisms that regulate this landscape remain poorly understood. CHD4, a component of the chromatin remodeling NuRD complex, directly binds H3K9me3, an epigenetic mark present at the Igh locus during CSR. We find that CHD4 is essential for early B cell development but is dispensable for the homeostatic maintenance of mature, naive B cells. However, loss of CHD4 in mature B cells impairs CSR because of suboptimal targeting of AID to the Igh locus. Additionally, we find that CHD4 represses p53 expression to promote B cell proliferation. This work reveals distinct roles for CHD4 in B cell development and CSR and links the H3K9me3 epigenetic mark with AID recruitment to the Igh locus., Graphical Abstract, In Brief Yen et al. demonstrate that CHD4, a component of the NuRD remodeling complex, is essential for early B cell development, represses p53 expression in mature B cells, and influences the recruitment of AID to DNA during class switch recombination.

Summary: The phosphatidylinositol 3-kinase (PI3K) signaling cascade downstream of the B cell receptor (BCR) signalosome is essential for B cell maturation. Proper signaling strength is maintained through the PI3K negative regulator phosphatase and tensin homolog (PTEN). Although a role for microRNA (miRNA)-dependent control of the PTEN-PI3K axis has been described, the contribution of individual miRNAs to the regulation of this crucial signaling modality in mature B lymphocytes remains to be elucidated. Our analyses reveal that ablation of miR-29 specifically in B lymphocytes results in an increase in PTEN expression and dampening of the PI3K pathway in mature B cells. This dysregulation has a profound impact on the survival of B lymphocytes and results in increased class switch recombination and decreased plasma cell differentiation. Furthermore, we demonstrate that ablation of one copy of Pten is sufficient to ameliorate the phenotypes associated with miR-29 loss. Our data suggest a critical role for the miR-29-PTEN-PI3K regulatory axis in mature B lymphocytes.

Phenotypic differences among substrains of laboratory mice due to spontaneous mutations or pre-existing genetic variation confound the interpretation of targeted mutagenesis experiments and contribute to challenges with reproducibility across institutions. Notably, C57BL/6 Hsd mice and gene-targeted mice that have been backcrossed to this substrain have been reported to harbor a duplication in exons 28 and 29 of Dock2. In this study, we demonstrate the presence of this Dock2 variant in the widely used Nod2−/− mice. Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is a cytosolic innate immune receptor associated with inflammatory bowel disease susceptibility. Consistent with a role of NOD2 in an immunological disorder, Nod2−/− mice bred at our institution displayed multiple B cell defects including deficiencies in recirculating B cells, marginal zone B cells, and B1a cells in vivo, as well as defects in class switch recombination in vitro. However, we found that these effects are due to the Dock2 variant and are independent of Nod2 deletion. Despite originating from the same gene-targeted founder mice, Nod2−/− mice from another source did not harbor the Dock2 variant or B cell defects. Finally, we show that Dock2−/− mice display the same B cell defects as mice harboring the Dock2 variant, confirming that the variant is a loss-of-function mutation and is sufficient to explain the alterations to the B cell compartment observed in Nod2−/− mice. Our findings highlight the effects of confounding mutations from widely used inbred strains on gene-targeted mice and reveal new functions of DOCK2 in B cells.

The overexpression of microRNA cluster miR-17-92 has been implicated in development of solid tumors and hematological malignancies. The role of miR-17-92 in lymphomagenesis has been extensively investigated; however, because of the developmental defects caused by miR-17-92 dysregulation, its ability to drive tumorigenesis has remained undetermined until recently. Here we demonstrate that overexpression of miR-17-92 in a limited number of hematopoietic cells is sufficient to cause B cell malignancies. In sum, our study provides a novel and physiologically relevant model that exposes the potent ability of miR-17-92 to act as a driver of tumourigenesis.

Phenotypic differences among substrains of laboratory mice due to spontaneous mutations or pre-existing genetic variation confound the interpretation of targeted mutagenesis experiments, and contribute to challenges with reproducibility across institutions. Notably, C57BL/6NHsd mice and gene-targeted mice that have been backcrossed to this substrain have been reported to harbor a duplication in exons 28 and 29 of Dock2. Here, we demonstrate the presence of this Dock2 variant in the widely used Nod2−/− mice. NOD2 is a cytosolic innate immune receptor that has been the subject of intense investigation due to its association with inflammatory bowel disease (IBD) susceptibility. Consistent with a role of NOD2 in an immunological disorder, Nod2−/− mice bred at our institution displayed multiple B cell defects including deficiencies in recirculating B cells, marginal zone B cells and B1a cells. However, we found that these effects are due to the Dock2 variant and are independent of Nod2 deletion. Despite originating from the same gene-targeted founder mice, Nod2−/− mice from another source did not harbor the Dock2 variant or B cell defects. Finally, we show that Dock2−/− mice display the same B cell defects as mice harboring the Dock2 variant, confirming that the variant is a loss-of-function mutation and is sufficient to explain the alterations to the B cell compartment observed in Nod2−/− mice. Our findings highlight the effects of confounding mutations from widely-used inbred strains on gene-targeted mice and reveal new functions of DOCK2 in B cells.

B lymphocytes are essential for an efficient immune response against a variety of pathogens. A large fraction of hematologic malignancies is of B cell origin, suggesting that the development and activation of B cells need to be tightly regulated. In recent years, increasing evidence has emerged demonstrating that microRNAs (miRNAs) – a class of non-coding RNAs that control gene expression – are involved in the regulation of B cell development and function. We provide here an overview of the current knowledge on the role of miRNAs and their relevant targets in B cell development, B cell activation, and B cell malignant transformation.

Th17 cells are a proinflammatory subset of effector T cells that have been implicated in the pathogenesis of asthma. Their production of the cytokine IL-17 is known to induce local recruitment of neutrophils, but the direct impact of IL-17 on the lung epithelium is poorly understood. In this study, we describe a novel mouse model of spontaneous IL-17–driven lung inflammation that exhibits many similarities to asthma in humans. We have found that STAT3 hyperactivity in T lymphocytes causes an expansion of Th17 cells, which home preferentially to the lungs. IL-17 secretion then leads to neutrophil infiltration and lung epithelial changes, in turn leading to a chronic inflammatory state with increased mucus production and decreased lung function. We used this model to investigate the effects of IL-17 activity on airway epithelium and identified CXCL5 and MIP-2 as important factors in neutrophil recruitment. The neutralization of IL-17 greatly reduces pulmonary neutrophilia, underscoring a key role for IL-17 in promoting chronic airway inflammation. These findings emphasize the role of IL-17 in mediating neutrophil-driven pulmonary inflammation and highlight a new mouse model that may be used for the development of novel therapies targeting Th17 cells in asthma and other chronic pulmonary diseases.

© 2016 The Author(s) B cell development is a tightly regulated process dependent on sequential rearrangements of immunoglobulin loci that encode the antigen receptor. To elucidate the role of microRNAs (miRNAs) in the orchestration of B cell development, we ablated all miRNAs at the earliest stage of B cell development by conditionally targeting the enzymes critical for RNAi in early B cell precursors. Absence of any one of these enzymes led to a block at the pro- to pre-B cell transition due to increased apoptosis and a failure of pre-B cells to proliferate. Expression of a Bcl2 transgene allowed for partial rescue of B cell development, however, the majority of the rescued B cells had low surface immunoglobulin expression with evidence of ongoing light chain editing. Our analysis revealed that miRNAs are critical for the regulation of the PTEN-AKT-FOXO1 pathway that in turn controls Rag expression during B cell development.

A salient feature of pancreatic ductal adenocarcinoma (PDAC) is an abundant fibroinflammatory response characterized by the recruitment of immune and mesenchymal cells and the consequent establishment of a protumorigenic microenvironment. Here, we report the prominent presence of B cells in human pancreatic intraepithelial neoplasia and PDAC lesions as well as in oncogenic Kras-driven pancreatic neoplasms in the mouse. The growth of orthotopic pancreatic neoplasms harboring oncogenic Kras was significantly compromised in B-cell–deficient mice (μMT), and this growth deficiency could be rescued by the reconstitution of a CD1dhiCD5+ B-cell subset. The protumorigenic effect of B cells was mediated by their expression of IL35 through a mechanism involving IL35-mediated stimulation of tumor cell proliferation. Our results identify a previously unrecognized role for IL35-producing CD1dhiCD5+ B cells in the pathogenesis of pancreatic cancer and underscore the potential significance of a B-cell/IL35 axis as a therapeutic target. Significance: This study identifies a B-cell subpopulation that accumulates in the pancreatic parenchyma during early neoplasia and is required to support tumor cell growth. Our findings provide a rationale for exploring B-cell–based targeting approaches for the treatment of pancreatic cancer. Cancer Discov; 6(3); 247–55. ©2015 AACR. See related commentary by Roghanian et al., p. 230. See related article by Lee et al., p. 256. See related article by Gunderson et al., p. 270. This article is highlighted in the In This Issue feature, p. 217

For two decades, Th1 cells have been implicated in the pathogenesis of chronic inflammatory diseases. This notion has been challenged by the discovery of IL-23 and Th17 cells as key players in mouse inflammatory disease models. Furthermore, recent genome wide association studies have found a strong association between spondylarthropathy (SpA, a group of chronic inflammatory rheumatisms) and the presence of single nucleotide polymorphisms at the IL-23R locus. Our aim is to study the role of the different CD4+ T cell subsets in SpA and to determine whether genetic variation at IL23R affects IL-23 signaling and CD4+ T cell function. To address these questions, we analyzed secretion of inflammatory cytokines and expression of Th17 and Th1 marker genes by CD4+ T cells after stimulation in the presence or absence of IL-23. We also genotyped three SNPs at the IL23R locus, highly associated with SpA. The minor allele of SNP rs1004819 is positively associated with disease (more frequent in the disease cohort). In contrast, the minor allele of SNPs rs11209026 and rs1343151 are “protective” (less frequent in the disease cohort). We found that CD4+ T cells from patients carrying the positively associated minor allele secreted more IFNg, IL17A and IL17F. In contrast, secretion of these cytokines was decreased in patients carrying a protective minor allele. Further support for critical role of these SNPs at IL23R locus on CD4+ T cell function comes from gene expression analysis. We observed that patients carrying the positively associated minor allele expressed higher levels of Th1 and Th17 marker genes whereas patients carrying a protective minor allele expressed lower levels of these genes. Our results suggest that genetic variation at the IL23R locus strongly affects CD4+ T cell function in spondylarthropathy, and provide a link between genotype and pathology.

Fogli, L. K., M. S. Sundrud, S. Goel, S. Bajwa, K. Jensen, E. Derudder, A. Sun, M. Coffre, C. Uyttenhove, J. Van Snick, M. Schmidt-Supprian, A. Rao, G. Grunig, J. Durbin, S. S. Casola, K. Rajewsky, and S. B. Koralov. 2013. T cell–derived IL-17 mediates epithelial changes in the airway and drives

Background Recent genome-wide association studies (GWAS) have revealed numerous genetic associations between specific single-nucleotide polymorphisms (SNPs) and spondyloarthritis (SpA), a highly heritable arthropathy. These studies highlighted the importance of non-HLA genes, in particular of key components of the IL-23 signaling pathway such as IL23R and IL12B, as well as genes associated with other pathways such as ERAP1 and ANTXR2. However, the link between these genes and the disease mechanisms remained unknown. Objectives The current challenge is to define associations between defined genetic variants and effector mechanisms implicated in disease pathogenesis. Here, we have investigated the link between genetic variants associated with spondyloarthritis (SpA) and the function of CD4+ effector T lymphocyte subsets, known mediators of chronic inflammatory disease. Methods To assess if genetic variants at IL23R and other genes in the IL-23/IL-17 pathway affect CD4+ T cell functions, we isolated CD4+ T cells from the peripheral blood of 49 SpA patients and stimulated them through the T cell receptor in vitro. We then analyzed the expression of genes associated with development and function of Th1 and Th17 cells. Genotyping was performed using pre-developed TaqMan allelic discrimination assays and immunophenotyping using standard flow cytometry panels. Results We found that selected disease-associated polymorphisms in genes involved in the IL-23 signaling pathway, such as IL23R, IL12B and CCR6, correlated with the expression levels of genes controlling differentiation and function of inflammatory Th1 and Th17 cells in SpA patients. In contrast, genetic variation at loci genetically linked to SpA, but not associated with the IL-23 pathway, had no measurable impact on the expression of inflammatory cytokines. To assess the global effect of multiple SNPs on CD4 effector functions, we correlated the numbers of susceptibility or protective alleles at molecules in the IL-23 pathway that each patient carried with the expression levels of Th1 and Th17 cytokine genes. Patients carrying predominantly alleles associated with increased disease risk expressed overall higher levels of effector cytokines. In contrast, patients carrying the highest number of protective alleles expressed the lowest levels of these cytokine genes. Conclusions Our data revealed a combinatorial control of T helper cell effector functions by SNPs at genes associated with the IL-23 signaling pathway in SpA patients and provide a framework to delineate the mechanisms by which genetic variants may contribute to SpA pathogenesis. We propose that genetic variation at genes in the IL-23/IL-17 axis tunes the homeostasis of CD4 effector cell populations, by affecting the frequencies of inflammatory T cell subsets. Disclosure of Interest None Declared

Distinct CD4+ T cell subsets are necessary for efficient immune responses against various pathogens. Among these, Th17 cells play a critical role in mucosal immunity. On the other hand, Th17 cells are pathogenic in several autoimmune disease models. In addition to IL-17A, Th17 cells also produce the IL-10-related cytokine IL-22. However, recent reports have suggested that human IL-22-producing CD4+ T cells may represent a distinct T helper cell subset indicating an unexpected level of heterogeneity of CD4+ T cell “lineages”. The goal of this study was determine the heterogeneity of inflammatory human CD4+ T cell subsets using single-cell techniques. Individual IL-17 or IFN-g-producing cells were isolated using cytokine secretion assays (CSA) and single-cell sorting. Single-cell gene expression profiling was performed by qRT-PCR using BioMark technology. We first determined which human CD4+ T cell populations secrete IL-17, IL-22, and IFN-g. Analysis of cytokine production at the single cell level revealed that only a small proportion of IL-22-producing cells also produced IL-17, whereas the major IL-22-secreting cell populations were a subset of Th1 cells and “Th22” cells that produce neither IL-17 nor IFN-γ. Finally, we identified a small population of CD4+ T cell producing the 3 cytokines. To define the molecular basis for the observed heterogeneity of inflammatory T cell subsets, we isolated individual IL-17A and IFN-g-producing CD4+ T cells using CSA and analyzed the expression of 48 “marker” genes for Th1, Th2, and Th17 subsets. Hierarchical clustering revealed that the gene expression profiles of Th1 and Th17 cells are distinct at the single-cell level. However, we observed large differences in the expression levels and a remarkable heterogeneity of the expression of marker genes for individual Th1 and Th17 cells. Our data reveal a striking heterogeneity of gene expression within inflammatory T cell populations, which may provide an explanation for the diverse functions of CD4+ T cell subsets in orchestrating immune responses.

Negative regulation of the immune system is of critical importance to prevent pathology. This level of regulation is compromised in autoimmunity and graft-versus-host disease, a life-threatening complication of hematopoietic stem cell transplantation. CD4+CD25+FOXP3+ regulatory T cells (Treg) are potent suppressors of these adverse immune reactions. Treg also play critical roles in the maintenance self-tolerance and the control of immune homeostasis. Recent reports have suggested that human Treg cells may not be a homogenous cell population. The aim of this study was to identify and characterize human Treg sub-populations. Treg subpopulations were isolated by cell sorting and characterized by immuno-phenotyping, functional assays and gene expression profiling. Single-cell gene expression profiling was performed by qRT-PCR using BioMark technology. We have identified three different subsets of Treg in human peripheral blood: CD25hiFOXP3hiCD127-CD45RA-HLADR+ (“activated” Treg), CD25hiFOXP3intCD45RA-HLADR- (“memory” Treg) and CD25hiFOXP3intCD127-CD45RA+HLADR- (“naive” Treg). We noted substantial differences in the expression of several Treg markers, such as FOXP3 and CTLA4, in the three subsets. Gene expression profiling combined with global pathway analysis revealed clearly distinct immune signatures. In particular, we found that memory Treg, but not naive or activated Treg, expressed transcripts encoding cytokines such as IL-17A, IL-22, IFN-γ, IL-10, and IL-4. Despite their heterogeneity, all three human Treg subsets suppressed the proliferation of effector cells in vitro and are already present at birth, although in different proportions. Single-cell gene expression profiling revealed substantial heterogeneity within the three Treg subsets, in particular within the memory Treg population. Of note, cytokine-expressing memory Treg did not downregulate FOXP3 and other Treg marker molecules. Current work addresses a potential “plasticity” and the ontogeny of this cell population. In conclusion, our data revealed a striking heterogeneity of the human Treg compartment, indicating that Treg may use multiple mechanisms to exert their immunoregulatory functions.