Microbial ligand-independent regulation of lymphopoiesis by NOD1.

Aberrant differentiation of progenitor cells in the hematopoietic system is known to severely impact host immune responsiveness. Here we demonstrate that NOD1, a cytosolic innate sensor of bacterial peptidoglycan, also functions in murine hematopoietic cells as a major regulator of both the generation and differentiation of lymphoid progenitors as well as peripheral T lymphocyte homeostasis. We further show that NOD1 mediates these functions by facilitating STAT5 signaling downstream of hematopoietic cytokines. In steady-state, loss of NOD1 resulted in a modest but significant decrease in numbers of mature T, B and natural killer cells. During systemic protozoan infection this defect was markedly enhanced, leading to host mortality. Lack of functional NOD1 also impaired T cell-dependent anti-tumor immunity while preventing colitis. These findings reveal that, in addition to its classical role as a bacterial ligand receptor, NOD1 plays an important function in regulating adaptive immunity through interaction with a major host cytokine signaling pathway.

A CTCF-binding site in the Mdm1-Il22-Ifng locus shapes cytokine expression profiles and plays a critical role in early Th1 cell fate specification.

Cytokine expression during T cell differentiation is a highly regulated process that involves long-range promoter-enhancer and CTCF-CTCF contacts at cytokine loci. Here, we investigated the impact of dynamic chromatin loop formation within the topologically associating domain (TAD) in regulating the expression of interferon gamma (IFN-γ) and interleukin-22 (IL-22); these cytokine loci are closely located in the genome and are associated with complex enhancer landscapes, which are selectively active in type 1 and type 3 lymphocytes. In situ Hi-C analyses revealed inducible TADs that insulated Ifng and Il22 enhancers during Th1 cell differentiation. Targeted deletion of a 17 bp boundary motif of these TADs imbalanced Th1- and Th17-associated immunity, both in vitro and in vivo, upon Toxoplasma gondii infection. In contrast, this boundary element was dispensable for cytokine regulation in natural killer cells. Our findings suggest that precise cytokine regulation relies on lineage- and developmental stage-specific interactions of 3D chromatin architectures and enhancer landscapes.

Developmental Acquisition of Regulomes Underlies Innate Lymphoid Cell Functionality.

Innate lymphoid cells (ILCs) play key roles in host defense, barrier integrity, and homeostasis and mirror adaptive CD4(+) T helper (Th) cell subtypes in both usage of effector molecules and transcription factors. To better understand the relationship between ILC subsets and their Th cell counterparts, we measured genome-wide chromatin accessibility. We find that chromatin in proximity to effector genes is selectively accessible in ILCs prior to high-level transcription upon activation. Accessibility of these regions is acquired in a stepwise manner during development and changes little after in vitro or in vivo activation. Conversely, dramatic chromatin remodeling occurs in naive CD4(+) T cells during Th cell differentiation using a type-2-infection model. This alteration results in a substantial convergence of Th2 cells toward ILC2 regulomes. Our data indicate extensive sharing of regulatory circuitry across the innate and adaptive compartments of the immune system, in spite of their divergent developing pathways.

MicroRNA-221 and -222 modulate intestinal inflammatory Th17 cell response as negative feedback regulators downstream of interleukin-23.

MicroRNAs are important regulators of immune responses. Here, we show miR-221 and miR-222 modulate the intestinal Th17 cell response. Expression of miR-221 and miR-222 was induced by proinflammatory cytokines and repressed by the cytokine TGF-ß. Molecular targets of miR-221 and miR-222 included Maf and Il23r, and loss of miR-221 and miR-222 expression shifted the transcriptomic spectrum of intestinal Th17 cells to a proinflammatory signature. Although the loss of miR-221 and miR-222 was tolerated for maintaining intestinal Th17 cell homeostasis in healthy mice, Th17 cells lacking miR-221 and miR-222 expanded more efficiently in response to IL-23. Both global and T cell-specific deletion of miR-221 and miR-222 rendered mice prone to mucosal barrier damage. Collectively, these findings demonstrate that miR-221 and miR-222 are an integral part of intestinal Th17 cell response that are induced after IL-23 stimulation to constrain the magnitude of proinflammatory response.

Rapid Enhancer Remodeling and Transcription Factor Repurposing Enable High Magnitude Gene Induction upon Acute Activation of NK Cells.

Innate immune responses rely on rapid and precise gene regulation mediated by accessibility of regulatory regions to transcription factors (TFs). In natural killer (NK) cells and other innate lymphoid cells, competent enhancers are primed during lineage acquisition, and formation of de novo enhancers characterizes the acquisition of innate memory in activated NK cells and macrophages. Here, we investigated how primed and de novo enhancers coordinate to facilitate high-magnitude gene induction during acute activation. Epigenomic and transcriptomic analyses of regions near highly induced genes (HIGs) in NK cells both in vitro and in a model of Toxoplasma gondii infection revealed de novo chromatin accessibility and enhancer remodeling controlled by signal-regulated TFs STATs. Acute NK cell activation redeployed the lineage-determining TF T-bet to de novo enhancers, independent of DNA-sequence-specific motif recognition. Thus, acute stimulation reshapes enhancer function through the combinatorial usage and repurposing of both lineage-determining and signal-regulated TFs to ensure an effective response.

Gata6+ Pericardial Cavity Macrophages Relocate to the Injured Heart and Prevent Cardiac Fibrosis.

Macrophages play an important role in structural cardiac remodeling and the transition to heart failure following myocardial infarction (MI). Previous research has focused on the impact of blood-derived monocytes on cardiac repair. Here we examined the contribution of resident cavity macrophages located in the pericardial space adjacent to the site of injury. We found that disruption of the pericardial cavity accelerated maladaptive post-MI cardiac remodeling. Gata6+ macrophages in mouse pericardial fluid contributed to the reparative immune response. Following experimental MI, these macrophages invaded the epicardium and lost Gata6 expression but continued to perform anti-fibrotic functions. Loss of this specialized macrophage population enhanced interstitial fibrosis after ischemic injury. Gata6+ macrophages were present in human pericardial fluid, supporting the notion that this reparative function is relevant in human disease. Our findings uncover an immune cardioprotective role for the pericardial tissue compartment and argue for the reevaluation of surgical procedures that remove the pericardium.

The Magnitude of IFN-γ Responses Is Fine-Tuned by DNA Architecture and the Non-coding Transcript of Ifng-as1.

Interferon gamma (IFN-γ), critical for host defense and tumor surveillance, requires tight control of its expression. Multiple cis-regulatory elements exist around Ifng along with a non-coding transcript, Ifng-as1 (also termed NeST). Here, we describe two genetic models generated to dissect the molecular functions of this locus and its RNA product. DNA deletion within the Ifng-as1 locus disrupted chromatin organization of the extended Ifng locus, impaired Ifng response, and compromised host defense. Insertion of a polyA signal ablated the Ifng-as1 full-length transcript and impaired host defense, while allowing proper chromatin structure. Transient knockdown of Ifng-as1 also reduced IFN-γ production. In humans, discordant expression of IFNG and IFNG-AS1 was evident in memory T cells, with high expression of this long non-coding RNA (lncRNA) and low expression of the cytokine. These results establish Ifng-as1 as an important regulator of Ifng expression, as a DNA element and transcribed RNA, involved in dynamic and cell state-specific responses to infection.

The Transcription Factor T-bet Limits Amplification of Type I IFN Transcriptome and Circuitry in T Helper 1 Cells.

Host defense requires the specification of CD4+ helper T (Th) cells into distinct fates, including Th1 cells that preferentially produce interferon-γ (IFN-γ). IFN-γ, a member of a large family of anti-pathogenic and anti-tumor IFNs, induces T-bet, a lineage-defining transcription factor for Th1 cells, which in turn supports IFN-γ production in a feed-forward manner. Herein, we show that a cell-intrinsic role of T-bet influences how T cells perceive their secreted product in the environment. In the absence of T-bet, IFN-γ aberrantly induced a type I IFN transcriptomic program. T-bet preferentially repressed genes and pathways ordinarily activated by type I IFNs to ensure that its transcriptional response did not evoke an aberrant amplification of type I IFN signaling circuitry, otherwise triggered by its own product. Thus, in addition to promoting Th1 effector commitment, T-bet acts as a repressor in differentiated Th1 cells to prevent abberant autocrine type I IFN and downstream signaling.

Divergent roles for STAT4 in shaping differentiation of cytotoxic ILC1 and NK cells during gut inflammation.

Natural killer (NK) cells and type 1 innate lymphoid cells (ILC1) require signal transducer and activator of transcription 4 (STAT4) to elicit rapid effector responses and protect against pathogens. By combining genetic and transcriptomic approaches, we uncovered divergent roles for STAT4 in regulating effector differentiation of these functionally related cell types. Stat4 deletion in Ncr1-expressing cells led to impaired NK cell terminal differentiation as well as to an unexpected increased generation of cytotoxic ILC1 during intestinal inflammation. Mechanistically, Stat4-deficient ILC1 exhibited upregulation of gene modules regulated by STAT5 in vivo and an aberrant effector differentiation upon in vitro stimulation with IL-2, used as a prototypical STAT5 activator. Moreover, STAT4 expression in NCR+ innate lymphocytes restrained gut inflammation in the dextran sulfate sodium-induced colitis model limiting pathogenic production of IL-13 from adaptive CD4+ T cells in the large intestine. Collectively, our data shed light on shared and distinctive mechanisms of STAT4-regulated transcriptional control in NK cells and ILC1 required for intestinal inflammatory responses.

Asymmetric Action of STAT Transcription Factors Drives Transcriptional Outputs and Cytokine Specificity.

Interleukin-6 (IL-6) and IL-27 signal through a shared receptor subunit and employ the same downstream STAT transcription proteins, but yet are ascribed unique and overlapping functions. To evaluate the specificity and redundancy for these cytokines, we quantified their global transcriptomic changes and determined the relative contributions of STAT1 and STAT3 using genetic models and chromatin immunoprecipitation-sequencing (ChIP-seq) approaches. We found an extensive overlap of the transcriptomes induced by IL-6 and IL-27 and few examples in which the cytokines acted in opposition. Using STAT-deficient cells and T cells from patients with gain-of-function STAT1 mutations, we demonstrated that STAT3 is responsible for the overall transcriptional output driven by both cytokines, whereas STAT1 is the principal driver of specificity. STAT1 cannot compensate in the absence of STAT3 and, in fact, much of STAT1 binding to chromatin is STAT3 dependent. Thus, STAT1 shapes the specific cytokine signature superimposed upon STAT3's action.

Molecular mechanisms regulating wound repair: Evidence for paracrine signaling from corneal epithelial cells to fibroblasts and immune cells following transient epithelial cell treatment with Mitomycin C.

In this paper, we use RNAseq to identify senescence and phagocytosis as key factors to understanding how mitomyin C (MMC) stimulates regenerative wound repair. We use conditioned media (CM) from untreated (CMC) and MMC treated (CMM) human and mouse corneal epithelial cells to show that corneal epithelial cells indirectly exposed to MMC secrete elevated levels of immunomodulatory proteins including IL-1α and TGFß1 compared to cells exposed to CMC. These factors increase epithelial and macrophage phagocytosis and promote ECM turnover. IL-1α supplementation can increase phagocytosis in control epithelial cells and attenuate TGFß1 induced αSMA expression by corneal fibroblasts. Yet, we show that epithelial cell CM contains factors besides IL-1α that regulate phagocytosis and αSMA expression by fibroblasts. Exposure to CMM also impacts the activation of bone marrow derived dendritic cells and their ability to present antigen. These in vitro studies show how a brief exposure to MMC induces corneal epithelial cells to release proteins and other factors that function in a paracrine way to enhance debris removal and enlist resident epithelial and immune cells as well as stromal fibroblasts to support regenerative and not fibrotic wound healing.

Homeobox transcription factor DLX4 is not necessary for skin development and homeostasis.

Dlx4 is a member of a family of homeobox genes with homology to Drosophila distal-less (dll) gene. We show that Dlx4 expression pattern partially overlaps with its cis-linked gene Dlx3 during mouse development as well as in neonatal and adult skin. In mice, Dlx4 is expressed in the branchial arches, embryonic limbs, digits, nose, hair follicle and in the basal and suprabasal layers of mouse interfollicular epidermis. We show that inactivation of Dlx4 in mice did not result in any overtly gross pathology. Skin development, homeostasis and response to TPA treatment were similar in mice with loss of Dlx4 compared to wild-type counterparts.

Identification of Fibroinflammatory and Fibrotic Transcriptomic Subsets of Human Cutaneous Sclerotic Chronic Graft-Versus-Host Disease.

Cutaneous sclerotic chronic graft-versus-host disease (cGVHD) is a common and highly morbid complication of allogeneic hematopoietic stem cell transplantation. Our goals were to identify signals active in the skin of patients with sclerotic cGVHD in an effort to better understand how to treat this manifestation and to explore the heterogeneity of the disease. We identified genes that are significantly upregulated in the skin of patients with sclerotic cGVHD (n = 17) compared with those in the skin of patients who underwent allogeneic hematopoietic stem cell transplantation without cutaneous cGVHD (n = 9) by bulk RNA sequencing. Sclerotic cGVHD was most associated with T helper 1, phagocytic, and fibrotic pathways. In addition, different transcriptomic groups of affected patients were discovered: those with fibrotic and inflammatory/T helper 1 gene expression (the fibroinflammatory group) and those with predominantly fibrotic/TGFß-associated expression (the fibrotic group). Further study will help elucidate whether these gene expression findings can be used to tailor treatment decisions. Multiple proteins encoded by highly induced genes in the skin (SFRP4, SERPINE2, COMP) were also highly induced in the plasma of patients with sclerotic cGVHD (n = 16) compared with those in plasma of control patients who underwent allogeneic hematopoietic stem cell transplantation without sclerotic cGVHD (n = 17), suggesting these TGFß and Wnt pathway mediators as candidate blood biomarkers of the disease.

CHOP-mediated IL-23 overexpression does not drive colitis in experimental spondyloarthritis.

HLA-B27 is a major risk factor for spondyloarthritis (SpA), yet the underlying mechanisms remain unclear. HLA-B27 misfolding-induced IL-23, which is mediated by endoplasmic reticulum (ER) stress has been hypothesized to drive SpA pathogenesis. Expression of HLA-B27 and human ß2m (hß2m) in rats (HLA-B27-Tg) recapitulates key SpA features including gut inflammation. Here we determined whether deleting the transcription factor CHOP (Ddit3-/-), which mediates ER-stress induced IL-23, affects gut inflammation in HLA-B27-Tg animals. ER stress-mediated Il23a overexpression was abolished in CHOP-deficient macrophages. Although CHOP-deficiency also reduced Il23a expression in immune cells isolated from the colon of B27+ rats, Il17a levels were not affected, and gut inflammation was not reduced. Rather, transcriptome analysis revealed increased expression of pro-inflammatory genes, including Il1a, Ifng and Tnf in HLA-B27-Tg colon tissue in the absence of CHOP, which was accompanied by higher histological Z-scores. RNAScope localized Il17a mRNA to the lamina propria of the HLA-B27-Tg rats and revealed similar co-localization with Cd3e (CD3) in the presence and absence of CHOP. This demonstrates that CHOP-deficiency does not improve, but rather exacerbates gut inflammation in HLA-B27-Tg rats, indicating that HLA-B27 is not promoting gut disease through ER stress-induced IL-23. Hence, CHOP may protect rats from more severe HLA-B27-induced gut inflammation.

RichPathR: a gene set enrichment analysis and visualization tool.

Gene set enrichment analysis (GSEA) is an important step for disease and drug discovery. Genomic, transcriptomics, proteomics and epigenetic analysis of tissue or cells generates gene lists that need to be further investigated in the known biological context. The advent of high-throughput technologies generates the vast number of gene lists that are up or down regulated together. One way of getting meaningful insights of the relationship of these genes is utilizing existing knowledge bases linking them with biological functions or phenotypes. Multiple public databases with annotated gene sets are available for GSEA, and enrichR is the most popular web application still requiring custom tools for large-scale mining. richPathR package is a collection of R functions that helps researchers carry out exploratory analysis and visualization of gene set enrichment using EnrichR.

CD271 activation prevents low to high-risk progression of cutaneous squamous cell carcinoma and improves therapy outcomes.

BACKGROUND: Cutaneous squamous cell carcinoma (cSCC) is the second most prevalent form of skin cancer, showing a rapid increasing incidence worldwide. Although most cSCC can be cured by surgery, a sizeable number of cases are diagnosed at advanced stages, with local invasion and distant metastatic lesions. In the skin, neurotrophins (NTs) and their receptors (CD271 and Trk) form a complex network regulating epidermal homeostasis. Recently, several works suggested a significant implication of NT receptors in cancer. However, CD271 functions in epithelial tumors are controversial and its precise role in cSCC is still to be defined. METHODS: Spheroids from cSCC patients with low-risk (In situ or Well-Differentiated cSCC) or high-risk tumors (Moderately/Poorly Differentiated cSCC), were established to explore histological features, proliferation, invasion abilities, and molecular pathways modulated in response to CD271 overexpression or activation in vitro. The effect of CD271 activities on the response to therapeutics was also investigated. The impact on the metastatic process and inflammation was explored in vivo and in vitro, by using zebrafish xenograft and 2D/3D models. RESULTS: Our data proved that CD271 is upregulated in Well-Differentiated tumors as compared to the more aggressive Moderately/Poorly Differentiated cSCC, both in vivo and in vitro. We demonstrated that CD271 activities reduce proliferation and malignancy marker expression in patient-derived cSCC spheroids at each tumor grade, by increasing neoplastic cell differentiation. CD271 overexpression significantly increases cSCC spheroid mass density, while it reduces their weight and diameter, and promotes a major fold-enrichment in differentiation and keratinization genes. Moreover, both CD271 overexpression and activation decrease cSCC cell invasiveness in vitro. A significant inhibition of the metastatic process by CD271 was observed in a newly established zebrafish cSCC model. We found that the recruitment of leucocytes by CD271-overexpressing cells directly correlates with tumor killing and this finding was further highlighted by monocyte infiltration in a THP-1-SCC13 3D model. Finally, CD271 activity synergizes with Trk receptor inhibition, by reducing spheroid viability, and significantly improves the outcome of photodynamic therapy (PTD) or chemotherapy in spheroids and zebrafish. CONCLUSION: Our study provides evidence that CD271 could prevent the switch between low to high-risk cSCC tumors. Because CD271 contributes to maintaining active differentiative paths and favors the response to therapies, it might be a promising target for future pharmaceutical development.

Chromatin Landscape Governing Murine Epidermal Differentiation.

Chromatin landscape and regulatory networks are determinants in lineage specification and differentiation. To define the temporospatial differentiation axis in murine epidermal cells in vivo, we generated datasets profiling expression dynamics (RNA sequencing), chromatin accessibility (assay for transposase-accessible chromatin using sequencing), architecture (Hi-C), and histone modifications (chromatin immunoprecipitation followed by sequencing) in the epidermis. We show that many differentially regulated genes are suppressed during the differentiation process, with superenhancers controlling differentiation-specific epigenomic changes. Our data shows the relevance of the Dlx/Klf/Grhl combinatorial regulatory network in maintaining correct temporospatial gene expression during epidermal differentiation. We determined differential open compartments, topologically associating domain score, and looping in the basal cell and suprabasal cell epidermal fractions, with the evolutionarily conserved epidermal differentiation complex region showing distinct suprabasal cell-specific topologically associating domain and loop formation that coincided with superenhancer sites. Overall, our study provides a global genome-wide resource of chromatin dynamics that define unrecognized regulatory networks and the epigenetic control of Dlx3-bound superenhancer elements during epidermal differentiation.

Risk of relapse of ANCA-associated vasculitis among patients homozygous for the proteinase 3 gene Val119Ile polymorphism.

BACKGROUND: The frequency of proteinase 3 gene (PRTN3) polymorphisms in patients with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is not fully characterised. We hypothesise that the presence of a PRTN3 gene polymorphism (single nucleotide polymorphism (SNP) rs351111) is relevant for clinical outcomes. METHODS: DNA variant calling for SNP rs351111 (chr.19:844020, c.355G>A) in PRTN3 gene assessed the allelic frequency in patients with PR3-AAV included in the Rituximab in ANCA-Associated Vasculitis trial. This was followed by RNA-seq variant calling to characterise the mRNA expression. We compared clinical outcomes between patients homozygous for PRTN3-Ile119 or PRTN3-Val119. RESULTS: Whole blood samples for DNA calling were available in 188 patients. 75 patients with PR3-AAV had the allelic variant: 62 heterozygous PRTN3-Val119Ile and 13 homozygous for PRTN3-Ile119. RNA-seq was available for 89 patients and mRNA corresponding to the allelic variant was found in 32 patients with PR3-AAV: 25 heterozygous PRTN3-Val119Ile and 7 homozygous for PRTN3-Ile119. The agreement between the DNA calling results and mRNA expression of the 86 patients analysed by both methods was 100%. We compared the clinical outcomes of 64 patients with PR3-AAV: 51 homozygous for PRTN3-Val119 and 13 homozygous for PRTN3-Ile119. The frequency of severe flares at 18 months in homozygous PRTN3-Ile119 was significantly higher when compared with homozygous PRTN3-Val119 (46.2% vs 19.6%, p=0.048). Multivariate analysis identified homozygous PR3-Ile119 as main predictor of severe relapse (HR 4.67, 95% CI 1.16 to 18.86, p=0.030). CONCLUSION: In patients with PR3-AAV, homozygosity for PRTN3-Val119Ile polymorphism appears associated with higher frequency of severe relapse. Further studies are necessary to better understand the association of this observation with the risk of severe relapse.

Constitutively active Lyn kinase causes a cutaneous small vessel vasculitis and liver fibrosis syndrome.

Neutrophilic inflammation is a hallmark of many monogenic autoinflammatory diseases; pathomechanisms that regulate extravasation of damaging immune cells into surrounding tissues are poorly understood. Here we identified three unrelated boys with perinatal-onset of neutrophilic cutaneous small vessel vasculitis and systemic inflammation. Two patients developed liver fibrosis in their first year of life. Next-generation sequencing identified two de novo truncating variants in the Src-family tyrosine kinase, LYN, p.Y508*, p.Q507* and a de novo missense variant, p.Y508F, that result in constitutive activation of Lyn kinase. Functional studies revealed increased expression of ICAM-1 on induced patient-derived endothelial cells (iECs) and of ß2-integrins on patient neutrophils that increase neutrophil adhesion and vascular transendothelial migration (TEM). Treatment with TNF inhibition improved systemic inflammation; and liver fibrosis resolved on treatment with the Src kinase inhibitor dasatinib. Our findings reveal a critical role for Lyn kinase in modulating inflammatory signals, regulating microvascular permeability and neutrophil recruitment, and in promoting hepatic fibrosis.

A central role for STAT5 in the transcriptional programing of T helper cell metabolism.

Activated lymphocytes adapt their metabolism to meet the energetic and biosynthetic demands imposed by rapid growth and proliferation. Common gamma chain (cγ) family cytokines are central to these processes, but the role of downstream signal transducer and activator of transcription 5 (STAT5) signaling, which is engaged by all cγ members, is poorly understood. Using genome-, transcriptome-, and metabolome-wide analyses, we demonstrate that STAT5 is a master regulator of energy and amino acid metabolism in CD4+ T helper cells. Mechanistically, STAT5 localizes to an array of enhancers and promoters for genes encoding essential enzymes and transporters, where it facilitates p300 recruitment and epigenetic remodeling. We also find that STAT5 licenses the activity of two other key metabolic regulators, the mTOR signaling pathway and the MYC transcription factor. Building on the latter, we present evidence for transcriptome-wide cooperation between STAT5 and MYC in both normal and transformed T cells. Together, our data provide a molecular framework for transcriptional programing of T cell metabolism downstream of cγ cytokines and highlight the JAK-STAT pathway in mediating cellular growth and proliferation.