IRF-4 deficiency reduces inflammation and kidney fibrosis after folic acid-induced acute kidney injury.

The chronic phase following toxin-induced acute kidney injury (AKI) is characterized by robust inflammation and progressive kidney fibrosis. Interferon regulatory factor 4 (IRF-4) is a type of multifunctional transcription factor that has been deeply linked to inflammation and fibrotic diseases. However, the role of IRF-4 in kidney damage and renal fibrosis after toxin-induced AKI remain to be explored. In this work, we examined the effect of IRF-4 deficiency on inflammation and kidney fibrosis in an AKI-chronic kidney disease (CKD) transition model induced by folic acid (FA) injury. We showed that FA treatment resulted in severe acute tubular injury followed by inflammatory reaction and interstitial fibrosis in wild-type mice. A sharp elevation of IRF-4 levels was observed in FA-injured kidneys. IRF-4 knockout led to a substantial reduction of extracellular matrix (ECM) proteins deposition and inhibited myofibroblasts transformation in the kidneys of mice subjected to FA treatment. In addition, IRF-4 ablation impaired F4/80+ macrophages and CD3+ T lymphocytes infiltration into the FA-injured kidneys. Loss of IRF-4 reduced the production of inflammatory molecules such as CXCL16, IL-18, IL-6, and TGF-ß1 in the kidneys in response to FA stress. Following FA injury, the kidneys of IRF-4 knockout mice had fewer bone marrow-derived myofibroblasts than wild-type controls. Moreover, IRF-4 disruption inhibited macrophages to myofibroblasts differentiation in the kidneys in response to FA stimuli. In vitro, IL-4 stimulated expression of α-smooth muscle actin and ECM proteins and promoted M2 macrophages to myofibroblasts transition in mouse bone marrow-derived monocytes, which was abolished in the absence of IRF-4. Thus, we identified an important role of IRF-4 in the pathogenesis of progressive CKD following FA-induced AKI.

Monoallelic IRF5 deficiency in B cells prevents murine lupus.

Gain-of-function polymorphisms in the transcription factor IFN regulatory factor 5 (IRF5) are associated with an increased risk of developing systemic lupus erythematosus. However, the IRF5-expressing cell type(s) responsible for lupus pathogenesis in vivo is not known. We now show that monoallelic IRF5 deficiency in B cells markedly reduced disease in a murine lupus model. In contrast, similar reduction of IRF5 expression in macrophages, monocytes, and neutrophils did not reduce disease severity. B cell receptor and TLR7 signaling synergized to promote IRF5 phosphorylation and increase IRF5 protein expression, with these processes being independently regulated. This synergy increased B cell-intrinsic IL-6 and TNF-α production, both key requirements for germinal center (GC) responses, with IL-6 and TNF-α production in vitro and in vivo being substantially lower with loss of 1 allele of IRF5. Mechanistically, TLR7-dependent IRF5 nuclear translocation was reduced in B cells from IRF5-heterozygous mice. In addition, we show in multiple lupus models that IRF5 expression was dynamically regulated in vivo with increased expression in GC B cells compared with non-GC B cells and with further sequential increases during progression to plasmablasts and long-lived plasma cells. Overall, a critical threshold level of IRF5 in B cells was required to promote disease in murine lupus.

A RUNX-CBFß-driven enhancer directs the Irf8 dose-dependent lineage choice between DCs and monocytes.

The transcription factor IRF8 is essential for the development of monocytes and dendritic cells (DCs), whereas it inhibits neutrophilic differentiation. It is unclear how Irf8 expression is regulated and how this single transcription factor supports the generation of both monocytes and DCs. Here, we identified a RUNX-CBFß-driven enhancer 56 kb downstream of the Irf8 transcription start site. Deletion of this enhancer in vivo significantly decreased Irf8 expression throughout the myeloid lineage from the progenitor stages, thus resulting in loss of common DC progenitors and overproduction of Ly6C+ monocytes. We demonstrated that high, low or null expression of IRF8 in hematopoietic progenitor cells promotes differentiation toward type 1 conventional DCs, Ly6C+ monocytes or neutrophils, respectively, via epigenetic regulation of distinct sets of enhancers in cooperation with other transcription factors. Our results illustrate the mechanism through which IRF8 controls the lineage choice in a dose-dependent manner within the myeloid cell system.

Cytosolic DNA sensing through cGAS and STING is inactivated by gene mutations in pangolins.

The release of DNA into the cytoplasm upon damage to the nucleus or during viral infection triggers an interferon-mediated defense response, inflammation and cell death. In human cells cytoplasmic DNA is sensed by cyclic GMP-AMP Synthase (cGAS) and Absent In Melanoma 2 (AIM2). Here, we report the identification of a "natural knockout" model of cGAS. Comparative genomics of phylogenetically diverse mammalian species showed that cGAS and its interaction partner Stimulator of Interferon Genes (STING) have been inactivated by mutations in the Malayan pangolin whereas other mammals retained intact copies of these genes. The coding sequences of CGAS and STING1 are also disrupted by premature stop codons and frame-shift mutations in Chinese and tree pangolins, suggesting that expression of these genes was lost in a common ancestor of all pangolins that lived more than 20 million years ago. AIM2 is retained in a functional form in pangolins whereas it is inactivated by mutations in carnivorans, the phylogenetic sister group of pangolins. The deficiency of cGAS and STING points to the existence of alternative mechanisms of controlling cytoplasmic DNA-associated cell damage and viral infections in pangolins.

IRF5 guides monocytes toward an inflammatory CD11c+ macrophage phenotype and promotes intestinal inflammation.

Mononuclear phagocytes (MNPs) are vital for maintaining intestinal homeostasis but, in response to acute microbial stimulation, can also trigger immunopathology, accelerating recruitment of Ly6Chi monocytes to the gut. The regulators that control monocyte tissue adaptation in the gut remain poorly understood. Interferon regulatory factor 5 (IRF5) is a transcription factor previously shown to play a key role in maintaining the inflammatory phenotype of macrophages. Here, we investigate the impact of IRF5 on the MNP system and physiology of the gut at homeostasis and during inflammation. We demonstrate that IRF5 deficiency has a limited impact on colon physiology at steady state but ameliorates immunopathology during Helicobacter hepaticus-induced colitis. Inhibition of IRF5 activity in MNPs phenocopies global IRF5 deficiency. Using a combination of bone marrow chimera and single-cell RNA-sequencing approaches, we examined the intrinsic role of IRF5 in controlling colonic MNP development. We demonstrate that IRF5 promotes differentiation of Ly6Chi monocytes into CD11c+ macrophages and controls the production of antimicrobial and inflammatory mediators by these cells. Thus, we identify IRF5 as a key transcriptional regulator of the colonic MNP system during intestinal inflammation.

Exacerbated Imiquimod-Induced Psoriasis-Like Skin Inflammation in IRF5-Deficient Mice.

Interferon regulatory factors (IRFs) play diverse roles in the regulation of the innate and adaptive immune responses in various diseases. In psoriasis, IRF2 is known to be involved in pathogenesis, while studies on other IRFs are limited. In this study, we investigated the role of IRF5 in psoriasis using imiquimod-induced psoriasis-like dermatitis. Although IRF5 is known to play a critical role in the induction of proinflammatory cytokines by immune cells, such as dendritic cells (DCs), macrophages, and monocytes, IRF5 deficiency unexpectedly exacerbated psoriasiform skin inflammation. The interferon-α and tumor necrosis factor-α mRNA expression levels were decreased, while levels of Th17 cytokines including IL-17, IL-22, and IL-23 were increased in IRF5-deficient mice. Furthermore, IL-23 expression in DCs from IRF5-deficient mice was upregulated both in steady state and after toll-like receptor 7/8 agonist stimulation. Importantly, the expression of IRF4, which is also important for the IL-23 production in DCs, was augmented in DCs from IRF5-deficient mice. Taken together, our results suggest that IRF5 deficiency induces the upregulation of IRF4 in DCs followed by augmented IL-23 production, resulting in the amplification of Th17 responses and the exacerbation of imiquimod-induced psoriasis-like skin inflammation. The regulation of IRF4 or IRF5 expression may be a novel therapeutic approach to psoriasis.

Microdeletion of the entire IRF6 gene in a Subsaharian African's family with Van der Woude syndrome.

Microdeletion of the entire interferon regulatory factory 6 (IRF 6) gene is a rare cause of Van der Woude syndrome (VDW) with only few cases reported in medical literature. Its occurrence in multiple affected members of a family is exceptional. The aim of this presentation was to describe a Central African family with typical VDW phenotype carrying an IRF6 gene deletion. Here we reported phenotype features of members of a Central African family with VDW syndrome consisting of labioalveolar cleft, depressions of the lower lip with labial fistulae (lip pits), submucosal clefts and cleft palate. Mutation analysis by means of multiplex ligation-dependent probe amplification and chromosomal microarray revealed a 374.070 kb, deletion encompassing the entire IRF6 gene in four affected family members. Microdeletion of the entire IRF6 gene causes the classical VDW syndrome phenotype.

The RIPK4-IRF6 signalling axis safeguards epidermal differentiation and barrier function.

The integrity of the mammalian epidermis depends on a balance of proliferation and differentiation in the resident population of stem cells1. The kinase RIPK4 and the transcription factor IRF6 are mutated in severe developmental syndromes in humans, and mice lacking these genes display epidermal hyperproliferation and soft-tissue fusions that result in neonatal lethality2-5. Our understanding of how these genes control epidermal differentiation is incomplete. Here we show that the role of RIPK4 in mouse development requires its kinase activity; that RIPK4 and IRF6 expressed in the epidermis regulate the same biological processes; and that the phosphorylation of IRF6 at Ser413 and Ser424 primes IRF6 for activation. Using RNA sequencing (RNA-seq), histone chromatin immunoprecipitation followed by sequencing (ChIP-seq) and assay for transposase-accessible chromatin using sequencing (ATAC-seq) of skin in wild-type and IRF6-deficient mouse embryos, we define the transcriptional programs that are regulated by IRF6 during epidermal differentiation. IRF6 was enriched at bivalent promoters, and IRF6 deficiency caused defective expression of genes that are involved in the metabolism of lipids and the formation of tight junctions. Accordingly, the lipid composition of the stratum corneum of Irf6-/- skin was abnormal, culminating in a severe defect in the function of the epidermal barrier. Collectively, our results explain how RIPK4 and IRF6 function to ensure the integrity of the epidermis and provide mechanistic insights into why developmental syndromes that are characterized by orofacial, skin and genital abnormalities result when this axis goes awry.

Irf5 deficiency in myeloid cells prevents necrotizing enterocolitis by inhibiting M1 macrophage polarization.

Necrotizing enterocolitis (NEC) is a life-threatening inflammatory disease in newborns, but the mechanisms remain unclear. Interferon regulatory factor 5 (IRF5) is a master regulator of macrophage function and is essential for proinflammatory M1 macrophage polarization. Our previous data indicated that M1 macrophages promote NEC injury. Here, we investigated whether IRF5 is involved in the pathogenesis of NEC. First, we found that IRF5 was upregulated in infiltrated macrophages in human neonates with NEC compared to controls. We further confirmed IRF5 upregulation in macrophages in experimental murine NEC and that the infiltrated macrophages were predominantly polarized into the M1 but not the M2 phenotype. Myeloid-specific deficiency of Irf5, which was associated with reduced M1 macrophage polarization and systematic inflammation, dramatically prevented experimental NEC. Moreover, we found that the ablation of Irf5 in myeloid cells markedly suppressed intestinal epithelial cell apoptosis and further prevented intestinal barrier dysfunction in experimental NEC. Bioinformatic and chromatin immunoprecipitation analysis further showed that IRF5 binds to the promoters of the M1 macrophage-associated genes Ccl4, Ccl5, Tnf, and Il12b. Overall, our study provides evidence that IRF5 participates in the pathogenesis of NEC, while the deletion of Irf5 in myeloid cells prevents NEC via inhibiting M1 macrophage polarization.

Inactivating Mutation in IRF8 Promotes Osteoclast Transcriptional Programs and Increases Susceptibility to Tooth Root Resorption.

This is the first study to our knowledge to report a novel mutation in the interferon regulatory factor 8 gene (IRF8G388S ) associated with multiple idiopathic tooth root resorption, a form of periodontal disease. The IRF8G388S variant in the highly conserved C-terminal motif is predicted to alter the protein structure, likely impairing IRF8 function. Functional assays demonstrated that the IRF8G388S mutant promoted osteoclastogenesis and failed to inhibit NFATc1-dependent transcriptional activation when compared with IRF8WT control. Further, similar to subjects with heterozygous IRF8G388S mutation, Irf8+/- mice exhibited increased osteoclast activity in the mandibular alveolar bone surrounding molar teeth. Immunohistochemistry illustrated increased NFATc1 expression in the dentoalveolar region of Irf8-/- and Irf8+/- mice when compared with Irf8+/+ controls. Genomewide analyses revealed that IRF8 constitutively bound to regulatory regions of several thousand genes in osteoclast precursors, and genetic aberration of IRF8 significantly enhanced many osteoclast-specific transcripts. Collectively, this study delineates the critical role of IRF8 in defining osteoclast lineage and osteoclast transcriptional program, which may help in better understanding of various osteoclast-mediated disorders, including periodontal disease. © 2019 American Society for Bone and Mineral Research.

IRF5 regulates unique subset of genes in dendritic cells during West Nile virus infection.

Pathogen recognition receptor (PRR) signaling is critical for triggering innate immune activation and the expression of immune response genes, including genes that impart restriction against virus replication. RIG-I-like receptors and TLRs are PRRs that signal immune activation and drive the expression of antiviral genes and the production of type I IFN leading to induction of IFN-stimulated genes, in part through the interferon regulatory factor (IRF) family of transcription factors. Previous studies with West Nile virus (WNV) showed that IRF3 and IRF7 regulate IFN expression in fibroblasts and neurons, whereas macrophages and dendritic cells (DCs) retained the ability to induce IFN-ß in the absence of IRF3 and IRF7 in a manner implicating IRF5 in PRR signaling actions. Here we assessed the contribution of IRF5 to immune gene induction in response to WNV infection in DCs. We examined IRF5-dependent gene expression and found that loss of IRF5 in mice resulted in modest and subtle changes in the expression of WNV-regulated genes. Anti-IRF5 chromatin immunoprecipitation with next-generation sequencing of genomic DNA coupled with mRNA analysis revealed unique IRF5 binding motifs within the mouse genome that are distinct from the canonical IRF binding motif and that link with IRF5-target gene expression. Using integrative bioinformatics analyses, we identified new IRF5 primary target genes in DCs in response to virus infection. This study provides novel insights into the distinct and unique innate immune and immune gene regulatory program directed by IRF5.

Depletion of MicroRNA-373 Represses the Replication of Hepatitis C Virus via Activation of Type 1 Interferon Response by Targeting IRF5.

PURPOSE: Hepatitis C virus (HCV) poses a risk of chronic liver disease and threatens a significant number of people worldwide. MicroRNAs (miRNAs) are linked to the regulation of hepatocarcinogenesis. Although miR-373 is required for HCV infection, the underlying mechanisms of miR-373 involvement in HCV replication remain elusive. MATERIALS AND METHODS: Quantitative reverse transcription PCR assays were performed to detect the abundances of miR-373 and HCV RNA either in Huh 7.5 cells or liver biopsy specimens with HCV infection. Luciferase assay was employed to probe the interactions between miR-373 and interferon regulatory factor 5 (IRF5). Western blot was conducted to investigate the effect of miR-373 and IRF5 on HCV replication and activation of type 1 interferon (IFN) response in JFH1-infected Huh 7.5 cells. RESULTS: HCV infection appeared to be caused by increased miR-373 expression. Addition of miR-373 promoted HCV RNA expression, while miR-373 depletion led to an inhibitive effect on HCV replication. Concordantly, IRF5, as a direct target, was limited by miR-373 in JFH1-infected Huh 7.5 cells. In addition, introduction of IRF5 protected HCV replication in the presence of abundant miR-373. Furthermore, the miR-373-mediated inhibitory effect on type 1 IFN response was ablated following IRF5 accumulation. CONCLUSION: miR-373 abrogation reduced HCV replication via activation of type 1 IFN responses by targeting IRF5 in JFH1-infected Huh 7.5 cells, suggesting a promising therapeutic for treating HCV infection.

Transcription Factor IRF4 Promotes CD8+ T Cell Exhaustion and Limits the Development of Memory-like T Cells during Chronic Infection.

During chronic stimulation, CD8+ T cells acquire an exhausted phenotype characterized by expression of inhibitory receptors, down-modulation of effector function, and metabolic impairments. T cell exhaustion protects from excessive immunopathology but limits clearance of virus-infected or tumor cells. We transcriptionally profiled antigen-specific T cells from mice infected with lymphocytic choriomeningitis virus strains that cause acute or chronic disease. T cell exhaustion during chronic infection was driven by high amounts of T cell receptor (TCR)-induced transcription factors IRF4, BATF, and NFATc1. These regulators promoted expression of inhibitory receptors, including PD-1, and mediated impaired cellular metabolism. Furthermore, they repressed the expression of TCF1, a transcription factor required for memory T cell differentiation. Reducing IRF4 expression restored the functional and metabolic properties of antigen-specific T cells and promoted memory-like T cell development. These findings indicate that IRF4 functions as a central node in a TCR-responsive transcriptional circuit that establishes and sustains T cell exhaustion during chronic infection.

Regulation of mononuclear phagocyte development by IRF8.

Mononuclear phagocytes, such as monocytes and dendritic cells (DCs), are essential for tissue homeostasis and immunity. In adults, these cells develop from hematopoietic stem cells via a common progenitor population. We have been investigating the mechanism underlying the development of mononuclear phagocytes from the viewpoint of gene expression control by transcription factors. Particularly, IRF8, the loss of which causes immunodeficiency and chronic myeloid leukemia-like neutrophilia in mice and humans, promotes the development of monocytes and DCs, while it limits neutrophil differentiation. IRF8 cooperates with the myeloid master transcription factor, PU.1, in mononuclear phagocyte progenitors. KLF4 and BATF3 serve as critical transcription factors downstream of IRF8 to induce the differentiation of monocytes and DCs, respectively. Conversely, IRF8 blocks the activity of the transcription factor C/EBPα to suppress the neutrophil differentiation program. Indeed, Irf8-/- mononuclear phagocyte progenitors do not efficiently generate monocytes and DCs and, instead, aberrantly give rise to a large number of neutrophils. Our recent data have begun to uncover the vital role of IRF8 in the establishment of distal enhancers in mononuclear phagocyte progenitors. These results place IRF8 as a central regulator of the development of monocytes and DCs.

Tumor Suppressor p53 Stimulates the Expression of Epstein-Barr Virus Latent Membrane Protein 1.

Epstein-Barr virus (EBV) is associated with multiple human malignancies. EBV latent membrane protein 1 (LMP1) is required for the efficient transformation of primary B lymphocytes in vitro and possibly in vivo The tumor suppressor p53 plays a seminal role in cancer development. In some EBV-associated cancers, p53 tends to be wild type and overly expressed; however, the effects of p53 on LMP1 expression is not clear. We find LMP1 expression to be associated with p53 expression in EBV-transformed cells under physiological and DNA damaging conditions. DNA damage stimulates LMP1 expression, and p53 is required for the stimulation. Ectopic p53 stimulates endogenous LMP1 expression. Moreover, endogenous LMP1 blocks DNA damage-mediated apoptosis. Regarding the mechanism of p53-mediated LMP1 expression, we find that interferon regulatory factor 5 (IRF5), a direct target of p53, is associated with both p53 and LMP1. IRF5 binds to and activates a LMP1 promoter reporter construct. Ectopic IRF5 increases the expression of LMP1, while knockdown of IRF5 leads to reduction of LMP1. Furthermore, LMP1 blocks IRF5-mediated apoptosis in EBV-infected cells. All of the data suggest that cellular p53 stimulates viral LMP1 expression, and IRF5 may be one of the factors for p53-mediated LMP1 stimulation. LMP1 may subsequently block DNA damage- and IRF5-mediated apoptosis for the benefits of EBV. The mutual regulation between p53 and LMP1 may play an important role in EBV infection and latency and its related cancers.IMPORTANCE The tumor suppressor p53 is a critical cellular protein in response to various stresses and dictates cells for various responses, including apoptosis. This work suggests that an Epstein-Bar virus (EBV) principal viral oncogene is activated by cellular p53. The viral oncogene blocks p53-mediated adverse effects during viral infection and transformation. Therefore, the induction of the viral oncogene by p53 provides a means for the virus to cope with infection and DNA damage-mediated cellular stresses. This seems to be the first report that p53 activates a viral oncogene; therefore, the discovery would be interesting to a broad readership from the fields of oncology to virology.

Interferon Regulatory Factor 5 Controls Necrotic Core Formation in Atherosclerotic Lesions by Impairing Efferocytosis.

BACKGROUND: Myeloid cells are central to atherosclerotic lesion development and vulnerable plaque formation. Impaired ability of arterial phagocytes to uptake apoptotic cells (efferocytosis) promotes lesion growth and establishment of a necrotic core. The transcription factor interferon regulatory factor (IRF)-5 is an important modulator of myeloid function and programming. We sought to investigate whether IRF5 affects the formation and phenotype of atherosclerotic lesions. METHODS: We investigated the role of IRF5 in atherosclerosis in 2 complementary models. First, atherosclerotic lesion development in hyperlipidemic apolipoprotein E-deficient (ApoE-/-) mice and ApoE-/- mice with a genetic deletion of IRF5 (ApoE-/-Irf5-/-) was compared and then lesion development was assessed in a model of shear stress-modulated vulnerable plaque formation. RESULTS: Both lesion and necrotic core size were significantly reduced in ApoE-/-Irf5-/- mice compared with IRF5-competent ApoE-/- mice. Necrotic core size was also reduced in the model of shear stress-modulated vulnerable plaque formation. A significant loss of CD11c+ macrophages was evident in ApoE-/-Irf5-/- mice in the aorta, draining lymph nodes, and bone marrow cell cultures, indicating that IRF5 maintains CD11c+ macrophages in atherosclerosis. Moreover, we revealed that the CD11c gene is a direct target of IRF5 in macrophages. In the absence of IRF5, CD11c- macrophages displayed a significant increase in expression of the efferocytosis-regulating integrin-ß3 and its ligand milk fat globule-epidermal growth factor 8 protein and enhanced efferocytosis in vitro and in situ. CONCLUSIONS: IRF5 is detrimental in atherosclerosis by promoting the maintenance of proinflammatory CD11c+ macrophages within lesions and controlling the expansion of the necrotic core by impairing efferocytosis.

Modelling IRF8 Deficient Human Hematopoiesis and Dendritic Cell Development with Engineered iPS Cells.

Human induced pluripotent stem (iPS) cells can differentiate into cells of all three germ layers, including hematopoietic stem cells and their progeny. Interferon regulatory factor 8 (IRF8) is a transcription factor, which acts in hematopoiesis as lineage determining factor for myeloid cells, including dendritic cells (DC). Autosomal recessive or dominant IRF8 mutations occurring in patients cause severe monocytic and DC immunodeficiency. To study IRF8 in human hematopoiesis we generated human IRF8-/- iPS cells and IRF8-/- embryonic stem (ES) cells using RNA guided CRISPR/Cas9n genome editing. Upon induction of hematopoietic differentiation, we demonstrate that IRF8 is dispensable for iPS cell and ES cell differentiation into hemogenic endothelium and for endothelial-to-hematopoietic transition, and thus development of hematopoietic progenitors. We differentiated iPS cell and ES cell derived progenitors into CD141+ cross-presenting cDC1 and CD1c+ classical cDC2 and CD303+ plasmacytoid DC (pDC). We found that IRF8 deficiency compromised cDC1 and pDC development, while cDC2 development was largely unaffected. Additionally, in an unrestricted differentiation regimen, IRF8-/- iPS cells and ES cells exhibited a clear bias toward granulocytes at the expense of monocytes. IRF8-/- DC showed reduced MHC class II expression and were impaired in cytokine responses, migration, and antigen presentation. Taken together, we engineered a human IRF8 knockout model that allows studying molecular mechanisms of human immunodeficiencies in vitro, including the pathophysiology of IRF8 deficient DC. Stem Cells 2017;35:898-908.

CRISPLD2 (LGL1) inhibits proinflammatory mediators in human fetal, adult, and COPD lung fibroblasts and epithelial cells.

Chronic lung disease of prematurity/bronchopulmonary dysplasia (BPD) is the leading cause of perinatal morbidity in developed countries. Inflammation is a prominent finding. Currently available interventions have associated toxicities and limited efficacy. While BPD often resolves in childhood, survivors of preterm birth are at risk for acquired respiratory disease in early life and are more likely to develop chronic obstructive pulmonary disease (COPD) in adulthood. We previously cloned Crispld2 (Lgl1), a glucocorticoid-regulated mesenchymal secretory protein that modulates lung branching and alveogenesis through mesenchymal-epithelial interactions. Absence of Crispld2 is embryonic lethal. Heterozygous Crispld2+/- mice display features of BPD, including distal airspace enlargement, disruption of elastin, and neonatal lung inflammation. CRISPLD2 also plays a role in human fetal lung fibroblast cell expansion, migration, and mesenchymal-epithelial signaling. This study assessed the effects of endogenous and exogenous CRISPLD2 on expression of proinflammatory mediators in human fetal and adult (normal and COPD) lung fibroblasts and epithelial cells. CRISPLD2 expression was upregulated in a lipopolysaccharide (LPS)-induced human fetal lung fibroblast line (MRC5). LPS-induced upregulation of the proinflammatory cytokines IL-8 and CCL2 was exacerbated in MRC5-CRISPLD2(knockdown) cells. siRNA suppression of endogenous CRISPLD2 in adult lung fibroblasts (HLFs) led to augmented expression of IL-8, IL-6, CCL2. LPS-stimulated expression of proinflammatory mediators by human lung epithelial HAEo- cells was attenuated by purified secretory CRISPLD2. RNA sequencing results from HLF-CRISPLD2(knockdown) suggest roles for CRISPLD2 in extracellular matrix and in inflammation. Our data suggest that suppression of CRISPLD2 increases the risk of lung inflammation in early life and adulthood.

IRF5 and IRF5 Disease-Risk Variants Increase Glycolysis and Human M1 Macrophage Polarization by Regulating Proximal Signaling and Akt2 Activation.

Interferon regulatory factor 5 (IRF5) regulates inflammatory M1 macrophage polarization, and disease-associated IRF5 genetic variants regulate pattern-recognition-receptor (PRR)-induced cytokines. PRR-stimulated macrophages and M1 macrophages exhibit enhanced glycolysis, a central mediator of inflammation. We find that IRF5 is needed for PRR-enhanced glycolysis in human macrophages and in mice in vivo. Upon stimulation of the PRR nucleotide binding oligomerization domain containing 2 (NOD2) in human macrophages, IRF5 binds RIP2, IRAK1, and TRAF6. IRF5, in turn, is required for optimal Akt2 activation, which increases expression of glycolytic pathway genes and HIF1A as well as pro-inflammatory cytokines and M1 polarization. Furthermore, pro-inflammatory cytokines and glycolytic pathways co-regulate each other. Rs2004640/rs2280714 TT/TT IRF5 disease-risk-carrier cells demonstrate increased IRF5 expression and increased PRR-induced Akt2 activation, glycolysis, pro-inflammatory cytokines, and M1 polarization relative to GG/CC carrier macrophages. Our findings identify that IRF5 disease-associated polymorphisms regulate diverse immunological and metabolic outcomes and provide further insight into mechanisms contributing to the increasingly recognized important role for glycolysis in inflammation.

Tick Salivary Sialostatin L Represses the Initiation of Immune Responses by Targeting IRF4-Dependent Transcription in Murine Mast Cells.

Coevolution of ticks and the vertebrate immune system has led to the development of immunosuppressive molecules that prevent immediate response of skin-resident immune cells to quickly fend off the parasite. In this article, we demonstrate that the tick-derived immunosuppressor sialostatin L restrains IL-9 production by mast cells, whereas degranulation and IL-6 expression are both unaffected. In addition, the expression of IL-1ß and IRF4 is strongly reduced in the presence of sialostatin L. Correspondingly, IRF4- or IL-1R-deficient mast cells exhibit a strong impairment in IL-9 production, demonstrating the importance of IRF4 and IL-1 in the regulation of the Il9 locus in mast cells. Furthermore, IRF4 binds to the promoters of Il1b and Il9, suggesting that sialostatin L suppresses mast cell-derived IL-9 preferentially by inhibiting IRF4. In an experimental asthma model, mast cell-specific deficiency in IRF4 or administration of sialostatin L results in a strong reduction in asthma symptoms, demonstrating the immunosuppressive potency of tick-derived molecules.