Interferon lambda promotes immune dysregulation and tissue inflammation in TLR7-induced lupus.
ABSTRACT: Type III IFN lambdas (IFN-?) have recently been described as important mediators of immune responses at barrier surfaces. However, their role in autoimmune diseases such as systemic lupus erythematosus (SLE), a condition characterized by aberrant type I IFN signaling, has not been determined. Here, we identify a nonredundant role for IFN-? in immune dysregulation and tissue inflammation in a model of TLR7-induced lupus. IFN-? protein is increased in murine lupus and IFN-? receptor (Ifnlr1) deficiency significantly reduces immune cell activation and associated organ damage in the skin and kidneys without effects on autoantibody production. Single-cell RNA sequencing in mouse spleen and human peripheral blood revealed that only mouse neutrophils and human B cells are directly responsive to this cytokine. Rather, IFN-? activates keratinocytes and mesangial cells to produce chemokines that induce immune cell recruitment and promote tissue inflammation. These data provide insights into the immunobiology of SLE and identify type III IFNs as important factors for tissue-specific pathology in this disease.
Project description:Interferon lambdas share important functional similarities with type I interferons, but their role in inflammation and autoimmune disease remains controversial and is not well studied. Here, we present the first evidence that interferon lambda is pathogenic and has nonredundant functions in TLR7-associated lupus inflammation. Most notably, we found that interferon lambda promotes systemic immune dysregulation through localized effects in the skin and kidneys. These data identify a novel role for interferon lambda in lupus immunobiology and tissue-specific pathology. Overall design: This dataset includes 6 scRNA samples. Immune cells were isolated from mouse spleen and human peripheral blood. Cells were treated with recombinant interferon alpha or interferon lambda for 4 hours. Single cell RNA seq analysis was subsequently performed to analyze responsiveness to stimulation with interferon alpha and interferon lambda cytokine across different immune cell clusters.
Project description:Type III interferons (IFN-lambdas(?)) are important cytokines that inhibit viruses and modulate immune responses by acting through a unique IFN-?R1/IL-10RB heterodimeric receptor. Until now, the primary antiviral function of IFN-?s has been proposed to be at anatomical barrier sites. Here, we examine the regulation of IFN-?R1 expression and measure the downstream effects of IFN-?3 stimulation in primary human blood immune cells, compared with lung or liver epithelial cells. IFN-?3 directly bound and upregulated IFN-stimulated gene (ISG) expression in freshly purified human B cells and CD8+ T cells, but not monocytes, neutrophils, natural killer cells, and CD4+ T cells. Despite similar IFNLR1 transcript levels in B cells and lung epithelial cells, lung epithelial cells bound more IFN-?3, which resulted in a 50-fold greater ISG induction when compared to B cells. The reduced response of B cells could be explained by higher expression of the soluble variant of IFN-?R1 (sIFN-?R1), which significantly reduced ISG induction when added with IFN-?3 to peripheral blood mononuclear cells or liver epithelial cells. T-cell receptor stimulation potently, and specifically, upregulated membrane-bound IFNLR1 expression in CD4+ T cells, leading to greater antiviral gene induction, and inhibition of human immunodeficiency virus type 1 infection. Collectively, our data demonstrate IFN-?3 directly interacts with the human adaptive immune system, unlike what has been previously shown in published mouse models, and that type III IFNs could be potentially utilized to suppress both mucosal and blood-borne viral infections.
Project description:Type I interferon (IFN), namely IFN- α, and B cell aberrations are long recognized in systemic lupus erythematosus (SLE) pathogenesis. Type I IFN receptor blockade has undergone clinical trials in SLE with varying degrees of success. Type III IFN (IFN-λ) produce a gene signature currently indistinguishable from that of type I in responsive cell types. IFN-λ are not blocked by type I IFN receptor blockade as they utilize a unique receptor (IFNLR1). Type III IFN are appreciated to have an important role in viral infection at epithelial barriers where IFNLR1 is strongly expressed. The effects of IFN-λ on immune cells remain understudied and are different between human and murine models. We have previously shown that human B cells can transcribe type I IFN genes after IFN-λ treatment including those associated with SLE. We have found that IFN-λ is detected in the serum of human SLE patients and correlates with IgD- CD27- CD21- CD24- (DN2) B cells, a compartment which contains CD11c+ age/autoimmunity B cells (ABC). ABC are a target of interest as recent studies suggest they are poised for plasma cell differentiation and enriched in autoreactivity and thus have the potential to contribute to SLE pathogenesis. Results: Naïve and DN cells display a prominent type I IFN gene expression profile in SLE. Transcript for type I, type II, and type III IFN receptors (IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, and IL10RB) are detected in HD and SLE B cells. CD11c+ CD21- frequency increased in DN compared to naïve B cells for SLE and HD (both p< 0.001). The mean and range of CD11c+ CD21- frequency was higher in SLE DN (30.7± 9.5%, mean±SEM; range 4.8-74.7%) compared to HD DN (7.6%±1.0%,3.6-9.4%). Increased IFNLR1 transcript correlated with CD11c+ CD21- B cell expansion (r2=0.922, p<0.0001). Increased pSTAT1 after IFN-α2 treatment is found in monocytes, T cells, and B cells but only in the B cells after IFN-λ1 treatment. Naïve, DN, switched, and unswitched memory HD B cells are responsive to type I and type III IFN, but demonstrated a higher pSTAT1 fold change with type I IFN treatment compared to type III IFN. In all B cell subsets, CD11c+ cells had a higher pSTAT1 fold change after IFN-λ1 stimulation than did CD11c- B cells. In HD with well-defined populations of CD11c+ CD21- DN cells, pSTAT1 fold change for IFN-λ approached that of IFN-α2. Conclusions: All human B cell subsets defined by CD27 and IgD respond to IFN-α and IFN-λ, but those expressing CD11c+ have increased responsiveness to IFN-λ. CD11c+ cells expand in SLE and associate with autoreactive plasma cell development. Thus, the role of IFN-λ may take on increased clinical significance in the setting type I IFN receptor blockade. These results suggest IFN-λ is an underappreciated driver of the IFN signature and B cell aberrations in SLE. Overall design: Patients meeting 1997 ACR systemic lupus erythematosus classification criteria (n = 8) and healthy donors (HD, n = 5) had blood drawn under IRB-approved protocol. The transcriptome of sorted IgD+ CD27- naïve and IgD- CD27- (DN) B cells was measured by bulk RNA sequencing for differential expression and gene set enrichment analysis. Cell phenotype and STAT1 phosphorylation (pSTAT1) in IFN-α2 and IFN-λ1 treated PBMC was measured by flow cytometry (n =10).
Project description:Interferon lambdas (IFN-?s; IFNL1-4) modulate immunity in the context of infections and autoimmune diseases, through a network of induced genes. IFN-?s act by binding to the heterodimeric IFN-? receptor (IFNLR), activating a STAT phosphorylation-dependent signaling cascade. Thereby hundreds of IFN-stimulated genes are induced, which modulate various immune functions via complex forward and feedback loops. When compared to the well-characterized IFN-? signaling cascade, three important differences have been discovered. First, the IFNLR is not ubiquitously expressed: in particular, immune cells show significant variation in the expression levels of and susceptibilities to IFN-?s. Second, the binding affinities of individual IFN-?s to the IFNLR varies greatly and are generally lower compared to the binding affinities of IFN-? to its receptor. Finally, genetic variation in the form of a series of single-nucleotide polymorphisms (SNPs) linked to genes involved in the IFN-? signaling cascade has been described and associated with the clinical course and treatment outcomes of hepatitis B and C virus infection. The clinical impact of IFN-? signaling and the SNP variations may, however, reach far beyond viral hepatitis. Recent publications show important roles for IFN-?s in a broad range of viral infections such as human T-cell leukemia type-1 virus, rotaviruses, and influenza virus. IFN-? also potentially modulates the course of bacterial colonization and infections as shown for Staphylococcus aureus and Mycobacterium tuberculosis. Although the immunological processes involved in controlling viral and bacterial infections are distinct, IFN-?s may interfere at various levels: as an innate immune cytokine with direct antiviral effects; or as a modulator of IFN-?-induced signaling via the suppressor of cytokine signaling 1 and the ubiquitin-specific peptidase 18 inhibitory feedback loops. In addition, the modulation of adaptive immune functions via macrophage and dendritic cell polarization, and subsequent priming, activation, and proliferation of pathogen-specific T- and B-cells may also be important elements associated with infectious disease outcomes. This review summarizes the emerging details of the IFN-? immunobiology in the context of the host immune response and viral and bacterial infections.
Project description:Increased type I interferon (IFN-I) production and IFN-stimulated gene (ISG) expression are linked to the pathogenesis of systemic lupus erythematosus (SLE). Although the mechanisms responsible for dysregulated IFN-I production in SLE remain unclear, autoantibody-mediated uptake of endogenous nucleic acids is thought to play a role. 2,6,10,14-tetramethylpentadecane (TMPD; also known as pristane) induces a lupus-like disease in mice characterized by immune complex nephritis with autoantibodies to DNA and ribonucleoproteins. We recently reported that TMPD also causes increased ISG expression and that the development of the lupus is completely dependent on IFN-I signaling (Nacionales, D.C., K.M. Kelly-Scumpia, P.Y. Lee, J.S. Weinstein, R. Lyons, E. Sobel, M. Satoh, and W.H. Reeves. 2007. Arthritis Rheum. 56:3770-3783). We show that TMPD elicits IFN-I production, monocyte recruitment, and autoantibody production exclusively through a Toll-like receptor (TLR) 7- and myeloid differentiation factor 88 (MyD88)-dependent pathway. In vitro studies revealed that TMPD augments the effect of TLR7 ligands but does not directly activate TLR7 itself. The effects of TMPD were amplified by the Y-linked autoimmune acceleration cluster, which carries a duplication of the TLR7 gene. In contrast, deficiency of Fcgamma receptors (FcgammaRs) did not affect the production of IFN-I. Collectively, the data demonstrate that TMPD-stimulated IFN-I production requires TLR7/MyD88 signaling and is independent of autoantibody-mediated uptake of ribonucleoproteins by FcgammaRs.
Project description:Interferon-? (IFN-?) is increased and plays an important role in the pathogenesis of systemic lupus erythematosus (SLE). Plasmacytoid dendritic cells (pDCs) are the main producer of IFN-?, but their IFN-? producing capacity has been shown to be unchanged or reduced when stimulated with a Toll-like receptor 9 (TLR9) agonist in patients with SLE compared to in healthy individuals. In this study, we investigated the IFN-?-producing capacity of lupus pDCs under different stimulation.pDCs from patients with SLE and healthy controls (HC) were stimulated with TLR9 or TLR7 agonist, and their IFN-? producing capacity was examined by intracellular cytokine staining and flow cytometry. The correlation of IFN-?-producing capacity with serum IFN-? levels and disease activity was assessed. The effect of in vitro IFN-? exposure on IFN-? production by pDCs was examined. Localization of TLR7 in cellular compartments in pDCs was investigated.The IFN-? producing capacity of pDCs was reduced after TLR9 stimulation, but increased when stimulated with a TLR7 agonist in SLE compared to in HC. IFN-? production by pDCs upon TLR9 stimulation was reduced and the percentage of IFN-?+pDC was inversely correlated with disease activity and serum IFN-? levels. However, the TLR7 agonist-induced IFN-? producing capacity of lupus pDCs was enhanced and correlated with disease activity and serum IFN-?. Exposure to IFN-? enhanced IFN-? production of TLR7-stimulated pDCs, but reduced that of pDCs activated with a TLR9 agonist. TLR7 localization was increased in late endosome/lysosome compartments in pDCs from SLE patients.These findings indicate that enhanced TLR7 responses of lupus pDCs, owing to TLR7 retention in late endosome/lysosome and exposure to IFN-?, are associated with the pathogenesis of SLE.
Project description:Systemic lupus erythematosus (SLE) is a multisystem, autoimmune disease that predominantly affects women. Previous findings that duplicated Toll-like receptor 7 (Tlr7) promotes lupus-like disease in male BXSB mice prompted us to evaluate TLR7 in human SLE. By using a candidate gene approach, we identified and replicated association of a TLR7 3'UTR SNP, rs3853839 (G/C), with SLE in 9,274 Eastern Asians (P(combined) = 6.5 x 10(-10)), with a stronger effect in male than female subjects [odds ratio, male vs. female = 2.33 (95% CI = 1.64-3.30) vs. 1.24 (95% CI = 1.14-1.34); P = 4.1 x 10(-4)]. G-allele carriers had increased TLR7 transcripts and more pronounced IFN signature than C-allele carriers; heterozygotes had 2.7-fold higher transcripts of G-allele than C-allele. These data established a functional polymorphism in type I IFN pathway gene TLR7 predisposing to SLE, especially in Chinese and Japanese male subjects.
Project description:Type III interferons (IFNs) (interleukin-28/29 or lambda interferon [IFN-lambda]) are cytokines with IFN-like activities. Here we show that several classes of viruses induce expression of IFN-lambda1 and -lambda2/3 in similar patterns. The IFN-lambdas were-unlike alpha/beta interferon (IFN-alpha/beta)-induced directly by stimulation with IFN-alpha or -lambda, thus identifying type III IFNs as IFN-stimulated genes. In vitro assays revealed that IFN-lambdas have appreciable antiviral activity against encephalomyocarditis virus (EMCV) but limited activity against herpes simplex virus type 2 (HSV-2), whereas IFN-alpha potently restricted both viruses. Using three murine models for generalized virus infections, we found that while recombinant IFN-alpha reduced the viral load after infection with EMCV, lymphocytic choriomeningitis virus (LCMV), and HSV-2, treatment with recombinant IFN-lambda in vivo did not affect viral load after infection with EMCV or LCMV but did reduce the hepatic viral titer of HSV-2. In a model for a localized HSV-2 infection, we further found that IFN-lambda completely blocked virus replication in the vaginal mucosa and totally prevented development of disease, in contrast to IFN-alpha, which had a more modest antiviral activity. Finally, pretreatment with IFN-lambda enhanced the levels of IFN-gamma in serum after HSV-2 infection. Thus, type III IFNs are expressed in response to most viruses and display potent antiviral activity in vivo against select viruses. The discrepancy between the observed antiviral activity in vitro and in vivo may suggest that IFN-lambda exerts a significant portion of its antiviral activity in vivo via stimulation of the immune system rather than through induction of the antiviral state.
Project description:Lambda interferons (IFN-?s) are a major component of the innate immune defense to viruses, bacteria, and fungi. In human liver, IFN-? not only drives antiviral responses, but also promotes inflammation and fibrosis in viral and non-viral diseases. Here we demonstrate that macrophages are primary responders to IFN-?, uniquely positioned to bridge the gap between IFN-? producing cells and lymphocyte populations that are not intrinsically responsive to IFN-?. While CD14+ monocytes do not express the IFN-? receptor, IFNLR1, sensitivity is quickly gained upon differentiation to macrophages in vitro. IFN-? stimulates macrophage cytotoxicity and phagocytosis as well as the secretion of pro-inflammatory cytokines and interferon stimulated genes that mediate immune cell chemotaxis and effector functions. In particular, IFN-? induced CCR5 and CXCR3 chemokines, stimulating T and NK cell migration, as well as subsequent NK cell cytotoxicity. Using immunofluorescence and cell sorting techniques, we confirmed that human liver macrophages expressing CD14 and CD68 are highly responsive to IFN-? ex vivo. Together, these data highlight a novel role for macrophages in shaping IFN-? dependent immune responses both directly through pro-inflammatory activity and indirectly by recruiting and activating IFN-? unresponsive lymphocytes.
Project description:The type III interferon (IFN) receptor is preferentially expressed by epithelial cells. It is made of two subunits: IFNLR1, which is specific to IFN-lambda (IFN-?) and IL10RB, which is shared by other cytokine receptors. Human hepatocytes express IFNLR1 and respond to IFN-?. In contrast, the IFN-? response of the mouse liver is very weak and IFNLR1 expression is hardly detectable in this organ. Here we investigated the IFN-? response at the cellular level in the mouse liver and we tested whether human and mouse hepatocytes truly differ in responsiveness to IFN-?. When monitoring expression of the IFN-responsive Mx genes by immunohistofluorescence, we observed that the IFN-? response in mouse livers was restricted to cholangiocytes, which form the bile ducts, and that mouse hepatocytes were indeed not responsive to IFN-?. The lack of mouse hepatocyte response to IFN-? was observed in different experimental settings, including the infection with a hepatotropic strain of influenza A virus which triggered a strong local production of IFN-?. With the help of chimeric mice containing transplanted human hepatocytes, we show that hepatocytes of human origin readily responded to IFN-? in a murine environment. Thus, our data suggest that human but not mouse hepatocytes are responsive to IFN-? in vivo. The non-responsiveness is an intrinsic property of mouse hepatocytes and is not due to the mouse liver micro-environment.