Faruk Sheikh

The expanded family of class II cytokines that share the IL-10 receptor-2 (IL-10R2) chain

Several novel interleukin (IL)‐10‐related cytokines have recently been discovered. These include IL‐22, IL‐26, and the interferon‐λ (IFN‐λ) proteins IFN‐λ1 (IL‐29), IFN‐λ2 (IL‐28A), and IFN‐λ3 (IL‐28B). The ligand‐binding chains for IL‐22, IL‐26, and IFN‐λ are distinct from that used by IL‐10; however, all of these cytokines use a common second chain, IL‐10 receptor‐2 (IL‐10R2; CRF2‐4), to assemble their active receptor complexes. Thus, IL‐10R2 is a shared component in at least four distinct class II cytokine‐receptor complexes. IL‐10 binds to IL‐10R1; IL‐22 binds to IL‐22R1; IL‐26 binds to IL‐20R1; and IFN‐λ binds to IFN‐λR1 (also known as IL‐28R). The binding of these ligands to their respective R1 chains induces a conformational change that enables IL‐10R2 to interact with the newly formed ligand‐receptor complexes. This in turn activates a signal‐transduction cascade that results in rapid activation of several transcription factors, particularly signal transducer and activator of transcription (STAT)3 and to a lesser degree, STAT1. Activation by IL‐10, IL‐22, IL‐26, or IFN‐λ can be blocked with neutralizing antibodies to the IL‐10R2 chain. Although IL‐10R2 is broadly expressed on a wide variety of tissues, only a subset of these tissues expresses the ligand‐binding R1 chains. The receptors for these cytokines are often present on cell lines derived from various tumors, including liver, colorectal, and pancreatic carcinomas. Consequently, the receptors for these cytokines may provide novel targets for inhibiting the growth of certain types of cancer.

Unique functions of the type II interleukin 4 receptor identified in mice lacking the interleukin 13 receptor α1 chain

The interleukin 4 receptor (IL-4R) is a central mediator of T helper type 2 (TH2)–mediated disease and associates with either the common γ-chain to form the type I IL-4R or with the IL-13R α1 chain (IL-13Rα1) to form the type II IL-4R. Here we used Il13ra1−/− mice to characterize the distinct functions of type I and type II IL-4 receptors in vivo. In contrast to Il4ra−/− mice, which have weak TH2 responses, Il13ra1−/− mice had exacerbated TH2 responses. Il13ra1−/− mice showed much less mortality after infection with Schistosoma mansoni and much more susceptibility to Nippostrongylus brasiliensis. IL-13Rα1 was essential for allergen-induced airway hyperreactivity and mucus hypersecretion but not for fibroblast or alternative macrophage activation. Thus, type I and II IL-4 receptors exert distinct effects on immune responses.

Interleukin-26: An IL-10-related cytokine produced by Th17 cells

IL-26 is classified as a member of the IL-10 cytokine family because it has limited sequence homology to IL-10 and the IL-10-related cytokines. The human IL-26 gene, IL26, is located on chromosome 12q15 between the genes for two other important class-2 cytokines, IFNG (IFN-γ) and IL22 (IL-22). IL-26 is often co-expressed with IL-22 by activated T cells, especially Th17 cells. It signals through a heterodimeric receptor complex composed of the IL-20R1 and IL-10R2 chains. IL-26 receptors are primarily expressed on non-hematopoietic cell types, particularly epithelial cells. Signaling through IL-26 receptor complexes results in the activation of STAT1 and STAT3 with subsequent induction of IL-26-responsive genes. The biological functions of IL-26 have only begun to be defined.

Interferon-lambda (IFN-λ) induces signal transduction and gene expression in human hepatocytes, but not in lymphocytes or monocytes

This study compared the ability of IFN‐α and IFN‐λ to induce signal transduction and gene expression in primary human hepatocytes, PBLs, and monocytes. IFN‐α drug products are widely used to treat chronic HCV infection; however, IFN‐α therapy often induces hematologic toxicities as a result of the broad expression of IFNARs on many cell types, including most leukocytes. rIFN‐λ1 is currently being tested as a potential alternative to IFN‐α for treating chronic HCV. Although IFN‐λ has been shown to be active on hepatoma cell lines, such as HepG2 and Huh‐7, its ability to induce responses in primary human hepatocytes or leukocytes has not been examined. We found that IFN‐λ induces activation of Jak/STAT signaling in mouse and human hepatocytes, and the ability of IFN‐λ to induce STAT activation correlates with induction of numerous ISGs. Although the magnitude of ISG expression induced by IFN‐λ in hepatocytes was generally lower than that induced by IFN‐α, the repertoire of regulated genes was quite similar. Our findings demonstrate that although IFN‐α and IFN‐λ signal through distinct receptors, they induce expression of a common set of ISGs in hepatocytes. However, unlike IFN‐α, IFN‐λ did not induce STAT activation or ISG expression by purified lymphocytes or monocytes. This important functional difference may provide a clinical advantage for IFN‐λ as a treatment for chronic HCV infection, as it is less likely to induce the leukopenias that are often associated with IFN‐α therapy.

An essential role for IFN-β in the induction of IFN-stimulated gene expression by LPS in macrophages.

TLR agonists such as LPS and poly(I:C) induce expression of type I IFNs, such as IFN‐α and ‐β, by macrophages. To examine the role of IFN‐β in the induction of ISGs by LPS, we compared the ability of LPS to induce ISGF3 activity and ISG expression in bone marrow–derived macrophages from WT and Ifnb1−/− mice. We found that LPS treatment activated ISGF3 and induced expression of ISGs such as Oas1, Mx1, Ddx58 (RIG‐I), and Ifih1 (MDA5) in WT macrophages, but not in macrophages derived from Ifnb1−/− mice or Ifnar1−/− mice. The inability of LPS to induce activation of ISGF3 and ISG expression in Ifnb1−/− macrophages correlated with the failure of LPS to induce activation of STAT1 and ‐2 in these cells. Consistent with these findings, LPS treatment also failed to induce ISG expression in bone marrow–derived macrophages from Stat2 KO mice. Although activation of ISGF3 and induction of ISG expression by LPS was abrogated in Ifnb1−/− and Ifnar1−/− macrophages, activation of NF‐κB and induction of NF‐κB‐responsive genes, such as Tnf (TNF‐α) and Il1b (IL‐1β), were not affected by deletion of either the IFN‐β or IFN‐αR1 genes. These findings demonstrate that induction of ISGF3 activity and ISG expression by LPS is critically dependent on intermediate production of IFN‐β and autocrine signaling through type I IFN receptors.

Distinct and overlapping genomic profiles and antiviral effects of Interferon-λ and -α on HCV-infected and noninfected hepatoma cells.

Summary.u2002 Recently, several SNPs in the region of the IL28B (IFN‐λ) gene have been associated with spontaneous clearance of hepatitis C virus (HCV) and enhanced cure rates for IFN‐alfa‐based therapies, suggesting a potential correlation between IFN‐λ and the ability to clear HCV. To understand the mechanism of IFN‐λ’s as compared to IFN‐α’s antiviral activity, we performed a comprehensive analysis of their anti‐HCV effects, whole genome transcriptome profiling with validation, and signalling of IFN‐α and IFN‐λ using J6/JFH‐1 and Huh7.5 cells in vitro. IFN‐λ and IFN‐α exhibited comparable anti‐HCV activity and gene expression profiles in Huh7.5 cells. While the majority of genes induced by IFN‐α and IFN‐λ were similar, IFN‐λ exhibits profound, but delayed kinetics of IFN‐stimulated genes (ISG) induction, while IFN‐α induced more rapid induction of ISGs. Furthermore, the increased induction of ISG expression by IFN‐λ correlated with up‐regulation of IFN‐λ receptor (IL‐28RA) expression and more prolonged activation of the Jak‐STAT signalling pathway. The findings from our comparative analysis of IFN‐α and IFN‐λ in HCV‐infected and noninfected cells support the clinical use of IFN‐λ as a potential alternative to IFN‐α in the treatment of chronic hepatitis C.

Induction of Uteroglobin-Related Protein 2 (Ugrp2) Gene Expression by the Th2 Cytokines IL-4 and IL-13

Uteroglobin-related proteins 1 and 2 (UGRP1 and -2) are thought to play important roles in inflammation and immunologic responses in the lung. In this study we demonstrate that IL-4 and IL-13 enhance Ugrp2 gene expression in the mouse transformed Clara cell line, mtCC, in a time- and dose-dependent manner. Addition of actinomycin D abrogated the IL-4- and IL-13-induced increase of Ugrp2 expression, demonstrating that this increase occurs at the transcriptional level. When mtCC cells were pretreated with IFN-γ before the addition of IL-4 or IL-13, IL-4- and 13-induced Ugrp2 mRNA increase was markedly decreased. IL-4 and IL-13 induced phosphorylation of STAT6 in mtCC cells, which binds to the proximal STAT-binding element (SBE) in the Ugrp2 gene promoter, leading to transcriptional activation of this gene. Mutations of the proximal SBE abrogated the binding of activated STAT6 to this site and the IL-4-induced increase in Ugrp2 gene promoter activity. IFN-γ-activated STAT1 binds to the same SBE in the Ugrp2 gene promoter to which STAT6 binds and decreases the binding of STAT6 to this site. Furthermore, an IL-4-induced increase in Ugrp2 expression was not observed in primary cultures of lung cells derived from STAT6-deficient mice. These results indicate that Ugrp2 expression is enhanced by IL-4 and IL-13 through STAT6 binding to the proximal SBE located in the Ugrp2 gene promoter.

IFN-λ4 Attenuates Antiviral Responses by Enhancing Negative Regulation of IFN Signaling

Type III IFNs are important mediators of antiviral immunity. IFN-λ4 is a unique type III IFN because it is produced only in individuals who carry a dG allele of a genetic variant rs368234815-dG/TT. Counterintuitively, those individuals who can produce IFN-λ4, an antiviral cytokine, are also less likely to clear hepatitis C virus infection. In this study, we searched for unique functional properties of IFN-λ4 that might explain its negative effect on hepatitis C virus clearance. We used fresh primary human hepatocytes (PHHs) treated with recombinant type III IFNs or infected with Sendai virus to model acute viral infection and subsequently validated our findings in HepG2 cell line models. Endogenous IFN-λ4 protein was detectable only in Sendai virus–infected PHHs from individuals with the dG allele, where it was poorly secreted but highly functional, even at concentrations < 50 pg/ml. IFN-λ4 acted faster than other type III IFNs in inducing antiviral genes, as well as negative regulators of the IFN response, such as USP18 and SOCS1. Transient treatment of PHHs with IFN-λ4, but not IFN-λ3, caused a strong and sustained induction of SOCS1 and refractoriness to further stimulation with IFN-λ3. Our results suggest unique functional properties of IFN-λ4 that can be important in viral clearance and other clinical conditions.

Oncostatin M Regulates Secretoglobin 3A1 and 3A2 Expression in a Bidirectional Manner

Secretoglobin (SCGB) 3A1 and 3A2 are members of the small molecular weight secretoglobin gene superfamily. SCGB3A1 is a tumor suppressor gene, whereas SCGB3A2 has anti-inflammatory properties. Both genes are mainly expressed in the lung and trachea in mice. Whether the expression and/or function of these two genes are related is not known. Here we show that the expression of SCGB3A1 and SCGB3A2 are bidirectionally regulated by oncostatin M (OSM) when examined in a mouse transformed Clara cell line (mtCC); SCGB3A1 is up-regulated by OSM, while SCGB3A2 is down-regulated in a time- and dose-dependent manner. OSM-activated STAT3/5, through binding to the STAT-binding element located at -201 to -209 bp in the mouse Scgb3a1 gene promoter, and the extracellular signal-regulated kinase (ERK)- and p38-mitogen-activated protein kinase (MAPK) pathways are responsible for the OSM-induced up-regulation of SCGB3A1 expression. On the other hand, the -113 to -273 bp region in the Scgb3a2 promoter appears to be responsible for the OSM induced down-regulation of the gene. No significant differences in the levels or patterns of specific DNA-binding proteins were found in the -113 to -273 bp region as determined by electrophoretic mobility shift assays. Neither the ERK- nor p38-MAPK pathways were involved in the OSM-induced reduction of Scgb3a2 promoter activity. These results suggest that OSM-induced suppression of SCGB3A2 expression is an indirect effect of OSM. Expression of the Clara cell marker, CYP2F2, was markedly decreased upon OSM treatment in parallel with the decrease of SCGB3A2 expression in mtCC cells. The differential regulation of Scgb3a1 and Scgb3a2 gene expression by OSM may explain the unique functions of these genes in the lung.

Influenza virus NS1 protein mutations at position 171 impact innate interferon responses by respiratory epithelial cells

The influenza virus NS1 protein interacts with a wide range of proteins to suppress the host cell immune response and facilitate virus replication. The amino acid sequence of the 2009 pandemic virus NS1 protein differed from sequences of earlier related viruses. The functional impact of these differences has not been fully defined. Therefore, we made mutations to the NS1 protein based on these sequence differences, and assessed the impact of these changes on host cell interferon (IFN) responses. We found that viruses with mutations at position 171 replicated efficiently but did not induce expression of interferon genes as effectively as wild-type viruses in A459 lung epithelial cells. The decreased ability of these NS1 mutant viruses to induce IFN gene and protein expression correlated with decreased activation of STAT1 and lower levels of IFN-stimulated gene (ISG) expression. These findings demonstrate that mutations at position 171 in the NS1 protein result in decreased expression of IFN and ISGs by A549 cells. Consequently, these viruses may be more virulent than the parental strains that do not contain mutations at position 171 in the NS1 protein.