Synthetic interleukin 22 (IL-22) signaling reveals biological activity of homodimeric IL-10 receptor 2 and functional cross-talk with the IL-6 receptor gp130.

Cytokine signaling is transmitted by cell-surface receptors that function as biological switches controlling mainly immune-related processes. Recently, we have designed synthetic cytokine receptors (SyCyRs) consisting of GFP and mCherry nanobodies fused to transmembrane and intracellular domains of cytokine receptors that phenocopy cytokine signaling induced by nonphysiological homo- and heterodimeric GFP-mCherry ligands. Interleukin 22 (IL-22) signals via both IL-22 receptor α1 (IL-22Rα1) and the common IL-10R2, belongs to the IL-10 cytokine family, and is critically involved in tissue regeneration. Here, IL-22 SyCyRs phenocopied native IL-22 signal transduction, indicated by induction of cytokine-dependent cellular proliferation, signal transduction, and transcriptome analysis. Whereas homodimeric IL-22Rα1 SyCyRs failed to activate signaling, homodimerization of the second IL-22 signaling chain, SyCyR(IL-10R2), which previously was considered not to induce signal transduction, led to induction of signal transduction. Interestingly, the SyCyR(IL-10R2) and SyCyR(IL-22Rα1) constructs could form functional heterodimeric receptor signaling complexes with the synthetic IL-6 receptor chain SyCyR(gp130). In summary, we have demonstrated that IL-22 signaling can be phenocopied by synthetic cytokine receptors, identified a functional IL-10R2 homodimeric receptor complex, and uncovered broad receptor cross-talk of IL-22Rα1 and IL-20R2 with gp130.

Expression of Interferons Lambda 3 and 4 Induces Identical Response in Human Liver Cell Lines Depending Exclusively on Canonical Signaling.

Lambda interferons mediate antiviral immunity by inducing interferon-stimulated genes (ISGs) in epithelial tissues. A common variant rs368234815TT/∆G creating functional gene from an IFNL4 pseudogene is associated with the expression of major ISGs in the liver but impaired clearance of hepatitis C. To explain this, we compared Halo-tagged and non-tagged IFNL3 and IFNL4 signaling in liver-derived cell lines. Transfection with non-tagged IFNL3, non-tagged IFNL4 and Halo-tagged IFNL4 led to a similar degree of JAK-STAT activation and ISG induction; however, the response to transfection with Halo-tagged IFNL3 was lower and delayed. Transfection with non-tagged IFNL3 or IFNL4 induced no transcriptome change in the cells lacking either IL10R2 or IFNLR1 receptor subunits. Cytosolic overexpression of signal peptide-lacking IFNL3 or IFNL4 in wild type cells did not interfere with JAK-STAT signaling triggered by interferons in the medium. Finally, expression profile changes induced by transfection with non-tagged IFNL3 and IFNL4 were highly similar. These data do not support the hypothesis about IFNL4-specific non-canonical signaling and point out that functional studies conducted with tagged interferons should be interpreted with caution.

The expression of ß-Defensin-2, IL-22, IL-22R1 and IL-10R2 in rat model of Klebsiella pneumonia and their correlation with histological grades.

Backgrounds and Aims:Klebsiella pneumoniae represents the most common opportunistic pathogen contributing to Klebsiella pneumonia in hospital-acquired infections. Klebsiella pneumonia has a rapidly progressive clinical course and multi-drug resistant (MDR). Identification of the effective biochemical markers is crucial for improving early diagnosis and treatment of Klebsiella pneumonia. The aims of our study are to 1) investigate the expression of ß-Defensin-2(rßD2), IL-22, IL-22R1 and IL-10R2 in Klebsiella pneumonia-infected rats and 2) their association with the histological grades of Klebsiella pneumonia.Methods and Materials: Fifty specific pathogen free (SPF) male SD rats were randomly divided into two groups: control group (treated with normal saline) and pneumonia group (treated with K. pneumoniae). All animals were sacrificed 1 h, 12 h, 1 d, 3 d, 5 d post infection. The severity and property of pneumonia was evaluated by histopathologic observation and pathogen identification. The mRNA expression of rßD2, IL-22, IL-22R1 and IL-10R2 was measured by RT-qPCR assay. The expression of rßD2 in rat lung tissue was determined by Western blot analysis, and the level of IL-22 in rat serum was determined by ELISA.Results: Histopathologic examination and bacterial counting of lung tissues confirmed the successful establishment of rat pneumonia model. The gene expression of rßD2, IL-22, IL-22R1 and IL-10R2 in pneumonia rats were significantly higher than those in healthy control mice (P < 0.05). The expression of rßD2 was correlated with histological grades of Klebsiella pneumonia and the level of IL-22. RT-qPCR results showed that the peak expression of IL-22R1 appeared earlier than IL-10R2 in rat pneumonia model.Conclusions: The expression of rßD2 and IL-22 was increased significantly at early stage in rat Klebsiella pneumonia model, suggesting that IL-22 and rßD2 might serve as potential biomarkers for the early diagnosis of Klebsiella pneumonia.

The Microarchitecture of Pancreatic Cancer as Measured by Diffusion-Weighted Magnetic Resonance Imaging Is Altered by T Cells with a Tumor Promoting Th17 Phenotype.

Diffusion-weighted magnetic resonance imaging (DW-MRI) is a diagnostic tool that is increasingly used for the detection and characterization of focal masses in the abdomen, among these, pancreatic ductal adenocarcinoma (PDAC). DW-MRI reflects the microarchitecture of the tissue, and changes in diffusion, which are reflected by changes in the apparent diffusion coefficient (ADC), are mainly attributed to variations in cellular density, glandular formation, and fibrosis. When analyzing the T cell infiltrates, we found an association of a tumor-promoting subpopulation, characterized by the expression of interleukin (IL) 21 and IL26, with high ADC values. Moreover, the presence of IL21+ and IL26+ positive T cells was associated with poor prognosis. Pancreatic cancers-but not healthy pancreatic tissue-expressed receptors for IL21 and IL26, a finding that could be confirmed in pancreatic cell lines. The functionality of these receptors was demonstrated in pancreatic tumor cell lines, which showed phosphorylation of ERK1/2 and STAT3 pathways in response to the respective recombinant interleukins. Moreover, in vitro data showed an increased colony formation of tumor cells. In summary, our data showed an association of IL21+ and IL26+ immune cell infiltration, increased ADC, and aggressive tumor disease, most likely due to the activation of the key cancer signaling pathways ERK1/2 and STAT3 and formation of tumor colonies.

The Effects of IFN-λ on Epithelial Barrier Function Contribute to Klebsiella pneumoniae ST258 Pneumonia.

IFN-λ and IL-22, cytokines that share the coreceptor IL-10RB, are both induced over the course of Klebsiella pneumoniae ST258 (KP35) pneumonia. IL-22 is known to protect mucosal barriers, whereas the effects of IFN-λ on the mucosa are not established. We postulated that IFN-λ plays a role in regulating the airway epithelial barrier to facilitate cellular trafficking to the site of infection. In response to IFN-λ, the transmigration of neutrophils across a polarized monolayer of airway epithelial cells was increased, consistent with diminished epithelial integrity. KP35 infection increased epithelial permeability, and pretreatment with IFN-λ amplified this effect and facilitated bacterial transmigration. These effects of IFN-λ were confirmed in vivo, in that mice lacking the receptor for IFN-λ (Ifnlr1-/-) were protected from bacteremia in a murine model of KP35 pneumonia. Conversely, the integrity of the epithelial barrier was protected by IL-22, with subsequent impairment of neutrophil and bacterial transmigration in vitro. Maximal expression of IL-22 in vivo was observed later in the course of infection than IFN-λ production, with high levels of IL-22 produced by recruited immune cells at 48 hours, consistent with a role in epithelial barrier recovery. The divergent and opposing expression of these two related cytokines suggests a regulated interaction in the host response to KP35 infection. A major physiological effect of IFN-λ signaling is a decrease in epithelial barrier integrity, which facilitates immune cell recruitment but also enables K. pneumoniae invasion.

IL-22 suppresses HSV-2 replication in human cervical epithelial cells.

Interleukin (IL)-22, a member of the IL-10 family, plays a role in antiviral immune responses to a number of viral infections. However, it is unclear whether IL-22 is involved in the mucosal immunity against herpes simplex virus 2 (HSV-2) infection in the female reproductive tract (FRT). In this study, we studied whether IL-22 could inhibit HSV-2 infection of human cervical epithelial cells (End1/E6E7 cells). We showed that End1/E6E7 cells express the functional IL-22 receptor complex (IL-22R1 and IL-10R2). When treated with IL-22, End1/E6E7 cells expressed the higher levels of IFN-stimulated genes (ISGs: ISG15, ISG56, OAS-1, OAS-2, and Mx2) than untreated cells. In addition, IL-22-treated cells produced higher levels of the tight junction proteins (ZO-1 and Occludin) than untreated cells. Mechanistically, IL-22 could activate the JAK/STAT signaling pathway by inducing the phosphorylation of STAT1 and STAT3. These observations indicate the potential of IL-22 as an anti-HSV-2 agent in the FRT mucosal innate immunity against HSV-2 infection.

IFN-λ4 inhibits HIV infection of macrophages through signalling of IFN-λR1/IL-10R2 receptor complex.

The recently discovered IFN-λ4 has been found to have antiviral activity against several viruses. However, it's unknown whether IFN-λ4 can inhibit HIV infection. Here, we show that IFN-λ4 could suppress HIV infection of macrophages. This IFN-λ4-mediated HIV inhibition was compromised by the antibodies against IFN-λ receptor complex, IFN-λR1/IL-10R2. IFN-λ4 enhanced the phosphorylation of STAT1, and induced antiviral interferon-stimulated genes. These findings indicated that IFN-λ4 can inhibit HIV via JAK/STAT signalling pathway.

Clinical features of interleukin 10 receptor gene mutations in children with very early-onset inflammatory bowel disease.

OBJECTIVES: In the present study, we studied a cohort of patients with very early onset inflammatory bowel disease (IBD) to determine the frequency of mutations in the interleukin 10 (IL10) receptor genes as a cause of early-onset IBD. METHODS: Sanger sequencing was performed to determine the presence of IL10 and/or IL10 receptor mutations in 17 patients with a diagnosis of very early onset IBD (disease onset <2 years of age in 15 patients, between 3 and 4 years in the other 2). Mutation screening was performed including all of the coding regions of the IL10, IL10RA, and IL10RB genes. We then compared the follow-up findings of the patients with IL10 receptor mutations in terms of demographic, clinical, laboratory, and treatment response properties with those of patients diagnosed as having very early onset IBD with no mutation. RESULTS: We identified 3 patients bearing mutations in the IL10 or IL10 receptor genes, including 1 mutation in IL10RB that has been described recently (c.G477A, p.Trp159*) and 2 novel mutations affecting the IL10RA gene (c.T192G, p.Tyr64 and c.T133G, p.Trp45Gly). Collectively, these mutations thus provided genetic etiology for 17.6% of the cohort under investigation. The presence of a family history of IBD and the clinical course of Crohn disease differed between patients with mutations in the IL-10 pathway and those without such mutations. Although perianal fistulas were found in all of the patients with IL10 receptor mutations, they were found in only 14.3% of those without such mutations. The lower values of weight-for-age and height-for-age z scores, necessity for more intensive therapy, achievement of longer periods until remission, and frequent relapses in the patients bearing mutations in the IL10 receptor genes all underlined the severity of the disease and its relatively poor response to treatment. CONCLUSIONS: In spite of the small number of patients with mutations affecting the IL-10 signaling pathway in our study, in all of the patients with IL10 receptor mutations, the disease onset occurs at an early age, the prognosis is poor, and the response to treatment is insufficient.

The structural network of Interleukin-10 and its implications in inflammation and cancer.

BACKGROUND: Inflammation has significant roles in all phases of tumor development, including initiation, progression and metastasis. Interleukin-10 (IL-10) is a well-known immuno-modulatory cytokine with an anti-inflammatory activity. Lack of IL-10 allows induction of pro-inflammatory cytokines and hinders anti-tumor immunity, thereby favoring tumor growth. The IL-10 network is among the most important paths linking cancer and inflammation. The simple node-and-edge network representation is useful, but limited, hampering the understanding of the mechanistic details of signaling pathways. Structural networks complete the missing parts, and provide details. The IL-10 structural network may shed light on the mechanisms through which disease-related mutations work and the pathogenesis of malignancies. RESULTS: Using PRISM (a PRotein Interactions by Structural Matching tool), we constructed the structural network of IL-10, which includes its first and second degree protein neighbor interactions. We predicted the structures of complexes involved in these interactions, thereby enriching the available structural data. In order to reveal the significance of the interactions, we exploited mutations identified in cancer patients, mapping them onto key proteins of this network. We analyzed the effect of these mutations on the interactions, and demonstrated a relation between these and inflammation and cancer. Our results suggest that mutations that disrupt the interactions of IL-10 with its receptors (IL-10RA and IL-10RB) and α2-macroglobulin (A2M) may enhance inflammation and modulate anti-tumor immunity. Likewise, mutations that weaken the A2M-APP (amyloid precursor protein) association may increase the proliferative effect of APP through preventing ß-amyloid degradation by the A2M receptor, and mutations that abolish the A2M-Kallikrein-13 (KLK13) interaction may lead to cell proliferation and metastasis through the destructive effect of KLK13 on the extracellular matrix. CONCLUSIONS: Prediction of protein-protein interactions through structural matching can enrich the available cellular pathways. In addition, the structural data of protein complexes suggest how oncogenic mutations influence the interactions and explain their potential impact on IL-10 signaling in cancer and inflammation.

IFN-υ and its receptor subunits, IFN-υR1 and IL10RB in mallard Anas platyrhynchos.

Type IV interferon (IFN) has been shown to be a cytokine with antiviral activity in fish and amphibian. But, it has not been cloned and characterized functionally in avian species. In this study, type IV IFN, IFN-υ, and its 2 possible receptors, IFN-υR1 and IL10RB, were identified from an avian species, the mallard (Anas platyrhynchos). Mallard IFN-υ has a 531 bp open reading frame (ORF), encoding 176 amino acids (aa), and has highly conserved features as reported in different species, with an N-terminal signal peptide and a predicted multi-helix structure. The IFN-υR1 and IL10RB contain 528 and 343 aa, respectively, with IFN-υR1 protein containing JAK1 and STAT binding sites, and IL10RB containing TYK2 binding site. These 2 receptor subunits also possess 3 domains, the N-terminal extracellular domain, the transmembrane domain, and the C-terminal intracellular domain. Expression analysis indicated that IFN-υ, IFN-υR1 and IL10RB were widely expressed in examined organs/tissues, with the highest level observed in pancreas, blood, and kidney, respectively. The expression of IFN-υ, IFN-υR1 and IL10RB in liver, spleen or kidney was significantly upregulated after stimulation with polyI:C. Furthermore, recombinant IFN-υ protein induced the expression of ISGs, and the receptor of IFN-υ was verified as IFN-υR1 and IL10RB using a chimeric receptor approach in HEK293 cells. Taken together, these results indicate that IFN-υ is involved in the host innate immune response in mallard.

In search of a function for human type III interferons: insights from inherited and acquired deficits.

The essential and redundant functions of human type I and II interferons (IFNs) have been delineated over the last three decades by studies of patients with inborn errors of immunity or their autoimmune phenocopies, but much less is known about type III IFNs. Patients with cells that do not respond to type III IFNs due to inherited IL10RB deficiency display no overt viral disease, and their inflammatory disease phenotypes can be explained by defective signaling via other interleukine10RB-dependent pathways. Moreover, patients with inherited deficiencies of interferon-stimulated gene factor 3 (ISGF-3) (STAT1, STAT2, IRF9) present viral diseases also seen in patients with inherited deficiencies of the type I IFN receptor (IFNAR1/2). Finally, patients with autoantibodies neutralizing type III IFNs have no obvious predisposition to viral disease. Current findings thus suggest that type III IFNs are largely redundant in humans. The essential functions of human type III IFNs, particularly in antiviral defenses, remain to be discovered.

IL-10-- and IL-20--expressing epithelial and inflammatory cells are increased in patients with ulcerative colitis.

Interleukin (IL)-20, a pro-inflammatory cytokine, is a recently discovered member of the IL-10 family. This cytokine has been described in inflammatory diseases such as psoriasis and asthma. However, IL-20 expression in ulcerative colitis (UC) patients has not been yet described. The aim of this study was to evaluate IL-20 and IL-10 gene and protein expression and their receptors in the mucosa from UC patients. Forty UC patients and 18 non-inflamed controls were studied. IL-10, IL-20, IL-10R1, IL-10R2, IL-20R1 and IL-20R2 gene expression was determined by real time RT-PCR in colonic biopsies. Protein expression was evaluated by immunohistochemistry. Patients in remission had significantly higher IL-10 gene expression in mucosa compared with active patients and controls. Conversely, IL-10R1/B gene expression was decreased in remission compared with active UC patients and controls. IL-20 gene expression was lower in colonic mucosa from UC patients in remission compared with controls and active patients. IL-20R1/B mRNA expression was higher in remission compared with active UC patients and controls. IHQ analysis showed an increased IL-10-, IL-20-, and IL-20R2-producing cells in active UC patients. IL-10-, IL-20- and IL-20R2-expressing epithelial and inflammatory cells were increased in active UC patients, meanwhile IL-20R1 was up-regulated only on inflammatory infiltrates vs. controls. This is the first depiction of the presence of IL-20 and its receptors in UC. Much remains to be learned however, about the pathogenic mechanisms that lead to IBD. This cytokine/receptor imbalance may be implicated in the pathogenesis of UC.

Identification and establishment of type IV interferon and the characterization of interferon-υ including its class II cytokine receptors IFN-υR1 and IL-10R2.

Interferons (IFNs) are critical soluble factors in the immune system and are composed of three types, (I, II and III) that utilize different receptor complexes IFN-αR1/IFN-αR2, IFN-γR1/IFN-γR2, and IFN-λR1/IL-10R2, respectively. Here we identify IFN-υ from the genomic sequences of vertebrates. The members of class II cytokine receptors, IFN-υR1 and IL-10R2, are identified as the receptor complex of IFN-υ, and are associated with IFN-υ stimulated gene expression and antiviral activity in zebrafish (Danio rerio) and African clawed frog (Xenopus laevis). IFN-υ and IFN-υR1 are separately located at unique and highly conserved loci, being distinct from all other three-type IFNs. IFN-υ and IFN-υR1 are phylogenetically clustered with class II cytokines and class II cytokine receptors, respectively. Therefore, the finding of this IFN ligand-receptor system may be considered as a type IV IFN, in addition to the currently recognized three types of IFNs in vertebrates.

Defective IL10 signaling defining a subgroup of patients with inflammatory bowel disease.

OBJECTIVES: Early onset inflammatory bowel diseases (EO-IBD) developing during the first year of life are likely to reflect inherited defects in key mechanism(s) controlling intestinal homeostasis, as recently suggested for interleukin 10 (IL10). Thus, we aimed to further elaborate the hypothesis of defective anti-inflammatory responses in patients with IBD. METHODS: The capacities of transforming growth factor ß (TGFß) and IL10 to inhibit proinflammatory cytokine production by monocyte-derived dendritic cells (MoDC) or peripheral blood cells (PBMC) was analyzed in 75 children with IBD, including 13 infants with EO-IBD (in whom autoimmune diseases or classical immunodeficiencies were ruled out). IL10 receptor-A/-B expression, STAT3 activation in response to IL6, IL10, IL21, IL22 were analyzed by FACS and western blotting. IL10RA and B genes were sequenced. The response to IL22 was tested in ileal/colonic tissue cultures. Tissue gene expression was analyzed by Taqman real-time polymerase chain reaction. RESULTS: Production of IL10 in response to bacterial motifs was normal in all IBD patients. In contrast to our original hypothesis, no defect of the anti-inflammatory potential of TGFß and IL10 was observed in children with IBD or EO-IBD except two infants who presented with granuloma-positive colitis at 3 months of life: no response to IL10 was observed secondary to mutations in the α (p.R262C) or ß (p.E141X) chain of IL10R, respectively, although a fully functional Jak-STAT3 pathway was present in both patients. When analyzing the regulation of intestinal bacterial clearance, we detected a defect in the patient with absent IL10 RB to upregulate protective transcripts in response to IL22, whereas all other EO-IBD patients, including the patient with an abnormal α chain, responded normally. CONCLUSIONS: Impaired IL10 signaling characterizes a subgroup of IBD patients, whereas the majority of children with severe IBD including EO forms normally produces and responds to IL10. Defective IL22 signaling may additionally impair intestinal epithelial clearance. Our data point out the complexity of IBD, which represent a group of distinct diseases with several pathogenetic abnormalities.

Ileal immune dysregulation in necrotizing enterocolitis: role of CD40/CD40L in the pathogenesis of disease.

OBJECTIVES: CD40, a co-stimulatory molecule, plays a critical role in coordinating enteric inflammatory immune responses. In necrotizing enterocolitis (NEC), upregulation of IL-10, a CD40-modulated cytokine, has been described, but the role of the IL-10 receptor (IL-10Rß), CD40, and its ligand CD40L in disease pathogenesis is unknown. The study herein investigates ileal expression of CD40, CD40L, and IL-10R in a rat model of NEC. SUBJECTS AND METHODS: NEC was induced in newborn rats using established methods of formula feeding, asphyxia, and cold stress. Expression of CD40, CD40L, IL-10Rß, and other proinflammatory molecules, including Toll-like receptor-4 (TLR-4) and IL-18, was assessed by immunoblotting. Tissue infiltration by macrophages, monocytes, and T cells was examined by confocal immunohistochemistry. RESULTS: Ileum from rat pups with NEC showed increased expression of TLR-4, IL-18, and IL-10Rß. Sections from both NEC and control pups demonstrated preservation of ileal cells expressing CD40/CD40L. The distal ileum of controls expressed both CD40 and CD40L; conversely, neither molecule was detected in ileal tissue from NEC pups. Additional studies showed that treatment with epidermal growth factor (EGF), previously shown to ameliorate the severity of NEC in an animal model, did not restore CD40 expression. CONCLUSIONS: Ileal cytokine dysregulation, manifested by decreased CD40/CD40L and increased IL-10Rß expression, may be involved in the pathogenesis of NEC. Dampened CD40 signaling may be related to enhanced IL-10 expression and a suppressed T-cell response to injury. We speculate that augmenting CD40-CD40L interactions may achieve a protective effect in this NEC model.

Tongue sole (Cynoglossus semilaevis) interleukin 10 receptors are involved in the immune response against bacterial infection.

Interleukin (IL)-10, an immune-regulatory cytokine, exerts various biological functions through interaction with IL-10 receptors. In teleost, very limited functional studies on IL-10 receptors have been documented. In this study, we reported the expression patterns of IL-10 receptor 1 (CsIL-10R1) and receptor 2 (CsIL-10R2) of tongue sole (Cynoglossus semilaevis) and examined their biological properties. The expression of CsIL-10R1 and CsIL-10R2 occurred in multiple tissues and were regulated by bacterial challenge. In vitro binding studies showed that recombinant extracellular region of CsIL-10R1 (rCsIL-10R1ex) rather than rCsIL-10R2ex could bind with rCsIL-10. Cellular study showed that both CsIL-10R1 and CsIL-10R2 were expressed on peripheral blood leukocytes (PBLs), and blockade of CsIL-10R1 or CsIL-10R2 by antibody could reduce inhibitory effect of CsIL-10 on ROS production of PBLs. When injected in vivo, anti-rCsIL-10R1 or anti-rCsIL-10R2 antibody dramatically promoted the expression of proinflammatory cytokines and suppressed bacterial dissemination in tongue sole tissues. Consistently, the overexpression of CsIL-10R1 or CsIL-10R2 significantly enhanced bacterial dissemination, and the overexpression of CsIL-10R1M bearing STAT3 site mutation reduced bacterial dissemination. Overall, these results demonstrate for the first time teleost IL-10 receptors play a negative role in antibacterial immunity and add insight into the function of CsIL-10 receptors.

Structure-based decoupling of the pro- and anti-inflammatory functions of interleukin-10.

Interleukin-10 (IL-10) is an immunoregulatory cytokine with both anti-inflammatory and immunostimulatory properties and is frequently dysregulated in disease. We used a structure-based approach to deconvolute IL-10 pleiotropy by determining the structure of the IL-10 receptor (IL-10R) complex by cryo-electron microscopy at a resolution of 3.5 angstroms. The hexameric structure shows how IL-10 and IL-10Rα form a composite surface to engage the shared signaling receptor IL-10Rß, enabling the design of partial agonists. IL-10 variants with a range of IL-10Rß binding strengths uncovered substantial differences in response thresholds across immune cell populations, providing a means of manipulating IL-10 cell type selectivity. Some variants displayed myeloid-biased activity by suppressing macrophage activation without stimulating inflammatory CD8+ T cells, thereby uncoupling the major opposing functions of IL-10. These results provide a mechanistic blueprint for tuning the pleiotropic actions of IL-10.

Interferon-lambda is functionally an interferon but structurally related to the interleukin-10 family.

Interferon-lambda (IFN-lambda) is an antiviral cytokine that signals through a distinct receptor complex, composed of the IFN-lambdaR1 and interleukin-10R2 (IL-10R2) receptor chains. We have determined the crystal structure of human IFN-lambda3 and characterized the interaction with its receptor complex through structure-based site-directed mutagenesis. The ability of IFN-lambda3 mutants to signal was determined by measuring the antiviral activity and induced STAT2 phosphorylation. In conclusion, our data show that, although IFN-lambda is functionally an interferon, it is clearly structurally related to members of the IL-10 family. In particular, we found an interesting similarity between IFN-lambda and IL-22, and we suggest that IFN-lambda and IL-22 possess parallel functions, protecting epithelial tissue against viral and bacterial infections, respectively.

Modulation of IL10 and Its Receptor Subunits in Normal and Progesterone-Prolonged Gestation in the Mouse.

These experiments aimed to understand the relationship between interleukin 10 (IL10), the IL10 receptor subunits, and progesterone (P4) at the time of parturition. We hypothesized that there is a biologic connection between IL10 and P4, supporting an immunomodulatory mechanism for the onset of labor. Using samples from control and P4-treated pregnant mice, we assessed the production of IL10 and its receptor subunits (IL10Rα and IL10Rß) in gestational tissues. After preliminary studies, P4-treated pregnant mice were compared with controls to assess for differences in IL10 and IL10 receptor subunit expression throughout gestation. To investigate the contribution of the P4 receptor at the onset of labor, we performed timed studies on pregnant mice after treatment with RU486. Samples collected included placentas, placentation sites, and maternal livers. IL10, IL10Rα, and IL10Rß levels were measured in homogenized tissue using ELISA assays; the cytokine results were normalized for homogenate protein concentration. Control mice delivered on gd 18-19, and P4 treatment prevented parturition to beyond gd 20, as expected. In treated mice, P4 not only prevented the anticipated nadir of IL10 at term, but maintained elevated levels of IL10 through gd 20 (p < 0.05). P4 also reversed the anticipated decrease of the IL10Rα, which was increased in P4-treated mice (p < 0.05). Treatment with RU486 did not modulate the expression of IL10 or IL10Rα, but showed a significant decrease in the level of IL10Rß (p < 0.05). Progesterone functions at least in part through the IL10 signaling pathway to prolong gestation.

Loss of IL-10 signaling in macrophages limits bacterial killing driven by prostaglandin E2.

Loss of IL-10 signaling in macrophages (Mφs) leads to inflammatory bowel disease (IBD). Induced pluripotent stem cells (iPSCs) were generated from an infantile-onset IBD patient lacking a functional IL10RB gene. Mφs differentiated from IL-10RB-/- iPSCs lacked IL-10RB mRNA expression, were unable to phosphorylate STAT3, and failed to reduce LPS induced inflammatory cytokines in the presence of exogenous IL-10. IL-10RB-/- Mφs exhibited a striking defect in their ability to kill Salmonella enterica serovar Typhimurium, which was rescuable after experimentally introducing functional copies of the IL10RB gene. Genes involved in synthesis and receptor pathways for eicosanoid prostaglandin E2 (PGE2) were more highly induced in IL-10RB-/- Mφs, and these Mφs produced higher amounts of PGE2 after LPS stimulation compared with controls. Furthermore, pharmacological inhibition of PGE2 synthesis and PGE2 receptor blockade enhanced bacterial killing in Mφs. These results identify a regulatory interaction between IL-10 and PGE2, dysregulation of which may drive aberrant Mφ activation and impaired host defense contributing to IBD pathogenesis.