CCR2 promotes monocyte recruitment and intestinal inflammation in mice lacking the interleukin-10 receptor.

Macrophages are a heterogeneous population of mononuclear phagocytes abundantly distributed throughout the intestinal compartments that adapt to microenvironmental specific cues. In adult mice, the majority of intestinal macrophages exhibit a mature phenotype and are derived from blood monocytes. In the steady-state, replenishment of these cells is reduced in the absence of the chemokine receptor CCR2. Within the intestine of mice with colitis, there is a marked increase in the accumulation of immature macrophages that demonstrate an inflammatory phenotype. Here, we asked whether CCR2 is necessary for the development of colitis in mice lacking the receptor for IL10. We compared the development of intestinal inflammation in mice lacking IL10RA or both IL10RA and CCR2. The absence of CCR2 interfered with the accumulation of immature macrophages in IL10R-deficient mice, including a novel population of rounded submucosal Iba1+ cells, and reduced the severity of colitis in these mice. In contrast, the absence of CCR2 did not reduce the augmented inflammatory gene expression observed in mature intestinal macrophages isolated from mice lacking IL10RA. These data suggest that both newly recruited CCR2-dependent immature macrophages and CCR2-independent residual mature macrophages contribute to the development of intestinal inflammation observed in IL10R-deficient mice.

Utilization of lipopolysaccharide challenge in cynomolgus macaques to assess IL-10 receptor antagonism.

The current era of drug discovery has been marked by a significant increase in the development of immune modulating agents to address a range of diseases such as cancer, chronic inflammation, and other conditions of dysregulated immunity. Non-clinical evaluation of these agents in animal models can be challenging, as the presence of an active immune state is often required in order to detect the effects of the test agent. Modulation of interleukin (IL)-10 signaling represents this type of situation in that altering IL-10 action in vivo can be difficult to appreciate in the absence of an ongoing immune response. The study presented here reports on the use of lipopolysaccharide (LPS) challenge in cynomolgus macaques to induce predictable inflammatory cytokine responses. The results showed that IL-10 receptor (IL-10R) blockade with an antagonist monoclonal antibody (mAb) dramatically enhanced the LPS-induced cytokine response, thus demonstrating in vivo pharmacologic activity of this immunomodulatory antibody. We submit that this approach could be applied to other cases where the intent of a candidate therapeutic is to modulate components of inflammatory cytokine responses.

Oral antibody to interleukin-10 receptor 2, but not interleukin-10 receptor 1, as an effective Eimeria species immunotherapy in broiler chickens.

Coccidiosis is a major gastrointestinal disease caused by several Eimeria species in floor raised chickens. Feeding an antibody to interleukin 10 (aIL-10) ameliorates the negative symptoms of coccidiosis in broilers, i.e., lack of weight gain, decreased feed conversion, and mortality. IL-10 signals by forming a ligand-receptor complex with IL-10 Receptor 1 (IL-10 R1) and IL-10 Receptor 2 (IL-10 R2). In this study, we hypothesize oral antibodies to the IL-10 receptors will neutralize the IL-10 signaling pathway equal to or better than aIL-10 to act as an oral anti-coccidiosis immunotherapy. A total of 5 sequential feed trials, set up as a 4 (diet antibody) × 2 (Eimeria challenge) factorial design, tested oral egg yolk antibodies to a total of 6 IL-10 R1 epitopes and 3 IL-10 R2 epitopes compared to a control antibody diet. A total of 10 pens of 5 chicks/pen/diet antibody/Eimeria challenge were housed for 21 d. On day 3 of age, chicks were either infected or not infected with a 10× dose of an Eimeria vaccine containing Eimeria acervulina, Eimeria tenella, and Eimeria maxima. Pen feed consumption and mean body weights were assessed weekly (d1, d7, d14, and d21); fecal oocyst shedding was assessed on day 10. Data were analyzed using a 2-way ANOVA. No significant interaction on chick weight was observed in chicks fed IL-10 R1 antibodies compared to chicks fed the control antibody was observed. In studies evaluating aIL-10 R2 oral antibodies, infected chicks fed aIL-10 R2: epitope 1 overcame the negative effects of Eimeria infection and had similar 21-d body weight to uninfected chicks (P4 = 0.07). We hypothesized that feeding oral antibodies to the IL-10 receptors would result in equivalent anti-coccidial benefits to aIL-10. However, none of the 6 antibodies to IL-10 R1 epitopes yielded any benefits during Eimeria infection compared to controls. A total of 2 oral antibodies to IL-10 R2 showed promising results equivalent to the aIL-10 immunotherapeutic. Immunofluorescence staining shows that the IL-10R2 significantly increases in abundance in response to Eimeria infection, whereas IL-10R1 does not.

A Novel Subset of Anti-Inflammatory CD138+ Macrophages Is Deficient in Mice with Experimental Lupus.

Dead cells accumulating in the tissues may contribute to chronic inflammation. We examined the cause of impaired apoptotic cell clearance in human and murine lupus. Dead cells accumulated in bone marrow from lupus patients but not from nonautoimmune patients undergoing myeloablation, where they were efficiently removed by macrophages (MΦ). Impaired apoptotic cell uptake by MΦ also was seen in mice treated i.p. with pristane (develop lupus) but not mineral oil (MO) (do not develop lupus). The inflammatory response to both pristane and MO rapidly depleted resident (Tim4+) large peritoneal MΦ. The peritoneal exudate of pristane-treated mice contained mainly Ly6Chi inflammatory monocytes; whereas in MO-treated mice, it consisted predominantly of a novel subset of highly phagocytic MΦ resembling small peritoneal MΦ (SPM) that expressed CD138+ and the scavenger receptor Marco. Treatment with anti-Marco-neutralizing Abs and the class A scavenger receptor antagonist polyinosinic acid inhibited phagocytosis of apoptotic cells by CD138+ MΦ. CD138+ MΦ expressed IL-10R, CD206, and CCR2 but little TNF-α or CX3CR1. They also expressed high levels of activated CREB, a transcription factor implicated in generating alternatively activated MΦ. Similar cells were identified in the spleen and lung of MO-treated mice and also were induced by LPS. We conclude that highly phagocytic, CD138+ SPM-like cells with an anti-inflammatory phenotype may promote the resolution of inflammation in lupus and infectious diseases. These SPM-like cells are not restricted to the peritoneum and may help clear apoptotic cells from tissues such as the lung, helping to prevent chronic inflammation.

Exploiting induced pluripotent stem cell-derived macrophages to unravel host factors influencing Chlamydia trachomatis pathogenesis.

Chlamydia trachomatis remains a leading cause of bacterial sexually transmitted infections and preventable blindness worldwide. There are, however, limited in vitro models to study the role of host genetics in the response of macrophages to this obligate human pathogen. Here, we describe an approach using macrophages derived from human induced pluripotent stem cells (iPSdMs) to study macrophage-Chlamydia interactions in vitro. We show that iPSdMs support the full infectious life cycle of C. trachomatis in a manner that mimics the infection of human blood-derived macrophages. Transcriptomic and proteomic profiling of the macrophage response to chlamydial infection highlighted the role of the type I interferon and interleukin 10-mediated responses. Using CRISPR/Cas9 technology, we generated biallelic knockout mutations in host genes encoding IRF5 and IL-10RA in iPSCs, and confirmed their roles in limiting chlamydial infection in macrophages. This model can potentially be extended to other pathogens and tissue systems to advance our understanding of host-pathogen interactions and the role of human genetics in influencing the outcome of infections.

IL-10 control of CD11c+ myeloid cells is essential to maintain immune homeostasis in the small and large intestine.

Although IL-10 promotes a regulatory phenotype of CD11c+ dendritic cells and macrophages in vitro, the role of IL-10 signaling in CD11c+ cells to maintain intestinal tolerance in vivo remains elusive. To this aim, we generated mice with a CD11c-specific deletion of the IL-10 receptor alpha (Cd11ccreIl10rafl/fl). In contrast to the colon, the small intestine of Cd11ccreIl10rafl/fl mice exhibited spontaneous crypt hyperplasia, increased numbers of intraepithelial lymphocytes and lamina propria T cells, associated with elevated levels of T cell-derived IFNγ and IL-17A. Whereas naive mucosal T-cell priming was not affected and oral tolerance to ovalbumin was intact, augmented T-cell function in the lamina propria was associated with elevated numbers of locally dividing T cells, expression of T-cell attracting chemokines and reduced T-cell apoptosis. Upon stimulation, intestinal IL-10Rα deficient CD11c+ cells exhibited increased activation associated with enhanced IL-6 and TNFα production. Following colonization with Helicobacter hepaticus Cd11ccreIl10rafl/fl mice developed severe large intestinal inflammation characterized by infiltrating T cells and increased levels of Il17a, Ifng, and Il12p40. Altogether these findings demonstrate a critical role of IL-10 signaling in CD11c+ cells to control small intestinal immune homeostasis by limiting reactivation of local memory T cells and to protect against Helicobacter hepaticus-induced colitis.

Monogenic autoinflammatory diseases: Cytokinopathies.

Rapid advances in genetics are providing unprecedented insight into functions of the innate immune system with identification of the mutations that cause monogenic autoinflammatory disease. Cytokine antagonism is profoundly effective in a subset of these conditions, particularly those associated with increased interleukin-1 (IL-1) activity, the inflammasomopathies. These include syndromes where the production of IL-1 is increased by mutation of innate immune sensors such as NLRP3, upstream signalling molecules such as PSTPIP1 and receptors or downstream signalling molecules, such as IL-1Ra. Another example of this is interferon (IFN) and the interferonopathies, with mutations in the sensors STING and MDA5, the upstream signalling regulator AP1S3, and a downstream inhibitor of IFN signalling, ISG15. We propose that this can be extended to cytokines such as IL-36, with mutations in IL-36Ra, and IL-10, with mutations in IL-10RA and IL-10RB, however mutations in sensors or upstream signalling molecules are yet to be described in these instances. Additionally, autoinflammatory diseases can be caused by multiple cytokines, for example with the activation of NF-κB/Rel, for which we propose the term Relopathies. This nosology is limited in that some cytokine pathways may be degenerate in their generation or execution, however provides insight into likely autoinflammatory disease candidates and the cytokines with which newly identified mutations may be associated, and therefore targeted.

Interleukin-10 receptor-1 expression in monocyte-derived antigen-presenting cell populations: dendritic cells partially escape from IL-10's inhibitory mechanisms.

Interleukin (IL)-10 is an important immunoregulatory cytokine that mediates its effects via a transmembrane receptor complex consisting of two different chains, IL-10R1 and IL-10R2. While IL-10R2 is ubiquitously expressed and does not bind IL-10 primarily, the expression of IL-10R1 determines cellular responsiveness. However, the current knowledge about the expression and regulation of IL-10R1 is still limited. Here we analyzed the expression of IL-10R1 on monocytic cells and demonstrated that human blood monocytes carried about 720 IL-10-binding sites on their surface. Compared with lymphocytes and various tissue cells and tissues, blood monocytes expressed the highest IL-10R1 levels. The in vitro differentiation of these cells into macrophages provoked a further increase of IL-10R1 surface expression. In contrast, their differentiation into myeloid dendritic cells (mDCs) resulted in reduced surface IL-10R1 levels. The different IL-10R1 levels expressed by monocyte-derived antigen-presenting cell populations were reflected in their different responsiveness toward IL-10. Importantly, also in vivo developed immature macrophages and mDCs showed different IL-10 sensitivity. These data suggest that, compared with monocytes and macrophages, mDCs partially escape from IL-10's inhibitory mechanisms by downregulating IL-10R1.

Anti-interleukin-10R1 monoclonal antibody in combination with bacillus Calmette--Guérin is protective against bladder cancer metastasis in a murine orthotopic tumour model and demonstrates systemic specific anti-tumour immunity.

Effective treatment of bladder cancer with bacillus Calmette-Guérin (BCG) depends on the induction of a T helper type (Th) 1 immune response. Interleukin (IL)-10 down-regulates the Th1 response and is associated with BCG failure. In this study, we investigated whether blocking IL-10 signalling could enhance the BCG-induced Th1 response and anti-tumour immunity in a murine orthotopic tumour model. Treatment with BCG and anti-IL-10 receptor 1 monoclonal antibody (anti-IL-10R1 mAb) increased the interferon (IFN)-γ to IL-10 ratio in both splenocyte cultures and urine. Mice bearing luciferase-expressing MB49 (MB49-Luc) tumours were treated and followed for tumour growth by bioluminescent imaging, bladder weight and histology. Mice treated with phosphate-buffered saline (PBS) (group 1), BCG plus control immunoglobulin (Ig)G1 (group 2) or BCG plus anti-IL-10R1 mAb (group 3) showed 0, 6 and 22% tumour regression, respectively. The mean bladder weight of group 3 mice was substantially lower than those of groups 1 and 2 mice. Remarkably, 36% of group 1 and 53% of group 2 mice but no group 3 mice developed lung metastasis (P = 0·02). To investigate the mechanisms underlying the effect of combination therapy, splenocytes were stimulated with S12 peptide (serine mutation at codon 12 of the K-ras oncogene) known to be expressed in MB49-Luc cells. Induction of ras mutation-specific IFN-γ and cytotoxicity was observed in mice treated with combination therapy. These observations indicate that BCG, in combination with anti-IL-10R1 mAb, induces enhanced anti-tumour immunity that is protective against lung metastasis. Anti-IL-10R1 mAb demonstrates systemic effects and may prove useful in clinical practice for treating bladder cancer in high-risk patients.

TLR4, IL10RA, and NOD2 mutation in paediatric Crohn's disease patients: an association with Mycobacterium avium subspecies paratuberculosis and TLR4 and IL10RA expression.

Mycobacterium avium subspecies paratuberculosis (MAP) has been implicated in the pathogenesis of Crohn's disease (CD). The role of CD susceptibility genes in association with these microbes is not known. Sixty-two early onset paediatric CD patients and 46 controls with known MAP status were analysed for an association with 34 single nucleotide polymorphisms (SNPs) from 18 CD susceptibility genes. Functional studies on peripheral blood mononuclear cells (PBMCs) were conducted on 17 CD patients with known CD mutations to assess IL-6, IL-10, and TNF-α expression upon stimulation with MAP precipitated protein derivative (PPD) and lipopolysaccharide (LPS). In addition, surface expression of IL10R and TLR4 on resting B cells, NK cells, T cells, and monocytes was assessed. A mutation in TLR4 (rs4986790) and IL10RA (rs22291130) was significantly associated with MAP-positive CD patients compared to MAP-negative CD patients (27.6 vs. 6.1 %, p = 0.021, and 62.1 vs. 33.3 %, p = 0.024, respectively). PPD and LPS significantly increased IL-6, IL-10, and TNF-α production in PBMCs. IL-10 and TNF-α production were significantly lower in a subgroup of CD patients (5/12) with a known NOD2 mutation. Receptor for IL-10 was significantly higher expressed on NK cells (CD56low) and on NK T cells harbouring a NOD2 mutations compared to wildtype cells (p = 0.031 and 0.005, respectively). TLR4 was significantly higher expressed on NK cells (CD56high) harbouring a NOD2 mutations compared to wildtype cells (p = 0.038).

Anti-interleukin-10R1 monoclonal antibody enhances bacillus Calmette-Guérin induced T-helper type 1 immune responses and antitumor immunity in a mouse orthotopic model of bladder cancer.

PURPOSE: Proper induction of the T-helper type 1 immune response is required for effective bacillus Calmette-Guérin immunotherapy for bladder cancer. Interleukin-10 down-regulates the T-helper 1 response and is associated with bacillus Calmette-Guérin failure. We investigated whether blocking interleukin-10 receptor 1 would enhance the bacillus Calmette-Guérin induced T-helper type 1 immune response and anti-bladder cancer immunity in a mouse model. MATERIALS AND METHODS: Splenocytes were incubated with bacillus Calmette-Guérin or bacillus Calmette-Guérin plus control IgG1, anti-interleukin-10 receptor 1 mAb or anti-interleukin-10 neutralizing mAb, followed by enzyme-linked immunosorbent assay of interferon-γ production. Bladder RNA was extracted after intravesical bacillus Calmette-Guérin plus intraperitoneal IgG1 or anti-interleukin-10 receptor 1 mAb and analyzed by reverse transcriptase and/or quantitative polymerase chain reaction. Urine was collected and analyzed by enzyme-linked immunosorbent assay. Mice bearing a luciferase expressing MB49 orthotopic tumor were treated with intravesical bacillus Calmette-Guérin plus intraperitoneal IgG1 or anti-interleukin-10 receptor 1 mAb. Tumor response was assessed by bioluminescent imaging and bladder weight measurement. RESULTS: Bacillus Calmette-Guérin plus anti-interleukin-10R1 mAb induced significantly higher interferon-γ production by splenocytes than bacillus Calmette-Guérin plus anti-interleukin-10 mAb. Bacillus Calmette-Guérin plus anti-interleukin-10 receptor 1 mAb also induced significantly higher interferon-γ mRNA and protein in bladder and urine, respectively, in a dose dependent manner. Treatment with phosphate buffered saline, bacillus Calmette-Guérin plus control IgG1 and bacillus Calmette-Guérin plus anti-interleukin-10 receptor 1 mAb showed a 0% tumor-free rate with a 20% death rate, a 20% tumor-free rate with a 20% death rate and a 40% tumor-free rate with a 0% death rate, respectively. Bladder weight also revealed the effect of anti-interleukin-10 receptor 1 mAb on the bacillus Calmette-Guérin induced bladder tumor response. CONCLUSIONS: Anti-interleukin-10 receptor 1 mAb enhanced the bacillus Calmette-Guérin induced T-helper type 1 immune response and anti-bladder cancer immunity. A humanized form of this mAb warrants future investigation for bacillus Calmette-Guérin treatment of bladder cancer.

Identification and molecular characterization of the interleukin-10 receptor 1 of the zebrafish (Danio rerio) and the goldfish (Carassius auratus L.).

This is the first report of the identification and molecular characterization of an interleukin-10 receptor 1 in bony fish. By gene synteny analysis, we identified the zebrafish interleukin-10 receptor 1 (IL10R1) and using this IL10R1 sequence, we cloned the goldfish IL10R1 cDNA transcript. The identified fish IL10R1 protein sequences had a putative JAK1 binding site, only one of the two STAT3 binding sites, that are present in all other vertebrates IL10R1 proteins as well as C-terminal serine rich areas, believed to be responsible for the anti-inflammatory properties of IL10R1. Phylogenetically, the fish IL10R1 proteins grouped independently of the amphibian, avian and mammalian IL10R1s. Quantitative gene expression analysis of the IL10R1 of zebrafish and goldfish revealed highest mRNA levels in the spleen tissues. High mRNA levels were also observed in the zebrafish muscle in contrast to low mRNA levels in the muscle of the goldfish. Moderate IL10R1 mRNA levels were seen in most other tissues examined and lowest gene expression was in the liver of both fish species. Goldfish monocytes stimulated with a recombinant goldfish interleukin-10 (rgIL-10) or with heat killed fish pathogens, Aeromonas salmonicida or Trypanosoma carassii, exhibited significantly reduced mRNA levels of the IL10R1. Furthermore, we produced a recombinant form of the goldfish IL10R1 (rgIL10R1) and using in vitro binding studies, demonstrated that the dimerized rgIL-10 specifically interacted with rgIL10R1. Our results suggest that interleukin-10 system has been highly conserved throughout evolution.

Collective action of hematopoietic cell subsets mediates anti-IL10R1 and CpG tumor immunity.

Based on the specificity of antigen recognition and the ability to generate long-lived memory responses, cancer immunotherapies primarily target tumor-associated T cells. Systemic administration of anti-IL-10R1 antibody in combination with local CpG administration has been shown to induce tumor regression in a T-cell-dependent manner. Here, we confirmed the anti-tumor efficacy of anti-IL-10R1 and CpG therapy in the highly aggressive B16F10 melanoma model. However, T cells were not required for tumor growth inhibition. Through cellular depletions and genetic models of leukocyte deficiency, we demonstrated that T, B, and NK cells, and neutrophils are not essential for anti-tumor efficacy. Nevertheless, hematopoietic cells as a whole are required for anti-IL-10R1- and CpG-induced tumor growth inhibition, suggesting that the collective action of multiple subsets of hematopoietic-derived cells is required for anti-tumor efficacy.

cDNA cloning, genomic structure, molecular characterization and mRNA expression analysis of the Pekin duck interleukin-10 receptor 1.

Interleukin-10 (IL-10) mediates its broad anti-inflammatory and immunoregulatory effects through two cell surface receptors by which binding to the IL-10 receptor 1 (IL-10R1) is the initial step that leads to recruitment of IL-10R2 and initiation of the ternary complex signal transduction cascade. The duck IL-10R1 (duIL-10R1) cDNA was obtained by using RT-PCR and 5'RACE. The deduced 574 amino acid protein has an amino acid identity of 62%, 27% and 28% with chicken, mouse and human IL-10R1, respectively. Comparison of the duIL-10R1 cDNA with duck genomic sequences revealed a seven exon-six intron structure of the duck IL-10R1 gene that shares a similar size with the respective exons 1-7 of the chicken and human IL-10R1 genes, but the avian genes are more compact. Promoter analysis identified putative binding sites for regulatory elements such as CCAAT enhancer binding protein-α, specificity protein 1 (Sp1), nuclear factor 1 (NF1), transcriptional regulatory protein Oct-1, nuclear factor (NF) κB and interferon-stimulated gene factor-3 (ISGF-3). A canonical TATA box was absent in proximity of the transcription initiation site, but a CpG island was present. Sequence analysis of the predicted duIL-10R1 protein revealed characteristic features of class-II cytokine receptors (CFR2) family members and a considerable degree of conservation of residues implicated in ligand binding across higher vertebrates. The predicted secondary structure of the duIL-10R1 extracellular domain is compatible with the two-subdomain structure of the human IL-10R1 protein established by its crystal structure. The 3D model structure shows conservation of the positions of conserved contact residues within four of the five ligand-binding loops. Within the cytoplasmic domain, residues implicated in signal transduction were conserved including two redundant peptide motifs GYXXQ essential for recruitment and activation of STAT3. DuIL-10R1 mRNA expression was most abundant in spleen, thymus, peripheral blood mononuclear cells (PBMCs) and lung. Mitogen stimulation of PBMCs transiently increased duIL-10R1 mRNA expression. Our observations suggest significant evolutionary conservation of the IL-10R1 genomic organization, protein structure and receptor function through the JAK/STAT signalling pathway across higher vertebrates.

Design and analysis of rhesus cytomegalovirus IL-10 mutants as a model for novel vaccines against human cytomegalovirus.

BACKGROUND: Human cytomegalovirus (HCMV) expresses a viral ortholog (CMVIL-10) of human cellular interleukin-10 (cIL-10). Despite only ∼26% amino acid sequence identity, CMVIL-10 exhibits comparable immunosuppressive activity with cIL-10, attenuates HCMV antiviral immune responses, and contributes to lifelong persistence within infected hosts. The low sequence identity between CMVIL-10 and cIL-10 suggests vaccination with CMVIL-10 may generate antibodies that specifically neutralize CMVIL-10 biological activity, but not the cellular cytokine, cIL-10. However, immunization with functional CMVIL-10 might be detrimental to the host because of its immunosuppressive properties. METHODS AND FINDINGS: Structural biology was used to engineer biologically inactive mutants of CMVIL-10 that would, upon vaccination, elicit a potent immune response to the wild-type viral cytokine. To test the designed proteins, the mutations were incorporated into the rhesus cytomegalovirus (RhCMV) ortholog of CMVIL-10 (RhCMVIL-10) and used to vaccinate RhCMV-infected rhesus macaques. Immunization with the inactive RhCMVIL-10 mutants stimulated antibodies against wild-type RhCMVIL-10 that neutralized its biological activity, but did not cross-react with rhesus cellular IL-10. CONCLUSION: This study demonstrates an immunization strategy to neutralize RhCMVIL-10 biological activity using non-functional RhCMVIL-10 antigens. The results provide the methodology for targeting CMVIL-10 in vaccine, and therapeutic strategies, to nullify HCMV's ability to (1) skew innate and adaptive immunity, (2) disseminate from the site of primary mucosal infection, and (3) establish a lifelong persistent infection.

Th17 cells express interleukin-10 receptor and are controlled by Foxp3⁻ and Foxp3+ regulatory CD4+ T cells in an interleukin-10-dependent manner.

T helper 17 (Th17) cells are important for host defense against extracellular microorganisms. However, they are also implicated in autoimmune and chronic inflammatory diseases, and as such need to be tightly regulated. The mechanisms that directly control committed pathogenic Th17 cells in vivo remain unclear. We showed here that IL-17A-producing CD4+ T cells expressed interleukin-10 receptor α (IL-10Rα) in vivo. Importantly, T cell-specific blockade of IL-10 signaling led to a selective increase of IL-17A+IFN-γ⁻ (Th17) and IL-17A+IFN-γ+ (Th17+Th1) CD4+ T cells during intestinal inflammation in the small intestine. CD4+Foxp3⁻ IL-10-producing (Tr1) cells and CD4+Foxp3+ regulatory (Treg) cells were able to control Th17 and Th17+Th1 cells in an IL-10-dependent manner in vivo. Lastly, IL-10 treatment of mice with established colitis decreased Th17 and Th17+Th1 cell frequencies via direct signaling in T cells. Thus, IL-10 signaling directly suppresses Th17 and Th17+Th1 cells.

IL-10 within the CNS is necessary for CD4+ T cells to mediate neuroprotection.

We have previously shown that immunodeficient mice exhibit significant facial motoneuron (FMN) loss compared to wild-type (WT) mice after a facial nerve axotomy. Interleukin-10 (IL-10) is known as a regulatory cytokine that plays an important role in maintaining the anti-inflammatory environment within the central nervous system (CNS). IL-10 is produced by a number of different cells, including Th2 cells, and may exert an anti-apoptotic action on neurons directly. In the present study, the role of IL-10 in mediating neuroprotection following facial nerve axotomy in Rag-2- and IL-10-deficient mice was investigated. Results indicate that IL-10 is neuroprotective, but CD4+ T cells are not the requisite source of IL-10. In addition, using real-time PCR analysis of laser microdissected brainstem sections, results show that IL-10 mRNA is constitutively expressed in the facial nucleus and that a transient, significant reduction of IL-10 mRNA occurs following axotomy under immunodeficient conditions. Dual labeling immunofluorescence data show, unexpectedly, that the IL-10 receptor (IL-10R) is constitutively expressed by facial motoneurons, but is selectively induced in astrocytes within the facial nucleus after axotomy. Thus, a non-CD4+ T cell source of IL-10 is necessary for modulating both glial and neuronal events that mediate neuroprotection of injured motoneurons, but only with the cooperation of CD4+ T cells, providing an avenue of novel investigation into therapeutic approaches to prevent or reverse motoneuron diseases, such as amyotrophic lateral sclerosis (ALS).

Human interleukin 10 receptor 1/IgG1-Fc fusion proteins: immunoadhesins for human IL-10 with therapeutic potential.

Interleukin 10 (IL-10) is produced by various types of human cancer, including malignant melanoma, and plays an important role in negative regulation of cell-mediated immune responses against tumors. We have developed chimeric molecules (immunoadhesins), combining the extracellular domain of human interleukin 10 receptor 1 (IL-10R1) with the Fc regions of human IgG1 heavy chain and investigated their capability of blocking the biological activities of human IL-10. Monomeric and dimeric immunoadhesins (IL-10R1/IgG1) constructs were tested for capturing human IL-10 and blocking its biological activities. Plasmid vectors that contained the IL-10 immunoadhesin constructs were directly transfected into human melanoma cell lines. Transfection of plasmid vectors into melanoma cell lines resulted in capturing of exogenously added as well as endogeneously produced IL-10. The supernatants obtained from an IL-10 non-producing melanoma cell line transfected with monomeric IL-10 immunoadhesin plasmids most efficiently captured exogenously added IL-10, compared to those obtained with the dimeric IL-10R1/IgG1 plasmid vector. Transfection of IL-10-producing melanoma cells with the monomeric IL-10 immunoadhesin plasmids totally captured endogenously produced IL-10 and enhanced T cell responses against allogeneic melanoma cells. Furthermore, purified monomeric IL-10 immunoadhesin protein showed IL-10 capturing efficacy compatible with that of IL-10-specific monoclonal antibodies. Collectively, these studies indicate that IL-10 immunoadhesins, especially in monomeric form, are potent inhibitors of biological activities of IL-10 and suggest that these molecules, alone or in conjunctions with other immunotherapeutic approaches, can be utilized for the immuno-targeting of IL-10 producing tumors.

Differential signaling of cmvIL-10 through common variants of the IL-10 receptor 1.

Human IL-10 (hIL-10) signaling is mediated by receptors consisting of two subunits, IL-10 receptor 1 (IL-10R1) and IL-10 receptor 2. Two common variants of the IL-10R1 (Ser 138 Gly (single-nucleotide polymorphism 3, SNP3) and Gly 330 Arg (SNP4)) are associated with diverse disease phenotypes. Viral homologs to hIL-10, such as cmvIL-10, utilize the same IL-10 receptor complex as part of viral immune evasion strategies. For the present study we hypothesized that IL-10R1 variants alter the ability of viral IL-10 to utilize the IL-10R1 signaling pathway. HeLa cell clones expressing different IL-10R1 haplotypes (WT or any variant) were incubated with hIL-10 or cmvIL-10. In cells expressing IL-10R1-WT, cmvIL-10 (both non-glycosylated- and HeLa-expressed) resulted in equal or slightly stronger STAT3 phosphorylation compared with hIL-10. In clones expressing IL-10R1-SNP3, IL-10R1-SNP4 or IL-10R1-SNP3+4, the cmvIL-10 showed significantly less STAT3 phosphorylation, especially when HeLa-expressed cytokines were used. Time course experiments demonstrated a slower kinetic of cmvIL-10 STAT3 activation through the variant IL-10R1. Similarly, IL-10R1 variants decreased the cmvIL-10-induced SOCS3 and signaling lymphocytic activation molecule mRNA expression. These data suggest that the IL-10R1 variants differentially reduce the signaling activity of cmvIL-10 and thereby may affect CMV's ability to escape from the host's immune surveillance.

Tourette's syndrome is not associated with interleukin-10 receptor 1 variants on chromosome 11q23.3.

Interleukin-10 receptor 1 (IL-10R1) single nucleotide polymorphisms, located on chromosome 11q23 - a strong candidate for linkage with Tourette's syndrome (TS) - have been investigated for association with TS. DNA of 77 patients with a DSM-IV (Diagnostic and Statistical Manual IV) diagnosis of TS and 250 healthy controls was genotyped. IL-10R1 was not associated with TS.