Disease severity in K/BxN serum transfer-induced arthritis is not affected by IL-33 deficiency

Introduction

Interleukin (IL)-33 is a cytokine of the IL-1 family, which signals through the ST2 receptor. Previous work suggested implication of the IL-33/ST2 axis in the pathogenesis of human and mouse arthritis. Here, we directly investigated the role of endogenous IL-33 in K/BxN serum transfer-induced arthritis by using IL-33 knockout (KO) mice.

Methods

Arthritis was induced by injection of complete K/BxN serum or purified IgG. Disease severity was monitored by clinical and histological scoring.

Results

K/BxN serum transfer induced pronounced arthritis with similar incidence and severity in IL-33 KO and wild-type (WT) mice. In contrast, disease development was significantly reduced in ST2 KO mice. IL-33 expression in synovial tissue was comparable in arthritic WT and ST2 KO mice, and absent in IL-33 KO mice. Transfer of purified arthritogenic IgG instead of complete K/BxN serum also resulted in similar arthritis severity in IL-33 KO and WT mice, excluding a contribution of IL-33 contained in the serum of donor mice to explain this result. We investigated additional potential confounding factors, including purity of genetic background, but the mechanisms underlying reduced arthritis in ST2 KO mice remained unclear.

Conclusions

The data obtained with IL-33 KO mice indicate that endogenous IL-33 is not required for the development of joint inflammation in K/BxN serum transfer-induced arthritis. On the contrary, arthritis severity was reduced in ST2 KO mice. This observation might relate to IL-33 independent effects of ST2, and/or reveal the existence of confounding variables affecting the severity of joint inflammation in these KO strains.     

Joint production of IL-22 participates in the initial phase of antigen-induced arthritis through IL-1β production

IntroductionRheumatoid arthritis (RA) is a chronic autoimmune disease characterized by neutrophil articular infiltration, joint pain and the progressive destruction of cartilage and bone. IL-22 is a key effector molecule that plays a critical role in autoimmune diseases. However, the function of IL-22 in the pathogenesis of RA remains controversial. In this study, we investigated the role of IL-22 in the early phase of antigen-induced arthritis (AIA) in mice.MethodsAIA was induced in C57BL/6, IL-22^−/−, ASC^−/− and IL-1R1^−/− immunized mice challenged intra-articularly with methylated bovine serum albumin (mBSA). Expression of IL-22 in synovial membranes was determined by RT-PCR. Articular hypernociception was evaluated using an electronic von Frey. Neutrophil recruitment and histopathological analyses were assessed in inflamed knee joint. Joint levels of inflammatory mediators and mBSA-specific IgG concentration in the serum were measured by ELISA.ResultsThe IL-22 mRNA expression and protein levels in synovial tissue were increased during the onset of AIA. In addition, pharmacological inhibition (anti-IL-22 antibody) and genetic deficiency (IL-22^−/− mice) reduced articular pain and neutrophil migration in arthritic mice. Consistent with these findings, recombinant IL-22 joint administration promoted articular inflammation per se in WT mice, restoring joint nociception and neutrophil infiltration in IL-22^−/− mice. Moreover, IL-22-deficient mice showed reduced synovitis (inflammatory cell influx) and lower joint IL-1β levels, whereas the production of IL-17, MCP-1/CCL2, and KC/CXCL1 and the humoral immune response were similar, compared with WT mice. Corroborating these results, the exogenous administration of IL-22 into the joints induced IL-1β production in WT mice and reestablished IL-1β production in IL-22^−/− mice challenged with mBSA. Additionally, IL-1R1^−/− mice showed attenuated inflammatory features induced by mBSA or IL-22 challenge. Articular nociception and neutrophil migration induced by IL-22 were also reduced in ASC^−/− mice.ConclusionsThese results suggest that IL-22 plays a pro-inflammatory/pathogenic role in the onset of AIA through an ASC-dependent stimulation of IL-1β production.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0759-2) contains supplementary material, which is available to authorized users.     

Interleukin-10 attenuation of collagen-induced arthritis is associated with suppression of interleukin-17 and retinoid-related orphan receptor γt production in macrophages and repression of classically activated macrophages

Introduction

Our objective in the present study was to determine the signaling pathway of interleukin 10 (IL-10) for modulating IL-17 expression in macrophages and the importance of this mediation in collagen-induced arthritis (CIA).

Methods

IL-10-knockout (IL-10^−/−) mice and wild-type (WT) mice were immunized with chicken type II collagen (CII) to induce arthritis. The expression levels of IL-17 and retinoid-related orphan receptor γt (RORγt) in macrophages and joint tissues of IL-10^−/− and WT mice were analyzed by enzyme-linked immunosorbent assay, quantitative RT-PCR (qRT-PCR) and Western blotting. The F4/80 macrophages and positive IL-17-producing macrophages in synovial tissues of the mice were determined by immunohistochemistry. The populations of classically activated macrophage (M1) and alternatively activated macrophage (M2) phenotypes were analyzed by flow cytometry. The expression of genes associated with M1 and M2 markers was analyzed by qRT-PCR.

Results

Compared to WT mice, IL-10^−/− mice had exacerbated CIA development, which was associated with increased production of T helper 17 cell (Th17)/Th1 proinflammatory cytokines and CII-specific immunoglobulin G2a antibody after CII immunization. Macrophages in IL-10^−/− mice had increased amounts of IL-17 and RORγt compared with the amounts in WT mice with CIA. Immunofluorescence microscopy showed that the number of IL-17-producing macrophages in synovial tissues was significantly higher in IL-10^−/− mice than in WT mice. IL-10 deficiency might promote macrophage polarization toward the proinflammatory M1 phenotype, which contributes to the rheumatoid arthritis inflammation response.

Conclusion

IL-10 inhibits IL-17 and RORγt expression in macrophages and suppresses macrophages toward the proinflammatory M1 phenotype, which is important for the role of IL-10 in mediating the pathogenesis of CIA.     

Synovial Fibroblasts Promote the Expression and Granule Accumulation of Tryptase via Interleukin-33 and Its Receptor ST-2 (IL1RL1)

A characteristic feature of tissue resident human mast cells (MCs) is their hTryptase-β-rich cytoplasmic granules. Mouse MC protease-6 (mMCP-6) is the ortholog of hTryptase-β, and we have shown that this tetramer-forming tryptase has beneficial roles in innate immunity but adverse roles in inflammatory disorders like experimental arthritis. Because the key tissue factors that control tryptase expression in MCs have not been identified, we investigated the mechanisms by which fibroblasts mediate the expression and granule accumulation of mMCP-6. Immature mouse bone marrow-derived MCs (mBMMCs) co-cultured with fibroblast-like synoviocytes (FLS) or mouse 3T3 fibroblasts markedly increased their levels of mMCP-6. This effect was caused by an undefined soluble factor whose levels could be increased by exposing FLS to tumor necrosis factor-α or interleukin (IL)-1β. Gene expression profiling of mBMMCs and FLS for receptor·ligand pairs of potential relevance raised the possibility that IL-33 was a sought after fibroblast-derived factor that promotes tryptase expression and granule maturation via its receptor IL1RL1/ST2. MCs lacking IL1RL1 exhibited defective fibroblast-driven tryptase accumulation, whereas recombinant IL-33 induced mMCP-6 mRNA and protein accumulation in wild-type mBMMCs. In agreement with these data, synovial MCs from IL1RL1-null mice exhibited a marked reduction in mMCP-6 expression. IL-33 is the first factor shown to modulate tryptase expression in MCs at the mRNA and protein levels. We therefore have identified a novel pathway by which mesenchymal cells exposed to inflammatory cytokines modulate the phenotype of local MCs to shape their immune responses.     

A Balanced IL-1β Activity Is Required for Host Response to Citrobacter rodentium Infection

Microbial sensing plays essential roles in the innate immune response to pathogens. In particular, NLRP3 forms a multiprotein inflammasome complex responsible for the maturation of interleukin (IL)-1β. Our aim was to delineate the role of the NLRP3 inflammasome in macrophages, and the contribution of IL-1β to the host defense against Citrobacter rodentium acute infection in mice. Nlrp3^−/− and background C57BL/6 (WT) mice were infected by orogastric gavage, received IL-1β (0.5 µg/mouse; ip) on 0, 2, and 4 days post-infection (DPI), and assessed on 6 and 10 DPI. Infected Nlrp3^−/− mice developed severe colitis; IL-1β treatments reduced colonization, abrogated dissemination of bacteria to mesenteric lymph nodes, and protected epithelial integrity of infected Nlrp3^−/− mice. In contrast, IL-1β treatments of WT mice had an opposite effect with increased penetration of bacteria and barrier disruption. Microscopy showed reduced damage in Nlrp3^−/− mice, and increased severity of disease in WT mice with IL-1β treatments, in particular on 10 DPI. Secretion of some pro-inflammatory plasma cytokines was dissipated in Nlrp3^−/− compared to WT mice. IL-1β treatments elevated macrophage infiltration into infected crypts in Nlrp3^−/− mice, suggesting that IL-1β may improve macrophage function, as exogenous administration of IL-1β increased phagocytosis of C. rodentium by peritoneal Nlrp3^−/− macrophages in vitro. As well, the exogenous administration of IL-1β to WT peritoneal macrophages damaged the epithelial barrier of C. rodentium-infected polarized CMT-93 cells. Treatment of Nlrp3^−/− mice with IL-1β seems to confer protection against C. rodentium infection by reducing colonization, protecting epithelial integrity, and improving macrophage activity, while extraneous IL-1β appeared to be detrimental to WT mice. Together, these findings highlight the importance of balanced cytokine responses as IL-1β improved bacterial clearance in Nlrp3^−/− mice but increased tissue damage when given to WT mice.     

Deletion of IL-33R (ST2) Abrogates Resistance to EAE in BALB/C Mice by Enhancing Polarization of APC to Inflammatory Phenotype

The administration of interleukin 33 and deletion of IL-33 receptor, ST2 molecule, affects the induction of autoimmunity in different experimental models of human autoimmune diseases. The aim of this study was to analyze the effect of ST2 deletion on the induction of experimental autoimmune encephalomyelitis (EAE) in resistant BALB/c mice. Mice were immunized with MOG_35–55 peptide or disease was induced by passive transfer of encephalitogenic singenic cells and EAE was clinically and histologically evaluated. Expression of intracellular inflammatory cytokines, markers of activation and chemokine receptors on lymphoid tissue and CNS infiltrating mononuclear cells was analyzed by flow cytometry. We report here that deletion of ST2^−/− molecule abrogates resistance of BALB/c mice to EAE induction based on clinical and histopathological findings. Brain and spinal cord infiltrates of ST2^−/− mice had significantly higher number of CD4⁺ T lymphocytes containing inflammatory cytokines compared to BALB/c WT mice. Adoptive transfer of ST2^−/− primed lymphocytes induced clinical signs of the disease in ST2^−/− as well as in WT mice. MOG_35–55 restimulated ST2^−/− CD4⁺ cells as well as ex vivo analyzed lymph node cells had higher expression of T-bet and IL-17, IFN-γ, TNF-α and GM-CSF in comparison with WT CD4⁺ cells. ST2^−/− mice had higher percentages of CD4⁺ cells expressing chemokine receptors important for migration to CNS in comparison with WT CD4⁺ cells. Draining lymph nodes of ST2^−/− mice contained higher percentage of CD11c⁺CD11b⁺CD8⁻ cells containing inflammatory cytokines IL-6 and IL-12 with higher expression of activation markers. Transfer of ST2^−/− but not WT dendritic cells induced EAE in MOG_35–55 immunized WT mice. Our results indicate that ST2 deficiency attenuates inherent resistance of BALB/c mice to EAE induction by enhancing differentiation of proinflammatory antigen presenting cells and consecutive differentiation of encephalitogenic T cells in the draining lymph node rather than affecting their action in the target tissue.     

Liposomal Targeting of Prednisolone Phosphate to Synovial Lining Macrophages during Experimental Arthritis Inhibits M1 Activation but Does Not Favor M2 Differentiation

Background

To determine the effects of liposomal targeting of prednisolone phosphate (Lip-PLP) to synovial lining macrophages on M1 and M2 polarization in vitro and during experimental arthritis.

Material and Methods

Experimental arthritis (antigen and immune complex induced) was elicited in mice and prednisolone containing liposomes were given systemically. Synovium was investigated using microarray analysis, RT-PCR and histology. Bone–marrow macrophages were stimulated towards M1 using LPS and IFNγ before treatment by PLP-liposomes. M1 and M2 markers were determined using RT-PCR.

Results

Microarray analysis of biopsies of inflamed synovium during antigen induced arthritis (AIA) showed an increased M1 signature characterized by upregulation of IL-1β, IL-6 and FcγRI starting from day 1 and lasting up until day 7 after arthritis induction. The M2 signature remained low throughout the 7 day course of arthritis. Treatment of AIA with intravenously delivered Lip-PLP strongly suppressed joint swelling and synovial infiltration whereas colloidal gold containing liposomes exclusively targeted the macrophages within the inflamed synovial intima layer. In vitro studies showed that Lip-PLP phagocytosed by M1 macrophages resulted in a suppression of the M1 phenotype and induction of M2 markers (IL-10, TGF-β, IL-1RII, CD163, CD206 and Ym1). In vivo, Lip-PLP treatment strongly suppressed M1 markers (TNF-α, IL-1β, IL-6, IL-12p40, iNOS, FcγRI, Ciita and CD86) after local M1 activation of lining macrophages with LPS and IFN-γ and during experimental AIA and immune complex arthritis (ICA). In contrast, M2 markers were not significantly upregulated in antigen-induced arthritis and down regulated in immune complex arthritis.

Conclusion

This study clearly shows that systemic treatment with PLP-liposomes selectively targets synovial lining macrophages and inhibits M1 activation. In contrast to in vitro findings, PLP-liposomes do not cause a shift of synovial lining macrophages towards M2.     

Hypoxia-Inducible Factor-1α and Interleukin 33 Form a Regulatory Circuit to Perpetuate the Inflammation in Rheumatoid Arthritis

Hyperplasia of synovial fibroblasts, infiltration with inflammatory cytokines, and tissue hypoxia are the major characteristics of rheumatoid arthritis (RA). Interleukin 33 (IL-33) is a newly identified inflammatory cytokine exacerbating the disease severity of RA. Hypoxia-inducible factor-1α (HIF-1α) showed increased expression in RA synovium and could regulate a number of inflammatory cytokine productions. Nevertheless, its correlation with IL-33 remains largely unknown. Here, we showed that elevated levels of IL-33 were demonstrated in RA patient synovial fluids, with upregulated expression of HIF-1α and IL-33 in the synovial fibroblasts. Knocking down HIF-1α compromised IL-33 expression in rheumatoid arthritis synovial fibroblasts (RASF), while enforcing HIF-1α expression in RASF substantially upregulated IL-33 levels. HIF-1α promoted the activation of the signalling pathways controlling IL-33 production, particularly the p38 and ERK pathways. Moreover, we showed for the first time that IL-33 in turn could induce more HIF-1α expression in RASF, thus forming a HIF-1α/IL-33 regulatory circuit that would perpetuate the inflammatory process in RA. Targeting this pathological pathway and HIF-1α may provide new therapeutic strategies for overcoming the persistent and chronic inflammatory disease.     

Caspase-1/ASC Inflammasome-Mediated Activation of IL-1β–ROS–NF-κB Pathway for Control of Trypanosoma cruzi Replication and Survival Is Dispensable in NLRP3?/? Macrophages

In this study, we have utilized wild-type (WT), ASC^−/−, and NLRP3^−/− macrophages and inhibition approaches to investigate the mechanisms of inflammasome activation and their role in Trypanosoma cruzi infection. We also probed human macrophages and analyzed published microarray datasets from human fibroblasts, and endothelial and smooth muscle cells for T. cruzi-induced changes in the expression genes included in the RT Profiler Human Inflammasome arrays. T. cruzi infection elicited a subdued and delayed activation of inflammasome-related gene expression and IL-1β production in mφs in comparison to LPS-treated controls. When WT and ASC^−/− macrophages were treated with inhibitors of caspase-1, IL-1β, or NADPH oxidase, we found that IL-1β production by caspase-1/ASC inflammasome required reactive oxygen species (ROS) as a secondary signal. Moreover, IL-1β regulated NF-κB signaling of inflammatory cytokine gene expression and, subsequently, intracellular parasite replication in macrophages. NLRP3^−/− macrophages, despite an inability to elicit IL-1β activation and inflammatory cytokine gene expression, exhibited a 4-fold decline in intracellular parasites in comparison to that noted in matched WT controls. NLRP3^−/− macrophages were not refractory to T. cruzi, and instead exhibited a very high basal level of ROS (>100-fold higher than WT controls) that was maintained after infection in an IL-1β-independent manner and contributed to efficient parasite killing. We conclude that caspase-1/ASC inflammasomes play a significant role in the activation of IL-1β/ROS and NF-κB signaling of cytokine gene expression for T. cruzi control in human and mouse macrophages. However, NLRP3-mediated IL-1β/NFκB activation is dispensable and compensated for by ROS-mediated control of T. cruzi replication and survival in macrophages.     

Cross-Talk between TLR4 and FcγReceptorIII (CD16) Pathways

Pathogen-pattern-recognition by Toll-like receptors (TLRs) and pathogen clearance after immune complex formation via engagement with Fc receptors (FcRs) represent central mechanisms that trigger the immune and inflammatory responses. In the present study, a linkage between TLR4 and FcγR was evaluated in vitro and in vivo. Most strikingly, in vitro activation of phagocytes by IgG immune complexes (IgGIC) resulted in an association of TLR4 with FcγRIII (CD16) based on co-immunoprecipitation analyses. Neutrophils and macrophages from TLR4 mutant (mut) mice were unresponsive to either lipopolysaccharide (LPS) or IgGIC in vitro, as determined by cytokine production. This phenomenon was accompanied by the inability to phosphorylate tyrosine residues within immunoreceptor tyrosine-based activation motifs (ITAMs) of the FcRγ-subunit. To transfer these findings in vivo, two different models of acute lung injury (ALI) induced by intratracheal administration of either LPS or IgGIC were employed. As expected, LPS-induced ALI was abolished in TLR4 mut and TLR4^−/− mice. Unexpectedly, TLR4 mut and TLR4^−/− mice were also resistant to development of ALI following IgGIC deposition in the lungs. In conclusion, our findings suggest that TLR4 and FcγRIII pathways are structurally and functionally connected at the receptor level and that TLR4 is indispensable for FcγRIII signaling via FcRγ-subunit activation.     

IL-17RA signaling amplifies antibody-induced arthritis

Objective

To investigate the role of IL-17RA signaling in the effector phase of inflammatory arthritis using the K/BxN serum-transfer model.

Methods

Wild-type and Il17ra^−/− mice were injected with serum isolated from arthritic K/BxN mice and their clinical score was recorded daily. Mice were also harvested on days 12 and 21 and ankles were analyzed for cytokine and chemokine mRNA expression by qPCR on day 12 and for bone and cartilage erosions by histology on day 21, respectively. The induction of cytokine and chemokine expression levels by IL-17A in synovial-like fibroblasts was also analyzed using qPCR.

Results

Il17ra^−/− mice were partially protected from clinical signs of arthritis and had markedly fewer cartilage and bone erosions. The expression of several pro-inflammatory mediators, including the chemokines KC/CXCL1, MIP-2/CXCL2, LIX/CXCL5 MIP-1γ/CCL9, MCP-3/CCL7, MIP-3α/CCL20, the cytokines IL-1β, IL-6, RANKL and the matrix metalloproteinases MMP2, MMP3, and MMP13 were decreased in the ankles of Il17ra^−/− mice compared to wild-type mice. Many of these proinflammatory genes attenuated in the ankles of Il17ra^−/− mice were shown to be directly induced by IL-17A in synovial fibroblasts in vitro.

Conclusions

IL-17RA signaling plays a role as an amplifier of the effector phase of inflammatory arthritis. This effect is likely mediated by direct activation of synovial fibroblasts by IL-17RA to produce multiple inflammatory mediators, including chemokines active on neutrophils. Therefore, interrupting IL-17RA signaling maybe a promising pharmacological target for the treatment of inflammatory arthritis.     

CD55 deposited on synovial collagen fibers protects from immune complex-mediated arthritis

IntroductionCD55, a glycosylphosphatidylinositol-anchored, complement-regulating protein (decay-accelerating factor), is expressed by fibroblast-like synoviocytes (FLS) with high local abundance in the intimal lining layer. We here explored the basis and consequences of this uncommon presence.MethodsSynovial tissue, primary FLS cultures, and three-dimensional FLS micromasses were analyzed. CD55 expression was assessed by quantitative polymerase chain reaction (PCR), in situ hybridization, flow cytometry, and immunohistochemistry. Reticular fibers were visualized by Gomori staining and colocalization of CD55 with extracellular matrix (ECM) proteins by confocal microscopy. Membrane-bound CD55 was released from synovial tissue with phospholipase C. Functional consequences of CD55 expression were studied in the K/BxN serum transfer model of arthritis using mice that in addition to CD55 also lack FcγRIIB (CD32), increasing susceptibility for immune complex-mediated pathology.ResultsAbundant CD55 expression seen in FLS of the intimal lining layer was associated with linearly oriented reticular fibers and was resistant to phospholipase C treatment. Expression of CD55 colocalized with collagen type I and III as well as with complement C3. A comparable distribution of CD55 was established in three-dimensional micromasses after ≥3 weeks of culture together with the ECM. CD55 deficiency did not enhance K/BxN serum-induced arthritis, but further exaggerated disease activity in Fcgr2b^−/− mice.ConclusionsCD55 is produced by FLS and deposited on the local collagen fiber meshwork, where it protects the synovial tissue against immune complex-mediated arthritis.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0518-4) contains supplementary material, which is available to authorized users.     

In Vivo Immune Evasion Mediated by the Herpes Simplex Virus Type 1 Immunoglobulin G Fc Receptor

Herpes simplex virus (HSV) glycoproteins gE and gI form an immunoglobulin G (IgG) Fc receptor (FcγR) that binds the Fc domain of human anti-HSV IgG and inhibits Fc-mediated immune functions in vitro. gE or gI deletion mutant viruses are avirulent, probably because gE and gI are also involved in cell-to-cell spread. In an effort to modify FcγR activity without affecting other gE functions, we constructed a mutant virus, NS-gE₃₃₉, that has four amino acids inserted into gE within the domain homologous to mammalian IgG FcγRs. NS-gE₃₃₉ expresses gE and gI, is FcγR⁻, and does not participate in antibody bipolar bridging since it does not block activities mediated by the Fc domain of anti-HSV IgG. In vivo studies were performed with mice because the HSV-1 FcγR does not bind murine IgG; therefore, the absence of an FcγR should not affect virulence in mice. NS-gE₃₃₉ causes disease at the skin inoculation site comparably to wild-type and rescued viruses, indicating that the FcγR⁻ mutant virus is pathogenic in animals. Mice were passively immunized with human anti-HSV IgG and then infected with mutant or wild-type virus. We postulated that the HSV-1 FcγR should protect wild-type virus from antibody attack. Human anti-HSV IgG greatly reduced viral titers and disease severity in NS-gE₃₃₉-infected animals while having little effect on wild-type or rescued virus. We conclude that the HSV-1 FcγR enables the virus to evade antibody attack in vivo, which likely explains why antibodies are relatively ineffective against HSV infection.     

Inhibition of Inflammatory Arthritis Using Fullerene Nanomaterials

Inflammatory arthritis (e.g. rheumatoid arthritis; RA) is a complex disease driven by the interplay of multiple cellular lineages. Fullerene derivatives have previously been shown to have anti-inflammatory capabilities mediated, in part, by their ability to prevent inflammatory mediator release by mast cells (MC). Recognizing that MC can serve as a cellular link between autoantibodies, soluble mediators, and other effector populations in inflammatory arthritis, it was hypothesized that fullerene derivatives might be used to target this inflammatory disease. A panel of fullerene derivatives was tested for their ability to affect the function of human skin-derived MC as well as other lineages implicated in arthritis, synovial fibroblasts and osteoclasts. It is shown that certain fullerene derivatives blocked FcγR- and TNF-α-induced mediator release from MC; TNF-α-induced mediator release from RA synovial fibroblasts; and maturation of human osteoclasts. MC inhibition by fullerene derivatives was mediated through the reduction of mitochondrial membrane potential and FcγR-mediated increases in cellular reactive oxygen species and NF-κB activation. Based on these in vitro data, two fullerene derivatives (ALM and TGA) were selected for in vivo studies using K/BxN serum transfer arthritis in C57BL/6 mice and collagen-induced arthritis (CIA) in DBA/1 mice. Dye-conjugated fullerenes confirmed localization to affected joints in arthritic animals but not in healthy controls. In the K/BxN moldel, fullerenes attenuated arthritis, an effect accompanied by reduced histologic inflammation, cartilage/bone erosion, and serum levels of TNF-α. Fullerenes remained capable of attenuating K/BxN arthritis in mast cell-deficient mice Cre-Master mice, suggesting that lineages beyond the MC represent relevant targets in this system. These studies suggest that fullerene derivatives may hold promise both as an assessment tool and as anti-inflammatory therapy of arthritis.     

Limited Anti-Inflammatory Role for Interleukin-1 Receptor Like 1 (ST2) in the Host Response to Murine Postinfluenza Pneumococcal Pneumonia

Interleukin-1 receptor like 1 (ST2) is a negative regulator of Toll-like receptor (TLR) signaling. TLRs are important for host defense during respiratory tract infections by both influenza and Streptococcus (S.) pneumoniae. Enhanced susceptibility to pneumococcal pneumonia is an important complication following influenza virus infection. We here sought to determine the role of ST2 in primary influenza A infection and secondary pneumococcal pneumonia. ST2 knockout (st2 ^−/−) and wild-type (WT) mice were intranasally infected with influenza A virus; in some experiments mice were infected 2 weeks later with S. pneumoniae. Both mouse strains cleared the virus similarly during the first 14 days of influenza infection and had recovered their weights equally at day 14. Overall st2^−/− mice tended to have a stronger pulmonary inflammatory response upon infection with influenza; especially 14 days after infection modest but statistically significant elevations were seen in lung IL-6, IL-1β, KC, IL-10, and IL-33 concentrations and myeloperoxidase levels, indicative of enhanced neutrophil activity. Interestingly, bacterial lung loads were higher in st2^−/− mice during the later stages of secondary pneumococcal pneumonia, which was associated with relatively increased lung IFN-γ levels. ST2 deficiency did not impact on gross lung pathology in either influenza or secondary S. pneumoniae pneumonia. These data show that ST2 plays a limited anti-inflammatory role during both primary influenza and postinfluenza pneumococcal pneumonia.     

Galectin-3 Ablation Enhances Liver Steatosis,but Attenuates Inflammation and IL-33-Dependent Fibrosis in Obesogenic Mouse Model of Nonalcoholic Steatohepatitis

The importance of Galectin-3 (Gal-3) in obesity-associated liver pathology is incompletely defined. To dissect the role of Gal-3 in fibrotic nonalcoholic steatohepatitis (NASH), Gal-3-deficient (LGALS3^−/−) and wild-type (LGALS3^+/+) C57Bl/6 mice were placed on an obesogenic high fat diet (HFD, 60% kcal fat) or standard chow diet for 12 and 24 wks. Compared to WT mice, HFD-fed LGALS3^−/− mice developed, in addition to increased visceral adiposity and diabetes, marked liver steatosis, which was accompanied with higher expression of hepatic PPAR-γ, Cd36, Abca-1 and FAS. However, as opposed to LGALS3^−/− mice, hepatocellular damage, inflammation and fibrosis were more extensive in WT mice which had an elevated number of mature myeloid dendritic cells, proinflammatory CD11b⁺Ly6C^hi monocytes/macrophages in liver, peripheral blood and bone marrow, and increased hepatic CCL2, F4/80, CD11c, TLR4, CD14, NLRP3 inflammasome, IL-1β and NADPH-oxidase enzymes mRNA expression. Thus, obesity-driven greater steatosis was uncoupled with attenuated fibrotic NASH in Gal-3-deficient mice. HFD-fed WT mice had a higher number of hepatocytes that strongly expressed IL-33 and hepatic CD11b⁺IL-13⁺ cells, increased levels of IL-33 and IL-13 and up-regulated IL-33, ST2 and IL-13 mRNA in liver compared with LGALS3^−/− mice. IL-33 failed to induce ST2 upregulation and IL-13 production by LGALS3^−/− peritoneal macrophages in vitro. Administration of IL-33 in vivo enhanced liver fibrosis in HFD-fed mice in both genotypes, albeit to a significantly lower extent in LGALS3^−/− mice, which was associated with less numerous hepatic IL-13-expressing CD11b⁺ cells. The present study provides evidence of a novel role for Gal-3 in regulating IL-33-dependent liver fibrosis.     

FcγR-driven Release of IL-6 by Macrophages Requires NOX2-dependent Production of Reactive Oxygen Species

Activation of the FcγR via antigen containing immune complexes can lead to the generation of reactive oxygen species, which are potent signal transducing molecules. However, whether ROS contribute to FcγR signaling has not been studied extensively. We set out to elucidate the role of NADPH oxidase-generated ROS in macrophage activation following FcγR engagement using antigen-containing immune complexes. We hypothesized that NOX2 generated ROS is necessary for propagation of downstream FcγR signaling and initiation of the innate immune response. Following exposure of murine bone marrow-derived macrophages (BMDMs) to inactivated Francisella tularensis (iFt)-containing immune complexes, we observed a significant increase in the innate inflammatory cytokine IL-6 at 24 h compared with macrophages treated with Ft LVS-containing immune complexes. Ligation of the FcγR by opsonized Ft also results in significant ROS production. Macrophages lacking the gp91^phox subunit of NOX2 fail to produce ROS upon FcγR ligation, resulting in decreased Akt phosphorylation and a reduction in the levels of IL-6 compared with wild type macrophages. Similar results were seen following infection of BMDMs with catalase deficient Ft that fail to scavenge hydrogen peroxide. In conclusion, our findings demonstrate that ROS participate in elicitation of an effective innate immune in response to antigen-containing immune complexes through FcγR.     

Optimal Attenuation of Experimental Autoimmune Encephalomyelitis by Intravenous Immunoglobulin Requires an Intact Interleukin-11 Receptor

Background

Intravenous immunoglobulin (IVIg) has been used to treat a variety of autoimmune disorders including multiple sclerosis (MS); however its mechanism of action remains elusive. Recent work has shown that interleukin-11 (IL-11) mRNAs are upregulated by IVIg in MS patient T cells. Both IVIg and IL-11 have been shown to ameliorate experimental autoimmune encephalomyelitis (EAE), an animal model of MS. The objective of this study was to determine whether the protective effects of IVIg in EAE occur through an IL-11 and IL-11 receptor (IL-11R)-dependent mechanism.

Methods

We measured IL-11 in the circulation of mice and IL-11 mRNA expression in various organs after IVIg treatment. We then followed with EAE studies to test the efficacy of IVIg in wild-type (WT) mice and in mice deficient for the IL-11 receptor (IL-11Rα^−/−). Furthermore, we evaluated myelin-specific Th1 and Th17 responses and assessed spinal cord inflammation and demyelination in WT and IL-11Rα^−/− mice, with and without IVIg treatment. We also examined the direct effects of mouse recombinant IL-11 on the production of IL-17 by lymph node mononuclear cells.

Results

IVIg treatment induced a dramatic surge (>1000-fold increase) in the levels of IL-11 in the circulation and a prominent increase of IL-11 mRNA expression in the liver. Furthermore, we found that IL-11Rα^−/− mice, unlike WT mice, although initially protected, were resistant to full protection by IVIg during EAE and developed disease with a similar incidence and severity as control-treated IL-11Rα^−/− mice, despite initially showing protection. We observed that Th17 cytokine production by myelin-reactive T cells in the draining lymph nodes was unaffected by IVIg in IL-11Rα^−/− mice, yet was downregulated in WT mice. Finally, IL-11 was shown to directly inhibit IL-17 production of lymph node cells in culture.

Conclusion

These results implicate IL-11 as an important immune effector of IVIg in the prevention of Th17-mediated autoimmune inflammation during EAE.