Interleukin-1 Receptor Antagonist Knockout Research Articles

IL-6 signaling drives self-renewal and alternative activation of adipose tissue macrophages.

Obesity is associated with chronic low-grade inflammation of adipose tissue (AT) and an increase of AT macrophages (ATMs) that is linked to the onset of type 2 diabetes. We have recently shown that neutralization of interleukin (IL)-6 in obese AT organ cultures inhibits proliferation of ATMs, which occurs preferentially in alternatively activated macrophage phenotype. In this study, we investigated AT biology and the metabolic phenotype of mice with myeloid cell-specific IL-6Rα deficiency (Il6ra Δmyel) after normal chow and 20 weeks of high-fat diet focusing on AT inflammation, ATM polarization and proliferation. Using organotypical AT culture and bone marrow derived macrophages (BMDMs) of IL-4Rα knockout mice (Il4ra -/-) we studied IL-6 signaling. Obese Il6ra Δmyel mice exhibited no differences in insulin sensitivity or histological markers of AT inflammation. Notably, we found a reduction of ATMs expressing the mannose receptor 1 (CD206), as well as a decrease of the proliferation marker Ki67 in ATMs of Il6ra Δmyel mice. Importantly, organotypical AT culture and BMDM data of Il4ra -/- mice revealed that IL-6 mediates a shift towards the M2 phenotype independent from the IL-6/IL-4Rα axis. Our results demonstrate IL-4Rα-independent anti-inflammatory effects of IL-6 on macrophages and the ability of IL-6 to maintain proliferation rates in obese AT.

Dendritic epidermal T cells (DETC) are not required for control of skin Staph infections

Abstract Staphylococcal bacteria are a common cause of skin infections in mice and humans. γδ T cells and the cytokine IL-17 are important in controlling these infections. To test roles for IL-17 in autoimmune Ets1 knockout (KO) mice, we generated double knockout (DKO) mice lacking both Ets1 and IL-17RA. We found that DKO mice develop worse autoimmune disease than Ets1 KO mice and are also susceptible to spontaneous Staphylococcus xylosus skin infections. While IL-17RA KO mice also develop skin lesions, these lesions are found at a higher rate and increased severity in DKO mice. Furthermore, exogenous infection of DKO skin with bioluminescent Staphylococcus aureus results in a failure to clear the bacteria, while IL-17RA KO mice are able to clear. Given that γδ T cells are important in controlling Staph skin infections, we examined these populations and found that both Ets1 KO and DKO mice lack the dendritic epidermal T cells (DETC), while other γδ T cell subsets are present. Further analysis showed that DETC progenitors are present in the fetal thymus of Ets1 KO mice, but DETC are missing from the skin of P5 and adult mice lacking Ets1. We hypothesized that loss of DETC (due to absence of Ets1) might cooperate with loss of IL-17 signaling (due to loss of IL-17RA) to create susceptibility to skin infections. To determine if DETC have a specific contribution to the skin immune response to Staphylococcal bacteria, we depleted DETC from wild-type mice using antibody against TCR Vγ5 followed by S. aureus infection. We found that DETC are not required to clear skin Staph infections, since depleted mice cleared with the same kinetics as non-depleted mice. Studies are in progress to further examine the roles of other γδ T cell populations in clearance of skin Staph infections. Supported by grant from NIH (R01 AI122720)

GABAergic neuronal IL-4R mediates T cell effect on memory

Mechanisms governing how immune cells and their derived molecules impact homeostatic brain function are still poorly understood. Here, we elucidate neuronal mechanisms underlying Tcell effects on synaptic function and episodic memory. Depletion of CD4 Tcells led to memory deficits and impaired long-term potentiation. Severe combined immune-deficient mice exhibited amnesia, which was reversible by repopulation with Tcells from wild-type but not from IL-4-knockout mice. Behaviors impacted by Tcells were mediated via IL-4 receptors expressed on neurons. Exploration of snRNA-seq of neurons participating in memory processing provided insights into synaptic organization and plasticity-associated pathways regulated by immune cells. IL-4Rα knockout in inhibitory (but not in excitatory) neurons was sufficient to impair contextual fear memory, and snRNA-seq from these mice pointed to IL-4-driven regulation of synaptic function in promoting memory. These findings provide new insights into complex neuroimmune interactions at the transcriptional and functional levels in neurons under physiological conditions.

Recruitment of M1 Macrophages May Not Be Critical for Protection against Colitis-Associated Tumorigenesis.

A close connection between inflammation and the risk of developing colon cancer has been suggested in the last few years. It has been estimated that patients diagnosed with some types of inflammatory bowel disease, such as ulcerative colitis or Crohn’s disease, have up to a 30% increased risk of developing colon cancer. However, there is also evidence showing that the activation of anti-inflammatory pathways, such as the IL-4 receptor-mediated pathway, may favor the development of colon tumors. Using an experimental model of colitis-associated colon cancer (CAC), we found that the decrease in tumor development in global IL4Rα knockout mice (IL4RαKO) was apparently associated with an inflammatory response mediated by the infiltration of M1 macrophages (F480+TLR2+STAT1+) and iNOS expression in colon tissue. However, when we developed mice with a specific deletion of IL4Rα in macrophages (LysMcreIL4Rα−/lox mice) and subjected them to CAC, it was found that despite presenting a large infiltration of M1 macrophages into the colon, these mice were as susceptible to colon-tumorigenesis as WT mice. These data suggest that in the tumor microenvironment the absence of IL4Rα expression on macrophages, as well as the recruitment of M1 macrophages, may not be directly associated with resistance to developing colon tumors. Therefore, it is possible that IL4Rα expression in other cell types, such as colonic epithelial cells, could have an important role in promoting the development of colitis-associated colon tumorigenesis.

S100A8/A9 is not essential for the development of inflammation and joint pathology in interleukin-1 receptor antagonist knockout mice

BackgroundExcessive osteoclast activity, which is strongly stimulated by pro-inflammatory mediators, results in bone and cartilage degeneration as central features of many arthritides. Levels of the alarmin S100A8/A9 and interleukin (IL)-1β are both increased in arthritis patients and correlate with disease activity and progression of tissue erosion. We previously presented S100A8/A9 as a good biomarker for joint inflammation and arthritis pathology under circumstances of high IL-1 signaling in mice that lack the gene encoding IL-1 receptor antagonist (Il1rn−/− mice). Here, we investigated whether S100A8/A9 is also actively involved in the development of joint inflammation and both cartilage and bone pathology under these conditions by comparing Il1rn−/− mice with mice that have an additional deficiency for S100a9 (Il1rn−/−XS100a9−/−).MethodsIl1rn−/−XS100a9−/− on a BALB/c background were obtained by crossing S100a9−/− mice and Il1rn−/− mice. Arthritis incidence and severity were macroscopically scored. Myeloid cell populations in the bone marrow and spleen were determined using flow cytometry. In vitro osteoclastogenesis of bone marrow cells was evaluated with TRAP staining. Microscopic joint inflammation, cartilage degeneration, and bone destruction were evaluated using histology of ankle joints of 12- and 20-week-old mice.ResultsMacroscopically scored arthritis severity was comparable between Il1rn−/− and Il1rn−/−XS100a9−/− mice. Inflammation, cartilage erosion, and bone erosion were clearly present in 12-week-old mice of both strains lacking Il1rn−/−, but not significantly different between Il1rn−/−XS100a9−/− and Il1rn−/−. Moreover, we observed that the numbers of neutrophils and monocytes were increased by the absence of Il1rn, which was affected by the absence of S100a9 only in the spleen but not in the bone marrow. In line with our other findings, the absence of S100a9 did not affect the osteoclastogenic potential of osteoclast precursors in the absence of Il1rn. Finally, in agreement with the findings in early arthritis development in 12-week-old mice, cartilage and bone erosion in 20-week-old mice was significantly higher in both Il1rn−/− strains, but the additional absence of S100a9 did not further affect tissue pathology.ConclusionS100A8/A9 deficiency does not significantly affect inflammation and joint destruction in mice with high IL1β signaling suggesting that S100A8/A9 is not essential for the development of arthritis under these conditions.

Experimental modulation of Interleukin 1 shows its key role in chronic kidney disease progression and anemia

Inflammation in chronic kidney disease (CKD) is mostly due to activation of the innate immune system, in which Interleukin-1 (IL-1) is a key player. Anemia of CKD may also be due to erythropoietin (EPO) resistance, clinically associated with inflammation. IL-1 receptor antagonist knockout (RaKO) mice show arthritis and excessive inflammation. Inhibition of IL-1 was shown to be beneficial in many inflammatory conditions, but its role in CKD and anemia is unknown. Here, we report that enhanced inflammation in RaKO mice with CKD provoked both higher degrees of renal insufficiency and anemia in comparison to wild-type CKD, in association with a downregulation of renal hypoxia inducible factor-2 (HIF2) as well as decreased bone marrow EPO-receptor (EPOR) and transferrin receptor (TFR). In contrast, administration of P2D7KK, an anti-IL1b monoclonal antibody, to CKD mice results in a lower grade of systemic inflammation, better renal function and blunted anemia. The latter was associated with upregulation of renal HIF-2α, bone marrow EPO-R and TFR. Altogether, this supports the key role of inflammation, and IL-1 particularly, in CKD progression and anemia. Novel treatments to reduce inflammation through this and other pathways, may improve renal function, attenuate the anemic state or increase the response to exogenous EPO.

Extracellular microvesicles that originated adipose tissue derived mesenchymal stem cells have the potential ability to improve rheumatoid arthritis on mice.

IntroductionMesenchymal stem cells (MSCs) are promising therapeutic tools in regenerative medicine. In particularly adipose tissue derived MSC (AMSC) has powerful potential for the therapeutics of rheumatoid arthritis (RA) because these cells can control immune balance. RA systemically occurs autoimmune disease. Interestingly, IL-1 receptor antagonist deficient (IL-1ra−/−) mice induce inflammation in joints like RA. In RA therapy, although AMSC improves the inflammation activity, it is little known to play roles of extracellular microvesicles (EV) for improvement of RA. To clarify the MSC-derived EVs are involved amelioration mechanisms for RA by themselves, we examined the functional effects of development for RA by AMSC-EVs.MethodsWe isolated AMSCs derived mice adipose tissue and purified EVs from the culture supernatant of AMSCs. To examine whether EVs can improve RA, we administrated EVs or AMSCs to IL-1ra knockout mice as RA model mice. We analyzed EVs-included factor by western blot methods and RA improvement effect by ELISA.ResultsIn this study, we showed that the swellings of joints on mice in wild type AMSC and that in AMSC-EVs decreased than that in IL-1ra−/− mice-AMSC-EVs and in none-treated. We detected IL-1ra expression in AMSC-EVs in wild type mice but not that in IL-1ra−/− mice. Proinflammatory cytokine expression changes in mice showed in AMSCs and AMSC-EVs, but no apparent differences cytokine expressions were detected in IL-1ra−/− mice.ConclusionsIn this study, we concluded that MSCs might improve RA by the transferring of factors such as IL-1ra, which are included their MSC derived- EVs.

Interleukin 1 is a key driver of inflammatory bowel disease-demonstration in a murine IL-1Ra knockout model.

Interleukin 1 (IL-1) is an important mediator of inflammation and tissue damage in inflammatory bowel disease (IBD). The balance between IL-1 and IL-1Ra as a natural inhibitor plays a vital role in a variety of diseases. Here, we investigated whether changes seen during IBD are induced spontaneously in mice lacking a functional IL-1rn gene. Histological staining was performed on the jejunum and ileum of BALB/c IL-1rn+/+ and IL-1rn-/- mice to characterize crypt-villus height, villus width, and number of goblet cells per villus. Pro-inflammatory cytokines, immune cell infiltration and matrix-degrading enzymes, together with the production of intestinal enzymes and the integrity of tight and adherent junction proteins were determined using immunohistochemistry. In the small intestine of BALB/c IL-1rn-/- mice the villus heights were significantly reduced; and in the ileum this was accompanied by a decrease in villi width. There was also an increase in goblet cell number and mucin production compared to wild-type mice. IL-1α and IL-1β immunopositivity were increased, whilst IL-1R1 expression was decreased in IL-1rn-/- mice. IL-15 and TNFα were also increased in older IL-1rn-/- mice. Increased polymorphonuclear and macrophage infiltration were seen in IL-1rn-/- mice, whilst expression of matrix-degrading enzymes and digestive enzymes were unchanged, except for dipeptidyl peptidase IV which was increased in younger IL-1rn-/- mice compared to wild type mice. The expression of tight and adhesion junctions were also dramatically decreased in IL-1rn-/- mice. In conclusion, IL-1rn-/- mice developed spontaneous abnormalities which displayed features associated with IBD, demonstrating a clear role for IL-1 in IBD.

Mice Lacking Alternatively Activated (M2) Macrophages Show Impairments in Restorative Sleep after Sleep Loss and in Cold Environment

The relationship between sleep, metabolism and immune functions has been described, but the cellular components of the interaction are incompletely identified. We previously reported that systemic macrophage depletion results in sleep impairment after sleep loss and in cold environment. These findings point to the role of macrophage-derived signals in maintaining normal sleep. Macrophages exist either in resting form, classically activated, pro-inflammatory (M1) or alternatively activated, anti-inflammatory (M2) phenotypes. In the present study we determined the contribution of M2 macrophages to sleep signaling by using IL-4 receptor α-chain-deficient [IL-4Rα knockout (KO)] mice, which are unable to produce M2 macrophages. Sleep deprivation induced robust increases in non-rapid-eye-movement sleep (NREMS) and slow-wave activity in wild-type (WT) animals. NREMS rebound after sleep deprivation was ~50% less in IL-4Rα KO mice. Cold exposure induced reductions in rapid-eye-movement sleep (REMS) and NREMS in both WT and KO mice. These differences were augmented in IL-4Rα KO mice, which lost ~100% more NREMS and ~25% more REMS compared to WTs. Our finding that M2 macrophage-deficient mice have the same sleep phenotype as mice with global macrophage depletion reconfirms the significance of macrophages in sleep regulation and suggests that the main contributors are the alternatively activated M2 cells.

Mesenchymal stem cells ameliorate experimental arthritis via expression of interleukin-1 receptor antagonist.

Human bone marrow-derived mesenchymal stem cells (MSCs) have been observed to inhibit arthritis in experimental animal models such as collagen-induced arthritis. However, the exact anti-inflammatory mechanisms remain poorly understood. Interleukin-1 receptor antagonist (IL-1Ra) is an anti-inflammatory cytokine produced by immune and stromal cells. We postulated that MSCs could produce IL-1Ra and attenuate experimental arthritis. In this study, 5x106 MSCs were injected into the peritoneal cavity of IL-1Ra knockout (IL-1RaKO) mice. MSCs reduced the severity of the arthritis by histology and decreased pro-inflammatory cytokine levels in IL-1RaKO mice. The ratio of splenic T helper 17 (Th17) cells to regulatory T cells (Treg) was significantly decreased in MSC-injected IL-1RaKO mice. Purified splenic CD4+ T cells from mice in each of the treatment groups were cultured under Th17 polarizing conditions and analyzed by flow cytometry. Less expansion of the Th17 population was observed in the MSC-treated group. Interestingly, MSCs expressed inducible IL-1Ra against inflammatory environmental stimuli. Human recombinant IL-1Ra could suppress Th17 cells differentiation under Th17 polarizing conditions. These results indicate that IL-1Ra expressed by MSCs can inhibit Th17 polarization and decrease the immune response in IL-1RaKO mice. Therefore, MSC-derived IL-1Ra may inhibit inflammation in IL-1RaKO mice via effects on Th17 differentiation.

β Cell-Specific Deletion of the IL-1 Receptor Antagonist Impairs β Cell Proliferation and Insulin Secretion

Interleukin-1 receptor antagonist (IL-1Ra) is elevated in the circulation during obesity and type 2 diabetes (T2D) but is decreased in islets from patients with T2D. The protective role of local IL-1Ra was investigated in pancreatic islet β cell (βIL-1Ra)-specific versus myeloid-cell (myeloIL-1Ra)-specific IL-1Ra knockout (KO) mice. Deletion of IL-1Ra in β cells, but not in myeloid cells, resulted in diminished islet IL-1Ra expression. Myeloid cells were not the main source of circulating IL-1Ra in obesity. βIL-1Ra KO mice had impaired insulin secretion, reduced β cell proliferation, and decreased expression of islet proliferation genes, along with impaired glucose tolerance. The key cell-cycle regulator E2F1 partly reversed IL-1β-mediated inhibition of potassium channel Kir6.2 expression and rescued impaired insulin secretion in IL-1Ra knockout islets. Our findings provide evidence for the importance of β cell-derived IL-1Ra for the local defense of β cells to maintain normal function and proliferation.

Neuropathological and behavioral sequelae in IL-1R1 and IL-1Ra gene knockout mice after soman (GD) exposure

Soman (GD) exposure results in status epilepticus (SE) that leads to neurodegeneration, neuroinflammation, and behavioral consequences including learning and memory deficits. The neuroinflammatory response is characterized by the upregulation of the pro-inflammatory cytokine, interleukin-1 (IL-1), which mediates the expression of other neurotoxic cytokines induced after GD exposure. However, the specific role of IL-1 signaling has not been defined in terms of the consequences of GD-induced SE. Therefore, the purpose of this study was to regulate IL-1 signaling and study the behavioral deficits and neurodegeneration that occur after convulsion onset. Wild type (WT), IL-1 receptor (IL-1R1) knockout (KO), and IL-1 receptor antagonist (IL-1Ra) KO mice were exposed to a convulsive dose of GD, and behavior was evaluated up to 18days later. Activity was studied using the Open Field, anxiety was assessed in the Zero Maze, and spatial learning and memory were evaluated with the Barnes Maze. The animals were euthanized at 24hours and 18days to determine neuropathology in the piriform cortex, amygdala, thalamus, and CA1, CA2/3, and CA4 regions of the hippocampus. Unlike the IL-1Ra KO, the IL-1R1 KO showed less neuropathology compared to WT at 24hours, but moderate to severe injury was found in all strains at 18days. Compared to their saline controls, the exposed WT mice were significantly more active in the Open Field, and the IL-1R1 KO strain showed reduced anxiety in the Zero Maze Test. Compared to WT mice, IL-1R1 and IL-1Ra KO mice had spatial learning and memory impairments in the Barnes Maze. Therefore, the IL-1 signaling pathway affects neurodegeneration and behavior after GD-induced convulsions.

The IL-4 receptor α has a critical role in bone marrow–derived fibroblast activation and renal fibrosis

Renal fibrosis is a common pathway leading to the progression of chronic kidney disease, and bone marrow-derived fibroblasts contribute significantly to the development of renal fibrosis. However, the signaling mechanisms underlying the activation of these fibroblasts are not completely understood. Here, we examined the role of IL-4 receptor α (IL-4Rα) in the activation of myeloid fibroblasts in two experimental models of renal fibrosis. Compared with wild-type mice, IL-4Rα knockout mice accumulated fewer bone marrow-derived fibroblasts and myofibroblasts in their kidneys. IL-4Rα deficiency suppressed the expression of α-smooth muscle actin, extracellular matrix proteins and the development of renal fibrosis. Furthermore, IL-4Rα deficiency inhibited the activation of signal transducer and activator of transcription 6 (STAT6) in the kidney. Moreover, wild-type mice engrafted with bone marrow cells from IL-4Rα knockout mice exhibited fewer myeloid fibroblasts in the kidney and displayed less severe renal fibrosis following ureteral obstructive injury compared with wild-type mice engrafted with wild-type bone marrow cells. Invitro, IL-4 activated STAT6 and stimulated expression of α-smooth muscle actin and fibronectin in mouse bone marrow monocytes. This was abolished in the absence of IL-4Rα. Thus, IL-4Rα plays an important role in bone marrow-derived fibroblast activation, resulting in extracellular matrix protein production and fibrosis development. Hence, the IL-4Rα/STAT6 signaling pathway may serve as a novel therapeutic target for chronic kidney disease.

The active metabolite of leflunomide, A77 1726, attenuates inflammatory arthritis in mice with spontaneous arthritis via induction of heme oxygenase-1

BackgroundLeflunomide is a low-molecular-weight compound that is widely used in the treatment of rheumatoid arthritis. Although leflunomide is thought to act through the inhibition of the de novo pyrimidine synthesis, the molecular mechanism of the drug remains largely unknown. We investigated the antiarthritis effects and mechanisms of action of the active metabolite of leflunomide, A77 1726, in interleukin-1 receptor antagonist-knockout (IL-1Ra-KO) mice.Methods14- to 15-week-old male IL-1Ra-KO mice were treated with 10 or 30 mg/kg A77 1726 via intraperitoneal injection three times per week for 6 weeks. The effects of A77 1726 on arthritis severities were assessed by clinical scoring and histological analysis. The serum concentrations of IL-1β, tumor necrosis factor-α (TNF-α), and malondialdehyde were measured by enzyme-linked immunosorbent assay. Histologic analysis of the joints was performed using Safranin O, and immunohistochemical staining. The frequencies of interleukin-17-producing CD4+ T (Th17) cells were analyzed by flow cytometry. Heme oxygenase-1 (HO-1) expression in splenic CD4+ T cells isolated from A77 1726-treated arthritis mice were assessed by western blotting.ResultsA77 1726 treatment induced heme oxygenase-1 (HO-1) in Jurkat cells and primary mouse T cells. Interestingly, A77 1726 inhibited Th17 cell differentiation. In vivo, A77 1726 reduced the clinical arthritis severity of histological inflammation and cartilage destruction. The joints isolated from A77 1726-treated mice showed decreased expression of inducible nitric oxide synthase, nitrotyrosine, TNF-α, and IL-1β. The serum levels of TNF-α, IL-1β, and malondialdehyde were also decreased in A77 1726-treated mice. Whereas the number of Th17 cells in spleens was decreased in A77 1726-treated arthritis mice, a significant increase in the number of Treg cells in spleens was observed. Interestingly, HO-1 expression was significantly higher in splenic CD4+ T cells isolated from A77 1726-treated mice compared with those from vehicle-treated mice, whereas HO-1 expression of splenic non-CD4+ T cells did not differ between groups.ConclusionThe inhibitory effects of A77 1726 on joint inflammation and oxidative stress in autoimmune arthritis may be associated with HO-1 induction in CD4+ T cells.

Role of the IL-1 Pathway in Dopaminergic Neurodegeneration and Decreased Voluntary Movement

Interleukin-1 (IL-1), a proinflammatory cytokine synthesized and released by activated microglia, can cause dopaminergic neurodegeneration leading to Parkinson’s disease (PD). However, it is uncertain whether IL-1 can act directly, or by exacerbating the harmful actions of other brain insults. To ascertain the role of the IL-1 pathway on dopaminergic neurodegeneration and motor skills during aging, we compared mice with impaired [caspase-1 knockout (casp1−/−)] or overactivated IL-1 activity [IL-1 receptor antagonist knockout (IL-1ra−/−)] to wild-type (wt) mice at young and middle age. Their motor skills were evaluated by the open-field and rotarod tests, and quantification of their dopamine neurons and activated microglia within the substantia nigra were performed by immunohistochemistry. IL-1ra−/− mice showed an age-related decline in motor skills, a reduced number of dopamine neurons, and an increase in activated microglia when compared to wt or casp1−/− mice. Casp1−/− mice had similar changes in motor skills and dopamine neurons, but fewer activated microglia cells than wt mice. Our results suggest that the overactivated IL-1 pathway occurring in IL-1ra−/− mice in the absence of inflammatory interventions (e.g., intracerebral injections performed in animal models of PD) increased activated microglia, decreased the number of dopaminergic neurons, and reduced their motor skills. Decreased IL-1 activity in casp1−/− mice did not yield clear protective effects when compared with wt mice. In summary, in the absence of overt brain insults, chronic activation of the IL-1 pathway may promote pathological aspects of PD per se, but its impairment does not appear to yield advantages over wt mice.

Clofazimine Biocrystal Accumulation in Macrophages Upregulates Interleukin 1 Receptor Antagonist Production To Induce a Systemic Anti-Inflammatory State.

Clofazimine (CFZ) is a poorly soluble antibiotic and anti-inflammatory drug indicated for the treatment of leprosy. In spite of its therapeutic value, CFZ therapy is accompanied by the formation of drug biocrystals that accumulate within resident tissue macrophages, without obvious toxicological manifestations. Therefore, to specifically elucidate the off-target consequences of drug bioaccumulation in macrophages, we compared the level of inflammasome activation in CFZ-accumulating organs (spleen, liver and lung) in mice after 2 and 8 weeks of CFZ treatment when the drug exists in soluble and insoluble (biocrystalline) forms, respectively. Surprisingly, the results showed a drastic reduction in caspase 1 and interleukin-1β (IL-1β) cleavage in the livers of mice treated with CFZ for 8 weeks (8-week-CFZ-treated mice) compared to 2-week-CFZ-treated and control mice, which was accompanied by a 3-fold increase in hepatic IL-1 receptor antagonist (IL-1RA) production and a 21-fold increase in serum IL-1RA levels. In the lung and spleen, IL-1β cleavage and tumor necrosis factor alpha expression were unaffected by soluble or biocrystal CFZ forms. Functionally, there was a drastic reduction of carrageenan- and lipopolysaccharide-induced inflammation in the footpads and lungs, respectively, of 8-week-CFZ-treated mice. This immunomodulatory activity of CFZ biocrystal accumulation was attributable to the upregulation of IL-1RA, since CFZ accumulation had minimal effect in IL-1RA knockout mice or 2-week-CFZ-treated mice. In conclusion, CFZ accumulation and biocrystal formation in resident tissue macrophages profoundly altered the host's immune system and prompted an IL-1RA-dependent, systemic anti-inflammatory response.

Interleukin-1 receptor antagonist knockout mice as a model of the inflammatory bowel disease

Interleukin-1 receptor antagonist knockout mice as a model of the inflammatory bowel disease

Mesenchymal Stem Cell-Derived Interleukin 1 Receptor Antagonist Promotes Macrophage Polarization and Inhibits B Cell Differentiation.

The role of interleukin 1 receptor antagonist (IL1RA) in mediating the immunosuppressive effect of mesenchymal stem/stromal cells (MSCs) has been reported in several studies. However, how MSC-derived IL1RA influences the host response has not been clearly investigated. We therefore derived MSCs from the bone marrow of IL1RA knockout mice and evaluated their immunosuppressive effect on different immune cell subsets. IL1RA deficient (IL1RA(-/-) ) or wild type (wt) MSCs inhibited to the same extend the proliferation of T lymphocytes. On the contrary, IL1RA(-/-) MSCs were less effective than wt MSCs to induce in vitro the macrophage polarization from M1 to M2 phenotype secreting IL10 and exerting a suppressive effect on CD4(+) T cells. Moreover compared with wt MSCs, IL1RA(-/-) MSCs did not efficiently support the survival of quiescent B lymphocytes and block their differentiation toward CD19(+) CD138(+) plasmablasts secreting IgG antibodies. The effectiveness of IL1RA secreted by MSCs in controlling inflammation was further shown in vivo using the collagen-induced arthritis murine model. MSCs lacking IL1RA expression were unable to protect mice from arthritic progression and even worsened clinical signs, as shown by higher arthritic score and incidence than control arthritic mice. IL1RA(-/-) MSCs were not able to decrease the percentage of Th17 lymphocytes and increase the percentage of Treg cells as well as decreasing the differentiation of B cells toward plasmablasts. Altogether, our results provide evidence of the key role of IL1RA secreted by MSCs to both control the polarization of macrophages toward a M2 phenotype and inhibit B cell differentiation in vivo.

Development of synthetic anti-cyclic citrullinated peptide antibody and its arthritogenic role.

This study was undertaken to develop a novel anti-citrullinated peptide antibody (ACPA) and to investigate its arthritogenicity in a collagen-induced arthritis (CIA) model. The novel ACPA, 12G1, was developed by injecting cyclic citrullinated antigen in mice and subsequently hybridizing the B cells producing citrullinated peptide-specific antibodies with a myeloma cell line. The arthritic joints of mice with CIA and collagen antibody-induced arthritis (CAIA) as well as interleukin-1 receptor antagonist (IL-1Ra) knockout (KO) mice were stained immunohistochemically using the 12G1 antibody. Confocal immunostaining was used to identify colocalization of 12G1 with various citrullinated proteins. 12G1 in the presence or absence of chelating beads was administered to CIA mice on days 21 and 28 after type II collagen (CII) immunization to investigate 12G1 arthritogenecity. 12G1 detected citrullinated proteins in the arthritic joints of all the experimental arthritis models used. Confocal immunostaining showed that 12G1 was colocalized with well-known citrullinated proteins, including vimentin, collagen, anti-immunoglobulin binding protein and fibronectin. Staining of citrullinated proteins using 12G1 was more diffuse in CIA mice compared with CAIA and IL-1Ra KO mice. 12G1 injection apparently acted as a booster of immunization in CIA mice in combination with a single CII immunization, with this effect being abolished when 12G1 was injected with chelating beads. The novel ACPA, 12G1, identified various citrullinated proteins in the arthritic joints of three experimental arthritis models. 12G1-treated mice developed arthritis following a single CII immunization, suggesting an arthritogenic potential for ACPA in CIA mice.

Interleukin-1 Receptor Antagonist Has a Novel Function in the Regulation of Matrix Metalloproteinase-13 Expression.

Interleukin-1 receptor antagonist (IL-1Ra) is an IL-1 family member, which binds to IL-1 receptors but does not induce any intracellular signaling. We addressed whether IL-1Ra has a novel function in regulation of the extracellular matrix or adhesion molecules. Polymerase chain reaction array analysis demonstrated a ~5-fold increase in matrix metalloproteinase 13 (MMP-13) mRNA expression of IL-1Ra siRNA-transfected Ca9-22 human oral squamous epithelial carcinoma cells compared with the control. In fact, MMP-13 mRNA and protein expression as well as its activity in IL-1Ra siRNA-transfected Ca9-22 cell lines were significantly higher than those in the control. IL-1Ra siRNA treatment resulted in strong elevation of MMP-13 expression, whereas addition of rhIL-1Ra (40 ng/ml) suppressed MMP-13 expression, suggesting that IL-1Ra had a specific effect on MMP-13 induction. IL-1Ra siRNA could potently suppress IL-1α. No significant difference was found between the MMP-13 mRNA expression of IL-1Ra siRNA-transfected cells and those treated with anti-IL-1α or anti-IL-1β antibodies. These results suggested that continuous supply of IL-1 had no effect on the induction of MMP-13 by IL-1Ra siRNA. Histopathological investigation of MMP-13 in periodontal tissue showed specific localization in the junctional epithelial cells of IL-1Ra knockout (KO) mice. Furthermore, infection with Aggregatibacter actinomycetemcomitans to establish an experimental periodontitis model resulted in predominant localization of MMP-13 along apical junctional epithelial cells. Laminin-5, which is degraded by MMP-13, was found in the internal basal lamina of wild-type mice, whereas the internal basal lamina of IL-1Ra KO mice did not show obvious laminin-5 localization. In particular, laminin-5 localization almost disappeared in the internal basal lamina of IL-1Ra KO mice infected with A. actinomycetemcomitans, suggesting that the suppression of IL-1Ra resulted in strong induction of MMP-13 that degraded laminin-5. In conclusion, IL-1Ra is associated with MMP-13 expression and has a novel function in such regulation without interference of the IL-1 signaling cascade.