Background and objectives The IL-36 family of cytokines comprises three agonists: IL-36α, IL-36β and IL-36γ, an antagonist: IL-36Ra, and IL-38: another potential IL-36 inhibitor. IL-36 agonists are highly expressed in skin and are involved in the pathogenesis of psoriasis, while antagonists limit uncontrolled inflammation. The expression and role of IL-36 cytokines in other chronic inflammatory diseases is currently debated. In this study, we compared the expression profile of IL-36 cytokines in psoriasis, RA and CD. Materials and methods Skin, joint and colon inflammation was induced in mice. Skin, synovial and colonic biopsies from patients respectively with psoriasis, RA or CD were collected. Synovial fluids from patients with RA were also collected. These samples were analysed for cytokines expression by RT-qPCR, ELISA, immunohistochemistry and confocal microscopy. The cell sources of IL-36 cytokines were confirmed in cell cultures after stimulation with inflammatory cytokines and TLR agonists by RT-qPCR. Results During imiquimod-induced mouse skin inflammation and in human psoriasis, expression of IL-36α, γ and IL-36Ra, but not IL-36β and IL-38, was induced and correlated with Th17 cytokines (IL-17A, IL-22…). In mice with collagen-induced arthritis and in the synovium of patients with RA, IL-36α, β, γ, IL-36Ra and IL-38 were all elevated and correlated with myeloid cytokines such as CCL3, CCL4 and MCSF, but not with Th17 cytokines. In mice with dextran sulfate sodium-induced colitis and in patients with CD, only IL-36α, γ and IL-38 were induced at a relatively low level. Only a minor subgroup of patients with RA (17–29%) or CD (25%) had an elevated IL-36 agonists/antagonists ratio, versus 93% of patients with psoriasis. By IHC and in cell cultures, the different IL-36 cytokines were produced at different levels by human keratinocytes, CD68+ inflammatory macrophages, dendritic/Langerhans cells, and CD79α+ plasmocytes. Conclusion Expression of the different IL-36 cytokines is differently regulated and their cell sources are distinct. This helps to explain the different expression profiles observed in three chronic inflammatory diseases and why only a minor subgroup of RA and CD patients have an elevated IL-36 agonists/antagonists ratio. Additional studies are necessary to better identify these patients and to investigate whether they would benefit from IL-36 neutralisation.

Allergic asthma is characterized by a strong Th2 response with inflammatory cell recruitment and structural changes in the lung. Papain is a protease allergen disrupting the airway epithelium triggering a rapid inflammation with eosinophilia mediated by innate lymphoid cell activation (ILC2) and leading to a Th2 immune response. In this study, we focused on inflammatory responses to a single exposure to papain and showed that intranasal administration of papain results in the recruitment of inflammatory cells, including neutrophils and eosinophils with a rapid production of IL‐1α, IL‐1β, and IL‐33. The inflammatory response is abrogated in the absence of IL‐1R1 and MyD88. To decipher the cell type(s) involved in MyD88‐dependent IL‐1R1/MyD88 signaling, we used new cell‐specific MyD88‐deficient mice and found that the deletion of MyD88 signaling in single cell types such as T cells, epithelial cells, CD11c‐positive or myeloid cells leads to only a partial inhibition compared to complete absence of MyD88, suggesting that several cell types contribute to the response. Importantly, the inflammatory response is largely ST2 and IL‐36R independent. In conclusion, IL‐1R1 signaling via MyD88 is critical for the first step of inflammatory response to papain.

Background and objectives The IL-36 family of cytokines comprises three agonists: IL-36α, IL-36β and IL-36γ, an antagonist: IL-36Ra, and IL-38: another potential IL-36 inhibitor. IL-36 agonists are highly expressed in skin and are involved in the pathogenesis of psoriasis, while antagonists limit uncontrolled inflammation. The expression and role of IL-36 cytokines in other chronic inflammatory diseases is currently debated. In this study, we compared the expression profile of IL-36 cytokines in psoriasis, RA and CD. Materials and methods Skin, joint and colon inflammation was induced in mice. Skin, synovial and colonic biopsies from patients respectively with psoriasis, RA or CD were collected. Synovial fluids from patients with RA were also collected. These samples were analysed for cytokines expression by RT-qPCR, ELISA, immunohistochemistry and confocal microscopy. The cell sources of IL-36 cytokines were confirmed in cell cultures after stimulation with inflammatory cytokines and TLR agonists by RT-qPCR. Results During imiquimod-induced mouse skin inflammation and in human psoriasis, expression of IL-36α, γ and IL-36Ra, but not IL-36β and IL-38, was induced and correlated with Th17 cytokines (IL-17A, IL-22…). In mice with collagen-induced arthritis and in the synovium of patients with RA, IL-36α, β, γ, IL-36Ra and IL-38 were all elevated and correlated with myeloid cytokines such as CCL3, CCL4 and MCSF, but not with Th17 cytokines. In mice with dextran sulfate sodium-induced colitis and in patients with CD, only IL-36α, γ and IL-38 were induced at a relatively low level. Only a minor subgroup of patients with RA (17–29%) or CD (25%) had an elevated IL-36 agonists/antagonists ratio, versus 93% of patients with psoriasis. By IHC and in cell cultures, the different IL-36 cytokines were produced at different levels by human keratinocytes, CD68 + inflammatory macrophages, dendritic/Langerhans cells, and CD79α + plasmocytes. Conclusion Expression of the different IL-36 cytokines is differently regulated and their cell sources are distinct. This helps to explain the different expression profiles observed in three chronic inflammatory diseases and why only a minor subgroup of RA and CD patients have an elevated IL-36 agonists/antagonists ratio. Additional studies are necessary to better identify these patients and to investigate whether they would benefit from IL-36 neutralisation.

Summary Interleukin (IL)-36α, IL-36β and IL-36γ are expressed highly in skin and are involved in the pathogenesis of psoriasis, while the antagonists IL-36Ra or IL-38, another potential IL-36 inhibitor, limit uncontrolled inflammation. The expression and role of IL-36 cytokines in rheumatoid arthritis (RA) and Crohn's disease (CD) is currently debated. Here, we observed that during imiquimod-induced mouse skin inflammation and in human psoriasis, expression of IL-36α, γ and IL-36Ra, but not IL-36β and IL-38 mRNA, was induced and correlated with IL-1β and T helper type 17 (Th17) cytokines (IL-17A, IL-22, IL-23, CCL20). In mice with collagen-induced arthritis and in the synovium of patients with RA, IL-36α, β, γ, IL-36Ra and IL-38 were all elevated and correlated with IL-1β, CCL3, CCL4 and macrophage colony-stimulating factor (M-CSF), but not with Th17 cytokines. In the colon of mice with dextran sulphate sodium-induced colitis and in patients with CD, only IL-36α, γ and IL-38 were induced at relatively low levels and correlated with IL-1β and IL-17A. We suggest that only a minor subgroup of patients with RA (17–29%) or CD (25%) had an elevated IL-36 agonists/antagonists ratio, versus 93% of patients with psoriasis. By immunohistochemistry, IL-36 cytokines were produced by various cell types in skin, synovium and colonic mucosa such as keratinocytes, CD68+ macrophages, dendritic/Langerhans cells and CD79α+ plasma cells. In primary cultures of monocytes or inflammatory macrophages (M1), IL-36β and IL-36Ra were produced constitutively, but IL-36α, γ and IL-38 were produced after lipopolysaccharide stimulation. These distinct expression profiles may help to explain why only subgroups of RA and CD patients have a potentially elevated IL-36 agonists/antagonists ratio.

IL-36 cytokines are members of the IL-1 family of cytokines that stimulate dendritic cells and T cells leading to enhanced T helper 1 responses in vitro and in vivo; however, their role in host defense has not been fully addressed thus far. The objective of this study was to examine the role of IL-36R signaling in the control of mycobacterial infection, using models of systemic attenuated M. bovis BCG infection and virulent aerogenic M. tuberculosis infection. IL-36γ expression was increased in the lung of M. bovis BCG infected mice. However, IL-36R deficient mice infected with M. bovis BCG showed similar survival and control of the infection as compared to wild-type mice, although their lung pathology and CXCL1 response were transiently different. While highly susceptible TNF-α deficient mice succumbed with overwhelming M. tuberculosis infection, and IL-1RI deficient mice showed intermediate susceptibility, IL-36R-deficient mice controlled the infection, with bacterial burden, lung inflammation and pathology, similar to wild-type controls. Therefore, IL-36R signaling has only limited influence in the control of mycobacterial infection.

IL-33 is a cytokine of the IL-1 family, which signals through the ST2 receptor. Previous studies emphasized a role for IL-33 in shaping innate and adaptive immune responses. IL-33 was also reported to modulate myelopoiesis and myeloid cell activity. In this article, we describe IL-33–overexpressing CMV/IL33 and LysM/IL33 mice, which display an inflammatory phenotype associated with growth retardation and paw swelling. The phenotype of CMV/IL33 mice is dependent on activation of the ST2 receptor and is characterized by extensive neutrophil infiltration into different organs, including the paws. Local or systemic levels of proinflammatory mediators such as IL-1β, Cxcl-1, G-CSF, and IL-6 are increased. CMV/IL-33 mice also suffer from anemia, thrombocytosis, and a marked dysregulation of myelopoiesis, leading to an important increase in myeloid cell production or accumulation in bone marrow (BM), spleen, and peripheral blood. Consistently, recombinant IL-33 induced proliferation of myeloid lineage cells in BM-derived granulocyte cultures, whereas IL-33 knockout mice exhibited minor deficiencies in spleen and BM myeloid cell populations. Our observations reveal a neutrophil-dominated inflammatory phenotype in IL-33–overexpressing CMV/IL33 and LysM/IL33 mice, and highlight important regulatory effects of IL-33 on myelopoiesis in vitro and in vivo, where excessive IL-33 signaling can translate into the occurrence of a myeloproliferative disorder.

Previous work suggested implication of the interleukin (IL)-1 family cytokine IL-33, signaling through its receptor ST2, in the pathogenesis of human and mouse arthritis. In this study, we directly investigated the role of endogenous IL-33 in antigen-induced arthritis (AIA) and collagen-induced arthritis (CIA) using IL-33 KO mice. AIA was induced by injection of methylated bovine serum albumin (mBSA) into knee joints of previously immunized mice. CIA was induced by immunization with bovine type II collagen. Disease severity was evaluated by clinical and histological scoring and cellular immune responses were assessed in cultured draining lymph node cells. Our results indicate that the development of AIA or CIA, as assessed by clinical or histological evaluation, is not impaired in IL-33 deficient mice. We did not observe any consistent modifications in humoral or cellular immune responses in IL-33 KO mice, although IL-33 deficiency enhanced antigen-specific IFN-γ production, proliferation or IgG2a titers in some experiments, suggesting that endogenous IL-33 may contribute to shaping the adaptive immune response. In conclusion, our data suggest that IL-33 plays a modifying rather than a pivotal role in disease development in two models of immune-mediated arthritis.

INTRODUCTION:Interleukin (IL)-36 refers to three related IL-1 family cytokines, IL-36α, IL-36β, and IL-36γ, that bind to the IL-36 receptor (IL-36R). IL-36 exerts proinflammatory effects in skin and lung and stimulates T cell responses. In the present study, we examined the expression and function of IL-36R and its ligands in experimental arthritis.METHODS:Collagen-induced arthritis (CIA), antigen-induced arthritis (AIA), and K/BxN serum transfer-induced arthritis were induced according to standard protocols. Messenger RNA levels for IL-36R and its ligands in the joints of mice with CIA were determined by RT-qPCR. Mice with CIA were injected with a blocking monoclonal anti-IL-36R, a blocking anti-IL-1RI, or their isotype-matched control antibodies at the time of arthritis onset. Anti-IL-36R or control antibodies were also injected at the time of AIA induction. Finally, IL-36R-deficient mice were examined in AIA and serum transfer-induced arthritis. The development and severity of arthritis were assessed by clinical and histological scoring.RESULTS:IL-36R, IL-36Ra and IL-36γ mRNA were detected in the joints of mice with CIA, but their levels did not correlate with arthritis severity. As opposed to anti-IL-1RI antibody treatment, the injection of an anti-IL-36R antibody was devoid of effect on the development and severity of CIA. The severity of joint inflammation and structural damage in AIA was also unaltered by anti-IL-36R antibody treatment. Finally, the severity of AIA and K/BxN serum transfer-induced arthritis was similar in IL-36R-deficient and wild-type mice.CONCLUSIONS:The development and severity of experimental arthritis are independent of IL-36R signaling.

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.

ABSTRACTThe proinflammatory activities of IL-1 are tightly controlled at different levels. IL-1R2 acts as a decoy receptor and has been shown to regulate the biological effects of IL-1 in vitro and in vivo. However, little is known about its natural expression in the mouse in physiologic and pathologic conditions. In this study, we examined IL-1R2 mRNA and protein expression in isolated cells and tissues in response to different stimulatory conditions. Data obtained using ex vivo CD11b+Ly6G+ peripheral blood cells and in vitro-differentiated CD11b+Ly6G+ BMG indicated that neutrophils are the major source of constitutively expressed IL-1R2 in the mouse. The expression of IL-1R2 on BMG and ex vivo Ly6G+ peripheral blood cells was highly up-regulated by HC. IL-1R2 pull-down experiments showed that mouse rIL-1β binds to BMG IL-1R2, whereas binding of IL-1Ra could not be detected. Furthermore, LPS treatment induced shedding of IL-1R2 from the neutrophil membrane in vitro and in vivo, executed mainly by ADAM17. Finally, in in vivo models of inflammation, including thioglycolate-induced acute peritonitis and acute lung injury, infiltrating Ly6G+ neutrophils, expressed IL-1R2. Our data show that in the mouse, neutrophils mainly express the decoy receptor IL-1R2 under naïve and inflammatory conditions. These data suggest that neutrophils may contribute to the resolution of acute inflammation.

IL-33 is the most recently described member of the IL-1 family of cytokines. Expression studies and data obtained with ST2 KO mice, ST2 blockade and recombinant IL-33 have suggested implication of the IL-33/ST2 axis in the pathogenesis of human and mouse arthritis. In the present study we investigated the role of endogenous IL-33 in K/BxN serum transfer-induced arthritis by using IL-33 KO mice and compared the results to those obtained using ST2 KO mice. Arthritis was induced in IL-33 KO and ST2 KO mice, backcrossed to the C57BL/6 background, or in WT C57BL/6 control mice, by injection of arthritogenic K/BxN serum or purified total IgG. The development of arthritis was followed by clinical scoring of the paws and confirmed by histological evaluation. Cytokine mRNA expression was assessed by RT-qPCR. Cytokine protein levels were monitored by ELISA or Milliplex. IL-33 mRNA and protein expression in synovial tissues, as well as circulating IL-33 levels, were similar in arthritic WT and ST2 KO mice, and absent in IL-33 KO mice. The incidence and severity of K/BxN serum transfer-induced arthritis was identical in IL-33 KO and WT mice, while disease development was significantly reduced in ST2 KO mice. Similar data were obtained when injecting purified arthritogenic total IgG instead of complete K/BxN serum, excluding a potential contribution of IL-33 contained in the serum of donor mice to explain this difference. IL-33 is expressed locally in the inflamed synovium and is detected in the circulation during serum transfer-induced arthritis. However IL-33 KO mice displayed identical arthritis development and severity as compared to WT controls. In contrast, disease severity was reduced in ST2 KO mice. The reasons for the difference between IL-33 KO and ST2 KO mice, which might suggest IL-33 independent effects of ST2, or reveal the existence of confounding variables in these KO strains, are currently under investigation.

Objectives To define the cell type (myeloid vs other cells) specific effect of interleukin 1 (IL-1) receptor antagonist (IL-1Ra) deficiency on the acute inflammatory phase of arthritis. Methods Arthritis was induced by K/BxN serum transfer in wild-type (WT), IL-1Ra-deficient (IL-1Ra−/−) and conditional knockout mice. In the latter, IL-1Ra production was specifically targeted in myeloid cells (IL-1RaΔM) or in both hepatocytes and myeloid cells (IL-1RaΔH+M). Arthritis severity was clinically evaluated and ankle sections were scored for synovial inflammation and cartilage erosion. Quantitative RT-PCR, western blot and immunohistochemical analyses measured expression, localisation and cellular sources of the different IL-1Ra isoforms in arthritic joints. Results Total and myeloid cell-specific IL-1Ra deficiency was associated with increased arthritis severity, although disease incidence was similar to that of WT mice. Increased clinical scores were associated with exacerbated synovial inflammation. All IL-1Ra isoforms, except for intracellular (ic)IL-1Ra2, were expressed in arthritic joints of WT mice. In contrast, production of secreted (s)IL-1Ra and icIL-1Ra3 isoforms was markedly decreased in arthritic joints of both IL-1RaΔM and IL-1RaΔH+M mice. Immunohistochemical and western blot analyses suggested that the icIL-1Ra1 isoform is produced primarily by synovial fibroblasts. Conclusion Myeloid cell-derived IL-1Ra, including both sIL-1Ra and icIL-1Ra3 isoforms, controls articular inflammation during the acute phase of K/BxN serum transfer-induced arthritis.

The interleukin (IL)-1 system comprises two agonists, IL-1α and IL-1β (IL-1), a specific receptor antagonist (IL-1Ra) and two receptors, type 1 and type 2 IL-1 receptor (IL-1R1, IL-1R2). As opposed to IL-1R1, IL-1R2 is incapable of signaling because it lacks the intracellular TIR-domain, which is required for signal transduction. Thus, IL-1R2 acts as a decoy receptor for IL-1. This system, together with IL-1Ra, regulates IL-1 activity. In vitro data, mainly obtained in human cells, demonstrated that IL-1R2 is expressed by B cells, monocytes and neutrophils and can be proteolytically cleaved from the plasma membrane and shed as a soluble form to bind circulating IL-1β. However, although anti-inflammatory activity of IL-1R2 has been described in overexpression studies, the expression of endogenous IL-1R2 in the mouse and its contribution to the control of inflammatory responses has been poorly investigated. The expression of IL-1R2 transcripts in mouse tissues and ex vivo-isolated or in vitro-differentiated immune cells was assessed by qRT-PCR. For further characterization of endogenously expressed IL-1R2 and its in vitro function, we used bone marrow-derived neutrophils (BMN) for qRT-PCR, extracellular fluorescence activated cell sorting (FACS) and immunoblot analyses. In addition, different mouse models of inflammation were investigated to confirm the expression of IL-1R2 in vivo. IL-1R2 mRNA levels are highly upregulated in lung tissue after LPS-administration. Furthermore, mRNA and protein data obtained using ex vivo Gr1+Ly6G+-peripheral blood cells and in vitro differentiated Gr1+Ly6G+-BMN indicated that, like in human, neutrophils are a major source of IL-1R2 in mouse. However, other mouse immune cells, including T and B lymphocytes, macrophages (both M1 and M2) and dendritic cells do not express detectable levels of the decoy receptor on their cell surface. The expression of IL-1R2 on BMN is regulated by the glucocorticoid hydrocortisone, which induces a strong upregulation of IL-1R2 mRNA and protein. In contrast, the expression of IL-1β but not that of IL-1Ra, is decreased by hydrocortisone, supporting its anti-inflammatory role. LPS treatment induces shedding of IL-1R2 from the membrane into the culture supernatant. We also demonstrate that IL-1R2 binds recombinant mouse IL-1β, but not IL-1Ra. Finally, in in vivo models of inflammation, including thioglycolate-induced acute peritonitis, acute lung injury and two models of arthritis, neutrophils infiltrating the site of inflammation express the decoy receptor IL-1R2. These data indicate that the decoy receptor IL-1R2 is mainly expressed on neutrophils among immune cells in the mouse. It can trap IL-1β and is present during inflammatory situations. Taken together, these results suggest that IL-1R2 plays an important role in regulating IL-1 activity in vivo.

The interleukin-1 (IL-1) superfamily of cytokines comprises a set of pivotal mediators of inflammation. Among them, the action of IL-36 cytokines in immune responses has remained elusive. In a recent study, we demonstrated a direct effect of IL-36 on immune cells. Here we show that, among T cells, the IL-36 receptor is predominantly expressed on naive CD4+ T cells and that IL-36 cytokines act directly on naive T cells by enhancing both cell proliferation and IL-2 secretion. IL-36β acts in synergy with IL-12 to promote Th1 polarization and IL-36 signaling is also involved in mediating Th1 immune responses to Bacillus Calmette-Guerin infection in vivo. Our findings point toward a critical function of IL-36 in the priming of Th1 cell responses in vitro, and in adaptive immunity in a model of mycobacterial infection in vivo.

GenBank entries for mouse Il33 reveal the existence of two transcripts, Il33a and Il33b, with different 5'UTRs but coding for the same protein. We investigated expression of these transcripts in different mouse organs and cell types in basal and inflammatory conditions. Il33a and Il33b mRNAs start with different noncoding first exons, transcribed from different promoter regions, which both contain a consensus TATA-like sequence. Constitutive Il33a mRNA expression was detected in mouse stomach, lung, spleen, and brain, whereas basal Il33b mRNA expression was observed only in the stomach. Expression of both transcripts increased after systemic LPS administration. In vitro, we observed high constitutive expression of Il33 transcripts in MEFs. Constitutive Il33a mRNA expression was observed also in BMDCs, where it was preferentially increased in response to poly(I:C), whereas LPS increased levels of Il33a and Il33b mRNA. In contrast, BMMs and Raw 264.7 cells did not express Il33 mRNA constitutively, and LPS stimulation selectively induced expression of Il33b mRNA in these cells. Our data indicate that the Il33 gene is expressed from two alternative promoters in the mouse and that the relative expression of Il33a and Il33b transcripts is cell type- and stimulus-dependent.

A new technique that uses gold immunochromatographic strips enhances the detection sensitivity by inducing the clustering of additional gold nanoparticles (AuNPs) around the immunogold particles immobilized on nitrocellulose strips. The additional AuNPs provide an intense signal that can be detected by the naked eye. The AuNPs were synthesized and conjugated to monoclonal antibodies using self-assembly. Other antibodies were immobilized in a defined detection zone on the nitrocellulose membrane. The detection principle is based on a “sandwich” immunoreaction, where gold-labeled antibodies serve as signal vehicles. To improve the sensitivity of the strips, we use a mixture of 1% HAuCl4 and 10 mmol L−1 NH2OH·HCl to “enlarge” the gold nanoparticles. The detecting limits of Avian influenza virus (AIV) and Newcastle disease virus (NDV) are significantly increased. Compared with commercial test strips, this method is 100-fold more sensitive. This method is easy to perform and can be carried out on-site in test laboratories.