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Background & Aims Liver macrophages can be involved in both pathogen clearance and/or pathogenesis. To get further insight on their role during chronic hepatitis B virus (HBV) infections, our aim was to phenotypically and functionally characterize in vivo and ex vivo the interplay between HBV, primary human liver macrophages (PLMs) and primary blood monocytes differentiated into pro-inflammatory or anti-inflammatory macrophages (M1-MDMs or M2-MDMs, respectively). Methods PLMs or primary blood monocytes, either ex vivo differentiated into M1-MDMs or M2-MDMs, were exposed to HBV and their activation followed by ELISA or quantitative reverse transcription PCR (RT-qPCR). Liver biopsies from HBV-infected patients were analysed by RT-qPCR or immunohistochemistry. Viral parameters in HBV-infected primary human hepatocytes and differentiated HepaRG cells were followed by ELISA, qPCR and RT-qPCR analyses. Results HBc protein was present within the macrophages of liver biopsies taken from HBV-infected patients. Macrophages from HBV-infected patients also expressed higher levels of anti-inflammatory macrophage markers than those from non-infected patients. Ex vivo exposure of naive PLMs to HBV led to reduced secretion of pro-inflammatory cytokines. Upon exposure to HBV or HBV-producing cells during differentiation and activation, M1-MDMs secreted less IL-6 and IL-1β, whereas M2-MDMs secreted more IL-10 when exposed to HBV during activation. Finally, cytokines produced by M1-MDMs, but not those produced by HBV-exposed M1-MDMs, decreased HBV infection of hepatocytes. Conclusions Altogether, our data strongly suggest that HBV modulates liver macrophage functions to favour the establishment of infection. Lay summary Hepatitis B virus modulates liver macrophage function in order to favour the establishment and likely maintenance of infection. It impairs the production of the antiviral cytokine IL-1β, while promoting that of IL-10 in the microenvironment. This phenotype can be recapitulated in naive liver macrophages or monocyte-derived-macrophages ex vivo by short exposure to the virus or cells replicating the virus, thus suggesting an "easy to implement" mechanism of inhibition.

International audience; Interleukin (IL)-2 is a pleiotropic cytokine that is necessary to prevent chronic inflammation in the gastrointestinal tract 1-4. The protective effects of IL-2 involve the generation, maintenance and function of regulatory T (T reg) cells 4-8 , and the use of low doses of IL-2 has emerged as a potential therapeutic strategy for patients with inflammatory bowel disease 9. However, the cellular and molecular pathways that control the production of IL-2 in the context of intestinal health are undefined. Here we show, in a mouse model, that IL-2 is acutely required to maintain T reg cells and immunological homeostasis throughout the gastrointestinal tract. Notably, lineage-specific deletion of IL-2 in T cells did not reduce T reg cells in the small intestine. Unbiased analyses revealed that, in the small intestine, group-3 innate lymphoid cells (ILC3s) are the dominant cellular source of IL-2, which is induced selectively by IL-1β. Macrophages in the small intestine produce IL-1β, and activation of this pathway involves MYD88-and NOD2-dependent sensing of the microbiota. Our loss-of-function studies show that ILC3-derived IL-2 is essential for maintaining T reg cells, immunological homeostasis and oral tolerance to dietary antigens in the small intestine. Furthermore, production of IL-2 by ILC3s was significantly reduced in the small intestine of patients with Crohn's disease, and this correlated with lower frequencies of T reg cells. Our results reveal a previously unappreciated pathway in which a microbiota-and IL-1β-dependent axis promotes the production of IL-2 by ILC3s to orchestrate immune regulation in the intestine. To determine whether IL-2 is constitutively required for the maintenance of T reg cells and immunological homeostasis in the intestine, we administered isotype-control or anti-IL-2 neutralizing antibodies every other day to adult mice for two weeks. Within this short time period, the neutralization of IL-2 promoted an enlargement of the spleen and mesenteric lymph nodes, and caused significant reductions of T reg cells and significant increases in the proliferation of CD4 + T cells throughout the gastrointestinal tract and associated lymphoid tissues, including the mesenteric lymph nodes, large intestine and small intestine (Extended Data Fig. 1a-g). Blockade of IL-2 resulted in significantly enhanced IFNγ production by CD4 + T cells in both the small and large intestine, as well as increased IL-17A production in the large intestine (Extended Data Fig. 1h-k). Previous studies have suggested that CD4 + T cells are the dominant cellular source of IL-2 1,2. Therefore, we generated mice with a lineage-specific deletion of IL-2 in T cells by crossing IL-2-floxed mice 10 with Lck cre mice. Lck cre Il2 f/f mice exhibited a complete loss of IL-2 protein staining in T cells, and we observed a significant reduction in the number of T reg cells, and an increase in CD4 + T cell proliferation and effector function in the mesenteric lymph nodes and large intestine (Extended Data Fig. 2a-g). By contrast, deletion of

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International audience; The aim of this work was to understand whether the nature of breast cancer cells could modify the nature of the dialog of mesenchymal stem cells (MSCs) with cancer cells. By treating MSCs with the conditioned medium of metastatic Estrogen-receptor (ER)-negative MDA-MB-231, or non-metastatic ER-positive MCF-7 breast cancer cells, we observed that a number of chemokines were produced at higher levels by MSCs treated with MDA-MB-231 conditioned medium (CM). MDA-MB-231 cells were able to induce NF-κB signaling in MSC cells. This was shown by the use of a NF-kB chemical inhibitor or an IκB dominant negative mutant, nuclear translocation of p65 and induction of NF-κB signature. Our results suggest that MDA-MB-231 cells exert their effects on MSCs through the secretion of IL-1β, that activates MSCs and induces the same chemokines as the MDA-MB-231CM. In addition, inhibition of IL-1β secretion in the MDA-MB-231 cells reduces the induced production of a panel of chemokines by MSCs, as well the motility of MDA-MB-231 cells. Our data suggest that aggressive breast cancer cells secrete IL-1β, which increases the production of chemokines by MSCs.

International audience; We report the clinical description and molecular dissection of a new fatal human inherited disorder characterized by chronic autoinflammation, invasive bacterial infections and muscular amylopectinosis. Patients from two kindreds carried biallelic loss-of-expression and loss-of-function mutations in HOIL1 (RBCK1), a component of the linear ubiquitination chain assembly complex (LUBAC). These mutations resulted in impairment of LUBAC stability. NF-κB activation in response to interleukin 1β (IL-1β) was compromised in the patients' fibroblasts. By contrast, the patients' mononuclear leukocytes, particularly monocytes, were hyper-responsive to IL-1β. The consequences of human HOIL-1 and LUBAC deficiencies for IL-1β responses thus differed between cell types, consistent with the unique association of autoinflammation and immunodeficiency in these patients. These data suggest that LUBAC regulates NF-κB–dependent IL-1β responses differently in different cell types.

International audience; Staphylococcus aureus is a major pathogen responsible for both nosocomial and community-acquired infections. Central to its virulence is its ability to secrete haemolysins, pore-forming toxins and cytolytic peptides. The large number of membrane-damaging toxins and peptides produced during S. aureus infections has hindered a precise understanding of their specific roles in diseases. Here, we used comprehensive libraries of recombinant toxins and synthetic cytolytic peptides, of S. aureus mutants and clinical strains to investigate the role of these virulence factors in targeting human macrophages and triggering IL-1β release. We found that the Panton Valentine leukocidin (PVL) is the major trigger of IL-1β release and inflammasome activation in primary human macrophages. The cytolytic peptides, δ-haemolysin and PSMα3; the pore-forming toxins, γ-haemolysin and LukDE; and β-haemolysin synergize with PVL to amplify IL-1β release, indicating that these factors cooperate with PVL to trigger inflammation. PVL(+) S. aureus causes necrotizing pneumonia in children and young adults. The severity of this disease is due to the massive recruitment of neutrophils that cause lung damage. Importantly, we demonstrate that PVL triggers IL-1β release in human alveolar macrophages. Furthermore, IL-1β released by PVL-intoxicated macrophages stimulates the secretion of the neutrophil attracting chemokines, IL-8 and monocyte chemotactic protein-1, by lung epithelial cells. Finally, we show that PVL-induced IL-8/monocyte chemotactic protein-1 release is abolished by the inclusion of IL-1 receptor antagonist (IL-1Ra) in a mixed culture of lung epithelial cells and macrophages. Together, our results identify PVL as the predominant S. aureus secreted factor for triggering inflammasome activation in human macrophages and demonstrate how PVL-intoxicated macrophages orchestrate inflammation in the lung. Finally, our work suggests that anakinra, a synthetic IL-1Ra, may be an effective therapeutic agent to reduce the massive neutrophils infiltration observed during necrotizing pneumonia and decrease the resulting host-mediated lung injury.

International audience; The mycotoxin enniatin B (EnnB) is predominantly produced by species of the Fusarium genera, and often found in grain. The cytotoxic effect of EnnB has been suggested to be related to its ability to form ionophores in cell membranes. The present study examines the effects of EnnB on cell death, differentiation, proliferation and pro-inflammatory responses in the murine monocyte-macrophage cell line RAW 264.7. Exposure to EnnB for 24 h caused an accumulation of cells in the G0/G1-phase with a corresponding decrease in cyclin D1. This cell cycle-arrest was possibly also linked to the reduced cellular ability to capture and internalize receptors as illustrated by the lipid marker ganglioside GM1. EnnB also increased the number of apoptotic, early apoptotic and necrotic cells, as well as cells with elongated spindle-like morphology. The Neutral Red assay indicated that EnnB induced lysosomal damage; supported by transmission electron microscopy (TEM) showing accumulation of lipids inside the lysosomes forming lamellar structures/myelin bodies. Enhanced levels of activated caspase-1 were observed after EnnB exposure and the caspase-1 specific inhibitor ZYVAD-FMK reduced EnnB-induced apoptosis. Moreover, EnnB increased the release of interleukin-1 beta (IL-1β) in cells primed with lipopolysaccharide (LPS), and this response was reduced by both ZYVAD-FMK and the cathepsin B inhibitor CA-074Me. In conclusion, EnnB was found to induce cell cycle arrest, cell death and inflammation. Caspase-1 appeared to be involved in the apoptosis and release of IL-1β and possibly activation of the inflammasome through lysosomal damage and leakage of cathepsin B.

International audience; Interleukin-1β (IL-1β), a key-cytokine in osteoarthritis, impairs TGFβ signaling through TβRII down-regulation by increasing its degradation. Here, we investigated the molecular mechanism that controls TßRII fate in IL-1ß treated cells. Chondrocytes were treated with IL-1ß in the presence of different inhibitors. TßRII and Cav-1 expression were assayed by Western blot and RT-PCR. We showed that IL-1ß-induced degradation of TßRII is dependent on proteasome and on its internalization in caveolae. In addition, IL-1ß enhances Cav-1 expression, a major constituent of lipid raft. In conclusion, we enlighten a new mechanism by which IL-1ß antagonizes TGFß pathway and propose a model of TßRII turnover regulation upon IL-1ß treatment.

International audience; BACKGROUND: Hippocampal damages, which are accompanied by inflammation, are among the main causes of epilepsy acquisition. We previously reported that chronic intracerebroventricular (i.c.v.) injection of lipopolysaccharide (LPS) modulates epileptogenesis in rats. There is a network of gap junction channels in the hippocampus that contribute to epileptogenesis. Gap junction channels are formed by oligomeric protein subunits called connexins (Cx). Astrocytic Cx43 and neuronal Cx36 are expressed in the hippocampus. In order to find out the possible role of gap junctions in seizure-modulating effect of LPS and neuroinflammation, we studied the effect of central administration of LPS on expression of Cx36 and Cx43 in rat hippocampus. METHODS: LPS, 2.5 mug/rat/day, was injected i.c.v. to male Wistar rats for 14 days. mRNA and protein abundance of Cx36, Cx43 and IL1-β were measured in rat hippocampus by real time-PCR, Western blot and ELISA techniques, at the beginning, in the middle, and at the end of the treatment period. RESULTS: IL1-β protein level was significantly increased 6 h after first injection of LPS. Cx36 and Cx43 mRNA expression did not alter during chronic administration of LPS. A selective decrease in Cx43 protein expression was observed after 7 injections of LPS. CONCLUSION: It is suggested that Cx43 containing gap junctions in the hippocampus is down-regulated in response to chronic injection of LPS. This event can inhibit propagation of toxic and noxious molecules to neighboring cells and modulate hippocampal excitability and epileptogenesis.

ObjectivesInterleukin (IL) 34 is a new cytokine implicated in macrophage differentiation and osteoclastogenesis. This study assessed IL-34 expression in the tissue of patients with rheumatoid arthritis (RA).MethodsImmunohistochemistry was performed in synovial biopsies from patients with RA (n=20), osteoarthritis (n=3) or other inflammatory arthritis (n=4). IL-34 was detected in the synovial fluid by ELISA and its messenger RNA expression was studied by quantitative PCR in rheumatoid synovial fibroblasts after stimulation by tumour necrosis factor α (TNFα) and IL-1β. Wild-type, jnk1−/−–jnk2−/− and nemo−/− murine fibroblasts and pharmacological inhibition were used to determine the involvement of nuclear factor kappa B (NF-κB) and JNK in that effect.ResultsIL-34 was expressed in 24/27 biopsies, with three samples from RA patients being negative. A significant association was found between IL-34 expression and synovitis severity. Levels of IL-34 and the total leucocyte count in synovial fluid were correlated. TNFα and IL-1β stimulated IL-34 expression by synovial fibroblasts in a dose/time-dependent manner through the NF-κB and JNK pathway.ConclusionThis work for the first time identifies IL-34 expression in the synovial tissue of patients with arthritis. This cytokine, as a downstream effector of TNFα and IL-1β, may contribute to inflammation and bone erosions in RA.

International audience; IL-33, a novel IL-1 family member, is crucially expressed and involved in pulmonary diseases, but its regulation in viral diseases such as influenza A virus (IAV) remains unclear. This study aimed to characterize the expression and release of IL-33 in lungs of IAV-infected mice in vivo and in murine respiratory epithelial cells (MLE-15) in vitro. Our results provide evidence of up-regulation of IL-33 mRNA in IAV-infected murine lungs, compared with noninfected control mice. The overexpression of IL-33 was positively correlated with a significant increase in mRNA encoding the proinflammatory cytokines TNF-α, IFN-γ, IL-1β, and IL-6, and was also associated with an increase in IFN-β mRNA. A profound overexpression of IL-33 protein was evident in IAV-infected murine lungs and bronchoalveolar lavages of influenza-infected mice, compared with low concentrations in naive lungs in vivo. Immunolocalization highlighted the cellular expression of IL-33 in alveolar epithelial and endothelial cells, along with increased infiltrate cells in virus-infected lungs. Further in vitro experiments showed an induction of IL-33 transcript-in MLE-15 cells and human epithelial cells (A549) infected with different strains of IAV in comparison with noninfected cells. In conclusion, our findings evidenced a profound expression of IL-33 in lungs during both in vivo and in vitro IAV infections, suggesting a role for IL-33 in virus-induced lung infections.

In breast carcinomas, patient survival seems to be negatively affected by the recruitment of regulatory T cells (Treg) within lymphoid aggregates by CCL22. However, the mechanisms underpinning this process, which may be of broader significance in solid tumors, have yet to be described. In this study, we determined how CCL22 production is controlled in tumor cells. In human breast carcinoma cell lines, CCL22 was secreted at low basal levels that were strongly increased in response to inflammatory signals [TNF-α, IFN-γ, and interleukin (IL)-1β], contrasting with CCL17. Primary breast tumors and CD45+ infiltrating immune cells appeared to cooperate in driving CCL22 secretion, as shown clearly in cocultures of breast tumor cell lines and peripheral blood mononuclear cells (PBMC) or their supernatants. We determined that monocyte-derived IL-1β and TNF-α are key players as monocyte depletion or neutralization of these cytokines attenuated secretion of CCL22. However, when purified monocytes were used, exogenous human IFN-γ was also required to generate this response suggesting a role for IFN-γ–producing cells within PBMCs. In this setting, we found that human IFN-γ could be replaced by the addition of (i) IL-2 or K562-activated natural killer (NK) cells or (ii) resting NK cells in the presence of anti-MHC class I antibody. Taken together, our results show a dialogue between NK and tumor cells leading to IFN-γ secretion, which in turn associates with monocyte-derived IL-1β and TNF-α to drive production of CCL22 by tumor cells and subsequent recruitment of Treg. As one validation of this conclusion in primary breast tumors, we showed that NK cells and macrophages tend to colocalize within tumors. In summary, our findings suggest that at early times during tumorigenesis, the detection of tumor cells by innate effectors (monocytes and NK cells) imposes a selection for CCL22 secretion that recruits Treg to evade this early antitumor immune response. Cancer Res; 71(19); 6143–52. ©2011 AACR.

International audience; Interleukin-33 (IL-33) is thought to be released during cellular death as an alarming cytokine during the acute phase of disease, but its regulation in vivo is poorly understood. We investigated the expression of IL-33 in two mouse models of acute hepatitis by administering either carbon tetrachloride (CCl(4) ) or concanavalin A (ConA). IL-33 was overexpressed in both models but with a stronger induction in ConA-induced hepatitis. IL-33 was weakly expressed in vascular and sinusoidal endothelial cells from normal liver and was clearly induced in CCl(4) -treated mice. Surprisingly, we found that hepatocytes strongly expressed IL-33 exclusively in the ConA model. CD1d knock-out mice, which are deficient in NKT cells and resistant to ConA-induced hepatitis, no longer expressed IL-33 in hepatocytes following ConA administration. Interestingly, invariant NKT (iNKT) cells adoptively transferred into ConA-treated CD1d KO mouse restored IL-33 expression in hepatocytes. This strongly suggests that NKT cells are responsible for the induction of IL-33 in hepatocytes.

International audience; Leptin is known to exert cardiodepressive effects and to induce left ventricular (LV) remodelling. Nevertheless, the autocrine and/or paracrine activities of this adipokine in the context of post-infarct dysfunction and remodelling have not yet been elucidated. Therefore, we have investigated the evolution of myocardial leptin expression following myocardial infarction (MI) and evaluated the consequences of specific cardiac leptin inhibition on subsequent LV dysfunction. Anaesthetized rats were subjected to temporary coronary occlusion. An antisense oligodesoxynucleotide (AS ODN) directed against leptin mRNA was injected intramyocardially along the border of the infarct 5 days after surgery. Cardiac morphometry and function were monitored by echocardiography over 11 weeks following MI. Production of myocardial leptin and pro-inflammatory cytokines interleukin (IL)-1β and IL-6 were assessed by ELISA. Our results show that (1) cardiac leptin level peaks 7 days after reperfused MI; (2) intramyocardial injection of leptin-AS ODN reduces early IL-1β and IL-6 overexpression and markedly protects contractile function. In conclusion, our findings demonstrate that cardiac leptin expression after MI could contribute to the evolution towards heart failure through autocrine and/or paracrine actions. The detrimental effect of leptin could be mediated by pro-inflammatory cytokines such as IL-1β and IL-6. Our data could constitute the basis of new therapeutic approaches aimed to improve post-MI outcome.

International audience; The pro-inflammatory cytokine IL-1β has been shown to promote angiogenesis. It can have a neurotoxic or neuroprotective effect. Here, we have studied the expression of IL-1β in vivo and the effect of the IL-1 receptor antagonist on choroidal neovascularization (CNV) and retinal degeneration (RD). IL-1β expression significantly increased after laser injury (real time PCR) in C57BL/6 mice, in the C57BL/6 Cx3cr1(-/-) model of age-related macular degeneration (enzyme-linked immunoabsorbent assay), and in albino Wistar rats and albino BALB Cx3cr1(+/+) and Cx3cr1(-/-) mice (enzyme-linked immunoabsorbent assay) after light injury. IL-1β was localized to Ly6G-positive, Iba1-negative infiltrating neutrophils in laser-induced CNV as determined by IHC. IL-1 receptor antagonist treatment significantly inhibited CNV but did not affect Iba1-positive macrophage recruitment to the injury site. IL-1β significantly increased endothelial cell outgrowth in aortic ring assay independently of vascular endothelial growth factor, suggesting a direct effect of IL-1β on choroidal endothelial cell proliferation. Inhibition of IL-1β in light- and laser-induced RD models did not alter photoreceptor degeneration in Wistar rats, C57BL/6 mice, or RD-prone Cx3cr1(-/-) mice. Our results suggest that IL-1β inhibition might represent a valuable and safe alternative to inhibition of vascular endothelial growth factor in the control of CNV in the context of concomitant photoreceptor degeneration as observed in age-related macular degeneration.

International audience; N-6 and n-3 polyunsaturated fatty acids (PUFAs) have been shown to prevent tissue release of inflammatory molecules. We have shown that a combination of n-6 and n-3 PUFAs is more efficient than single supplementations on the long-term consequences of intraocular pressure elevation. We hypothesized that such an association is also more effective during early retinal stress by modifying retinal proinflammatory prostaglandin and cytokine productions. Rats were supplemented for 3 months with n-6 PUFAs, n-3 PUFAs, or both n-6 and n-3 PUFAs. After 3 months, a surgical elevation of intraocular pressure was induced. Retinal morphometry and glial cell activation were evaluated 24 hours after laser treatment. The retinal levels of prostaglandin E(1) (PGE(1)) and prostaglandin E(2) (PGE(2)) and the messenger RNA levels of interleukin-1β, interleukin-6, and tumor necrosis factor-α were measured. Retinal glial cell activation after laser treatment was partly prevented by dietary n-6, n-3, and n-6 and n-3 PUFAs. Retinal PGE(1) was unaffected by the laser treatment or by the diet. Dietary n-6 and/or n-3 PUFAs prevented the increase in PGE(2) levels observed in laser-treated retinas without affecting the induction of interleukin-1β, interleukin-6, and tumor necrosis factor-α messenger RNAs. This study shows that not only a combination of n-6 and n-3 PUFAs but also single supplementations can preserve the retina from early glial cell activation and PGE(2) release. The protective effect is not mediated by changes in cytokine expression but may be related to modifications in retinal prostaglandin metabolism.

We have shown previously that peripheral lymph node-resident retinoic acid receptor-related orphan receptor γt+ NK1.1− invariant NKT (iNKT) cells produce IL-17A independently of IL-6. In this study, we show that the concomitant presence of IL-1 and IL-23 is crucial to induce a rapid and sustained IL-17A/F and IL-22 response by these cells that requires TCR–CD1d interaction and partly relies on IL-23–mediated upregulation of IL-23R and IL-1R1 expression. We further show that IL-1 and IL-23 produced by pathogen-associated molecular pattern-stimulated dendritic cells induce this response from NK1.1− iNKT cells in vitro, involving mainly TLR2/4-signaling pathways. Finally, we found that IL-17A production by these cells occurs very early and transiently in vivo in response to heat-killed bacteria. Overall, our study indicates that peripheral lymph node NK1.1− iNKT cells could be a source of innate Th17-related cytokines during bacterial infections and supports the hypothesis that they are able to provide an efficient first line of defense against bacterial invasion.

BackgroundIdiopathic pulmonary fibrosis is a devastating as yet untreatable disease. We demonstrated recently the predominant role of the NLRP3 inflammasome activation and IL-1β expression in the establishment of pulmonary inflammation and fibrosis in mice.MethodsThe contribution of IL-23 or IL-17 in pulmonary inflammation and fibrosis was assessed using the bleomycin model in deficient mice.ResultsWe show that bleomycin or IL-1β-induced lung injury leads to increased expression of early IL-23p19, and IL-17A or IL-17F expression. Early IL-23p19 and IL-17A, but not IL-17F, and IL-17RA signaling are required for inflammatory response to BLM as shown with gene deficient mice or mice treated with neutralizing antibodies. Using FACS analysis, we show a very early IL-17A and IL-17F expression by RORγt(+) γδ T cells and to a lesser extent by CD4αβ(+) T cells, but not by iNKT cells, 24 hrs after BLM administration. Moreover, IL-23p19 and IL-17A expressions or IL-17RA signaling are necessary to pulmonary TGF-β1 production, collagen deposition and evolution to fibrosis.ConclusionsOur findings demonstrate the existence of an early IL-1β-IL-23-IL-17A axis leading to pulmonary inflammation and fibrosis and identify innate IL-23 and IL-17A as interesting drug targets for IL-1β driven lung pathology.

Inflammatory bowel diseases (IBD) are characterized during their active phase by polymorphonuclear leukocyte (PMNL) transepithelial migration. The efflux of PMNL into the mucosa is associated with the production of proinflammatory cytokines and the release of ATP from damaged and necrotic cells. The expression and function of purinergic P2X7receptor (P2X7R) in intestinal epithelial cells (IEC) and its potential role in the “cross talk” between IEC and PMNL have not been explored. The aims of the present study were 1) to examine P2X7R expression in IEC (T84 cells) and in human intestinal biopsies; 2) to detect any changes in P2X7R expression in T84 cells during PMNL transepithelial migration, and during the active and quiescent phases of IBD; and 3) to test whether P2X7R stimulation in T84 monolayers can induce caspase-1 activation and IL-1β release by IEC. We found that a functional ATP-sensitive P2X7R is constitutively expressed at the apical surface of IEC T84 cells. PMNL transmigration regulates dynamically P2X7R expression and alters its distribution from the apical to basolateral surface of IEC during the early phase of PMNL transepithelial migration in vitro. P2X7R expression was weak in intestinal biopsies obtained during the active phase of IBD. We show that activation of epithelial P2X7R is mandatory for PMNL-induced caspase-1 activation and IL-1β release by IEC. Overall, these changes in P2X7R function may serve to tailor the intensity of the inflammatory response and to prevent IL-1β overproduction and inflammatory disease.

Victoni, Tatiana Coelho, Fernando Rodrigues Soares, Alexandre Learth de Freitas, Andressa Secher, Thomas Guabiraba, Rodrigo Erard, Francois de Oliveira-Filho, Ricardo Martins Vargaftig, B Boris Lauvaux, Gregoire Kamal, Mamdouh A Ryffel, Bernhard Moser, Rene Tavares-de-Lima, Wothan Germany Med Microbiol Immunol. 2010 Feb;199(1):35-42. doi: 10.1007/s00430-009-0134-5. Epub 2009 Nov 26.; International audience; Innate immune responses against microorganisms may be mediated by Toll-like receptors (TLRs). Intestinal ischemia-reperfusion (i-I/R) leads to the translocation of bacteria and/or bacterial products such as endotoxin, which activate TLRs leading to acute intestinal and lung injury and inflammation observed upon gut trauma. Here, we investigated the role of TLR activation by using mice deficient for the common TLR adaptor protein myeloid differentiation factor 88 (MyD88) on local and remote inflammation following intestinal ischemia. Balb/c and MyD88(-/-) mice were subjected to occlusion of the superior mesenteric artery (45 min) followed by intestinal reperfusion (4 h). Acute neutrophil recruitment into the intestinal wall and the lung was significantly diminished in MyD88(-/-) after i-I/R, which was confirmed microscopically. Diminished neutrophil recruitment was accompanied with reduced concentration of TNF-alpha and IL-1beta level. Furthermore, diminished microvascular leak and bacteremia were associated with enhanced survival of MyD88(-/-) mice. However, neither TNF-alpha nor IL-1beta neutralization prevented neutrophil recruitment into the lung but attenuated intestinal inflammation upon i-I/R. In conclusion, our data demonstrate that disruption of the TLR/MyD88 pathway in mice attenuates acute intestinal and lung injury, inflammation, and endothelial damage allowing enhanced survival.

Lung emphysema and fibrosis are severe complications of chronic obstructive pulmonary disease, and uncontrolled protease activation may be involved in the pathogenesis. Using experimental elastase-induced acute inflammation, we demonstrate here that inflammation and development of emphysema is IL-1R1 and Toll/IL-1R signal transduction adaptor MyD88 dependent; however, TLR recognition is dispensable in this model. Elastase induces IL-1β, TNF-α, keratinocyte-derived chemokine, and IL-6 secretion and neutrophil recruitment in the lung, which is drastically reduced in the absence of IL-1R1 or MyD88. Further, tissue destruction with emphysema and fibrosis is attenuated in the lungs of IL-1R1- and MyD88-deficient mice. Specific blockade of IL-1 by IL-1R antagonist diminishes acute inflammation and emphysema. Finally, IL-1β production and inflammation are reduced in mice deficient for the NALP3 inflammasome component apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and we identified uric acid, which is produced upon elastase-induced lung injury, as an activator of the NALP3/ASC inflammasome. In conclusion, elastase-mediated lung pathology depends on inflammasome activation with IL-1β production. IL-1β therefore represents a critical mediator and a possible therapeutic target of lung inflammation leading to emphysema.

International audience; BACKGROUND: Pulmonary artery remodeling triggered by alveolar hypoxia is considered the main mechanism of pulmonary hypertension (PH) in COPD patients. We hypothesized that the risk for PH in COPD is increased by an elevation in the proinflammatory cytokines interleukin (IL)-6, monocyte chemoattractant protein-1 (MCP-1), and IL-1beta, as well as by specific genetic polymorphisms of these cytokines. METHODS: We assessed cytokine plasma levels and the polymorphisms G(-174)C IL-6, C(-511)T IL-1beta, and A(-2518)G MCP-1 in 148 COPD patients (recruited at two centers) with right heart catheterization data and 180 control subjects including smokers and nonsmokers. Human pulmonary artery smooth muscle cells (PA-SMCs) were cultured for IL-6 messenger RNA assays under normoxic and hypoxic conditions. RESULTS: Patients with PH (mean pulmonary artery pressure [PAP], >or= 25 mm Hg) had lower Pao(2) and higher plasma IL-6 values than those without PH; there were no differences in terms of pulmonary function test results or CT scan emphysema scores. Plasma IL-6 correlated with mean PAP (r = 0.39; p < 0.001) and was included in a multiple stepwise regression analysis, with mean PAP as the dependent variable. In patients with the IL-6 GG genotype, the mean PAP value was significantly higher and PH was more common than in CG or CC patients (adjusted odds ratio, 4.32; 95% confidence interval, 1.96 to 9.54). Exposure to 4 h of hypoxia led to an about twofold increase in IL-6 messenger RNA in cultured human PA-SMCs. CONCLUSIONS: Inflammation, most likely involving IL-6, may contribute substantially to PH complicating COPD.

International audience; RATIONALE: Lung injury leads to pulmonary inflammation and fibrosis through myeloid differentiation primary response gene 88 (MyD88) and the IL-1 receptor 1 (IL-1R1) signaling pathway. The molecular mechanisms by which lung injury triggers IL-1beta production, inflammation, and fibrosis remain poorly understood. OBJECTIVES: To determine if lung injury depends on the NALP3 inflammasome and if bleomycin (BLM)-induced lung injury triggers local production of uric acid, thereby activating the NALP3 inflammasome in the lung. Methods: Inflammation upon BLM administration was evaluated in vivo in inflammasome-deficient mice. Pulmonary uric acid accumulation, inflammation, and fibrosis were analyzed in mice treated with the inhibitor of uric acid synthesis or with uricase, which degrades uric acid. MEASUREMENTS AND MAIN RESULTS: Lung injury depends on the NALP3 inflammasome, which is triggered by uric acid locally produced in the lung upon BLM-induced DNA damage and degradation. Reduction of uric acid levels using the inhibitor of uric acid synthesis allopurinol or uricase leads to a decrease in BLM-induced IL-1beta production, lung inflammation, repair, and fibrosis. Local administration of exogenous uric acid crystals recapitulates lung inflammation and repair, which depend on the NALP3 inflammasome, MyD88, and IL-1R1 pathways and Toll-like receptor (TLR)2 and TLR4 for optimal inflammation but are independent of the IL-18 receptor. CONCLUSIONS: Uric acid released from injured cells constitutes a major endogenous danger signal that activates the NALP3 inflammasome, leading to IL-1beta production. Reducing uric acid tissue levels represents a novel therapeutic approach to control IL-1beta production and chronic inflammatory lung pathology.

OBJECTIVE—Obesity is characterized by an overgrowth of adipose tissue that leads to the formation of hypoxic areas within this tissue. We investigated whether this phenomenon could be responsible for insulin resistance by studying the effect of hypoxia on the insulin signaling pathway in adipocytes. RESEARCH DESIGN AND METHODS—The hypoxic signaling pathway was modulated in adipocytes from human and murine origins through incubation under hypoxic conditions (1% O2) or modulation of hypoxia-inducible factor (HIF) expression. Insulin signaling was monitored through the phosphorylation state of several key partners of the pathway and glucose transport. RESULTS—In both human and murine adipocytes, hypoxia inhibits insulin signaling as revealed by a decrease in the phosphorylation of insulin receptor. In 3T3-L1 adipocytes, this inhibition of insulin receptor phosphorylation is followed by a decrease in the phosphorylation state of protein kinase B and AS160, as well as an inhibition of glucose transport in response to insulin. These processes were reversible under normoxic conditions. The mechanism of inhibition seems independent of protein tyrosine phosphatase activities. Overexpression of HIF-1α or -2α or activation of HIF transcription factor with CoCl2 mimicked the effect of hypoxia on insulin signaling, whereas downregulation of HIF-1α and -2α by small interfering RNA inhibited it. CONCLUSIONS—We have demonstrated that hypoxia creates a state of insulin resistance in adipocytes that is dependent upon HIF transcription factor expression. Hypoxia could be envisioned as a new mechanism that participates in insulin resistance in adipose tissue of obese patients.

International audience; The Candida albicans gpi7/gpi7 null mutant strain (Deltagpi7), which is affected in glycosylphosphatidylinositol (GPI) anchor biosynthesis, showed a reduced virulence following systemic infection of C57BL/6 mice. In vitro production of TNF-alpha, IL-6 and IL-1beta by macrophages in response to Deltagpi7 cells was significantly increased as compared to control (wild type GPI7/GPI7 and revertant gpi7/GPI7) cells; this probably contributes to the enhanced recruitment of neutrophils to the peritoneal cavity in response to Deltagpi7 cells. Survival of knockout mice for Toll-like receptor (TLR) 2 and TLR4 following intravenous injection of Deltagpi7 cells showed no significant differences as compared to C57BL/6 mice. In vitro production of TNF-alpha by macrophages and neutrophil recruitment were significantly inhibited in TLR2-/- mice in response to control yeast strains. Interestingly both TNF-alpha production and neutrophil recruitment in response to Deltagpi7 were significantly increased in all three types of mice, with no differences among them, and laminarin failed to inhibit this increased production of TNF-alpha. These results indicate that the enhanced proinflammatory response to Deltagpi7 does not involve recognition through TLR2, TLR4 nor dectin-1. Therefore, complete GPI anchors confer surface properties that are involved in modulation of cytokine production by macrophages in response to C. albicans.

International audience; Mycotoxins are fungal secondary metabolites responsible of food-mediated intoxication in animals and humans. Deoxynivalenol, ochratoxin A and patulin are the best known enteropathogenic mycotoxins able to alter intestinal functions resulting in malnutrition, diarrhea, vomiting and intestinal inflammation in vivo. Although their effects on intestinal barrier and transport activities have been extensively characterized, the mechanisms responsible for their pro-inflammatory effect are still poorly understood. Here we investigated if mycotoxin-induced intestinal inflammation results from a direct and/or indirect pro-inflammatory activity of these mycotoxins on human intestinal epithelial cells, using differentiated Caco-2 cells as model and interleukin 8 (IL-8) as an indicator of intestinal inflammation. Deoxynivalenol was the only mycotoxin able to directly increase IL-8 secretion (10- to 15-fold increase). We also investigated if these mycotoxins could indirectly stimulate IL-8 secretion through: (i) a modulation of the action of pro-inflammatory molecules such as the interleukin-1beta (IL-1beta), and/or (ii) an increase in the transepithelial passage of non-invasive commensal Escherichia coli. We found that deoxynivalenol, ochratoxin A and patulin all potentiated the effect of IL-1beta on IL-8 secretion (ranging from 35% to 138% increase) and increased the transepithelial passage of commensal bacteria (ranging from 12- to 1544-fold increase). In addition to potentially exacerbate established intestinal inflammation, these mycotoxins may thus participate in the induction of sepsis and intestinal inflammation in vivo. Taken together, our results suggest that the pro-inflammatory activity of enteropathogenic mycotoxins is mediated by both direct and indirect effects.

International audience; Previous studies have demonstrated the early appearance of inflammatory cytokines in the systemic circulation after thermal injury both in humans and animals. The aim of this study was to evaluate the time course of several cytokines, IL-6, TNF-alpha and IL-1beta in serum, lung, liver and brain of severely burned rats during the first week after thermal injury. Cytokine measurements were performed by enzyme-linked immunosorbent assay (ELISA). The comparison between the sham-burned animals and animals with third-degree burns on 20% or 40% of their total body surface area allowed for the study of the inflammatory process relative to the size of the injury. Serum IL-6 levels, which were undetectable in sham-treated animals, peaked during the first hours after injury and were proportionate to the size of the area burned. After a few days, IL-6 increased once more, but only in the most severely burned rats. In lung, liver and brain, low but measurable basal levels of TNF-alpha and IL-1 were detected in sham-burned animals. Strikingly, IL-1beta levels remained significantly elevated in the lung after injury in animals having 20% and 40% burned skin area. Unexpectedly, both TNF-alpha and IL-1beta production decreased gradually in liver and brain after burn injury. Also, the inflammatory response after a burn injury appeared to be biphasic. The first period corresponded to the early release of IL-6 into the circulation, proportional to the severity of the injury. After a few days, a second period was marked by the extension of the inflammatory processes from the injured area to the rest of the body, particularly to lung, which could be considered as at potential risk of involvement in severely burned patients.

Acute cigarette smoke exposure of the airways (two cigarettes twice daily for three days) induces acute inflammation in mice. In this study, we show that airway inflammation is dependent on Toll-like receptor 4 and IL-1R1 signaling. Cigarette smoke induced a significant recruitment of neutrophils in the bronchoalveolar space and pulmonary parenchyma, which was reduced in TLR4-, MyD88-, and IL-1R1-deficient mice. Diminished neutrophil influx was associated with reduced IL-1, IL-6, and keratinocyte-derived chemokine levels and matrix metalloproteinase-9 activity in the bronchoalveolar space. Further, cigarette smoke condensate (CSC) induced a macrophage proinflammatory response in vitro, which was dependent on MyD88, IL-1R1, and TLR4 signaling, but not attributable to LPS. Heat shock protein 70, a known TLR4 agonist, was induced in the airways upon smoke exposure, which probably activates the innate immune system via TLR4/MyD88, resulting in airway inflammation. CSC-activated macrophages released mature IL-1β only in presence of ATP, whereas CSC alone promoted the TLR4/MyD88 signaling dependent production of IL-1α and pro-IL-1β implicating cooperation between TLRs and the inflammasome. In conclusion, acute cigarette exposure results in LPS-independent TLR4 activation, leading to IL-1 production and IL-1R1 signaling, which is crucial for cigarette smoke induced inflammation leading to chronic obstructive pulmonary disease with emphysema.

The molecular mechanisms of acute lung injury resulting in inflammation and fibrosis are not well established. Here we investigate the roles of the IL-1 receptor 1 (IL-1R1) and the common adaptor for Toll/IL-1R signal transduction, MyD88, in this process using a murine model of acute pulmonary injury. Bleomycin insult results in expression of neutrophil and lymphocyte chemotactic factors, chronic inflammation, remodeling, and fibrosis. We demonstrate that these end points were attenuated in the lungs of IL-1R1- and MyD88-deficient mice. Further, in bone marrow chimera experiments, bleomycin-induced inflammation required primarily MyD88 signaling from radioresistant resident cells. Exogenous rIL-1beta recapitulated a high degree of bleomycin-induced lung pathology, and specific blockade of IL-1R1 by IL-1 receptor antagonist dramatically reduced bleomycin-induced inflammation. Finally, we found that lung IL-1beta production and inflammation in response to bleomycin required ASC, an inflammasome adaptor molecule. In conclusion, bleomycin-induced lung pathology required the inflammasome and IL-1R1/MyD88 signaling, and IL-1 represented a critical effector of pathology and therapeutic target of chronic lung inflammation and fibrosis.

In a previous report (Cebo et al. J Biol Chem 276 (2001) 5685-5691), it was established that biologically active recombinant human IL-1alpha and IL-1beta had different carbohydrate-binding properties. IL-1alpha recognized a di-antennary N-glycan with two alpha2-3-linked sialic acid residues, whereas IL-1beta recognized the GM(4), a alpha2-3-linked sialylated glycosphingolipid. These different carbohydrate-binding properties of two interleukins binding to the same receptor (IL-1R) could explain why these molecules had different biological effects and cell specificities. Molecular modeling of the ligands and in silico docking experiments defined putative carbohydrate-recognition domains localized in the same area of the two molecules, a domain different from that defined as the type I IL-1R binding domain. The calculated pattern of hydrogen bonding and of van der Waals interactions fulfilled the essential features observed for calcium-independent lectins (mammalian, viral or bacterial). The analysis of the same domain of the third members of this family of molecules, the IL-1R-antagonist, indicated it did not fulfill the criteria for carbohydrate-recognition domains. It is proposed that its role as a pure antagonist is due to the absence of lectin activity and consequently explained its inability to associate IL-1R with other surface molecular complexes necessary for signaling.

Treatment of rat glomerular mesangial cell (GMC) cultures with pancreatic secreted phospholipase A 2 (sPLA 2 -IB) results in an enhanced expression of sPLA 2 -IIA and COX-2, possibly via binding to its specific M-type sPLA 2 receptor. In the current study, we have investigated the expression and regulation of sPLA 2 -IB and its receptor during glomerulonephritis (GN). In vivo we used the well-established rat model of anti-Thy 1.1 GN (anti-Thy 1.1-GN) to study the expression of sPLA 2 -IB and the M-type sPLA 2 receptor by immunohistochemistry. In addition, in vitro we determined the interkeukin (IL)-1 β -regulated mRNA and protein expression in primary rat glomerular mesangial and endothelial cells as well as in rat peripheral blood leukocytes (PBLs). Shortly after induction of anti-Thy 1.1-GN, sPLA 2 -IB expression was markedly upregulated in the kidney at 6–24h. Within glomeruli, the strongest sPLA 2 -IB protein expression was detected on infiltrated granulocytes and monocytes. However, at the same time, the M-type receptor was also markedly upregulated on resident glomerular cells. In vitro , the most prominent cytokine-stimulated secretion of sPLA 2 -IB was observed in monocytes isolated from rat PBLs. Treating glomerular endothelial cells (GECs) with cytokines elicited only weak sPLA 2 -IB expression, but treatment of these cells with exogenous sPLA 2 -IB resulted in a marked expression of the endogenous sPLA 2 -IB. Mesangial cells did not express sPLA 2 -IB at all. The M-type sPLA 2 receptor protein was markedly upregulated on cytokine-stimulated mesangial and endothelial cells as well as on lymphocytes and granulocytes. During anti-Thy 1.1 rat GN, sPLA 2 -IB and the M-type sPLA 2 receptor are induced as primary downstream genes stimulated by inflammatory cytokines. Subsequently, both sPLA 2 -IB and the M-type sPLA 2 receptor are involved in the autocrine and paracrine amplification of the inflammatory process in different resident and infiltrating cells.

International audience; Reactive oxygen species (ROS) are regulators of redox-sensitive cell signaling pathways. In osteoarthritis, human interleukin-1beta is implicated in cartilage destruction through an ROS-dependent matrix metalloproteinase production. To determine the molecular source of ROS production in the human IL-1beta (hIL-1beta)-sensitive chondrocyte immortalized cell line C-20/A4, transfected cells were constructed that overexpress NAD(P)H oxidases. First, RT-PCR analysis showed that the C-20/A4 cell line expressed Nox2, Nox4, p22( phox ), and p67( phox ), but not p47( phox ). It was found that ROS production by C-20/A4 chondrocytes does not depend on PMA and ionomycin activation. This indicates that Nox2 was not involved in the production of ROS. In C- 20/A4 cells that overexpress Nox4, hIL-1beta stimulated ROS production three times more than the normal production of C-20/A4 cells. Moreover, there was a fourfold increase in the production of collagenase (MMP-1) by chondrocytes that overexpress Nox4. Interestingly, MMP-1 production in cells that overexpress Nox2 was not sensitive to hIL-1beta. These data suggest that under hIL-1beta stimulation, C-20/A4 chondrocytes produce MMP-1 through a Nox4-mediated, ROS-dependent pathway.