PKC,ERK/p38 MAP kinases and NF‐κB targeted signalling play a role in the expression and release of IL‐1β and CXCL8 in Porphyromonas gingivalis‐infected THP1 cells

Porphyromonas gingivalis is a keystone pathogen in periodontitis and is gaining importance in cardiovascular pathogenesis. Protease‐activated receptors (PARs), toll‐like receptors (TLRs) and nucleotide‐binding oligomerization domain (NOD) on monocytes recognize the structural components on P. gingivalis, inducing inflammatory intermediates. Here, we elucidate the modulation of PARs, TLRs, NODs, and the role of MAPK and NF‐κB in IL‐1β and CXCL8 release. THP1 cells were stimulated with P. gingivalis wild‐type W50 and its isogenic gingipain mutants: Rgp mutant E8 and Kgp mutant K1A. We observed modulation of PARs, TLRs, NOD, IL‐1β and CXCL8 expression by P. gingivalis. Gingipains hydrolyse IL‐1β and CXCL8, which is more evident for IL‐1β accumulation at 24 h. Inhibition of PKC (protein kinase C), p38 and ERK (extracellular signal‐regulated kinases) partially reduced P. gingivalis‐induced IL‐1β at 6 h, whereas PKC and ERK reduced CXCL8 at both 6 and 24 h. Following NF‐κB inhibition, P. gingivalis‐induced IL‐1β and CXCL8 were completely suppressed to basal levels. Overall, TLRs, PARs and NOD possibly act in synergy with PKC, MAPK ERK/p38 and NF‐κB in P. gingivalis‐induced IL‐1β and CXCL8 release from THP1 cells. These pro‐inflammatory cytokines could affect leucocytes in circulation and exacerbate other vascular inflammatory conditions such as atherosclerosis.     

In Vitro Cytokine Responses to Periodontal Pathogens: Generalized Aggressive Periodontitis is Associated with Increased IL‐6 Response to Porphyromonas gingivalis

Generalized aggressive periodontitis (GAgP) is an inflammatory condition resulting in destruction of tooth‐supporting tissues. We examined the production of IL‐1β, IL‐6, tumour necrosis factor (TNF)‐α, IL‐12 and IL‐10 in cultures of peripheral mononuclear cells (MNC) from 10 patients with GAgP and 10 controls stimulated with periodontal pathogens or a control antigen, tetanus toxoid (TT) in the presence of autologous serum. The pathogens used were Porphyromonas gingivalis, Prevotella intermedia and Fusobacterium nucleatum, either as type strains or bacteria isolated from the participants’ inherent oral flora. The P. gingivalis ‐induced production of IL‐6 was approximately 2.5‐fold higher in patients with GAgP than in healthy controls (P < 0.05), while the corresponding TNF‐α production was non‐significantly elevated. IL‐1β production induced by P. gingivalis, as all cytokine responses induced by Pr. intermedia, F. nucleatum and TT was similar in the two groups. A reduced IL‐12p70 response to Pr. intermedia and F. nucleatum was observed in smokers compared to non‐smoking patients (P < 0.02). To assess the role of serum factors in the elevated IL‐6 response to P. gingivalis, MNC from two donors free of disease were stimulated with this bacterium in the presence of the various patient and control sera. An elevated IL‐6 and TNF‐α response was observed in the presence of patient sera (P < 0.01 and P < 0.04, respectively). The data suggest that an exaggerated production of IL‐6 occurs in GAgP, and that pro‐inflammatory serum factors play an essential role in the response.     

Influence of lipopolysaccharide and interleukin‐6 on RANKL and OPG expression and release in human periodontal ligament cells

Recent research into periodontal disease pathology focuses on the role of receptor activator of nuclear factor‐κB ligand (RANKL) and osteoprotegerin (OPG) in periodontal bone destruction processes. RANKL regulates the differentiation of osteoclast by binding to its specific receptor RANK, while OPG inhibits the differentiation of osteoclasts by binding RANKL and therefore preventing RANKL to bind RANK. The aim of the present study was to investigate the influence of Porphyromonas gingivalis lipopolysaccharide (LPS) and interleukin‐6 (IL‐6) on RANKL and OPG expression and release in periodontal ligament (PDL) cells. Human PDL cells were stimulated for 48 h with purified P. gingivalis LPS and IL‐6. OPG and sRANKL release were assessed by using enzyme‐linked immunosorbent assay technique. OPG and RANKL expression was quantitatively measured by using the real‐time PCR technique. Whereas P. gingivalis LPS induced sRANKL release, expression was only slightly increased, IL‐6 did not show an effect on RANKL expression or release. In conclusion the data demonstrate that stimulation of PDL cells with P. gingivalis LPS leads to an increased release of sRANKL, rather than increased RANKL expression. Through this action, P. gingivalis LPS may exert its biological effect on osteoclast formation and bone resorption.     

Porphyromonas gingivalis B cell Antigen Epitope Vaccine,pIRES‐ragB'‐mGITRL,Promoted RagB‐Specific Antibody Production and Tfh Cells Expansion

The outer membrane protein RagB is one of the major virulence factors of Porphyromonas gingivalis (P. gingivalis). To prevent periodontitis and associated systemic diseases induced by P. gingivalis, we built B cell antigen epitope vaccine characterized by pIRES‐ragB'‐mGITRL to induce a protective immune responses. The B cell antigen epitope and scrambled peptide of ragB were predicted, cloned into pIRES and constructed pIRES‐ragB', pIRES‐scrambled epitopes and pIRES‐ragB'‐mGITRL. pIRES‐ragB'‐mGITRL was transfected into COS‐7 cells. Subsequently, the 6‐week‐old female BALB/c mice were challenged by P. gingivalis following three time immunization by pIRES, pIRES‐ragB', pIRES‐scrambled epitopes and pIRES‐ragB'‐mGITRL. The levels of RagB‐specific antibody in the serum and Tfh cells in the spleen were measured by ELISA and flow cytometry, respectively. And higher levels of RagB‐specific IgG were produced in the immunized mice with pIRES‐ragB'‐mGITRL. Additionally, the number of Tfh cells was also expanded and lesions were diminished in pIRES‐ragB'‐mGITRL mice comparing with control groups. Our results clearly demonstrated that P. gingivalis B cell antigen epitope vaccine, pIRES‐ragB'‐mGITRL, could induce protective immune responses. Furthermore, our data also indicated that pIRES‐ragB'‐mGITRL was a potential therapeutic vaccine against P. gingivalis.     

Differential expression of immunoregulatory genes in monocytes in response to Porphyromonas gingivalis and Escherichia coli lipopolysaccharide

Porphyromonas gingivalis lipopolysaccharide (LPS) (strain W50) interacts with Toll‐like receptor 2 (TLR‐2) leading to cytokine expression and inflammation, and thereby plays a key role in the pathogenesis of periodontal disease. The aims of this study were to investigate gene expression of key regulatory mediators of innate immune responses in a human monocytic cell line (THP‐1) to P. gingivalis LPS and to compare these results with those obtained using the TLR‐4 ligand, Escherichia coli LPS. Custom‐made Taqman low‐density arrays were used for expression profiling of 45 different cytokine‐related genes. Both types of LPS highly up‐regulated interleukin (IL)‐1α and IL‐1β, IL‐18 receptor (IL‐18R), IL‐18R accessory protein and IL‐1 family (IL‐1F)9. Expression levels of IL‐1F6, IL‐1F7 and caspase‐1 were unaltered by either LPS. Genes for tumour necrosis factor‐α, IL‐6, leukaemia inhibitory factor and IL‐32 were also highly induced by both LPS. For a subset of genes, including CXC chemokine ligand 5 (CXCL5), expression was induced only by E. coli LPS or was up‐regulated more highly by E. coli compared with P. gingivalis LPS in THP‐1 monocytes. A similar expression pattern was also observed in dendritic cells. Analysis of signalling pathways which lead to CXCL5 expression indicated that the mechanisms underpinning the differential responses did not involve the recruitment of different adaptor proteins by TLR‐2 and TLR‐4, and therefore occur downstream of the receptor–adaptor complex. We conclude that differences in signalling pathways activated by TLR‐2 and TLR‐4 ligands lead to differential innate immune responses which may be important in polymicrobial diseases such as periodontal disease.     

Pannexin‐1‐dependent caspase‐1 activation and secretion of IL‐1β is regulated by zinc

Inflammatory processes induced by IL‐1β are critical for host defence responses, but are also implicated in disease. Zinc deficiency is a common consequence of, or contributor to, human inflammatory disease. However, the molecular mechanisms through which zinc contributes to inflammatory disease remain largely unknown. We report here that zinc metabolism regulates caspase‐1 activation and IL‐1β secretion. One of the endogenous mediators of IL‐1β secretion is adenosine triphosphate, acting via the P2X7‐receptor and caspase‐1 activation in cells primed with an inflammatory stimulus such as LPS. We show that this process is selectively abolished by a brief pre‐treatment with the zinc chelator N,N,N′,N′‐tetrakis‐(2‐pyridylmethyl) ethylene diamine (TPEN). These effects on IL‐1β secretion were independent of rapid changes in free zinc within the cell, not a direct effect on caspase‐1 activity, and upstream of caspase‐1 activation. TPEN did however inhibit the activity of pannexin‐1, a hemi‐channel critical for adenosine triphosphate and nigericin‐induced IL‐1β release. These data provide new insights into the mechanisms of caspase‐1 activation and how zinc metabolism contributes to inflammatory mechanisms.     

NTPDase1 governs P2X7‐dependent functions in murine macrophages

P2X₇ receptor is an adenosine triphosphate (ATP)‐gated ion channel within the multiprotein inflammasome complex. Until now, little is known about regulation of P2X₇ effector functions in macrophages. In this study, we show that nucleoside triphosphate diphosphohydrolase 1 (NTPDase1)/CD39 is the dominant ectonucleotidase expressed by murine peritoneal macrophages and that it regulates P2X₇‐dependent responses in these cells. Macrophages isolated from NTPDase1‐null mice (Entpd1^?/?) were devoid of all ADPase and most ATPase activities when compared with WT macrophages (Entpd1^+/+). Entpd1^?/? macrophages exposed to millimolar concentrations of ATP were more susceptible to cell death, released more IL‐1β and IL‐18 after TLR2 or TLR4 priming, and incorporated the fluorescent dye Yo‐Pro‐1 more efficiently (suggestive of increased pore formation) than Entpd1^+/+ cells. Consistent with these observations, NTPDase1 regulated P2X₇‐associated IL‐1β release after synthesis, and this process occurred independently of, and prior to, cytokine maturation by caspase‐1. NTPDase1 also inhibited IL‐1β release in vivo in the air pouch inflammatory model. Exudates of LPS‐injected Entpd1^?/? mice had significantly higher IL‐1β levels when compared with Entpd1^+/+ mice. Altogether, our studies suggest that NTPDase1/CD39 plays a key role in the control of P2X₇‐dependent macrophage responses.     

Expression and regulation of interleukin-33 in human monocytes

Interleukin‐33 (IL‐33) is an IL‐1 family cytokine that has a role in regulating T helper type 2 cytokines and mast cell development. Expression of IL‐33 is also associated with chronic inflammatory conditions such as rheumatoid arthritis. However, there is little information regarding IL‐33 in myeloid cell immune responses, which are important in immunity and inflammation. We therefore investigated the expression, intracellular location and regulation of myeloid cell IL‐33 by lipopolysaccharide (LPS) from Escherichia coli and the periodontal pathogen Porphyromonas gingivalis. We detected IL‐33 messenger RNA in the human promonocytic cell line THP‐1, in monocytes derived from these cells and in primary human monocytes. However, IL‐33 was not expressed in primary monocyte‐derived dendritic cells. Stimulation of monocytes with E. coli LPS (Toll‐like receptor 4 agonist) and LPS from P. gingivalis (Toll‐like receptor 2 agonist) up‐regulated IL‐33 at both the messenger RNA and protein levels but IL‐1β and tumour necrosis factor‐α had no effect. The IL‐33 protein was mainly found in the cytoplasm of monocytes with no evidence of nuclear translocation in stimulated cells. Furthermore, no IL‐33 secretion was detected after stimulation with LPS and/or ATP. These data indicate that the function, if any, of IL‐33 in activated monocytes is primarily intracellular. Interestingly, immunofluorescence analysis indicated that IL‐33 was sequestered in the nucleus of monocytes undergoing apoptosis but released into the extracellular milieu by LPS‐stimulated cells in which necrosis had been induced by freeze–thawing. Therefore, this endorses the view that IL‐33 may function as an ‘alarmin’ and have a role in signalling cellular damage and inflammatory disease pathogenesis through release from damaged or necrotic cells.     

Staphylococcus epidermidis from prosthetic joint infections induces lower IL‐1β release from human neutrophils than isolates from normal flora

The aim of this study was to test the hypothesis that Staphylococcus epidermidis isolated from prosthetic joint infections (PJIs) differs from S. epidermidis isolated from normal flora in terms of its capacity to induce activation of caspase‐1 and release of IL‐1β in human neutrophils. The amount of active caspase‐1 was determined over 6 h by detecting Ac‐YVAD‐AMC fluorescence in human neutrophils incubated with S. epidermidis isolates from PJIs (ST2) or normal flora. The amount of IL‐1β was detected by ELISA in neutrophil supernatants after 6 h of incubation. Mean IL‐1β release was lower after incubation with S. epidermidis from PJIs compared to isolates from normal flora, but no statistically significant difference was found in active caspase‐1. Substantial inter‐individual differences in both active caspase‐1 and IL‐1β were noted. These results suggest that evasion of innate immune response, measured as reduced capacity to induce release of IL‐1β from human neutrophils, might be involved in the predominance of ST2 in S. epidermidis PJIs, but that other microbe‐related factors are probably also important.     

Expression and regulation of the NALP3 inflammasome complex in periodontal diseases

Periodontitis is an infectious process characterized by inflammation affecting the supporting structures of the teeth. Porphyromonas gingivalis is a major oral bacterial species implicated in the pathogenesis of periodontitis. Processing of interleukin (IL)‐1 family cytokines is regulated by an intracellular innate immune response system, known as the NALP3 [nacht domain‐, leucine‐rich repeat‐, and pyrin domain (PYD)‐containing protein 3] inflammasome complex. The aim of the present study was to investigate by quantitative real‐time polymerase chain reaction (PCR) the mRNA expression of NALP3, its effector molecule apoptosis associated speck‐like protein (ASC), its putative antagonist NLRP2 (NLR family, PYD‐containing protein 2), IL‐1β and IL‐18 (i) in gingival tissues from patients with gingivitis (n = 10), chronic periodontitis (n = 18), generalized aggressive periodontitis (n = 20), as well as in healthy subjects (n = 20), (ii) in vitro in a human monocytic cell line (Mono‐Mac‐6), in response to P. gingivalis challenge for 6 h. The clinical data indicate that NALP3 and NLRP2, but not ASC, are expressed at significantly higher levels in the three forms of inflammatory periodontal disease compared to health. Furthermore, a positive correlation was revealed between NALP3 and IL‐1β or IL‐18 expression levels in these tissues. The in vitro data demonstrate that P. gingivalis deregulates the NALP3 inflammasome complex in Mono‐Mac‐6 cells by enhancing NALP3 and down‐regulating NLRP2 and ASC expression. In conclusion, this study reveals a role for the NALP3 inflammasome complex in inflammatory periodontal disease, and provides a mechanistic insight to the host immune responses involved in the pathogenesis of the disease by demonstrating the modulation of this cytokine‐signalling pathway by bacterial challenge.     

Association of IL‐1β +3954 C/T and IL‐10‐1082 G/A Cytokine Gene Polymorphisms with Susceptibility to Tuberculosis

Tuberculosis (TB) constitutes the major cause of death due to infectious diseases. Cytokines play a major role in defence against Mycobacterium tuberculosis infection. Polymorphisms in the genes encoding various cytokines have been associated with tuberculosis susceptibility. Household contacts (HHC) are at increased risk of developing the disease. In this study, we examined the association of IL‐1β and IL‐10 cytokine gene polymorphisms with risk of developing tuberculosis in TB patients, their HHC and healthy controls (HC) using JavaStat and SPSS. Multifactor dimensionality reduction (MDR) analyses were performed to explore the potential gene–gene interactions. The genotype and allele frequencies of IL‐1β +3954C/T polymorphism did not vary significantly between TB patients and HC. GG (P < 0.005, OR = 0.219 and 95% CI = 0.059–0.735) and GA (P < 0.0001, OR = 2.938 and 95% CI = 1.526–5.696) genotypes of IL‐10‐1082 G/A polymorphism were found to be significantly associated with patients versus HC. HHC with CC (P < 0.03, OR = 1.833 and 95% CI = 1.1–3.35) genotype in IL‐1β and GA (P < 0.0001, OR = 4.612 and 95% CI = 2.225–9.702) genotype in IL‐10 were at increased risk of developing tuberculosis. MDR tests revealed high‐risk genotypes in IL‐1β and IL‐10 based on the association model. Our results demonstrate that the polymorphisms of IL‐1β and IL‐10 genes may be valuable markers to predict the risk for the development of TB in household contacts.     

Association of Disease Severity with IL‐1 levels in Methotrexate‐treated Psoriasis Patients

Interleukin‐1 plays a key role in inflammation and keratinocyte activation. It is an important mediator in the initiation and maintenance of psoriatic plaques and may represent an attractive therapeutic target. The aim of this study is to evaluate the effect of Methotrexate (MTX) on IL‐1 α and IL‐1 β levels in both plasma and skin biopsy of patients with psoriasis and to investigate their association with clinical disease activity. Forty‐five control subjects and 58 patients with psoriasis were recruited for this study. The patients were treated with 7.5 mg of MTX per week for 12 weeks. Folic acid was given at 5 mg once daily except on the day of MTX for 12 weeks. Blood samples and lesional skin biopsy were taken. Disease severity was assessed by Psoriasis Area Severity Index (PASI) score. IL‐1 levels in plasma and skin biopsy were analysed using ELISA. PASI score declined significantly (P < 0.001) from day 0 to 12 weeks of MTX treatment. IL‐1 α level in plasma and skin biopsy was reduced at day 0 sample and elevated significantly (P < 0.001) after MTX treatment. IL‐1β level in plasma and skin biopsy was higher at day 0 sample and reduced significantly (P < 0.001) after MTX treatment. IL‐1α levels and PASI score showed inverse correlation score before and after treatment with MTX. Whereas IL‐1β levels showed positive correlation before and after treatment with MTX. Decreasing IL‐1β levels by MTXs in psoriasis may block the Th17 differentiation. This shows the therapeutic effect of MTX in controlling the immunopathogenesis of psoriasis.     

Reverse plasticity: TGF‐β and IL‐6 induce Th1‐to‐Th17‐cell transdifferentiation in the gut

Th17 cells are a heterogeneous population of pro‐inflammatory T cells that have been shown to mediate immune responses against intestinal bacteria. Th17 cells are highly plastic and can transdifferentiate to Th1/17 cells or unconventional Th1 cells, which are highly pathogenic in animal models of immune‐mediated diseases such as inflammatory bowel diseases. A recent European Journal of Immunology article by Liu et al. (Eur. J. Immunol. 2015. 45:1010–1018) showed, surprisingly, that Th1 cells have a similar plasticity, and could transdifferentiate to Th17 cells. Thus, IFN‐γ‐producing Th1 effector cells specific for an intestinal microbial antigen were shown to acquire IL‐17‐producing capacities in the gut in a mouse model of colitis, and in response to TGF‐β and IL‐6 in vitro. TGF‐β induced Runx1, and together with IL‐6 was shown to render the ROR‐γt and IL‐17 promoters in Th1 cells accessible for Runx1 binding. In this commentary, we discuss how this unexpected plasticity of Th1 cells challenges our view on the generation of Th1/17 cells with the capacity to co‐produce IL‐17 and IFN‐γ, and consider possible implications of this Th1‐to‐Th17‐cell conversion for therapies of inflammatory bowel diseases and protective immune responses against intracellular pathogens.     

Virulence‐dependent induction of interleukin‐10‐producing‐tolerogenic dendritic cells by Mycobacterium tuberculosis impedes optimal T helper type 1 proliferation

Mycobacterium tuberculosis inhibits optimal T helper type 1 (Th1) responses during infection. However, the precise mechanisms by which virulent M. tuberculosis limits Th1 responses remain unclear. Here, we infected dendritic cells (DCs) with the virulent M. tuberculosis strain H37Rv or the attenuated strain H37Ra to investigate the phenotypic and functional alterations in DCs and resultant T‐cell responses. H37Rv‐infected DCs suppressed Th1 responses more strongly than H37Ra‐infected DCs. Interestingly, H37Rv, but not H37Ra, impaired DC surface molecule expression (CD80, CD86 and MHC class II) due to prominent interleukin‐10 (IL‐10) production while augmenting the expression of tolerogenic molecules including PD‐L1, CD103, Tim‐3 and indoleamine 2,3‐dioxygenase on DCs in a multiplicity‐of‐infection (MOI) ‐dependent manner. These results indicate that virulent M. tuberculosis drives immature DCs toward a tolerogenic phenotype. Notably, the tolerogenic phenotype of H37Rv‐infected DCs was blocked in DCs generated from IL‐10^−/− mice or DCs treated with an IL‐10‐neutralizing monoclonal antibody, leading to restoration of Th1 polarization. These findings suggest that IL‐10 induces a tolerogenic DC phenotype. Interestingly, p38 mitogen‐activated protein kinase (MAPK) activation predominantly mediates IL‐10 production; hence, H37Rv tends to induce a tolerogenic DC phenotype through expression of tolerogenic molecules in the p38 MAPK–IL‐10 axis. Therefore, suppressing the tolerogenic cascade in DCs is a novel strategy for stimulating optimal protective T‐cell responses against M. tuberculosis infection.     

The role of toll‐like and protease‐activated receptors and associated intracellular signaling in Porphyromonas gingivalis‐infected gingival fibroblasts

Porphyromonas gingivalis, which is considered a keystone agent in periodontitis, has evolved elaborate mechanisms to grow and survive in a hostile milieu. The gingival fibroblast is the major cell type in the gingiva and is considered to be important in the periodontitis‐associated inflammation. As a part of the innate immune response, they produce cytokines such as CXCL8 and interleukin (IL)‐6 which are believed to contribute to the destruction of the tooth‐supporting tissues. This study investigates how the expression of protease‐activated receptors (PAR1, PAR2) and toll‐like receptors (TLR2, TLR4) changes with P. gingivalis exposure and how silencing of one receptor affects the expression of the other receptors. The importance of protein kinase C (PKC) and p38 in the regulation of CXCL8 and IL‐6 was also examined. Receptors were knockdown with small‐interfering RNA. PKC or p38 was blocked prior to stimulation with P. gingivalis. Fibroblasts were able to compensate for PAR1 knockdown with increased expression of PAR2. PKC and p38 were involved in the regulation of P. gingivalis‐induced CXCL8 and IL‐6. Our results indicate that PAR1 and PAR2 could be implicated in periodontitis and that PKC and P38 play a role in the inflammatory response in P. gingivalis‐infected gingival fibroblasts.     

Protein tyrosine phosphatase PTPN22 regulates IL‐1β dependent Th17 responses by modulating dectin‐1 signaling in mice

A single nucleotide polymorphism within the PTPN22 gene is a strong genetic risk factor predisposing to the development of multiple autoimmune diseases. PTPN22 regulates Syk and Src family kinases downstream of immuno‐receptors. Fungal β‐glucan receptor dectin‐1 signals via Syk, and dectin‐1 stimulation induces arthritis in mouse models. We investigated whether PTPN22 regulates dectin‐1 dependent immune responses. Bone marrow derived dendritic cells (BMDCs) generated from C57BL/6 wild type (WT) and Ptpn22^?/? mutant mice, were pulsed with OVA_323‐339 and the dectin‐1 agonist curdlan and co‐cultured in vitro with OT‐II T‐cells or adoptively transferred into OT‐II mice, and T‐cell responses were determined by immunoassay. Dectin‐1 activated Ptpn22^?/? BMDCs enhanced T‐cell secretion of IL‐17 in vitro and in vivo in an IL‐1β dependent manner. Immunoblotting revealed that compared to WT, dectin‐1 activated Ptpn22^?/? BMDCs displayed enhanced Syk and Erk phosphorylation. Dectin‐1 activation of BMDCs expressing Ptpn22^R619W (the mouse orthologue of human PTPN22^R620W) also resulted in increased IL‐1β secretion and T‐cell dependent IL‐17 responses, indicating that in the context of dectin‐1 Ptpn22^R619W operates as a loss‐of‐function variant. These findings highlight PTPN22 as a novel regulator of dectin‐1 signals, providing a link between genetically conferred perturbations of innate receptor signaling and the risk of autoimmune disease.     

Evaluation of peroxisome proliferator‐activated receptor agonists on interleukin‐5‐induced eosinophil differentiation

Peroxisome proliferator‐activated receptor (PPAR) agonists have been suggested as novel therapeutics for the treatment of inflammatory lung disease, such as allergic asthma. Treatment with PPAR agonists has been shown to inhibit airway eosinophilia in murine models of allergic asthma, which can occur through several mechanisms including attenuated generation of chemoattractants (e.g. eotaxin) and decreased eosinophil migrational responses. In addition, studies report that PPAR agonists can inhibit the differentiation of several cell types. To date, no studies have examined the effects of PPAR agonists on interleukin‐5 (IL‐5) ‐induced eosinophil differentiation from haemopoietic progenitor cells. Non‐adherent mononuclear cells or CD34⁺ cells isolated from the peripheral blood of allergic subjects were grown for 2 weeks in Methocult^® cultures with IL‐5 (10 ng/ml) and IL‐3 (25 ng/ml) in the presence of 1–1000 nm PPARα agonist (GW9578), PPARβ/δ agonist (GW501516), PPARγ agonist (rosiglitazone) or diluent. The number of eosinophil/basophil colony‐forming units (Eo/B CFU) was quantified by light microscopy. The signalling mechanism involved was assessed by phosphoflow. Blood‐extracted CD34⁺ cells cultured with IL‐5 or IL‐5 + IL‐3 formed Eo/B CFU, which were significantly inhibited by rosiglitazone (100 nm , P < 0·01) but not GW9578 or GW501516. In addition, rosglitazone significantly inhibited IL‐5‐induced phosphorylation of extracellular signal‐regulated kinase 1/2. We observed an inhibitory effect of rosiglitazone on eosinophil differentiation in vitro, mediated by attenuation of the extracellular signal‐regulated kinase 1/2 signalling pathway. These findings indicate that the PPARγ agonist can attenuate tissue eosinophilia by interfering with local differentiative responses.     

Suppressive and pro‐inflammatory roles for IL‐4 in the pathogenesis of experimental drug‐induced liver injury

The pathogenesis of immune‐mediated drug‐induced liver injury (DILI) following halogenated anesthetics, carbamazepine or alcohol has not been fully elucidated. Detecting cytochrome P450 2E1 (CYP2E1) IgG4 auto‐antibodies in anesthetic DILI patients suggests a role for IL‐4 in this hapten‐mediated process. We investigated IL‐4‐mediated mechanisms using our model of experimental DILI induced by immunizing BALB/c (WT) and IL‐4^?/? (KO) mice with S100 liver proteins covalently modified by a trifluoroacetyl chloride (TFA) hapten formed following halogenated anesthetic metabolism by CYP2E1. WT mice developed more hepatitis, TFA and S100 antibodies (p<0.01), as well as T‐cell proliferation to CYP2E1 and TFA (p<0.01) than KO mice. Additionally, WT CD4⁺ T cells adoptively transferred hepatitis to naïve Rag^?/? mice (p<0.01). Pro‐inflammatory cytokines were expectedly decreased in TFA hapten‐stimulated KO splenocyte supernatants (p<0.001); however, IL‐2 and IFN‐γ (p<0.05), as well as IL‐6 and IL‐10 (p<0.001) levels were elevated in CYP2E1‐stimulated KO splenocyte supernatants, suggesting dual IL‐4‐mediated pro‐inflammatory and regulatory responses. Anti‐IL‐10 administered to KO mice increased hepatitis, TFA and CYP2E1 antibodies in KO mice confirming a critical role for IL‐4. This is the first demonstration of dual roles for IL‐4 in the pathogenesis of immune‐mediated DILI by suppressing auto‐antigen‐induced regulatory responses while promoting hapten‐induced pro‐inflammatory responses.