The IL-1 Receptor Is Required to Maintain Neutrophil Viability and Function During Aspergillus fumigatus Airway Infection.

Aspergillus fumigatus airway infections are associated with increased rates of hospitalizations and declining lung function in patients with chronic lung disease. While the pathogenesis of invasive A. fumigatus infections is well studied, little is known about the development and progression of airway infections. Previous studies have demonstrated a critical role for the IL-1 cytokines, IL-1α and IL-1ß in enhancing pulmonary neutrophil recruitment during invasive aspergillosis. Here we use a mouse model of A. fumigatus airway infection to study the role of these IL-1 cytokines in immunocompetent mice. In the absence of IL-1 receptor signaling, mice exhibited reduced numbers of viable pulmonary neutrophils and increased levels of neutrophil apoptosis during fungal airway infection. Impaired neutrophil viability in these mice was associated with reduced pulmonary and systemic levels of G-CSF, and treatment with G-CSF restored both neutrophil viability and resistance to A. fumigatus airway infection. Taken together, these data demonstrate that IL-1 dependent G-CSF production plays a key role for host resistance to A. fumigatus airway infection through suppressing neutrophil apoptosis at the site of infection.

Role of IL-36γ/IL-36R Signaling in Corneal Innate Defense Against Candida albicans Keratitis.

Purpose: Interleukin (IL)-36 cytokines have been shown to play either beneficial or detrimental roles in the infection of mucosal tissues in a pathogen-dependent manner, but their involvement in fungal keratitis remains elusive. We herein investigated their expression and function in mediating corneal innate immunity against Candida albicans infection. Methods: Gene expression in mouse corneas with or without C. albicans infection was determined by regular RT- and real-time (q)-PCR, Western blot analysis, ELISA or proteome profile assay. The severity of C. albicans keratitis was assessed using clinical scoring, bacterial counting, and myeloperoxidase (MPO) activity as an indicator of neutrophil infiltration. IL36R knockout mice and IL-33-specific siRNA were used to assess the involvement IL-33 signaling in C. albicans-infected corneas. B6 CD11c-DTR mice and clodronate liposomes were used to define the involvement of dendritic cells (DCs) and macrophages in IL-36R signaling and C. albicans keratitis, respectively. Results: IL-36γ were up-regulated in C57BL6 mouse corneas in response to C. albicans infection. IL-36 receptor-deficient mice display increased severity of keratitis, with a higher fungal load, MPO, and IL-1ß levels, and lower soluble sIL-1Ra and calprotectin levels. Exogenous IL-36γ prevented fungal keratitis pathogenesis with lower fungal load and MPO activity, higher expression of sIL-1Ra and calprotectin, and lower expression of IL-1ß, at mRNA or protein levels. Protein array analysis revealed that the expression of IL-33 and REG3G were related to IL-36/IL36R signaling, and siRNA downregulation of IL-33 increased the severity of C. albicans keratitis. Depletion of dendritic cells or macrophages resulted in severe C. albicans keratitis and yet exhibited minimal effects on exogenous IL-36γ-induced protection against C. albicans infection in B6 mouse corneas. Conclusions: IL-36/IL36R signaling plays a protective role in fungal keratitis by promoting AMP expression and by suppressing fungal infection-induced expression of proinflammatory cytokines in a dendritic cell- and macrophage-independent manner.

Contributions of MyD88-dependent receptors and CD11c-positive cells to corneal epithelial barrier function against Pseudomonas aeruginosa.

Previously we reported that corneal epithelial barrier function against Pseudomonas aeruginosa was MyD88-dependent. Here, we explored contributions of MyD88-dependent receptors using vital mouse eyes and confocal imaging. Uninjured IL-1R (-/-) or TLR4 (-/-) corneas, but not TLR2 (-/-), TLR5 (-/-), TLR7 (-/-), or TLR9 (-/-), were more susceptible to P. aeruginosa adhesion than wild-type (3.8-fold, 3.6-fold respectively). Bacteria adherent to the corneas of IL-1R (-/-) or TLR5 (-/-) mice penetrated beyond the epithelial surface only if the cornea was superficially-injured. Bone marrow chimeras showed that bone marrow-derived cells contributed to IL-1R-dependent barrier function. In vivo, but not ex vivo, stromal CD11c+ cells responded to bacterial challenge even when corneas were uninjured. These cells extended processes toward the epithelial surface, and co-localized with adherent bacteria in superficially-injured corneas. While CD11c+ cell depletion reduced IL-6, IL-1ß, CXCL1, CXCL2 and CXCL10 transcriptional responses to bacteria, and increased susceptibility to bacterial adhesion (>3-fold), the epithelium remained resistant to bacterial penetration. IL-1R (-/-) corneas also showed down-regulation of IL-6 and CXCL1 genes with and without bacterial challenge. These data show complex roles for TLR4, TLR5, IL-1R and CD11c+ cells in constitutive epithelial barrier function against P. aeruginosa, with details dependent upon in vivo conditions.

Role of MyD88-adaptor-like gene polymorphism rs8177374 in modulation of malaria severity in the Pakistani population

Abstract Introduction: The present study was designed to investigate the association between rs8177374 polymorphism and malaria symptoms due to exposure of Plasmodium vivax and Plasmodium falciparum. Materials and methods: A total of 454 samples were included in the study (228 malaria patients and 226 healthy individuals). Malaria patients, divided into P. vivax and P. falciparum groups on the basis of the causative species of Plasmodium, were categorized into mild and severe on the basis of clinical outcomes according to WHO criteria. Healthy individuals were used as controls. Allele specific PCR based strategy was used for the identification of rs8177374 SNP. Results: MyD88-adaptor-like gene polymorphism was associated with susceptibility to malaria (p < 0.001). C allele frequency (0.74) was higher in the population compared to T allele frequency (0.26). CT genotype increased the susceptibility of malaria (OR: 2.661; 95% CI: 1.722-4.113) and was positively associated with mild malaria (OR: 5.609; 95% CI: 3.479-9.044, p = 0.00). On the other hand, CC genotype was associated with severe malaria (OR: 3.116; 95% CI: 1.560-6.224, p = 0.00). P. vivax infection rate was higher in CT genotype carriers compared to other genotypes (OR: 3.616; 95% CI: 2.219-5.894, p < 0.001). Conclusion: MyD88-adaptor-like/TIR domain containing adaptor protein polymorphism for single nucleotide polymorphism rs8177374 is related with the susceptibility of malaria.

Interleukin 1α and the inflammatory process.

Inflammation occurs after disruption of tissue homeostasis by cell stress, injury or infection and ultimately involves the recruitment and retention of cells of hematopoietic origin, which arrive at the affected sites to resolve damage and initiate repair. Interleukin 1α (IL-1α) and IL-1ß are equally potent inflammatory cytokines that activate the inflammatory process, and their deregulated signaling causes devastating diseases manifested by severe acute or chronic inflammation. Although much attention has been given to understanding the biogenesis of IL-1ß, the biogenesis of IL-1α and its distinctive role in the inflammatory process remain poorly defined. In this review we examine key aspects of IL-1α biology and regulation and discuss its emerging importance in the initiation and maintenance of inflammation that underlie the pathology of many human diseases.

Interleukin-1 Family Cytokines in Liver Diseases.

The gene encoding IL-1 was sequenced more than 30 years ago, and many related cytokines, such as IL-18, IL-33, IL-36, IL-37, IL-38, IL-1 receptor antagonist (IL-1Ra), and IL-36Ra, have since been identified. IL-1 is a potent proinflammatory cytokine and is involved in various inflammatory diseases. Other IL-1 family ligands are critical for the development of diverse diseases, including inflammatory and allergic diseases. Only IL-1Ra possesses the leader peptide required for secretion from cells, and many ligands require posttranslational processing for activation. Some require inflammasome-mediated processing for activation and release, whereas others serve as alarmins and are released following cell membrane rupture, for example, by pyroptosis or necroptosis. Thus, each ligand has the proper molecular process to exert its own biological functions. In this review, we will give a brief introduction to the IL-1 family cytokines and discuss their pivotal roles in the development of various liver diseases in association with immune responses. For example, an excess of IL-33 causes liver fibrosis in mice via activation and expansion of group 2 innate lymphoid cells to produce type 2 cytokines, resulting in cell conversion into pro-fibrotic M2 macrophages. Finally, we will discuss the importance of IL-1 family cytokine-mediated molecular and cellular networks in the development of acute and chronic liver diseases.

IL-1R/TLR2 through MyD88 Divergently Modulates Osteoclastogenesis through Regulation of Nuclear Factor of Activated T Cells c1 (NFATc1) and B Lymphocyte-induced Maturation Protein-1 (Blimp1).

Toll-like receptors (TLR) and the receptor for interleukin-1 (IL-1R) signaling play an important role in bacteria-mediated bone loss diseases including periodontitis, rheumatoid arthritis, and osteomyelitis. Recent studies have shown that TLR ligands inhibit the receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation from un-committed osteoclast precursors, whereas IL-1 potentiates RANKL-induced osteoclast formation. However, IL-1R and TLR belong to the same IL-1R/TLR superfamily, and activate similar intracellular signaling pathways. Here, we investigate the molecular mechanisms underlying the distinct effects of IL-1 and Porphyromonas gingivalis lipopolysaccharide (LPS-PG) on RANKL-induced osteoclast formation. Our results show that LPS-PG and IL-1 differentially regulate RANKL-induced activation of osteoclast genes encoding Car2, Ctsk, MMP9, and TRAP, as well as expression of NFATc1, a master transcription factor of osteoclastogenesis. Regulation of osteoclast genes and NFATc1 by LPS-PG and IL-1 is dependent on MyD88, an important signaling adaptor for both TLR and IL-1R family members. Furthermore, LPS-PG and IL-1 differentially regulate RANKL-costimulatory receptor OSCAR (osteoclast-associated receptor) expression and Ca(2+) oscillations induced by RANKL. Moreover, LPS-PG completely abrogates RANKL-induced gene expression of B lymphocyte-induced maturation protein-1 (Blimp1), a global transcriptional repressor of anti-osteoclastogenic genes encoding Bcl6, IRF8, and MafB. However, IL-1 enhances RANKL-induced blimp1 gene expression but suppresses the gene expression of bcl6, irf8, and mafb. Our study reveals the involvement of multiple signaling molecules in the differential regulation of RANKL-induced osteoclastogenesis by TLR2 and IL-1 signaling. Understanding the signaling cross-talk among TLR, IL-1R, and RANK is critical for identifying therapeutic strategies to control bacteria-mediated bone loss.

Gedunin Binds to Myeloid Differentiation Protein 2 and Impairs Lipopolysaccharide-Induced Toll-Like Receptor 4 Signaling in Macrophages.

Recognition of bacterial lipopolysaccharide (LPS) by innate immune system is mediated by the cluster of differentiation 14/Toll-like receptor 4/myeloid differentiation protein 2 (MD-2) complex. In this study, we investigated the modulatory effect of gedunin, a limonoid from species of the Meliaceae family described as a heat shock protein Hsp90 inhibitor, on LPS-induced response in immortalized murine macrophages. The pretreatment of wild-type (WT) macrophages with gedunin (0.01-100 µM, noncytotoxic concentrations) inhibited LPS (50 ng/ml)-induced calcium influx, tumor necrosis factor-α, and nitric oxide production in a concentration-dependent manner. The selective effect of gedunin on MyD88-adapter-like/myeloid differentiation primary response 88- and TRIF-related adaptor molecule/TIR domain-containing adapter-inducing interferon-ß-dependent signaling pathways was further investigated. The pretreatment of WT, TIR domain-containing adapter-inducing interferon-ß knockout, and MyD88 adapter-like knockout macrophages with gedunin (10 µM) significantly inhibited LPS (50 ng/ml)-induced tumor necrosis factor-α and interleukin-6 production, at 6 hours and 24 hours, suggesting that gedunin modulates a common event between both signaling pathways. Furthermore, gedunin (10 µM) inhibited LPS-induced prostaglandin E2 production, cyclooxygenase-2 expression, and nuclear factor κB translocation into the nucleus of WT macrophages, demonstrating a wide-range effect of this chemical compound. In addition to the ability to inhibit LPS-induced proinflammatory mediators, gedunin also triggered anti-inflammatory factors interleukin-10, heme oxygenase-1, and Hsp70 in macrophages stimulated or not with LPS. In silico modeling studies revealed that gedunin efficiently docked into the MD-2 LPS binding site, a phenomenon further confirmed by surface plasmon resonance. Our results reveal that, in addition to Hsp90 modulation, gedunin acts as a competitive inhibitor of LPS, blocking the formation of the Toll-like receptor 4/MD-2/LPS complex.

Unified Modeling of Familial Mediterranean Fever and Cryopyrin Associated Periodic Syndromes.

Familial mediterranean fever (FMF) and Cryopyrin associated periodic syndromes (CAPS) are two prototypical hereditary autoinflammatory diseases, characterized by recurrent episodes of fever and inflammation as a result of mutations in MEFV and NLRP3 genes encoding Pyrin and Cryopyrin proteins, respectively. Pyrin and Cryopyrin play key roles in the multiprotein inflammasome complex assembly, which regulates activity of an enzyme, Caspase 1, and its target cytokine, IL-1ß. Overproduction of IL-1ß by Caspase 1 is the main cause of episodic fever and inflammatory findings in FMF and CAPS. We present a unifying dynamical model for FMF and CAPS in the form of coupled nonlinear ordinary differential equations. The model is composed of two subsystems, which capture the interactions and dynamics of the key molecular players and the insults on the immune system. One of the subsystems, which contains a coupled positive-negative feedback motif, captures the dynamics of inflammation formation and regulation. We perform a comprehensive bifurcation analysis of the model and show that it exhibits three modes, capturing the Healthy, FMF, and CAPS cases. The mutations in Pyrin and Cryopyrin are reflected in the values of three parameters in the model. We present extensive simulation results for the model that match clinical observations.

Pseudomonas fluorescens alters the intestinal barrier function by modulating IL-1ß expression through hematopoietic NOD2 signaling.

BACKGROUND: Ileal Crohn's disease is related to NOD2 mutations and to a gut barrier dysfunction. Pseudomonas fluorescens has also been associated with ileal Crohn's disease. The aim of this study was to determine the impact of P. fluorescens on the paracellular permeability in ileum and Peyer's patches. METHODS: To explore this question, in vivo and ex vivo experiments were performed in wild-type, Nod2, Nod2, and IL-1R mice together with in vitro analyses using the Caco-2 (epithelial) and the THP-1 (monocyte) human cell lines. RESULTS: Pseudomonas fluorescens increased the paracellular permeability of the intestinal mucosa through the secretion of IL-1ß by the immune cell populations and the activation of myosin light chain kinase in the epithelial cells. Induction of the IL-1ß pathway required the expression of Nod2 in the hematopoietic compartment, and muramyl dipeptide (a Nod2 ligand) had an inhibitory effect. CONCLUSIONS: Pseudomonas fluorescens thus alters the homeostasis of the epithelial barrier function by a mechanism similar to that previously observed for Yersinia pseudotuberculosis. This work further documents a putative role of psychrotrophic bacteria in Crohn's disease.

Biphasic effects of luteolin on interleukin-1ß-induced cyclooxygenase-2 expression in glioblastoma cells.

Success in developing therapeutic approaches to target brain tumor-associated inflammation in patients has been limited. Given that the inflammatory microenvironment is a hallmark signature of solid tumor development, anti-inflammatory targeting strategies have been envisioned as preventing glioblastoma initiation or progression. Consumption of foods from plant origin is associated with reduced risk of developing cancers, a chemopreventive effect that is, in part, attributed to their high content of phytochemicals with potent anti-inflammatory properties. We explored whether luteolin, a common flavonoid in many types of plants, may inhibit interleukin (IL)-1ß function induction of the inflammation biomarker cyclooxygenase (COX)-2. We found that IL-1ß triggered COX-2 expression in U-87 glioblastoma cells and synergized with luteolin to potentiate or inhibit that induction in a biphasic manner. Luteolin pretreatment of cells inhibited IL-1ß-mediated phosphorylation of inhibitor of κB, nuclear transcription factor-κB (NF-κB) p65, extracellular signal-regulated kinase-1/2, and c-Jun amino-terminal kinase in a concentration-dependent manner. Luteolin also inhibited AKT phosphorylation and survivin expression, while it triggered both caspase-3 cleavage and expression of glucose-regulated protein 78. These effects were all potentiated by IL-1ß, in part through increased nuclear translocation of NF-κB p65. Finally, luteolin was able to reduce IL-1 receptor gene expression, and treatment with IL-1 receptor antagonist or gene silencing of IL-1 receptor prevented IL-1ß/luteolin-induced COX-2 expression. Our results document a novel adaptive cellular response to luteolin, which triggers anti-survival and anti-inflammatory mechanisms that contribute to the chemopreventive properties of this diet-derived molecule.

Acute increases in intramuscular inflammatory cytokines are necessary for the development of mechanical hypersensitivity in a mouse model of musculoskeletal sensitization.

Musculoskeletal pain is a widespread health problem in the United States. Back pain, neck pain, and facial pain are three of the most prevalent types of chronic pain, and each is characterized as musculoskeletal in origin. Despite its prevalence, preclinical research investigating musculoskeletal pain is limited. Musculoskeletal sensitization is a preclinical model of muscle pain that produces mechanical hypersensitivity. In a rodent model of musculoskeletal sensitization, mechanical hypersensitivity develops at the hind paws after injection of acidified saline (pH 4.0) into the gastrocnemius muscle. Inflammatory cytokines contribute to pain during a variety of pathologies, and in this study we investigate the role of local, intramuscular cytokines in the development of mechanical hypersensitivity after musculoskeletal sensitization in mice. Local intramuscular concentrations of interleukin-1ß (IL-1), IL-6 and tumor necrosis factor-α (TNF) were quantified following injection of normal (pH 7.2) or acidified saline into the gastrocnemius muscle. A cell-permeable inhibitor was used to determine the impact on mechanical hypersensitivity of inhibiting nuclear translocation of the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) prior to musculoskeletal sensitization. The role of individual cytokines in mechanical hypersensitivity following musculoskeletal sensitization was assessed using knockout mice lacking components of the IL-1, IL-6 or TNF systems. Collectively, our data demonstrate that acidified saline injection increases intramuscular IL-1 and IL-6, but not TNF; that intramuscular pre-treatment with an NF-κB inhibitor blocks mechanical hypersensitivity; and that genetic manipulation of the IL-1 and IL-6, but not TNF systems, prevents mechanical hypersensitivity following musculoskeletal sensitization. These data establish that actions of IL-1 and IL-6 in local muscle tissue play an acute regulatory role in the development of mechanical hypersensitivity following musculoskeletal sensitization.

Obesity, inflammation, and lung injury (OILI): the good.

Obesity becomes pandemic, predisposing these individuals to great risk for lung injury. In this review, we focused on the anti-inflammatories and addressed the following aspects: adipocytokines and obesity, inflammation and other mechanisms, adipocytokines and lung injury in obesity bridged by inflammation, and potential therapeutic targets. To sum up, the majority of evidence supported that adiponectin, omentin, and secreted frizzled-related protein 5 (SFRP5) were reduced significantly in obesity, which is associated with increased inflammation, indicated by increase of TNF α and IL-6, through activation of toll-like receptor (TLR4) and nuclear factor light chain κ B (NF- κ B) signaling pathways. Administration of these adipocytokines promotes weight loss and reduces inflammation. Zinc-α 2-glycoprotein (ZAG), vaspin, IL-10, interleukin-1 receptor antagonist (IL-1RA), transforming growth factor ß (TGF-ß1), and growth differentiation factor 15 (GDF15) are also regarded as anti-inflammatories. There were controversial reports. Furthermore, there is a huge lack of studies for obesity related lung injury. The effects of adiponectin on lung transplantation, asthma, chronic obstructive pulmonary diseases (COPD), and pneumonia were anti-inflammatory and protective in lung injury. Administration of IL-10 agonist reduces mortality of acute lung injury in rabbits with acute necrotizing pancreatitis, possibly through inhibiting proinflammation and strengthening host immunity. Very limited information is available for other adipocytokines.

Cell death triggered by Yersinia enterocolitica identifies processing of the proinflammatory signal adapter MyD88 as a general event in the execution of apoptosis.

Many pathogenic microorganisms have evolved tactics to modulate host cell death or survival pathways for establishing infection. The enteropathogenic bacterium Yersinia enterocolitica deactivates TLR-induced signaling pathways, which triggers apoptosis in macrophages. In this article, we show that Yersinia-induced apoptosis of human macrophages involves caspase-dependent cleavage of the TLR adapter protein MyD88. MyD88 was also cleaved when apoptosis was mediated by overexpression of the Toll-IL-1R domain-containing adapter inducing IFN-ß in epithelial cells. The caspase-processing site was mapped to aspartate-135 in the central region of MyD88. MyD88 is consequently split by caspases in two fragments, one harboring the death domain and the other the Toll-IL-1R domain. Caspase-3 was identified as the protease that conferred the cleavage of MyD88 in in vitro caspase assays. In line with a broad role of caspase-3 in the execution of apoptosis, the processing of MyD88 was not restricted to Yersinia infection and to proapoptotic Toll-IL-1R domain-containing adapter inducing IFN-ß signaling, but was also triggered by staurosporine treatment. The cleavage of MyD88 therefore seems to be a common event in the advanced stages of apoptosis, when caspase-3 is active. We propose that the processing of MyD88 disrupts its scaffolding function and uncouples the activation of TLR and IL-1Rs from the initiation of proinflammatory signaling events. The disruption of MyD88 may consequently render dying cells less sensitive to proinflammatory stimuli in the execution phase of apoptosis. The cleavage of MyD88 could therefore be a means of conferring immunogenic tolerance to apoptotic cells to ensure silent, noninflammatory cell demise.

Effect evaluation of interleukin-1 receptor antagonist nanoparticles for mesenchymal stem cell transplantation.

AIM: To study the efficacy of marrow mesenchymal stem cells (MSCs) transplantation combined with interleukin-1 receptor antagonist (IL-1Ra) for acute liver failure (ALF). METHODS: Chinese experimental miniature swine were randomly divided into four groups (n = 7), and all animals were given D-galactosamine (D-gal) to induce ALF. Group A animals were then injected with 40 mL saline via the portal vein 24 h after D-gal induction; Group B animals were injected with 2 mg/kg IL-1Ra via the ear vein 18 h, 2 d and 4 d after D-gal induction; Group C received approximately 1 × 10(8) green fluorescence protein (GFP)-labeled MSCs (GFP-MSCs) suspended in 40 mL normal saline via the portal vein 24 h after D-gal induction; Group D animals were injected with 2 mg/kg IL-1Ra via the ear vein 18 h after D-gal induction, MSCs transplantation was then carried out at 24 h after D-gal induction, and finally 2 mg/kg IL-1Ra was injected via the ear vein 1 d and 3 d after surgery as before. Liver function, serum inflammatory parameters and pathological changes were measured and the fate of MSCs was determined. RESULTS: The optimal efficiency of transfection (97%) was achieved at an multiplicity of infection of 80, as observed by fluorescence microscopy and flow cytometry (FCM). Over 90% of GFP-MSCs were identified as CD44+ CD90+ CD45- MSCs by FCM, which indicated that most GFP-MSCs retained MSCs characteristics. Biochemical assays, the levels of serum inflammatory parameters and histological results in Group D all showed a significant improvement in liver injury compared with the other groups (P < 0.05). The number of GFP-MSCs in Group D was also greater than that in Group B, and the long-term cell proliferation rate was also better in Group D than in the other groups. CONCLUSION: MSCs transplantation is useful in ALF, IL-1Ra plays an important role in alleviating the inflammatory condition, and combination therapy with MSCs transplantation and IL-1Ra is a promising treatment for ALF.

Spinal toll-like receptor signaling and nociceptive processing: regulatory balance between TIRAP and TRIF cascades mediated by TNF and IFNß.

Toll-like receptors (TLRs) play a pivotal role in inflammatory processes, and individual TLRs have been investigated in nociception. We examined overlapping and diverging roles of spinal TLRs and their associated adaptor proteins in nociceptive processing. Intrathecal (IT) TLR2, TLR3, or TLR4 ligands (-L) evoked persistent (7-day) tactile allodynia (TA) that was abolished in respective TLR-deficient mice. Using Tnf(-/-) mice, we found that IT TLR2 and TLR4 TA was tumor necrosis factor (TNF) dependent, whereas TLR3 was TNF-independent. In Toll-interleukin 1 receptor (TIR) domain-containing adaptor protein (TIRAP; Tirap(-/-)) mice (downstream to TLR2 and TLR4), allodynia after IT TLR2-L and TLR4-L was abolished. Unexpectedly, in TIR-domain-containing adapter-inducing interferon-ß (Trif(lps2)) mice (downstream of TLR3 and TLR4), TLR3-L allodynia was abrogated, but intrathecal TLR4-L produced a persistent increase (>21days) in TA. Consistent with a role for interferon (IFN) ß (downstream to TIR-domain-containing adapter-inducing IFNß [TRIF]) in regulating recovery after IT TLR4-L, prolonged allodynia was noted in Ifnar1(-/-) mice. Further, IT IFNß given to Trif(lps2) mice reduced TLR4 allodynia. Hence, spinal TIR domain-containing adaptor protein (TIRAP) and TRIF cascades differentially lead to robust TA by TNF-dependent and independent pathways, whereas activation of TRIF modulated processing through type I IFN receptors. Based on these results, we believe that processes leading to the activation of these spinal TLRs initiate TNF-dependent and -independent cascades, which contribute to the associated persistent pain state. In addition, TRIF pathways are able to modulate the TNF-dependent pain state through IFNß.

CNS response to a second pro-inflammatory event depends on whether the primary demyelinating lesion is active or resolved.

Interleukin-1ß (IL-1ß) is considered to be one of the most important mediators in the pathogenesis of inflammatory diseases, particularly in neurodegenerative diseases such as multiple sclerosis (MS). MS is a chronic inflammatory disease characterized by demyelination and remyelination events, with unpredictable relapsing and remitting episodes that seldom worsen MS lesions. We proposed to study the effect of a unique component of the inflammatory process, IL-1ß, and evaluate its effect in repeated episodes, similar to the relapsing-remitting MS pathology. Using adenoviral vectors, we developed a model of focal demyelination/remyelination triggered by the chronic expression of IL-1ß. The long-term expression of IL-1ß in the striatum produced blood-brain barrier (BBB) breakdown, demyelination, microglial/macrophage activation, and neutrophil infiltration but no overt neuronal degeneration. This demyelinating process was followed by complete remyelination of the area. This simple model allows us to study demyelination and remyelination independently of the autoimmune and adaptive immune components. Re-exposure to this cytokine when the first inflammatory response was still unresolved generated a lesion with decreased neuroinflammation, demyelination, axonal injury and glial response. However, a second long-term expression of IL-1ß when the first lesion was resolved could not be differentiated from the first event. In this study, we demonstrated that the response to a second inflammatory stimulus varies depending on whether the initial lesion is still active or has been resolved. Considering that anti-inflammatory treatments have shown little improvement in MS patients, studies about the behavior of specific components of the inflammatory process should be taken into account to develop new therapeutic tools.

Inflammasome-derived IL-1ß regulates the production of GM-CSF by CD4(+) T cells and γδ T cells.

Recent findings have demonstrated an indispensable role for GM-CSF in the pathogenesis of experimental autoimmune encephalomyelitis. However, the signaling pathways and cell populations that regulate GM-CSF production in vivo remain to be elucidated. Our work demonstrates that IL-1R is required for GM-CSF production after both TCR- and cytokine-induced stimulation of immune cells in vitro. Conventional αß and γδ T cells were both identified to be potent producers of GM-CSF. Moreover, secretion of GM-CSF was dependent on IL-1R under both IL-12- and IL-23-induced stimulatory conditions. Deficiency in IL-1R conferred significant protection from experimental autoimmune encephalomyelitis, and this correlated with reduced production of GM-CSF and attenuated infiltration of inflammatory cells into the CNS. We also find that GM-CSF production in vivo is not restricted to a defined CD4(+) T cell lineage but is rather heterogeneously expressed in the effector CD4(+) T cell population. In addition, inflammasome-derived IL-1ß upstream of IL-1R is a critical regulator of GM-CSF production by T cells during priming, and the adapter protein, MyD88, promotes GM-CSF production in both αß and γδ T cells. These findings highlight the importance of inflammasome-derived IL-1ß and the IL-1R/MyD88 signaling axis in the regulation of GM-CSF production.

Interleukin-1 receptor role in the viability of corneal myofibroblasts.

The purpose of this study was to investigate the role of interleukin-1 (IL-1) in modulating myofibroblast viability in mouse corneas with stromal opacity. Twenty-four female B6; 129S1-Il1r1tm1Roml/J homozygous IL-1RI knockout mice and 24 control B6129SF2/J mice were included in this study. Each mouse had opacity-generating irregular phototherapeutic keratectomy (PTK) performed with an excimer laser in one eye. Groups of 8 mice from each group were euthanized at one month, three months and six months after surgery and the eyes cryo-preserved. The contralateral eye served as unwounded control. Immunohistochemistry was performed for α-smooth muscle actin (SMA) in central sections of all corneas. The TUNEL assay for apoptosis was performed on 8 sections of four eyes from each group. No SMA+ cells were detected in the stroma of unwounded control or knockout corneas. SMA+ myofibroblast density was significantly higher (p < 0.001) in the IL-1RI knockout group than in the control group at one month, three and six months after irregular PTK. Mean TUNEL+ stromal cells in the anterior 50 µm of stroma was significantly lower in the IL-1RI knockout group compared to the control group at six months after irregular PTK (p = 0.04). These results corroborate the findings of recent in vitro work that demonstrated an antagonistic effect of TGFß and IL-1 on myofibroblast viability, and found that IL-1-triggered myofibroblast apoptosis was suppressed by TGFß. Thus, IL-1 is an important modulator of myofibroblast viability during corneal wound healing.

Novel TNF-α receptor 1 antagonist treatment attenuates arterial inflammation and intimal hyperplasia in mice.

AIM: Tumor necrosis factor receptor 1 (TNFR1) participates importantly in arterial inflammation in genetically altered mice; however it remains undetermined whether a selective TNFR1 antagonist inhibits arterial inflammation and intimal hyperplasia. This study aimed to determine the effect and mechanism of a novel TNFR1 antagonist in the suppression of arterial inflammation. METHODS: We investigated intimal hyperplasia in IL-1 receptor antagonist-deficient mice two weeks after inducing femoral artery injury in an external vascular cuff model. All mice received intraperitoneal injections of TNFR1 antagonist (PEG-R1antTNF) or normal saline twice daily for 14 days. RESULTS: PEG-R1antTNF treatment yielded no adverse systemic effects, and we observed no significant differences in serum cholesterol or blood pressure in either group; however, selective PEG-R1antTNF treatment significantly reduced intimal hyperplasia (19,671±4,274 vs. 11,440±3,292 µm(2); p=0.001) and the intima/media ratio (1.86±0.43 vs. 1.34±0.36; p=0.029), compared with saline injection. Immunostaining revealed that PEG-R1antTNF inhibits Nuclear factor-κB (NF-κB), suppressing smooth muscle cell (SMC) proliferation and decreasing chemokine and adhesion molecule expression, and thus decreasing intimal hyperplasia and inflammation. CONCLUSIONS: Our data suggest that PEG-R1antTNF suppresses SMC proliferation and inflammation by inhibiting NF-κB. This study highlights the potential therapeutic benefit of selective TNFR1 antagonist therapy in preventing intimal hyperplasia and arterial inflammation.