Expression Of IL-1RI Research Articles

Abstract 6813: Immunosuppressive monocytes suppress CART19 functions through modulation of the IL-1 pathway

Abstract CD19-directed chimeric antigen receptor T (CART19) cell therapy has shown remarkable outcomes in B cell malignancies and was FDA approved in multiple indications, but durable remissions are limited to ~40%. Inhibitory myeloid cells in the tumor microenvironment have been found to suppress T cell expansion and contribute to CART19 failure. Here, we studied interactions between monocytes, CART19, and tumor cells to understand the impact of monocytes on CART19 effector functions. First, CD28-costimulated CART19 (CART19-28ζ) generated in the lab from healthy donors were cocultured with JeKo-1, a CD19+ mantle cell lymphoma cell line, and freshly isolated monocytes or ex vivo differentiated M2-like macrophages. M2-like macrophages were generated by incubating fresh monocytes with rhGM-CSF followed by coculturing with JeKo-1. CART19 antigen-specific proliferation was significantly inhibited by M2-like macrophages but not fresh monocytes (JeKo-1+CART19 vs JeKo-1+M2+CART19, p=0.00503). Transwell assays indicated that suppression from M2-like macrophages was not contact-dependent. IL-1 receptor antagonist (IL-1ra) was significantly elevated in M2-like macrophage coculture supernatant (JeKo-1+Mono+CART19 vs JeKo-1+M2+CART19, p=0.0292) and intracellularly in M2-like macrophages after coculture (90%+). We therefore hypothesized that M2-like macrophages inhibit CART19 by secreting IL-1ra, which blocks IL-1 signaling in CART19. We interrogated the role of IL-1ra in CART19. JeKo-1 was cocultured with CART19, supplemented with rhIL-1β only, IL-1β + IL-1ra, or IL-1β +IL-1ra + IL-1ra neutralizing antibody (neuAb). CART19 antigen-specific proliferation was improved by IL-1β (PBS vs IL-1β, p<0.0001). IL-1ra inhibited IL-1β-dependent CART19 proliferation (IL-1β vs IL-1β+IL1ra p=0.0006), which was restored by IL-1ra neutralization (IL-1β+IL-1ra vs IL-1β+IL-1ra+IL-1ra neuAb, p=0.0215). Additionally, expression of IL-1RI, the receptor of IL-1β and IL-1ra, was measured on T cells after coculturing CART19, JeKo-1, and M2-like macrophages. M2-like macrophages downregulated IL-1RI expression on CART cells, which is a potential mechanism of blocking CART response to IL-1β, resulting in further suppression of CART proliferation (JeKo-1+M2+CART19 vs JeKo-1+CART19, p=0.001). Next, we assessed the impact of IL-1ra on CART cell functions in the presence of M2-like macrophage in a mantle cell lymphoma xenograft model. NOD-SCID-γ−/- (NSG) mice were subcutaneously injected with human macrophages and luciferase+ JeKo-1 cells. Upon tumor engraftment by bioluminescent imaging, mice were randomized to treatment with CART19 + IL-1ra neuAb or control IgG for 3 weeks. CART19 + IL-1ra neuAb led to improved antitumor activity (IL-1ra neuAb vs control IgG, p=0.0033). Overall, our study revealed a role of IL-1ra in macrophage-induced CART inhibition mediated by blocking IL-1β signaling through IL-1RI. Citation Format: Kun Yun, Reona Sakemura, Truc Huynh, Claudia Manriquez Roman, Olivia Sirpilla, Carli Stewart, James Girsch, Ekene Ogbodo, Ismail Can, Jennifer Feigin, Long Mai, Hong Xia, Brooke Kimball, Makena Rodriguez, Lionel Kankeu Fonkoua, Mehrdad Hefazi, Michael Ruff, Elizabeth Siegler, Saad Kenderian. Immunosuppressive monocytes suppress CART19 functions through modulation of the IL-1 pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6813.

Endometrial expression of members of the IL-1 family: their involvement in delayed conception of dairy cows

The cytokines of the interleukin-1 (IL-1) family are closely involved in the resolution of inflammation in cows with metritis and endometritis. However, little is known about the role of these cytokines beyond uterine regression in the absence of disease, especially around conception. Thus, the aim of this study was to examine the gene and protein expression of IL-1α, IL-1β, IL-1RI, IL-1RII and IL-1Ra in endometrial biopsies previous to conception, to evaluate the possible association of these cytokines with delayed conception in dairy cows. Gene and protein expression levels were evaluated by real-time PCR and immunohistochemistry, respectively. The gene expression levels of cytokines were not associated with the duration of the period to conception following parturition. However, high protein expression of IL-1β and low protein expression of IL-1Ra were significantly associated with early conception. These results suggest that an imbalanced protein expression of IL-1β and IL-1Ra in the endometrium of dairy cows could be part of the maternal immune response mechanism necessary to propitiate early conception and probably to maintain pregnancy.

Effect of Restraint Stress on Pain Sensitivity, Spinal Trigeminal Nucleus Neurons, and Astrocytes in the Masseter Area of Rats.

To explore the changes of pain sensitivity (PS) in the masseter area (MA) in the rat model of psychological stress and the mechanism of action between spinal nucleus neurons and astrocytes in the trigeminal ganglion. The 40 Sprague-Dawley rats were randomly divided into control group (no treatment), group A (restraint stress (RS) 1 d), group B (RS 7 d), and group C (RS 14 d). The body weight growth rates (WGR) of rats in each group were compared and the difference of CORT and ACTH in serum was analyzed by ELISA. The open field test and the elevated “cross” maze test were adopted to detect the behavioral changes of rats. Finally, pain threshold of the MA in rats, the activation amount of brain tissue medulla oblongata parts astrocytes markers Glial fibrillary acidic protein (GFAP), and the protein expression of IL-1β and IL-1RI were detected. The results showed the WGR at 7 d and 14 d was greatly lower than control group (P < 0.01). In addition, the activity level and serum CORT and ACTH levels AND mean pain threshold in the MA of groups B and C were greatly lower than control group (P < 0.05). The activation rate of GFRP in group C (P < 0.01) and the protein expression of IL-1β and IL-1RI (P < 0.05) in rat trigeminal ganglion astrocytes of groups B and C was greatly higher than control group, indicating the increase of RS time, the release of IL-1β and IL-1RI can activate neurons and astrocytes in spinal trigeminal nucleus (STN) nerve and increase the PS of the MA.

Role of macrophage-mediated Toll-like receptor 4\u2013interleukin-1R signaling in ectopic tongue pain associated with tooth pulp inflammation

BackgroundThe existence of referred pain and ectopic paresthesia caused by tooth pulp inflammation may make definitive diagnosis difficult and cause misdiagnosis or mistreatment; thus, elucidation of that molecular mechanism is urgent. In the present study, we investigated the mechanisms underlying ectopic pain, especially tongue hyperalgesia, after tooth pulp inflammation.MethodsA rat model with mandibular first molar tooth pulp exposure was employed. Tooth pulp exposure-induced heat and mechanical-evoked tongue hypersensitivity was measured, and immunohistochemical staining for Iba1, a marker of active macrophages, IL-1β, IL-1 type I receptor (IL-1RΙ), and toll-like receptor 4 in the trigeminal ganglion was performed. In addition, we investigated the effects of injections of liposomal clodronate Clophosome-A (LCCA), a selective macrophage depletion agent, lipopolysaccharide from Rhodobacter sphaeroides (LPS-RS, a toll-like receptor 4 antagonist), IL-1β, or heat shock protein 70 (Hsp70, a selective agonist of toll-like receptor 4), to examine changes in tongue hypersensitivity and in the regulation of IL-1RΙ, toll-like receptor 4, and transient receptor potential vanilloid 1 (TRPV1) biosynthesis.ResultsAt day 1 after tooth pulp exposure, obvious tooth pulp inflammation was observed. Tooth pulp exposure-induced heat and mechanical tongue hypersensitivity was observed from days 1 to 3 after tooth pulp exposure. The production of IL-1β in activated macrophages and toll-like receptor 4 and IL-1RΙ expression were significantly increased in trigeminal ganglion neurons innervating the tongue following tooth pulp exposure. Intra-trigeminal ganglion injection of LCCA significantly suppressed tongue hypersensitivity; however, toll-like receptor 4 and IL-1RΙ expression in trigeminal ganglion neurons innervating the tongue was not significantly altered. Intra-trigeminal ganglion injection of LPS-RS significantly suppressed tongue hypersensitivity and reduced IL-1RΙ expression in the trigeminal ganglion neurons innervating the tongue following tooth pulp exposure. Intra-trigeminal ganglion injection of recombinant Hsp70 significantly promoted tongue hypersensitivity and increased IL-1RI expression in trigeminal ganglion neurons innervating the tongue in naive rats. Furthermore, intra-trigeminal ganglion injection of recombinant IL-1β led to tongue hypersensitivity and enhanced TRPV1 expression in trigeminal ganglion neurons innervating the tongue in naive rats.ConclusionsThe present findings suggest that the neuron-macrophage interaction mediated by toll-like receptor 4 and IL-1RI activation in trigeminal ganglion neurons affects the pathogenesis of abnormal tongue pain following tooth pulp inflammation via IL-1RI and TRPV1 signaling in the trigeminal ganglion. Further research may contribute to the establishment of new therapeutic and diagnostic methods.

356 IL-1β induces rapid secretion of the antimicrobial protein IL-26 from Th17 cells

Th17 cells play a fundamental role in both immunity and autoimmunity at mucosal surfaces, including skin. Recent work has implicated the Th17 cytokine IL-26 as directly antimicrobial to extracellular and intracellular bacteria, including Mycobacterium leprae, the causative agent of leprosy. IL-26 protein expression was greater in skin lesions from patients with the resistant vs. progressive form of the disease. To characterize the mechanism of IL-26 induction, we examined the kinetics of IL-26 secretion by PBMCs exposed to Mycobacterium leprae sonicate, which revealed significant release at 3 hours, reminiscent of an innate response. Pattern recognition receptor ligands induced IL-26 secretion from PBMC, which was mediated by IL-1β. Recombinant IL-1β alone was sufficient to stimulate IL-26 release from PBMC and memory CD4+ T cells in the absence of T cell receptor (TCR) activation. IL-1β stimulated IL-26 secretion more rapidly from memory CD4+ T cells as compared to TCR activation with crosslinking antibodies. Further investigation revealed that IL-1RI expression was necessary for IL-1β induced IL-26 from memory Th17 cells. IL-1β stimulation did not lead to secretion of other Th17 cytokines, unlike TCR activation. RNA sequencing of IL-1β treated IL-1RI+ Th17 cells revealed enrichment for NF-κB regulated genes. Inhibition of NF-κB signaling with Bay 11-7082 abrogated IL-26 production in response to either stimulus. Finally, supernatants from IL-1β treated memory T cells showed antimicrobial activity against E. coli in an IL-26 dependent manner. Together, these results identify IL-1RI+ Th17 cells as an antimicrobial Th17 subpopulation with the ability to rapidly respond to IL-1β and induce IL-26 to kill extracellular bacteria.

Follicular structures of cows with cystic ovarian disease present altered expression of cytokines.

Ovulation is considered an inflammatory, cytokine-mediated event. Cytokines, which are recognized as growth factors with immunoregulatory properties, are involved in many cellular processes at the ovarian level. In this sense, cytokines affect fertility and are involved in the development of different ovarian disorders such as bovine cystic ovarian disease (COD). Because it has been previously demonstrated that ovarian cells represent both sources and targets of cytokines, the aim of this study was to examine the expression of several cytokines, including IL-1β, IL-1RA, IL-1RI, IL-1RII, IL-4 and IL-8, in ovarian follicular structures from cows with spontaneous COD. The protein expression of these cytokines was evaluated by immunohistochemistry. Additionally, IL-1β, IL-4 and IL-8 concentrations in follicular fluid (FF) and serum were determined by enzyme-linked immunosorbent assay (ELISA). In granulosa and theca cells, IL-1RI, IL-1RII, IL-1RA and IL-4 expression levels were higher in cystic follicles than in the control dominant follicles. The serum and FF concentrations of IL-1β and IL-4 showed no differences between groups, whereas IL-8 concentration was detected only in FF of cysts from cows with COD. The FF and serum concentrations of IL-1β and IL-8 showed no significant differences, whereas IL-4 concentration was higher in FF than in serum in both the control and COD groups. These results evidenced an altered expression of cytokines in ovaries of cows with COD that could contribute to the pathogenesis of this disease.

Expression and function of IL-1 receptor type 2 (IL-1RII) regulating human Th17 responses

Abstract Th17 cells are a unique subset of proinflammatory cells conferring protection against extracellular bacteria and fungi, but have been also implicated in the pathogenesis of many autoimmune diseases. IL-1β is critical for the differentiation, expansion, in vivo survival, and effector function of Th17 cells. Among components of IL-1 receptor (IL-1R), decoy IL-1RII is a negative regulator of functional IL-1RI-mediated IL-1β signaling. Given its preferential expression on Treg cells, IL-1RII has been suggested to play a role for controlling the Th17 and Treg balance. However, little is known about a molecular mechanism underlying IL-1RII expression and its contribution to modulation of Th17 responses. Here, we found that IL-1RI and IL-1RII expression were rapidly induced by memory CD4+ T cells after stimulation with TCR alone. Cyclosporin A reduced IL-1RII expression, whereas an augmented FOXP3 was associated with enhanced IL-1RII expression. This finding was corroborated by in vitro assays using NFAT inhibitor, dNP2-VIVIT and an inhibitor of FOXP3/NFAT1 interaction, peptide FOXP3 393–403. Moreover, reporter assay suggests that NFAT/FOXP3 binding sites in IL-1RII promoter are critical for its transcription. At day 2 post-stimulation, there were three distinct subsets; IL-1RI+IL-1RII−, IL-1RI+IL-1RII+, and IL- 1RI−IL-1RII− CD4+ T cells, having unique features including cytokine production profile, the expression of Tregassociated genes, and responsiveness to IL-1β, suggesting a pivotal regulatory role of IL-1RII for human Th17 responses. Our findings will be beneficial to understanding a modulatory mechanism on Th17 response and IL- 1β-mediated IL-1R signaling contributing to pathogenesis of autoimmune diseases.

High Expression of IL-1RI and EP₂ Receptors in the IL-1β/COX-2 Pathway, and a New Alternative to Non-Steroidal Drugs-Osthole in Inhibition COX-2.

Background: Osthole (7-methoxy-8-isopentenylcoumarin) is natural coumarin isolated from the fruit of Cnidium monnieri (L.) Cusson, which is commonly used in medical practice of traditional Chinese medicine (TCM) in various diseases including allergies and asthma disorders. Purpose: Osthole was tested for the anti-histamine, anti-allergic, and inhibitory effects of COX-2 (cyclooxygenase-2) in children with diagnosed allergies. Additionally, we hypothesize that stated alterations in children with diagnosed allergies including increased expression of interleukin 1-β receptor type 1 (IL-1 type I) and E-prostanoid (EP) 2 receptors, as well as raised expression, production, and activity of COX-2 and IL-1β in incubated medium are approximately connected. Furthermore, we establish the mechanisms included in the changed regulation of the COX-2 pathway and determine whether osthole may be COX-2 inhibitor in peripheral blood mononuclear cells (PBMCs). Method: PBMCs were obtained from peripheral blood of healthy children (control, n = 28) and patients with diagnosed allergies (allergy, n = 30). Expression of the autocrine loop components regulating PGE2 production and signaling namely IL-1 type I receptor (IL-1RI), cyclooksygenaze-2 (COX-2), E-prostanoid (EP) 2, and also histamine receptor-1 (HRH-1) was assessed at baseline and after stimulation with histamine, osthole, and a mixture of histamine/osthole 1:2 (v/v). This comprised the expression of histamine receptor 1 (HRH-1), IL-1RI, COX-2, EP2 receptor, and the secretion of IL-1β and COX-2 in cultured media and sera. Results: Compared with control group, basal mRNA expression levels of HRH-1, IL-1RI, COX-2, and EP2 were higher in the allergy group. Histamine-induced EP2 and COX-2 expression mRNA levels were also increased. Conclusions: Osthole successively inhibits PGE2 and COX-2 mRNA expression. Furthermore, osthole reduces the secretion of COX-2 protein in signaling cellular mechanisms. Changed EP2 expression in children with allergies provides higher IL-1RI induction, increasing IL-1β capacity to increase COX-2 expression. This effects in higher PGE2 production, which in turn increases its capability to induce IL-1RI.

Innate lymphoid cells in the upper airways: importance of CD117 and IL-1RI expression.

Although type 1, 2 and 3 innate lymphoid cells (ILC1s, ILC2s and ILC3s, respectively) are emerging as important cell populations regulating tissue homeostasis, remodelling and inflammation, a vast majority of our knowledge stems from in vitro and murine experiments, and requires thorough confirmation in human diseases.Relative levels of ILCs were evaluated by means of flow cytometry in freshly resected human upper airways mucosa of patients with chronic rhinosinusitis without nasal polyps (CRSsNP) and with nasal polyps (CRSwNP), taking into account the patient's clinical parameters and disease comorbidities.We report that the CD117 and interleukin-receptor type I (IL-1RI) expression status of human ILC2s depends on the local tissue environment. Only CD117+ IL-1RI+ ILC2s, exclusively present in CRSwNP, possess an interrelationship with type 2 T-helper cell cytokine and eosinophil levels in human upper airway mucosa. In CRSsNP, mainly CD117-IL-1RI- ILC2s are increased, yielding lower eosinophilia in this disease despite the high levels of ILC2s.These data unveil that the CD117- and CD117+ fractions within the native human ILC2 population are not a random phenomenon, in contrast to what could be concluded from in vitro data, and that the IL-1RI expression is not ubiquitous in ILC2s in vivo in humans, which cannot be assessed via in vitro and murine experiments.

Altered expression of IL-1β, IL-1RI, IL-1RII, IL-1RA and IL-4 could contribute to anovulation and follicular persistence in cattle

Cystic ovarian disease (COD) is an important cause of infertility in dairy cattle. The main signs of this infertility are ovulation failure and follicular persistence. The aim of this study was to examine the expression of the cytokines IL-1β, IL-1RI, IL-1RII, IL-1RA and IL-4 in ovarian follicular structures at different times of persistence in a model of follicular persistence induced by prolonged administration of progesterone in dairy cows. Protein expression of IL-1β, IL-1RI, IL-1RII, IL-1RA and IL-4 was evaluated by immunohistochemistry. Additionally, IL-1β and IL-4 concentrations in follicular fluid and serum were determined by ELISA. In granulosa cells, IL1-RII and IL-4 expression was higher in follicles with different persistence times than in the control dominant follicles. IL-1RA expression was higher in persistent follicles of the P15 group (15 days of follicular persistence) than in those of the control group. In theca cells, IL-1RII expression was higher in persistent follicles of the P0 group (expected time of ovulation) than in dominant follicles from the control group (p < .05) and the other persistence groups, whereas IL-4 expression was higher in persistent follicles of groups P0 and P15 than in the dominant follicles of the control group (p < .05). Differences between serum and follicular fluid within each group were detected only in P0 for IL-1β, and in the control, P10 and P15 groups for IL-4 (p < .05). These results complement previous results, evidencing that early development of COD in cows is concurrent with an altered expression of cytokines in different ovarian follicular structures and may contribute to the follicular persistence and ovulation failure found in cattle with follicular cysts.

In vivo effects of GnRH on expression of interleukin 1 (IL-1) system members in bovine preovulatory follicles and the influence of IL-1β on cumulus-oocyte complexes cultured in vitro

Abstract The purpose of this study was to determine the effects of in vivo GnRH treatment on mRNA expression of IL-1β system in bovine granulosa cells from preovulatory follicles and to analyze the in vitro effect of gonadotropins on the IL-1β system gene expression in cumulus cells. Moreover, the additive effect of IL-1β on cumulus expansion, gene expression and ultrastructural integrity of COCs cultured in vitro was evaluated. In vivo studies in granulosa cells demonstrated that IL-1RA mRNA levels were increased after 24 h of GnRH treatment. Similarly, the presence of gonadotropins increased the levels of mRNAs for IL-1R1 and IL-1RA in cumulus cells after 24 h of culture in vitro. The presence of IL-1β in culture medium did not influence cumulus expansion, but it increased the expression of mRNAs for IL-1β, IL-1RA, HAS2 and iNOS after COC culture. In addition, the ultrastructure of COCs cultured in vitro in the presence of IL-1β was maintained. In conclusion, IL-1RA mRNA is upregulated in granulosa cells at later stages of the ovulatory process in cattle. In cumulus cells, mRNA expression of IL-1RI and IL-1RA is induced by gonadotropins and IL-1β maintains the ultrastructure of COC cultured in vitro.

Activated IL-1RI Signaling Pathway Induces Th17 Cell Differentiation via Interferon Regulatory Factor 4 Signaling in Patients with Relapsing-Remitting Multiple Sclerosis.

IL-1β plays a crucial role in the differentiation of human Th17 cells. We report here that IL-1RI expression is significantly increased in both naive and memory CD4+ T cells derived from relapsing-remitting multiple sclerosis (RR MS) patients in comparison to healthy controls. Interleukin 1 receptor (IL-1R)I expression is upregulated in the in vitro-differentiated Th17 cells from RR MS patients in comparison to the Th1 and Th2 cell subsets, indicating the role of IL-1R signaling in the Th17 cell differentiation in RR MS. When IL-1RI gene expression was silenced using siRNA, human naive CD4+ T cells cultured in the presence of Th17-polarizing cytokines had a significantly decreased expression of interleukin regulatory factor 4 (IRF4), RORc, IL-17A, IL-17F, IL-21, IL-22, and IL-23R genes, confirming that IL-1RI signaling induces Th17 cell differentiation. Since IL-1R gene expression silencing inhibited IRF4 expression and Th17 differentiation, and IRF4 gene expression silencing inhibited Th17 cell differentiation, our results indicate that IL-1RI induces human Th17 cell differentiation in an IRF4-dependant manner. Our study has identified that IL-1RI-mediated signaling pathway is constitutively activated, leading to an increased Th17 cell differentiation in IRF4-dependent manner in patients with RR MS.

TLR4-Upregulated IL-1β and IL-1RI Promote Alveolar Macrophage Pyroptosis and Lung Inflammation through an Autocrine Mechanism.

Acute lung injury (ALI) is a major component of multiple organ dysfunction syndrome (MODS) following pulmonary infection. Alveolar macrophages (AM) are at the center of the pathogenesis of the development of ALI. Interleukin-1β (IL-1β) is one of the key pro-inflammatory mediators, and its maturation is tightly controlled by the formation and activation of the inflammasome. The biological effects of IL-1β are mediated through IL-1 receptor (IL-1R). In this study, we investigated the influence of LPS-induced IL-1β release and IL-1RI upregulation on the development of lung inflammation. We demonstrated that in AM, LPS-TLR4 signaling not only activates Nlrp3 inflammasome activation and subsequent release of IL-1β, but also up-regulates IL-1RI expression on AM surface through MyD88 and NF-κB dependent signaling. The upregulated IL-1RI, therefore, sensitizes AM to IL-1β and results in pyroptosome formation, which in turn leads to AM pyroptosis, a type of caspase-1-dependent inflammatory cell death. We further showed that AM pyroptosis exaggerates lung inflammation. The present study demonstrates a novel mechanism underlying LPS-induced innate immunity; that is, a secondary upregulation of IL-1β-IL-1RI signaling is responsible for AM pyroptosis and augmented lung injury in response to LPS.

Bronchopulmonary C-fibers' IL1RI contributes to the prolonged apneic response to intra-atrial injection of capsaicin by prenatal nicotinic exposure in rat pups

Prenatal nicotinic exposure (PNE) as a SIDS model reportedly sensitizes bronchopulmonary C-fibers (PCFs), contributing to the prolonged PCF-mediated apnea in rat pups, but the relevant mechanisms are not fully understood. Pulmonary IL-1β upregulated by cigarette smoke is known to stimulate or sensitize PCFs acting via IL-1 type I receptor (IL1RI) and inhibit inspiration frequency. Because of its upregulation observed in SIDS victims, we hypothesized that PNE increased pulmonary IL-1β release and IL1RI expression in pulmonary C-neurons via action on α7 nicotinic acetylcholine receptors (α7nAChR) to induce the prolonged PCF-mediated apnea. IL-1β in BALF and IL1RI in the nodose/jugular (N/J) ganglion and vagal pulmonary C-neurons retrogradely-traced were compared between Ctrl (saline) and PNE pups and among the vehicle-treated Ctrl and PNE and methyllycaconitine (a selective α7nAChR antagonist)-treated PNE pups. The effect of IL-1RI blockade (IL-1Ra) on the PCF-mediated apnea was also compared between Ctrl and PNE pups. PNE significantly elevated IL-1β in BALF and upregulated IL1RI gene and protein expression in N/J ganglia and gene in vagal pulmonary C-neurons. All of these responses were eliminated by pretreatment with blockade of α7nAChR. In addition, the prolonged PCF-mediated apnea in PNE pups was significantly shortened by right atrial bolus injection of IL-1Ra. We conclude that PNE enhances pulmonary IL-1β release and PCF IL1RI expression acting via α7nAChR in contributing to sensitization of PCFs and prolongation of the PCF-mediated apneic response.

Hippocampal distribution of IL-1β and IL-1RI following lithium-pilocarpine-induced status epilepticus in the developing rat.

The contribution of Interleukin-1β (IL-1β) to neuronal injury induced by status epilepticus (SE) in the immature brain remains unclear. The goal of this study was to determine the hippocampal expression of IL-1β and its type 1 receptor (IL-1RI) following SE induced by the lithium-pilocarpine model in fourteen-days-old rat pups; control animals were given an equal volume of saline instead of the convulsant. IL-1β and IL-1RI mRNA hippocampal levels were assessed by qRT-PCR 6 and 24 h after SE or control conditions. IL-1β and IL-1RI expression was detected in the dorsal hippocampus by immunohistochemical procedures; Fluoro-Jade B staining was carried out in parallel sections in order to detect neuronal cell death. IL-1β mRNA expression was increased 6 h following SE, but not at 24 h; however IL-1RI mRNA expression was unaffected when comparing with the control group. IL-1β and IL-1RI immunoreactivity was not detected in control animals. IL-1β and IL-1RI were expressed in the CA1 pyramidal layer, the dentate gyrus granular layer and the hilus 6 h after SE, whereas injured cells were detected 24 h following seizures. Early expression of IL-1β and IL-1RI in the hippocampus could be associated with SE-induced neuronal cell death mechanisms in the developing rat.

Progesterone modulates pro-inflammatory cytokine expression profile after spinal cord injury: Implications for neuropathic pain

Neuropathic pain is a frequent complication of spinal cord injury (SCI), still refractory to conventional treatment. Glial cell activation and cytokine production contribute to the pathology of central neuropathic syndromes. In this study we evaluated the effects of progesterone, a neuroactive steroid, on pain development and the spinal expression of IL-1β, its receptors (IL-1RI and IL-1RII) and antagonist (IL-1ra), IL-6 and TNFα, and NR1 subunit of NMDAR. Our results show that progesterone, by modulating the expression of pro-inflammatory cytokines and neuronal IL-1RI/NR1 colocalization, emerges as a promising agent to prevent chronic pain after SCI.

Low E-prostanoid 2 receptor levels and deficient induction of the IL-1β/IL-1 type I receptor/COX-2 pathway: Vicious circle in patients with aspirin-exacerbated respiratory disease

We hypothesized that the 2 reported alterations in aspirin-exacerbated respiratory disease (AERD), reduced expression/production of COX-2/prostaglandin (PG) E2 and diminished expression of E-prostanoid (EP) 2 receptor, are closely linked. We sought to determine the mechanisms involved in the altered regulation of the COX pathway in patients with AERD. Fibroblasts were obtained from nasal mucosa; samples of control subjects (NM-C, n=8) and from nasal polyps from patients with aspirin-exacerbated respiratory disease (NP-AERD, n=8). Expression of the autocrine loop components regulating PGE2 production and signaling, namely IL-1 type I receptor (IL-1RI), COX-2, microsomal prostaglandin E synthase 1 (mPGES-1), and EP receptors, was assessed at baseline and after stimulation with IL-1β, PGE2, and specific EP receptor agonists. Compared with NM-C fibroblasts, basal expression levels of IL-1RI and EP2 receptor were lower in NP-AERD fibroblasts. IL-1β-induced IL-1RI, COX-2, and mPGES-1 expression levels were also lower in these cells. Levels of IL-1RI positively correlated with COX-2 and mPGES-1 expression in both NM-C and NP-AERD fibroblasts. Incubation with either exogenous PGE2 or selective EP2 agonist significantly increased expression of IL-1RI in NM-C fibroblasts and had hardly any effect on NP-AERD fibroblasts. Alterations in IL-1RI, COX-2, and mPGES-1 expression that were found in NP-AERD fibroblasts were corrected when EP2 receptor expression was normalized by transfection of NP-AERD fibroblasts. Altered expression of EP2 in patients with AERD contributes to deficient induction of IL-1RI, reducing the capacity of IL-1β to increase COX-2 and mPGES-1 expression, which results in low PGE2 production. This impairment in the generation of PGE2 subsequently reduces its ability to induce IL-1RI.

A4.22 Syndecan-4 controls interleukin (IL)-1 receptor trafficking and IL-1 signalling in chronic destructive arthritis

Background and objectives Syndecan-4 (sdc4) is a transmembrane heparan sulfate (HS) proteoglycan. We have shown previously that sdc4 blockade protects from osteoarthritis like changes in mice. Here, sdc4 has been implicated in the modulation of IL-1 mediated Erk signalling and regulation of MMP-3 expression in fibroblasts by modulating IL-1 receptor trafficking. Hence, we hypothesised that sdc4 blockade reduces cytokine signalling in RA synovial fibroblasts. Materials and methods Evaluating the impact of sdc4 on the RA phenotype in vivo , hTNFα transgenic (hTNFtg), sdc4 knockout (sdc4 -/- ) and hTNFtg/sdc4 -/- as well as hTNFtg mice treated with sdc4 blocking antibody were histological analysed. To quantify the histomorphometrical changes toluidin-blue stained sections were analysed for cartilage destaining and erosion. Furthermore, immunohistological stainings for MMP-3 and the IL-1 receptor (IL-1RI) were performed. MMP-3 production was analysed via ELISA after TNFα or IL-1 stimulation and the influence on the Erk signalling pathway was evaluated by Western blot analysis in cells lacking sdc4, IL-1RI or both proteins. Additionally, the expression and presentation of TNF and IL-1RI was assessed via quantitative RT-PCR and FACS analysis. Influence of IL-1 stimulation on sdc4 complex formation was characterised via crosslinking and subsequent Western blot detection. Results The loss of sdc4 or its antibody-mediated inhibition reduced the severity of chronic destructive arthritis in mice by impairing IL-1 responsiveness of resident fibroblast-like cells. RT-PCR revealed, that neither the expression of IL-1RI nor TNF receptor was altered in cells lacking sdc4 compared to wild type fibroblasts, whereas FACS analysis and histological stainings showed that the inhibition of sdc4 largely abolished the IL-1R presentation on fibroblasts in vitro and in vivo . We demonstrated that IL-1 directly binds to sdc4 and in an IL-1R independent manner leads to its dimerisation. Strikingly, IL-1 induced dimerisation of sdc4 and its loss diminished caveolin vesicle-mediated trafficking of IL-1RI. Hence, Western Blot analysis of Erk phosphorylation showed reduced IL-1 induced Erk1/2 phosphorylation in sdc4 -/- compared to wild type fibroblasts. The absence of IL1RI prevented IL-1 mediated Erk1/2 phosphorylation completely. Focusing at MMP-3 levels as read-out for the activation of the Erk-pathway, we found lower MMP-3 production upon IL-1 stimulation in sdc4 -/- compared to wild type controls, while IL-1RI -/- cells did not respond at all. MMP-3 production was not altered upon TNFα stimulation. Conclusion We could show that the loss plus the antibody-mediated sdc4 blockade reduced IL-1RI presentation and thereby IL-1 signalling in firboblasts, which constitutes a novel function of sdc4 and might be of great value for RA treatment.

Different Retinal Expression Patterns of IL-1α, IL-1β, and Their Receptors in a Rat Model of Type 1 STZ-Induced Diabetes.

Diabetic retinopathy (DR), a common complication of diabetes, remains a major cause of blindness among population. Considerable amounts of evidences suggest the involvement of inflammatory process in this pathology. Increased levels of proinflammatory cytokines, including interleukin-1β (IL-1β), were found in the vitreous of diabetic patients and in the retina of diabetic rats. However, in this context, no attention has been given to the other main IL-1 family members: IL-1α, two transmembrane receptors IL-1RI and IL-1RII and the natural antagonist receptor IL-1Ra. Despite that they actively participate in the IL-1-mediated inflammation process, their implication in DR has not been described. Thus, we investigated by Western blot and confocal laser scanning microscopy analysis the effect of hyperglycemia on expression of IL-1 family members in retinal layers, using an in vivo model of type 1 diabetes. It was induced in adult rats by intraperitoneal injection of streptozotocin (STZ). Exposure to hyperglycemia induces a significant increase in the protein expression of IL-1β, IL-1RI, IL-RII and IL-1Ra but not of IL-1α. Moreover, high glucose alters their distribution pattern in the rat's retinal layers. Among these latter, the most compromised are the photoreceptor, the inner plexiform and ganglion cell layers. These findings support previous data demonstrating the involvement of inflammation in DR and suggest new pharmacological approaches for the treatment of this pathology.

The Effect of Tanshinone-IIa on IL-1β Induced-Thrombocytosis in an Immune Vasculitis Model

Inflammation cytokines may be involved in the pathogenesis of thrombocytosis with vasculitis. Our previous study showed that inflammation cytokine IL-1β plays an important role on in-vitro megakaryopoiesis (Yang M et al, Br J Haematol 2000). The role of IL-1β and Tanshinone IIA (TIIA) (Isolated from Danshen, Radix Salviae Miltiorrhiza Bge) on platelets and megakaryocytes (MKs) in immune vasculitis model was investigated in this study. Rabbit model with immune vasculitis was established by injection (iv) of BSA. After treatment with BSA for 7 days, the platelet count, platelet aggregation and the expression of AnnexinⅤ were significantly increased in this vasculitis model group compared with normal control group (n=7). IL-1β levels was also significantly higher in vasculitis model. There were positive correlations between platelet count and IL-1β levels (R=0.65), platelet aggregation and IL-1β levels (R=0.60). Treatment with TIIA (5 mg/kg/day, iv) and aspirin significantly decreased all these parameters. MKs and CFU-MK number were also significantly increased in vasculitis group as compared to normal group. Treatment with TIIA and aspirin significantly reduced the number of MKs and CFU-MK in this model. Study further demonstrated that IL-1β alone or in combination with TPO induced in-vitro CFU-MK formation. The mRNA of of IL-1 type I and type II receptors (IL-1 RI and RII) were detected in cultured MK (CD61+ CD41+) cells. The expression of IL-1 RI and RII was also confirmed by immunofluorescence staining in bone marrow MKs. Moreover, the IL-1R bloker can reduced IL-1β induced megakaryopoiesis. TIIA on in-vitro megakaryopoiesis was also investigated. TIIA at 10-30 ug/ml significantly inhibited CFU-MK formation. TIIA also induced the apoptosis of MKs in a dose dependent manner by Anexin V assay. Caspase 3 assay showed the activation of Caspase 3 increased from 5% to 16%. Using JC-1 assay found that the depolarized cells increased from 9% to 17% suggesting the involvement of intrinsic apoptotic pathway. IL-1β may play an important role in the thrombocytosis of immune vasculitis by inducing megakaryopoiesis. TIIA has anti-platelet effect in this model which may be mediated via inhibiting IL-1β induced-megakaryopoiesis. DisclosuresNo relevant conflicts of interest to declare.