Minocycline inhibition of microglial rescues nigrostriatal dopaminergic neurodegeneration caused by mutant alpha-synuclein overexpression.

Studies indicate that mutant α-synuclein (mαSyn) is involved in the pathogenesis of Parkinson's disease (PD). The mαSyn expression leads to the loss of dopaminergic neurons in the substantia nigra (SN) and consequent motor dysfunctions. Additionally, studies found that PD was accompanied by extensive neuroinflammation of SN. However, it remains unclear as to whether microglia participate in the mαSyn pathology. This issue is addressed by using AAV-mα-Syn (A30P-A53T) to overexpress the human mαSyn in the SN in view of establishing the PD model. Subsequently, minocycline (Mino) was used to inhibit microglia activity, and an interleukin-1 receptor (IL-1R1) antagonist was used to hinder the IL-1R1 function. Finally, immunohistochemistry was used to analyze phosphorylated αSyn (Ser129) and TH-positive cells in the SN. Dopamine levels were analyzed by high performance liquid chromatography. mαSyn overexpression in the SN induced motor dysfunction, decreased striatal dopamine levels, and increased pathological αSyn 12 weeks after AAV injection. The data demonstrated that inhibiting microglial activation or hindering IL-1R1 reversed the persistent motor deficits, neurodegeneration of the nigrostriatal dopaminergic system, and development of Lewy body pathology caused by human mαSyn overexpression in the SN. Additionally, these findings indicate that neuroinflammation promotes the loss of neuronal cells.

Molecular expression patterns in the synovium and their association with advanced symptomatic knee osteoarthritis.

OBJECTIVE: Osteoarthritis (OA) is a major source of knee pain. Mechanisms of OA knee pain are incompletely understood but include synovial pathology. We aimed to identify molecular expression patterns in the synovium associated with symptomatic knee OA. DESIGN: Snap frozen synovia were from people undergoing total knee replacement (TKR) for advanced OA, or from post-mortem (PM) cases who had not sought help for knee pain. Associations with OA symptoms were determined using discovery and validation samples, each comprising TKR and post mortem (PM) cases matched for chondropathy (Symptomatic or Asymptomatic Chondropathy). Associations with OA were determined by comparing age matched TKR and PM control cases. Real-time quantitative PCR for 96 genes involved in inflammation and nerve sensitisation used TaqMan® Array Cards in discovery and validation samples, and protein expression for replicated genes was quantified using Luminex bead assay. RESULTS: Eight genes were differentially expressed between asymptomatic and symptomatic chondropathy cases and replicated between discovery and validation samples (P<0.05 or >3-fold change). Of these, matrix metalloprotease (MMP)-1 was also increased whereas interleukin-1 receptor 1 (IL1R1) and vascular endothelial growth factor (VEGF) were decreased at the protein level in the synovium of symptomatic compared to asymptomatic chondropathy cases. MMP1 protein expression was also increased in OA compared to PM controls. CONCLUSION: Associations of symptomatic OA may suggest roles of MMP1 expression and IL1R1 and VEGF pathways in OA pain. Better understanding of which inflammation-associated molecules mediate OA pain should inform refinement of existing therapies and development of new treatments.

A Critical Role of the IL-1ß-IL-1R Signaling Pathway in Skin Inflammation and Psoriasis Pathogenesis.

The IL-1 signaling pathway has been shown to play a critical role in the pathogenesis of chronic, autoinflammatory skin diseases such as psoriasis. However, the exact cellular and molecular mechanisms have not been fully understood. Here, we show that IL-1ß is significantly elevated in psoriatic lesional skin and imiquimod-treated mouse skin. In addition, IL-1R signaling appears to correlate with psoriasis disease progression and treatment response. IL-1 signaling in both dermal γδ T cells and other cells such as keratinocytes is essential to an IMQ-induced skin inflammation. IL-1ß induces dermal γδ T cell proliferation and IL-17 production in mice. In addition, IL-1ß stimulates keratinocytes to secrete chemokines that preferentially chemoattract peripheral CD27- CCR6+IL-17 capable of producing γδ T cells (γδT17). Further studies showed that endogenous IL-1ß secretion is regulated by skin commensals to maintain dermal γδT17 homeostasis in mice. Mouse skin associated with Corynebacterium species, bacteria enriched in human psoriatic lesional skin, has increased IL-1ß and dermal γδT17 cell expansion. Thus, the IL-1ß-IL-1R signaling pathway may contribute to skin inflammation and psoriasis pathogenesis via the direct regulation of dermal IL-17-producing cells and stimulation of keratinocytes for amplifying inflammatory cascade.

Interleukin-1 receptor type 1 is overexpressed in neurons but not in glial cells within the rat superficial spinal dorsal horn in complete Freund adjuvant-induced inflammatory pain.

BACKGROUND: All known biological functions of the pro-inflammatory cytokine interleukin-1ß (IL-1ß) are mediated by type 1 interleukin receptor (IL-1R1). IL-1ß-IL-1R1 signaling modulates various neuronal functions including spinal pain processing. Although the role of IL-1ß in pain processing is generally accepted, there is a discussion in the literature whether IL-1ß exerts its effect on spinal pain processing by activating neuronal or glial IL-1R1. To contribute to this debate, here we investigated the expression and cellular distribution of IL-1R1 in the superficial spinal dorsal horn in control animals and also in inflammatory pain. METHODS: Experiments were performed on rats and wild type as well as IL-1R1-deficient mice. Inflammatory pain was evoked by unilateral intraplantar injection of complete Freund adjuvant (CFA). The nociceptive responsiveness of control and CFA-treated animals were tested daily for withdrawal responses to mechanical and thermal stimuli before and after CFA injection. Changes in the expression of 48 selected genes/mRNAs and in the quantity of IL-1R1 protein during the first 3 days after CFA injection were measured with the TaqMan low-density array method and Western blot analysis, respectively. The cellular localization of IL-1R1 protein was investigated with single and double staining immunocytochemical methods. RESULTS: We found a six times and two times increase in IL-1R1 mRNA and protein levels, respectively, in the dorsal horn of CFA-injected animals 3 days after CFA injection, at the time of the summit of mechanical and thermal allodynia. Studying the cellular distribution of IL-1R1, we found an abundant expression of IL-1R1 on the somatodendritic compartment of neurons and an enrichment of the receptor in the postsynaptic membranes of some excitatory synapses. In contrast to the robust neuronal localization, we observed only a moderate expression of IL-1R1 on astrocytes and a negligible one on microglial cells. CFA injection into the hind paw caused a remarkable increase in the expression of IL-1R1 in neurons, but did not alter the glial expression of the receptor. CONCLUSION: The results suggest that IL-1ß exerts its effect on spinal pain processing primarily through neuronal IL-1R1, but it can also interact in some extent with IL-1R1 expressed by astrocytes.

Expression density of receptors to IL-1ß in atopic dermatitis.

Interleukin 1 (IL-1 ß) and the system for regulation of its biological effects play an important role in the development and behavior of inflammatory processes in atopic dermatitis. Notably, cells that are actively involved in the pathological process have altered expression of cytokine receptors. However, standard evaluation of cells by flow cytometry measures only the percentage of cells expressing the appropriate marker, which is not enough for a full assessment of these changes. The aim of this study was to investigate changes in the expression of IL-1ß cytokine receptors in patients with atopic dermatitis by both percentage of cells with receptors in various subsets and the absolute number of membrane-bound receptors themselves. It was found that an increase or decrease in the percentage of cells expressing the receptors in subsets of immune cells in patients with atopic dermatitis was not associated with a change in the number of receptors on the cell surface. Moreover, the changes in the percentage of cells and the number of receptors may occur in different directions, as shown for IL-1R2 expression on B cells and IL-1R1 expression for monocytes. Changes in the parameters of IL-1ß receptor expressions are associated with disease severity index SCORAD in atopic dermatitis. These findings underline the importance of studying the density of cytokine receptor expression in the pathology.

Genetic Cell Ablation Reveals Clusters of Local Self-Renewing Microglia in the Mammalian Central Nervous System.

During early embryogenesis, microglia arise from yolk sac progenitors that populate the developing central nervous system (CNS), but how the tissue-resident macrophages are maintained throughout the organism's lifespan still remains unclear. Here, we describe a system that allows specific, conditional ablation of microglia in adult mice. We found that the microglial compartment was reconstituted within 1 week of depletion. Microglia repopulation relied on CNS-resident cells, independent from bone-marrow-derived precursors. During repopulation, microglia formed clusters of highly proliferative cells that migrated apart once steady state was achieved. Proliferating microglia expressed high amounts of the interleukin-1 receptor (IL-1R), and treatment with an IL-1R antagonist during the repopulation phase impaired microglia proliferation. Hence, microglia have the potential for efficient self-renewal without the contribution of peripheral myeloid cells, and IL-1R signaling participates in this restorative proliferation process.

IL-1R1 is expressed on both Helios(+) and Helios(-) FoxP3(+) CD4(+) T cells in the rheumatic joint.

Synovial fluid from rheumatic joints displays a well-documented enrichment of forkhead box protein 3 (FoxP3)(+) regulatory T cells (tissue Tregs ). However, we have previously demonstrated that the mere frequency of FoxP3 expressing cells cannot predict suppressive function. Instead, extrinsic factors and the functional heterogeneity of FoxP3(+) Tregs complicate the picture. Here, we investigated FoxP3(+) Tregs from blood and synovial fluid of patients with rheumatic disease in relation to Helios expression by assessing phenotypes, proliferative potential and cytokine production by flow cytometry. Our aim was to investigate the discriminatory potential of Helios when studying FoxP3(+) Tregs in an inflammatory setting. We demonstrate that the majority of the synovial FoxP3(+) CD4(+) T cells in patients with inflammatory arthritis expressed Helios. Helios(+) FoxP3(+) Tregs displayed a classical Treg phenotype with regard to CD25 and cytotoxic T lymphocyte-associated antigen (CTLA)-4 expression and a demethylated Treg -specific demethylated region (TSDR). Furthermore, Helios(+) FoxP3(+) T cells were poor producers of the effector cytokines interferon (IFN)-γ and tumour necrosis factor (TNF), as well as of the anti-inflammatory cytokine interleukin (IL)-10. The less abundant Helios(-) FoxP3(+) T cell subset was also enriched significantly in the joint, displayed a overlapping phenotype to the double-positive Treg cells with regard to CTLA-4 expression, but differed by their ability to secrete IL-10, IFN-γ and TNF upon T cell receptor (TCR) cross-linking. We also demonstrate a striking enrichment of IL-1R1 expression in synovial CD4(+) T cells that was restricted to the CD25-expressing FoxP3 population, but independent of Helios. IL-1R1 expression appears to define a tissue Treg cell phenotype together with the expression of CD25, glucocorticoid-induced TNF receptor family-related gene (GITR) and CTLA-4.

Lipopolysaccharide stimulates platelets through an IL-1ß autocrine loop.

LPS activates platelets through TLR4, aiding productive sepsis, with stimulated splicing and translation of stored heteronuclear pro-IL-1ß RNA. Although the IL-1R type 1 (IL-1R1) receptor for IL-1 shares downstream components with the TLR4 receptor, platelets are not known to express IL-1R1, nor are they known to respond to this cytokine. We show by flow cytometry and Western blotting that platelets express IL-1R1, and that IL-1ß and IL-1α stimulate heteronuclear I-1ß splicing and translation of the newly made mRNA in platelets. Platelets also respond to the IL-1ß they make, which is exclusively associated with shed microparticles. Specific blockade of IL-1R1 with IL-1R antagonist suppressed platelet stimulation by IL-1, so IL-1ß stimulates its own synthesis in an autocrine signaling loop. Strikingly, IL-1R antagonist inhibition, pharmacologic or genetic suppression of pro-IL-1ß processing to active cytokine by caspase-1, or blockade of de novo protein synthesis also blocked LPS-induced IL-1ß mRNA production. Robust stimulation of platelets by LPS therefore also required IL-1ß amplification. Activated platelets made IL-1ß in vivo as IL-1ß rapidly accumulated in occluded murine carotid arteries by posttranscriptional RNA splicing unique to platelets. We conclude that IL-1ß is a platelet agonist, that IL-1ß acts through an autocrine stimulatory loop, that an IL-1ß autocrine loop is required to amplify platelet activation by LPS, and that platelets immobilized in occlusive thrombi are activated over time to produce IL-1ß. IL-1 is a new platelet agonist that promotes its own synthesis, connecting thrombosis with immunity.

In-vivo extravasation induces the expression of interleukin 1 receptor type 1 in human neutrophils.

In order to address neutrophil activation during inflammation we assessed the expression of interleukin 1 receptor type 1 (IL-1R1) following in-vivo extravasation. Extravasated neutrophils were collected from 11 healthy study subjects by a skin chamber technique and compared to neutrophils in peripheral blood. Expression of IL-1R1 was assessed by microarray, quantitative polymerase chain reaction (qPCR), Western blot, flow cytometry, enzyme linked immunosorbent assay (ELISA) and immunoelectron microscopy (iEM). IL-1R1 was induced following extravasation, demonstrated by both gene array and qPCR. Western blot demonstrated an increased expression of IL-1R1 in extravasated leucocytes. This was confirmed further in neutrophils by flow cytometry and iEM that also demonstrated an increased intracellular pool of IL-1R1 that could be mobilized by N-formyl-methionine-leucine-phenylalanine (fMLP). Stimulation of peripheral neutrophils with IL-1 resulted in transcription of NFκB and a number of downstream chemokines and the corresponding chemokines were also induced following in-vivo extravasation. The present results demonstrate that IL-1R1 is induced following extravasation and exists on the neutrophil surface, as well as in a mobile intracellular pool. Furthermore, neutrophils express functional IL-1R1 as demonstrated by the induction of chemokines following IL-1 stimulation. The results indicate a potential role for IL-1 in the activation of neutrophils at inflammatory sites.

Endometrioid ovarian cancer and endometriotic cells exhibit the same alteration in the expression of interleukin-1 receptor II: to a link between endometriosis and endometrioid ovarian cancer.

AIM: Endometrioid carcinoma of the ovary is the third most common type of epithelial ovarian cancer. Endometrioid tumors as well as endometriotic implants are characterized by the presence of epithelial cells, stromal cells, or a combination of booth, that resemble the endometrial cells, suggesting a possible endometrial origin of these tumors. Th1 cytokines including interleukin (IL)-1 have been reported to be involved in both endometriosis and ovarian carcinogenesis. We assessed the expression of receptors of IL-1 (IL-1RI and IL-1RII, the signal transducer and the specific inhibitor of IL-1, respectively) in cells of the most common subtypes of ovarian cancer compared to endometrial cells. MATERIAL & METHODS: IL1-Rs expression was analyzed at the levels of the protein and mRNA using immunofluorescent and real-time polymerase chain reaction methods, respectively. RESULTS: We showed that endometrioid cells exhibit a specific decrease of IL-1RII expression, whereas IL-1RI was constantly expressed in all studied cell subtypes. CONCLUSION: As already reported in endometriotic cells, endometrioid ovarian cancer cells exhibit the same alteration in the expression of IL-1RII, a key protector against tumorigenic effects of IL-1. Our findings highlight a common signature between endometrioid ovarian cancer and implants of endometriosis, which needs to be fully explored.

[Expression of recombinant extracellular region of human interleukin-1 receptor type I in Pichia pastoris].

OBJECTIVE: To construct pPICZalphaA-soluble interleukin-1 receptor type I (sIL-1RI) recombinant expression vector containing the gene fragment encoding the extracellular domain of sIL-1RI for its expression in Pichia pastoris. METHODS: sIL-1RI gene was amplified by RT-PCR and inserted into the yeast expression vector pPICZalphaA by digestion ligation. The recombinant plasmid pPICZalphaA-sIL1RI was transformed into E.coli Stb13, and the positive clones were analyzed by PCR and DNA sequencing. The pPICZalphaA-sIL1RI recombinant plasmid was electroporated into GS115 cells and the transformants were analyzed by PCR. After phenotype identification, the recombinant strains were induced by methanol to express the target protein, which was analyzed by Western blotting of the cell extract and supernatant. RESULTS: The recombinant plasmid pPICZalphaA-sIL-1RI was constructed successfully, and the results of Western blotting showed that yeast induced by methanol expressed a protein of about 39 kD. CONCLUSION: sIL-1RI protein has been successfully expressed in P.pastoris expression system, which provides the basis for further study of sIL-1RI.

Sensory pulpal nerve fibres and trigeminal ganglion neurons express IL-1RI: a potential mechanism for development of inflammatory hyperalgesia.

AIM: To localize interleukin-1 receptor type I (IL-1RI) in rat dental pulp and trigeminal ganglion (TG) and to test the hypothesis that pulpal inflammation increases neuronal expression of IL-1RI. METHODOLOGY: Female Wistar rats were subjected to unilateral pulp exposures in the maxillary and mandibular first molars, whereas the contralateral jaws served as untreated controls. Seven days later the animals were transcardiacally perfused and the jaws and the TGs were removed and prepared for immunohistochemistry. Immunoreactivity for IL-1RI was examined alone (DAB) and together with calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), CD31 or CD34 by multiple-labelling immunofluorescence. Quantification of IL-1RI-immunoreactive (-IR) cells in the maxillary and mandibular division of the ganglion was performed in parasagittal immunoreacted sections of the right and left TGs. Data were analysed with Mann-Whitney Rank Sum test (P < 0.05). RESULTS: Interleukin-1 receptor type I was found on sensory (CGRP-IR) and sympathetic (NPY-IR) nerve fibres and on blood vessels (CD31- and CD34-IR) in the dental pulp. It was also localized on sensory neurons and axons in the TG. Pulpal inflammation significantly increased the expression of IL-1RI in the TG (P < 0.001). CONCLUSIONS: The localization of IL-1RI on sensory nerve fibres and its up-regulation in TG neurons during pulpal inflammation may imply a direct effect of IL-1 in pulpal nociception. The presence of IL-1RI on sympathetic nerve fibres and on blood vessels may indicate a vasoactive role of the same cytokine in the pulp.

Initiation of dopaminergic differentiation of Nurr1(-) mesencephalic precursor cells depends on activation of multiple mitogen-activated protein kinase pathways.

Interleukin-1 (IL-1) plays a pivotal role in terminal dopaminergic differentiation of midbrain-derived neural precursor cells already committed to the mesencephalic dopaminergic phenotype (named mdNPCs for mesencephalic dopaminergic neural precursor cells). Here we characterized the molecular events in long-term expanded rat nuclear receptor related-1(-) (Nurr1(-)) mdNPCs in response to IL-1beta during their terminal dopaminergic specification. We showed that IL-1beta induced a rapid induction of mRNA of dopaminergic key fate-determining transcription factors, such as Nurr1 and Pitx3, and a subsequent increase of tyrosine hydroxylase protein as an early marker for dopaminergic neurons in vitro. These effects of IL-1beta were specific for mdNPCs and were not observed in striatal neural precursor cells (NPCs). Surprisingly, IL-1beta did not activate the NF-kappaB pathway or the transcription factor activating protein 1 (AP-1), but inhibition of nuclear translocation of NF-kappaB by SN50 facilitated IL-1beta-induced Nurr1 expression and dopaminergic differentiation of mdNPCs. Incubation of mdNPCs with IL-1beta led to a rapid phosphorylation of ERK1/2 and p38 mitogen-activated protein (MAP) kinases within 1 to 3 hours, whereas Jun kinase was not phosphorylated in response to IL-1beta. Consistently, inhibition of the ERK1/2 pathway or p38 MAP kinase blocked Nurr1 upregulation and further dopaminergic specification of mdNPCs, but not differentiation into MAP2ab(+) neurons. IL-1 receptor antagonist did not block early dopaminergic differentiation events, suggesting that the effects of IL-1beta are not mediated through activation of IL-1 receptor type I. Our results indicate that induction of terminal dopaminergic specification of Nurr1(-) mdNPCs by IL-1beta depends on activation of the ERK1/2 and p38 MAP kinase pathway.

Impaired counter-regulation of interleukin-1 by the soluble IL-1 receptor type II in patients with chronic liver disease.

OBJECTIVE: To assess the production of the endogenous IL-1 modulators IL-1 receptor antagonist (IL-1Ra), type I and II soluble IL-1 receptors (IL-1sRI and II) in patients with chronic liver disease (CLD). MATERIAL AND METHODS: Plasma levels of IL-1beta (IL-1beta) and IL-1 modulators were assessed in 126 CLD patients and 39 healthy controls. IL-1sRII was also measured in the supernatants of primary hepatocyte cultures. RESULTS: Plasma IL-1sRI and IL-1Ra levels were significantly higher in cirrhotic CLD patients than in non-cirrhotic CLD patients and in controls. Levels did not depend on the etiology of CLD. Likewise, plasma IL-1beta levels were elevated in CLD patients compared with those in controls. In contrast, IL-1sRII levels did not differ between CLD patients and controls. Cultures of human primary hepatocytes showed that IL-1sRII is induced by IL-1beta, but not IL-6. CONCLUSIONS: In cirrhotic CLD patients elevated plasma IL-1beta is not counteracted by endogenous levels of IL-1sRII, whereas high IL-1sRI is expected to neutralize the naturally occurring antagonist IL-1Ra, resulting in a dysregulation of the IL-1 system that might enhance pro-inflammatory activity of IL-1.

Imbalance in the expression of the activating type I and the inhibitory type II interleukin 1 receptors in endometriosis.

BACKGROUND: The ectopic establishment and progression of endometrial tissue is dependent upon its interaction with and responsiveness to the stimuli present in its new environment. Immune cell-derived cytokines, such as interleukin 1 (IL1), may alone or in concert with estrogens enhance the capability of ectopic endometrial cells to implant and develop into the host tissue. The objective of this study was to further evaluate the expression and significance of IL1 receptor type I (IL1R1), the signalling receptor that mediates cell activation by IL1, and IL1 receptor type II (IL1R2), a potent and specific down-regulator of IL1 action, in normal compared to endometriotic/endometrial tissues. METHODS: Techniques included immunohistochemistry, immunofluorescent staining, ELISA, western blotting and endometriotic cell culture transfection. RESULTS: Our study showed an imbalance in the expression of IL1R1 and IL1R2 in eutopic, and particularly in ectopic, endometrial tissues of women with endometriosis. Actually, a decreased IL1R2 expression is predominant in the eutopic and ectopic endometrium of women with endometriosis when compared with normal women, whereas a concomitant increase in IL1R1 expression occurs in ectopic endometrial tissue in comparison to eutopic endometrial tissue of normal or endometriotic women, particularly in the initial and most active implants. Transfection of endometriotic cells with a cDNA coding for IL1R2 resulted in a significant decrease in IL1-induced secretion of vascular endothelial cell growth factor and monocyte chemotactic protein 1. CONCLUSIONS: IL1R1/IL1R2 imbalance may amplify endometrial cell responsiveness to IL1 and represent a key mechanism underlying the ability of these cells to implant and develop into host tissues.