Benzydamine plays a role in limiting inflammatory pain induced by neuronal sensitization.

Benzydamine is an active pharmaceutical compound used in the oral care pharmaceutical preparation as NSAID. Beside from its anti-inflammatory action, benzydamine local application effectively reliefs pain showing analgesic and anaesthetic properties. Benzydamine mechanism of action has been characterized on inflammatory cell types and mediators highlighting its capacity to inhibit pro-inflammatory mediators' synthesis and release. On the other hand, the role of benzydamine as neuronal excitability modulator has not yet fully explored. Thus, we studied benzydamine's effect over primary cultured DRG nociceptors excitability and after acute and chronic inflammatory sensitization, as a model to evaluate relative nociceptive response. Benzydamine demonstrated to effectively inhibit neuronal basal excitability reducing its firing frequency and increasing rheobase and afterhyperpolarization amplitude. Its effect was time and dose-dependent. At higher doses, benzydamine induced changes in action potential wavelength, decreasing its height and slightly increasing its duration. Moreover, the compound reduced neuronal acute and chronic inflammatory sensitization. It inhibited neuronal excitability mediated either by an inflammatory cocktail, acidic pH or high external KCl. Notably, higher potency was evidenced under inflammatory sensitized conditions. This effect could be explained either by modulation of inflammatory and/or neuronal sensitizing signalling cascades or by direct modulation of proalgesic and action potential firing initiating ion channels. Apparently, the compound inhibited Nav1.8 channel but had no effect over Kv7.2, Kv7.3, TRPV1 and TRPA1. In conclusion, the obtained results strengthen the analgesic and anti-inflammatory effect of benzydamine, highlighting its mode of action on local pain and inflammatory signalling.

Design and validation of neuronal exocytosis blocking peptides as potential novel antiperspirants.

Thermoregulation and heat dissipation by sweat production and evaporation are vital for human survival. However, hyperhidrosis or excessive perspiration might affect people's quality of life by causing discomfort and stress. The prolonged use of classical antiperspirants, anticholinergic medications or botulinum toxin injections for persistent hyperhidrosis might produce diverse side effects that limit their clinical use. Inspired by botox molecular mode of action, we used an in silico molecular modelling approach to design novel peptides to target neuronal acetylcholine exocytosis by interfering with the Snapin-SNARE complex formation. Our exhaustive design rendered the selection of 11 peptides that decreased calcium-dependent vesicle exocytosis in rat DRG neurons, reducing αCGRP release and TRPV1 inflammatory sensitization. The most potent peptides were palmitoylated peptides SPSR38-4.1 and SPSR98-9.1 that significantly suppressed acetylcholine release in vitro in human LAN-2 neuroblastoma cells. Noteworthy, local acute and chronic administration of SPSR38-4.1 peptide significantly decreased, in a dose-dependent manner, pilocarpine-induced sweating in an in vivo mouse model. Taken together, our in silico approach lead to the identification of active peptides able to attenuate excessive sweating by modulating neuronal acetylcholine exocytosis, and identified peptide SPSR38-4.1 as a promising new antihyperhidrosis candidate for clinical development.

DD04107-Derived neuronal exocytosis inhibitor peptides: Evidences for synaptotagmin-1 as a putative target.

The analgesic peptide DD04107 (Pal-EEMQRR-NH2) and its acetylated analogue inhibit α-calcitonin gene-related peptide (α-CGRP) exocytotic release from primary sensory neurons. Examining the crystal structure of the SNARE-Synaptotagmin-1(Syt1) complex, we hypothesized that these peptides could inhibit neuronal exocytosis by binding to Syt1, hampering at least partially its interaction with the SNARE complex. To address this hypothesis, we first interrogate the role of individual side-chains on the inhibition of α-CGRP release, finding that E1, M3, Q4 and R6 residues were crucial for activity. CD and NMR conformational analysis showed that linear peptides have tendency to adopt α-helical conformations, but the results with cyclic analogues indicated that this secondary structure is not needed for activity. Isothermal titration calorimetry (ITC) measurements demonstrate a direct interaction of some of these peptides with Syt1-C2B domain, but not with Syt7-C2B region, indicating selectivity. As expected for a compound able to inhibit α-CGRP release, cyclic peptide derivative Pal-E-cyclo[EMQK]R-NH2 showed potent in vivo analgesic activity, in a model of inflammatory pain. Molecular dynamics simulations provided a model consistent with KD values for the interaction of peptides with Syt1-C2B domain, and with their biological activity. Altogether, these results identify Syt1 as a potential new analgesic target.

A capsaicinoid-based soft drug, AG1529, for attenuating TRPV1-mediated histaminergic and inflammatory sensory neuron excitability.

TRPV1, a member of the transient receptor potential (TRP) family, is a nonselective calcium permeable ion channel gated by physical and chemical stimuli. In the skin, TRPV1 plays an important role in neurogenic inflammation, pain and pruritus associated to many dermatological diseases. Consequently, TRPV1 modulators could represent pharmacological tools to respond to important patient needs that still represent an unmet medical demand. Previously, we reported the design of capsaicinoid-based molecules that undergo dermal deactivation (soft drugs), thus preventing their long-term dermal accumulation. Here, we investigated the pharmacological properties of the lead antagonist, 2-((4-hydroxy-2-iodo-5-methoxybenzyl) amino)-2-oxoethyl dodecanoate (AG1529), on heterologously expressed human TRPV1 (hTRPV1), on nociceptor excitability and on an in vivo model of acute pruritus. We report that AG1529 competitively blocked capsaicin-evoked activation of hTRPV1 with micromolar potency, moderately affected pH-induced gating, and did not alter voltage- and heat-mediated responses. AG1529 displays modest receptor selectivity as it mildly blocked recombinant hTRPA1 and hTRPM8 channels. In primary cultures of rat dorsal root ganglion (DRG) neurons, AG1529 potently reduced capsaicin-evoked neuronal firing. AG1529 exhibited lower potency on pH-evoked TRPV1 firing, and TRPA1-elicited nociceptor excitability. Furthermore, AG1529 abolished histaminergic and inflammation mediated TRPV1 sensitization in primary cultures of DRG neurons. Noteworthy, dermal wiping of AG1529, either in an acetone-based formulation or in an anhydrous ointment, dose-dependently attenuated acute histaminergic itch in a rodent model. This cutaneous anti-pruritic effect was devoid of the normal nocifensive action evoked by the burning sensation of capsaicin. Taken together, these preclinical results unveil the mode of action of AG1529 on TRPV1 channels and substantiate the tenet that this capsaicinoid-based soft drug is a promising candidate for drug development as a topical anti-pruritic and anti-inflammatory medication.

A Novel Agonist of the Type 1 Lysophosphatidic Acid Receptor (LPA1), UCM-05194, Shows Efficacy in Neuropathic Pain Amelioration.

Neuropathic pain (NP) is a complex chronic pain state with a prevalence of almost 10% in the general population. Pharmacological options for NP are limited and weakly effective, so there is a need to develop more efficacious NP attenuating drugs. Activation of the type 1 lysophosphatidic acid (LPA1) receptor is a crucial factor in the initiation of NP. Hence, it is conceivable that a functional antagonism strategy could lead to NP mitigation. Here we describe a new series of LPA1 agonists among which derivative (S)-17 (UCM-05194) stands out as the most potent and selective LPA1 receptor agonist described so far (Emax = 118%, EC50 = 0.24 µM, KD = 19.6 nM; inactive at autotaxin and LPA2-6 receptors). This compound induces characteristic LPA1-mediated cellular effects and prompts the internalization of the receptor leading to its functional inactivation in primary sensory neurons and to an efficacious attenuation of the pain perception in an in vivo model of NP.

Targeting Transient Receptor Potential Vanilloid 1 (TRPV1) Channel Softly: The Discovery of Passerini Adducts as a Topical Treatment for Inflammatory Skin Disorders.

Despite being an old molecule, capsaicin is still a hot topic in the scientific community, and the development of new capsaicinoids is a promising pharmacological approach in the management of skin disorders related to inflammation and pruritus. Here we report the synthesis and the evaluation of capsaicin soft drugs that undergo deactivation by the hydrolyzing activity of skin esterases. The implanting of an ester group in the lipophilic moiety of capsaicinoids by the Passerini multicomponent reaction affords both agonists and antagonists that retain transient receptor potential vanilloid 1 channel (TRPV1) modulating activity and, at the same time, are susceptible to hydrolysis. The most promising antagonist identified shows in vivo anti-nociceptive activity on pruritus and hyperalgesia without producing hyperthermia, thus validating it as novel treatment for dermatological conditions that implicate TRPV1 channel dysfunction.

Bradykinin Induces TRPV1 Exocytotic Recruitment in Peptidergic Nociceptors.

Transient receptor potential vanilloid I (TRPV1) sensitization in peripheral nociceptors is a prominent phenomenon that occurs in inflammatory pain conditions. Pro-algesic agents can potentiate TRPV1 activity in nociceptors through both stimulation of its channel gating and mobilization of channels to the neuronal surface in a context dependent manner. A recent study reported that ATP-induced TRPV1 sensitization in peptidergic nociceptors involves the exocytotic release of channels trafficked by large dense core vesicles (LDCVs) that cargo alpha-calcitonin gene related peptide alpha (αCGRP). We hypothesized that, similar to ATP, bradykinin may also use different mechanisms to sensitize TRPV1 channels in peptidergic and non-peptidergic nociceptors. We found that bradykinin notably enhances the excitability of peptidergic nociceptors, and sensitizes TRPV1, primarily through the bradykinin receptor 2 pathway. Notably, bradykinin sensitization of TRPV1 in peptidergic nociceptors was significantly blocked by inhibiting Ca(2+)-dependent neuronal exocytosis. In addition, silencing αCGRP gene expression, but not substance P, drastically reduced bradykinin-induced TRPV1 sensitization in peptidergic nociceptors. Taken together, these findings indicate that bradykinin-induced sensitization of TRPV1 in peptidergic nociceptors is partially mediated by the exocytotic mobilization of new channels trafficked by αCGRP-loaded LDCVs to the neuronal membrane. Our findings further imply a central role of αCGRP peptidergic nociceptors in peripheral algesic sensitization, and substantiate that inhibition of LDCVs exocytosis is a valuable therapeutic strategy to treat pain, as it concurrently reduces the release of pro-inflammatory peptides and the membrane recruitment of thermoTRP channels.

Calcium Entry Through Thermosensory Channels.

ThermoTRPs are unique channels that mediate Na(+) and Ca(2+) currents in response to changes in ambient temperature. In combination with their activation by other physical and chemical stimuli, they are considered key integrators of environmental cues into neuronal excitability. Furthermore, roles of thermoTRPs in non-neuronal tissues are currently emerging such as insulin secretion in pancreatic ß-cells, and links to cancer. Calcium permeability through thermoTRPs appears a central hallmark for their physiological and pathological activities. Moreover, it is currently being proposed that beyond working as a second messenger, Ca(2+) can function locally by acting on protein complexes near the membrane. Interestingly, thermoTRPs can enhance and expand the inherent plasticity of signalplexes by conferring them temperature, pH and lipid regulation through Ca(2+) signalling. Thus, unveiling the local role of Ca(2+) fluxes induced by thermoTRPs on the dynamics of membrane-attached signalling complexes as well as their significance in cellular processes, are central issues that will expand the opportunities for therapeutic intervention in disorders involving dysfunction of thermoTRP channels.

αCGRP is essential for algesic exocytotic mobilization of TRPV1 channels in peptidergic nociceptors.

Proalgesic sensitization of peripheral nociceptors in painful syndromes is a complex molecular process poorly understood that involves mobilization of thermosensory receptors to the neuronal surface. However, whether recruitment of vesicular thermoTRP channels is a general mechanism underlying sensitization of all nociceptor types or is subtype-specific remains controversial. We report that sensitization-induced Ca(2+)-dependent exocytotic insertion of transient receptor potential vanilloid 1 (TRPV1) receptors to the neuronal plasma membrane is a mechanism specifically used by peptidergic nociceptors to potentiate their excitability. Notably, we found that TRPV1 is present in large dense-core vesicles (LDCVs) that were mobilized to the neuronal surface in response to a sensitizing insult. Deletion or silencing of calcitonin-gene-related peptide alpha (αCGRP) gene expression drastically reduced proalgesic TRPV1 potentiation in peptidergic nociceptors by abrogating its Ca(2+)-dependent exocytotic recruitment. These findings uncover a context-dependent molecular mechanism of TRPV1 algesic sensitization and a previously unrecognized role of αCGRP in LDCV mobilization in peptidergic nociceptors. Furthermore, these results imply that concurrent secretion of neuropeptides and channels in peptidergic C-type nociceptors facilitates a rapid modulation of pain signaling.

Trafficking of ThermoTRP Channels.

ThermoTRP channels (thermoTRPs) define a subfamily of the transient receptor potential (TRP) channels that are activated by changes in the environmental temperature, from noxious cold to injurious heat. Acting as integrators of several stimuli and signalling pathways, dysfunction of these channels contributes to several pathological states. The surface expression of thermoTRPs is controlled by both, the constitutive and regulated vesicular trafficking. Modulation of receptor surface density during pathological processes is nowadays considered as an interesting therapeutic approach for management of diseases, such as chronic pain, in which an increased trafficking is associated with the pathological state. This review will focus on the recent advances trafficking of the thermoTRP channels, TRPV1, TRPV2, TRPV4, TRPM3, TRPM8 and TRPA1, into/from the plasma membrane. Particularly, regulated membrane insertion of thermoTRPs channels contributes to a fine tuning of final channel activity, and indeed, it has resulted in the development of novel therapeutic approaches with successful clinical results such as disruption of SNARE-dependent exocytosis by botulinum toxin or botulinomimetic peptides.

Neurotrophins, endocannabinoids and thermo-transient receptor potential: a threesome in pain signalling.

Because of the social and economic costs of chronic pain, there is a growing interest in unveiling the cellular and molecular mechanisms underlying it with the aim of developing more effective medications. Pain signalling is a multicomponent process that involves the peripheral and central nervous systems. At the periphery, nociceptor sensitisation by pro-inflammatory mediators is a primary step in pain transduction. Although pain is multifactorial at cellular and molecular levels, it is widely accepted that neurotrophin (TrkA, p75NTR, Ret and GFRs), cannabinoid (CB1 and CB2), and thermo-transient receptor potential (TRPs; TRPV1, TRPA1 and TRPM8) receptors play a pivotal role. They form a threesome for which endocannabinoids appear to be a first line of defence against pain, while neurotrophins and thermoTRPs are the major generators of painful signals. However, endocannabinoids may exhibit nociceptive activity while some neurotrophins may display anti-nociception. Accordingly, a clear-cut knowledge of the modulation and context-dependent function of these signalling cascades, along with the molecular and dynamic details of their crosstalk, is critical for understanding and controlling pain transduction. Here, the recent progress in this fascinating topic, as well as the tantalizing questions that remain unanswered, will be discussed. Furthermore, we will underline the need for using a systems biology approach (referred to as systems pain) to uncover the dynamics and interplay of these intricate signalling cascades, taking into consideration the molecular complexity and cellular heterogeneity of nociceptor populations. Nonetheless, the available information confirms that pharmacological modulation of this signalling triad is a highly valuable therapeutic strategy for effectively treating pain syndromes.

Role of the transient receptor potential vanilloid 1 in inflammation and sepsis.

The transient receptor potential vanilloid 1 (TRPV1) is a thermoreceptor that responds to noxious temperatures, as well as to chemical agonists, such as vanilloids and protons. In addition, its channel activity is notably potentiated by proinflammatory mediators released upon tissue damage. The TRPV1 contribution to sensory neuron sensitization by proalgesic agents has signaled this receptor as a prime target for analgesic and anti-inflammatory drug intervention. However, TRPV1 antagonists have notably failed in clinical and preclinical studies because of their unwanted side effects. Recent reports have unveiled previously unrecognized anti-inflammatory and protective functions of TRPV1 in several diseases. For instance, this channel has been suggested to play an anti-inflammatory role in sepsis. Therefore, the use of potent TRPV1 antagonists as a general strategy to treat inflammation must be cautiously considered, given the deleterious effects that may arise from inhibiting the population of channels that have a protective function. The use of TRPV1 antagonists may be limited to treating those pathologies where enhanced receptor activity contributes to the inflamed state. Alternatively, therapeutic paradigms, such as reduction of inflammatory-mediated increase of receptor expression in the cell surface, may be a better strategy to prevent abrogation of the TRPV1 subpopulation involved in anti-inflammatory and protective processes.

Tumor necrosis factor-interleukin-17 interplay induces S100A8, interleukin-1ß, and matrix metalloproteinases, and drives irreversible cartilage destruction in murine arthritis: rationale for combination treatment during arthritis.

OBJECTIVE: To examine whether synovial interleukin-17 (IL-17) expression promotes tumor necrosis factor (TNF)-induced joint pathologic processes in vivo, and to analyze the surplus ameliorative value of neutralizing IL-17 in addition to TNF during collagen-induced arthritis (CIA). METHODS: Adenoviral vectors were used to induce overexpression of IL-17 and/or TNF in murine knee joints. In addition, mice with CIA were treated, at different stages of arthritis, with soluble IL-17 receptor (sIL-17R), TNF binding protein (TNFBP), or the combination. RESULTS: Overexpression of IL-17 and TNF resulted in joint inflammation and bone erosion in murine knees. Interestingly, IL-17 strikingly enhanced both the joint-inflammatory and joint-destructive capacity of TNF. Further analysis revealed a strongly enhanced up-regulation of S100A8, IL-1ß, and matrix metalloproteinase (MMP) messenger RNA, only when both TNF and IL-17 were present. Moreover, the increase in irreversible cartilage destruction was not merely the result of enhanced inflammation, but also was associated with a direct synergistic effect of these cytokines in the joint. S100A9 deficiency in mice protected against IL-17/TNF-induced expression of cartilage NITEGE neoepitopes. During established arthritis, the combination of sIL-17R and TNFBP was more effective than the anticytokine treatments alone, and significantly inhibited further joint inflammation and cartilage destruction. CONCLUSION: Local synovial IL-17 expression enhances the role of TNF in joint destruction. Synergy between TNF and IL-17 in vivo results in striking exaggeration of cartilage erosion, in parallel with a synergistic up-regulation of S100A8, IL-1ß, and erosive MMPs. Moreover, neutralizing IL-17 in addition to TNF further improves protection against joint damage and is still effective during late-stage CIA. Therefore, compared with anti-TNF alone, combination blocking of TNF and IL-17 may have additional therapeutic value for the treatment of destructive arthritis.

New strategies to develop novel pain therapies: addressing thermoreceptors from different points of view.

One approach to develop successful pain therapies is the modulation of dysfunctional ion channels that contribute to the detection of thermal, mechanical and chemical painful stimuli. These ion channels, known as thermoTRPs, promote the sensitization and activation of primary sensory neurons known as nociceptors. Pharmacological blockade and genetic deletion of thermoTRP have validated these channels as therapeutic targets for pain intervention. Several thermoTRP modulators have progressed towards clinical development, although most failed because of the appearance of unpredicted side effects. Thus, there is yet a need to develop novel channel modulators with improved therapeutic index. Here, we review the current state-of-the art and illustrate new pharmacological paradigms based on TRPV1 that include: (i) the identification of activity-dependent modulators of this thermoTRP channel; (ii) the design of allosteric modulators that interfere with protein-protein interaction involved in the functional coupling of stimulus sensing and gate opening; and (iii) the development of compounds that abrogate the inflammation-mediated increase of receptor expression in the neuronal surface. These new sites of action represent novel strategies to modulate pathologically active TRPV1, while minimizing an effect on the TRPV1 subpopulation involved in physiological and protective roles, thus increasing their potential therapeutic use.

The CO-releasing molecule CORM-3 protects against articular degradation in the K/BxN serum transfer arthritis model.

Carbon monoxide-releasing molecules can counteract inflammatory responses. The aim of this study was to investigate whether tricarbonylchloro(glycinate)ruthenium (II) (CORM-3) is able to control the effector phase of experimental arthritis. Arthritis was induced in C57Black-6 mice by an intraperitoneal injection of serum from arthritic K/BxN mice. CORM-3 was administered intraperitoneally at 10 mg/kg/day (5 mg/kg twice a day) from days 0 to 10 and animals were sacrificed on day 11. Serum levels of osteocalcin and prostanoids were measured by enzyme-linked immunosorbent assay and radioimmunoassay. Gene expression was determined by real-time PCR. Histological analysis was performed and protein expression was examined by immunohistochemistry. Treatment with CORM-3 reduced the macroscopic score in hind paws, the migration of inflammatory cells and erosion of cartilage and bone. CORM-3 increased the levels of osteocalcin in the serum and reduced PGD2 levels, whereas PGE2 and 6-ketoPGF1alpha were not affected. In synovial tissues, we also observed a significant reduction in gene expression of interleukin-1beta, receptor activator of nuclear factor kappaB ligand (RANKL), matrix metalloproteinase (MMP)-9 and MMP-13. CORM-3 induced HO-1 expression in joint tissues but inhibited high mobility group box 1 (HMGB1), hematopoietic-prostaglandin D2 synthase (H-PGDS) and lipocalin-type prostaglandin D2 synthase (L-PGDS), as well as RANKL and intercellular adhesion molecule-1. COX-2 expression was not affected by CORM-3 treatment. We have shown that CORM-3 decreases the inflammatory response and protects against the degradation of cartilage and bone in the arthritic mice. Pharmacological CO delivery represents a novel strategy to regulate the effector phase of arthritis.

Bypassing pathogen-induced inflammasome activation for the regulation of interleukin-1beta production by the fungal pathogen Candida albicans.

BACKGROUND: Interleukin (IL)-1beta has an important role in antifungal defense mechanisms. The inflammasome is thought to be required for caspase-1 activation and processing of the inactive precursor pro-IL-1beta. The aim of the present study was to investigate the pathways of IL-1beta production induced by Candida albicans in human monocytes. METHODS: Human mononuclear cells were stimulated with C. albicans or mutant strains defective in mannosylation or chitin. Receptors were blocked with specific antagonists, and the IL-1beta concentration was measured. RESULTS: Human primary monocytes produce bioactive IL-1beta when stimulated with C. albicans. The transcription of IL-1beta was induced through mannose receptor (MR), Toll-like receptor (TLR) 2, and dectin-1 but not through TLR4 and TLR9. N-mannan-linked residues, chitin, and beta-glucan from C. albicans are important for IL-1beta stimulation. Surprisingly, processing and secretion of IL-1beta in monocytes did not require pathogen-mediated inflammasome activation, because of the constitutive activation of caspase-1 and the capability of monocytes to release endogenous adenosine-5'-triphosphate. CONCLUSIONS: This study is the first dissection of the molecular mechanisms of IL-1beta production by a fungal pathogen. Transcription through mannan/chitin/MR and beta-glucan/dectin-1/TLR2 induces production of IL-1beta by C. albicans in human monocytes, whereas processing of IL-1beta is mediated by constitutively active caspase-1.

Differential requirement for the activation of the inflammasome for processing and release of IL-1beta in monocytes and macrophages.

The processing of pro-interleukin-1beta depends on activation of caspase-1. Controversy has arisen whether Toll-like receptor (TLR) ligands alone can activate caspase-1 for release of interleukin-1beta (IL-1beta). Here we demonstrate that human blood monocytes release processed IL-1beta after a one-time stimulation with either TLR2 or TLR4 ligands, resulting from constitutively activated caspase-1 and release of endogenous adenosine triphosphate. The constitutive activation of caspase-1 depends on the inflammasome components, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and NALP3, but in monocytes caspase-1 activation is uncoupled from pathogen-associated molecular pattern recognition. In contrast, macrophages are unable to process and release IL-1beta solely by TLR ligands and require a second adenosine triphosphate stimulation. We conclude that IL-1beta production is differentially regulated in monocytes and macrophages, and this reflects their separate functions in host defense and inflammation.

Interleukin-1 drives pathogenic Th17 cells during spontaneous arthritis in interleukin-1 receptor antagonist-deficient mice.

OBJECTIVE: Interleukin-1 receptor antagonist-deficient (IL-1Ra-/-) mice spontaneously develop an inflammatory and destructive arthritis due to unopposed excess IL-1 signaling. In this study, the role of Th17 cells and the effect of neutralization of IL-17, IL-1, and tumor necrosis factor alpha (TNFalpha) were investigated in this IL-1-driven murine arthritis model. METHODS: T cells isolated from IL-1Ra-/- and wild-type (WT) mice were stained for IL-17 and interferon-gamma, with results assessed by fluorescence-activated cell sorting analysis. To investigate the contribution of IL-1 and IL-17 in further progression of arthritis in this model, mice were treated with neutralizing antibodies after the onset of arthritis. RESULTS: Compared with WT mice, IL-1Ra-/- mice had similar levels of Th1 cells but clearly enhanced levels of Th17 cells; this increase in the number of Th17 cells was evident even before the onset of arthritis, in young, nonarthritic IL-1Ra-/- mice. The percentage of Th17 cells increased even more after the onset of arthritis and, similar to the serum levels and local messenger RNA levels of IL-17, the percentage of IL-17+ Th17 cells clearly correlated with the severity of arthritis. Anti-IL-17 treatment prevented any further increase in inflammation and bone erosion, whereas blocking of TNFalpha after the onset of arthritis had no effect. In contrast, neutralization of IL-1 resulted in a complete suppression of arthritis. Interestingly, this anti-IL-1 treatment also significantly reduced the percentage of IL-17+ Th17 cells in the draining lymph nodes of these arthritic mice. CONCLUSION: Increased levels of Th17 cells can be detected in IL-1Ra-/- mice even preceding the onset of arthritis. In addition, the results of cytokine-blocking studies demonstrated that IL-17 contributes to the inflammation and bone erosion in this model, which suggests that IL-1 is the driving force behind the IL-17-producing Th17 cells.

Stimulation of TLR2 and TLR4 differentially skews the balance of T cells in a mouse model of arthritis.

TLRs may contribute to the progression of rheumatoid arthritis through recognition of microbial or host-derived ligands found in arthritic joints. Here, we show that TLR2 and TLR4, but not TLR9, are involved in the pathogenesis of autoimmune arthritis and play distinct roles in the regulation of T cells and cytokines. We investigated the involvement of TLR2, TLR4, and TLR9 in the progression of arthritis using IL-1 receptor antagonist-knockout (IL1rn-/-) mice, which spontaneously develop an autoimmune T cell-mediated arthritis. Spontaneous onset of arthritis was dependent on TLR activation by microbial flora, as germ-free mice did not develop arthritis. Clinical and histopathological evaluation of IL1rn-/-Tlr2-/- mice revealed more severe arthritis, characterized by reduced suppressive function of Tregs and substantially increased IFN-gamma production by T cells. IL1rn-/-Tlr4-/- mice were, in contrast, protected against severe arthritis and had markedly lower numbers of Th17 cells and a reduced capacity to produce IL-17. A lack of Tlr9 did not affect the progression of arthritis. While any therapeutic intervention targeting TLR2 still seems complicated, the strict position of TLR4 upstream of a number of pathogenic cytokines including IL-17 provides an interesting potential therapeutic target for rheumatoid arthritis.