Hyperactivation of sympathetic nerves drives depletion of melanocyte stem cells.

Empirical and anecdotal evidence has associated stress with accelerated hair greying (formation of unpigmented hairs)1,2, but so far there has been little scientific validation of this link. Here we report that, in mice, acute stress leads to hair greying through the fast depletion of melanocyte stem cells. Using a combination of adrenalectomy, denervation, chemogenetics3,4, cell ablation and knockout of the adrenergic receptor specifically in melanocyte stem cells, we find that the stress-induced loss of melanocyte stem cells is independent of immune attack or adrenal stress hormones. Instead, hair greying results from activation of the sympathetic nerves that innervate the melanocyte stem-cell niche. Under conditions of stress, the activation of these sympathetic nerves leads to burst release of the neurotransmitter noradrenaline (also known as norepinephrine). This causes quiescent melanocyte stem cells to proliferate rapidly, and is followed by their differentiation, migration and permanent depletion from the niche. Transient suppression of the proliferation of melanocyte stem cells prevents stress-induced hair greying. Our study demonstrates that neuronal activity that is induced by acute stress can drive a rapid and permanent loss of somatic stem cells, and illustrates an example in which the maintenance of somatic stem cells is directly influenced by the overall physiological state of the organism.

COVID-19-related hyperglycemia is associated with infection of hepatocytes and stimulation of gluconeogenesis.

Occurrence of hyperglycemia upon infection is associated with worse clinical outcome in COVID-19 patients. However, it is still unknown whether SARS-CoV-2 directly triggers hyperglycemia. Herein, we interrogated whether and how SARS-CoV-2 causes hyperglycemia by infecting hepatocytes and increasing glucose production. We performed a retrospective cohort study including patients that were admitted at a hospital with suspicion of COVID-19. Clinical and laboratory data were collected from the chart records and daily blood glucose values were analyzed to test the hypothesis on whether COVID-19 was independently associated with hyperglycemia. Blood glucose was collected from a subgroup of nondiabetic patients to assess pancreatic hormones. Postmortem liver biopsies were collected to assess the presence of SARS-CoV-2 and its transporters in hepatocytes. In human hepatocytes, we studied the mechanistic bases of SARS-CoV-2 entrance and its gluconeogenic effect. SARS-CoV-2 infection was independently associated with hyperglycemia, regardless of diabetic history and beta cell function. We detected replicating viruses in human hepatocytes from postmortem liver biopsies and in primary hepatocytes. We found that SARS-CoV-2 variants infected human hepatocytes in vitro with different susceptibility. SARS-CoV-2 infection in hepatocytes yields the release of new infectious viral particles, though not causing cell damage. We showed that infected hepatocytes increase glucose production and this is associated with induction of PEPCK activity. Furthermore, our results demonstrate that SARS-CoV-2 entry in hepatocytes occurs partially through ACE2- and GRP78-dependent mechanisms. SARS-CoV-2 infects and replicates in hepatocytes and exerts a PEPCK-dependent gluconeogenic effect in these cells that potentially is a key cause of hyperglycemia in infected patients.

Cigarette smoke induces miR-132 in Th17 cells that enhance osteoclastogenesis in inflammatory arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by joint destruction and severe morbidity. Cigarette smoking (CS) can exacerbate the incidence and severity of RA. Although Th17 cells and the Aryl hydrocarbon receptor (AhR) have been implicated, the mechanism by which CS induces RA development remains unclear. Here, using transcriptomic analysis, we show that microRNA-132 is specifically induced in Th17 cells in the presence of either AhR agonist or CS-enriched medium. miRNA-132 thus induced is packaged into extracellular vesicles produced by Th17 and acts as a proinflammatory mediator increasing osteoclastogenesis through the down-regulation of COX2. In vivo, articular knockdown of miR-132 in murine arthritis models reduces the number of osteoclasts in the joints. Clinically, RA patients express higher levels of miR-132 than do healthy individuals. This increase is further elevated by cigarette smoking. Together, these results reveal a hitherto unrecognized mechanism by which CS could exacerbate RA and further advance understanding of the impact of environmental factors on the pathogenesis of chronic inflammatory diseases.

CASP4/11 Contributes to NLRP3 Activation and COVID-19 Exacerbation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection triggers activation of the NLRP3 inflammasome, which promotes inflammation and aggravates severe COVID-19. Here, we report that SARS-CoV-2 induces upregulation and activation of human caspase-4/CASP4 (mouse caspase-11/CASP11), and this process contributes to NLRP3 activation. In vivo infections performed in transgenic hACE2 humanized mice, deficient or sufficient for Casp11, indicate that hACE2 Casp11-/- mice were protected from disease development, with the increased pulmonary parenchymal area, reduced clinical score of the disease, and reduced mortality. Assessing human samples from fatal cases of COVID-19, we found that CASP4 was expressed in patient lungs and correlated with the expression of inflammasome components and inflammatory mediators, including CASP1, IL1B, IL18, and IL6. Collectively, our data establish that CASP4/11 promotes NLRP3 activation and disease pathology, revealing a possible target for therapeutic interventions for COVID-19.

Enoxaparin improves COVID-19 by reducing Neutrophils Extracellular Traps (NETs) production.

BACKGROUND: COVID-19 causes consequences such as imbalance of the immune system and thrombotic events. During the infection process, NETs in excess induce a pro-inflammatory response and disseminated intravascular coagulation. We evaluated the role of enoxaparin as a potential inhibitor of NETs. METHODS: K18-hACE2 animals infected with the SARS-CoV-2 virus and a group of 23 individuals admitted to the hospital with COVID-19 treated with enoxaparin or without treatment and controls without the disease were included. RESULTS: Enoxaparin decreased the levels of NETs, reduced the signs of the disease and mitigated lung damage in the animals infected with SARS-CoV-2. These effects were partially associated with prevention of SARS-CoV-2 entry and NETs synthesis. Clinical data revealed that treatment with enoxaparin decreased the levels of inflammatory markers, the levels of NETs in isolated neutrophils and the organ dysfunction. CONCLUSION: This study provides evidence for the beneficial effects of enoxaparin in COVID-19 in addition to its anticoagulant role.

Gasdermin D inhibition prevents multiple organ dysfunction during sepsis by blocking NET formation.

Multiple organ dysfunction is the most severe outcome of sepsis progression and is highly correlated with a worse prognosis. Excessive neutrophil extracellular traps (NETs) are critical players in the development of organ failure during sepsis. Therefore, interventions targeting NET release would likely effectively prevent NET-based organ injury associated with this disease. Herein, we demonstrate that the pore-forming protein gasdermin D (GSDMD) is active in neutrophils from septic humans and mice and plays a crucial role in NET release. Inhibition of GSDMD with disulfiram or genic deletion abrogated NET formation, reducing multiple organ dysfunction and sepsis lethality. Mechanistically, we demonstrate that during sepsis, activation of the caspase-11/GSDMD pathway controls NET release by neutrophils during sepsis. In summary, our findings uncover a novel therapeutic use for disulfiram and suggest that GSDMD is a therapeutic target to improve sepsis treatment.

Targeting neutrophils extracellular traps (NETs) reduces multiple organ injury in a COVID-19 mouse model.

BACKGROUND: COVID-19 is characterized by severe acute lung injury, which is associated with neutrophil infiltration and the release of neutrophil extracellular traps (NETs). COVID-19 treatment options are scarce. Previous work has shown an increase in NETs release in the lung and plasma of COVID-19 patients suggesting that drugs that prevent NETs formation or release could be potential therapeutic approaches for COVID-19 treatment. METHODS: Here, we report the efficacy of NET-degrading DNase I treatment in a murine model of COVID-19. SARS-CoV-2-infected K18-hACE2 mice were performed for clinical sickness scores and lung pathology. Moreover, the levels of NETs were assessed and lung injuries were by histopathology and TUNEL assay. Finally, the injury in the heart and kidney was assessed by histopathology and biochemical-specific markers. RESULTS: DNase I decreased detectable levels of NETs, improved clinical disease, and reduced lung, heart, and kidney injuries in SARS-CoV-2-infected K18-hACE2 mice. Furthermore, our findings indicate a potentially deleterious role for NETs lung tissue in vivo and lung epithelial (A549) cells in vitro, which might explain part of the pathophysiology of severe COVID-19. This deleterious effect was diminished by the treatment with DNase I. CONCLUSIONS: Together, our results support the role of NETs in COVID-19 immunopathology and highlight NETs disruption pharmacological approaches as a potential strategy to ameliorate COVID-19 clinical outcomes.

Sepsis-Induced Immunosuppression Is Marked by an Expansion of a Highly Suppressive Repertoire of FOXP3+ T-Regulatory Cells Expressing TIGIT.

BACKGROUND: Although the literature shows that an increase in both the number and suppressive function of CD4+forkhead box P3 (FOXP3)+ T-regulatory cells (Tregs) during sepsis contributes to an immunosuppressed state, little is known about the identity of these cells. METHODS: Using the sepsis mouse model of cecal ligation and puncture (CLP), we analyzed the frequency and molecular signature of the T-cell immunoglobulin and ITIM domain (TIGIT)+ and TIGIT- Treg subsets, using flow cytometry and quantitative polymerase chain reaction. In addition, ST2-/- and signal transducer and activator of transcription 6 (STAT6)-/- mice were submitted to CLP or recombinant interleukin 33 (IL-33) treatment to investigate the mechanism whereby TIGIT+ Tregs differentiate during sepsis. RESULTS: Sepsis was marked by the sustained expansion of the highly suppressive TIGIT+ Treg subset, which expresses Helios, neuropilin 1, and high levels of Tnfrsf18 and Pdcd1 at 15 days after CLP. The increase in TIGIT+ Tregs was accompanied by higher susceptibility to nosocomial bacteria challenge, suggesting their association with post sepsis immunosuppression. Mechanistically, we found that the ST2 deletion abrogated the expansion of the TIGIT+ Treg subset during sepsis. Furthermore, treatment with recombinant IL-33 resulted in the expansion of TIGIT+ Tregs depending on the STAT6 and M2 macrophages. CONCLUSIONS: These findings demonstrated that only the TIGIT+ Tregs remain stably expanded at the late phase of sepsis. Moreover, the expansion of TIGIT+ Tregs is dependent on the IL-33/ST2/STAT6/M2 macrophage axis.

The NLRP3 inflammasome is involved with the pathogenesis of Mayaro virus.

Mayaro virus (MAYV) is an arbovirus that circulates in Latin America and is emerging as a potential threat to public health. Infected individuals develop Mayaro fever, a severe inflammatory disease characterized by high fever, rash, arthralgia, myalgia and headache. The disease is often associated with a prolonged arthralgia mediated by a chronic inflammation that can last months. Although the immune response against other arboviruses, such as chikungunya virus (CHIKV), dengue virus (DENV) and Zika virus (ZIKV), has been extensively studied, little is known about the pathogenesis of MAYV infection. In this study, we established models of MAYV infection in macrophages and in mice and found that MAYV can replicate in bone marrow-derived macrophages and robustly induce expression of inflammasome proteins, such as NLRP3, ASC, AIM2, and Caspase-1 (CASP1). Infection performed in macrophages derived from Nlrp3-/-, Aim2-/-, Asc-/-and Casp1/11-/-mice indicate that the NLRP3, but not AIM2 inflammasome is essential for production of inflammatory cytokines, such as IL-1ß. We also determined that MAYV triggers NLRP3 inflammasome activation by inducing reactive oxygen species (ROS) and potassium efflux. In vivo infections performed in inflammasome-deficient mice indicate that NLRP3 is involved with footpad swelling, inflammation and pain, establishing a role of the NLRP3 inflammasome in the MAYV pathogenesis. Accordingly, we detected higher levels of caspase1-p20, IL-1ß and IL-18 in the serum of MAYV-infected patients as compared to healthy individuals, supporting the participation of the NLRP3-inflammasome during MAYV infection in humans.

Ablation of sensory nerves favours melanoma progression.

The tumour mass is composed not only of heterogeneous neoplastic cells, but also a variety of other components that may affect cancer cells behaviour. The lack of detailed knowledge about all the constituents of the tumour microenvironment restricts the design of effective treatments. Nerves have been reported to contribute to the growth and maintenance of numerous tissues. The effects of sensory innervations on tumour growth remain unclear. Here, by using state-of-the-art techniques, including Cre/loxP technologies, confocal microscopy, in vivo-tracing and chemical denervation, we revealed the presence of sensory nerves infiltrating within the melanoma microenvironment, and affecting cancer progression. Strikingly, melanoma growth in vivo was accelerated following genetic ablation or chemical denervation of sensory nerves. In humans, a retrospective analysis of melanoma patients revealed that increased expression of genes related to sensory nerves in tumours was associated with better clinical outcomes. These findings suggest that sensory innervations counteract melanoma progression. The emerging knowledge from this research provides a novel target in the tumour microenvironment for therapeutic benefit in cancer patients.

Monosodium urate crystal interleukin-1ß release is dependent on Toll-like receptor 4 and transient receptor potential V1 activation.

OBJECTIVE: The present study aimed to elucidate the mechanisms involved in MSU-induced IL-1ß release in a rodent animal model of acute gout arthritis. METHODS: Painful (mechanical and thermal hypersensitivity, ongoing pain and arthritis score) and inflammatory (oedema, plasma extravasation, cell infiltration and IL-1ß release) parameters were assessed several hours after intra-articular injection of MSU (100 µg/articulation) in wild-type or knockout mice for Toll-like receptor 4 (TLR4), inducible nitric oxide synthase (iNOS), transient receptor potential (TRP) V1 and the IL-1 receptor (IL-1R). Also, wild-type animals were treated with clodronate, lipopolysaccharide from Rhodobacter sphaeroides (LPS-RS) (TLR4 antagonist), spleen tyrosine kinase (SYK) inhibitor (iSYK), aminoguanidine (AMG, an iNOS inhibitor) or SB366791 (TRPV1 antagonist). Nitrite/nitrate and IL-1ß levels were measured on the synovial fluid of wild-type mice, 2 h after intra-articular MSU injections, or medium from macrophages stimulated for MSU (1000 µg) for 2 h. RESULTS: Intra-articular MSU injection caused robust nociception and severe inflammation from 2 up to 6 h after injection, which were prevented by the pre-treatment with clodronate, LPS-RS, iSYK, AMG and SB366791, or the genetic ablation of TLR4, iNOS, TRPV1 or IL-1R. MSU also increased nitrite/nitrate and IL-1ß levels in the synovial fluid, which was prevented by clodronate, LPS-RS, iSYK and AMG, but not by SB366791. Similarly, MSU-stimulated peritoneal macrophages released nitric oxide, which was prevented by LPS-RS, iSYK and AMG, but not by SB366791, and released IL-1ß, which was prevented by LPS-RS, iSYK, AMG and SB366791. CONCLUSION: Our data indicate that MSU may activate TLR4, SYK, iNOS and TRPV1 to induce the release of IL-1ß by macrophages, triggering nociception and inflammation during acute gout attack.

S100A9 plays a pivotal role in a mouse model of herpetic neuralgia via TLR4/TNF pathway.

Herpetic neuralgia is a painful condition following herpes zoster disease, which results from Varicella-zoster virus reactivation in the dorsal or trigeminal sensory ganglia. Nevertheless, the pathophysiological mechanisms involved in herpetic neuralgia are not well understood. Recently, we identified, that neuroimmune-glia interactions in the sensory ganglion is a critical mechanism for the development of herpetic neuralgia. Here, we investigate the contribution of S100A9, a well-known pro-inflammatory molecule produced by myeloid cells, for the development of herpetic neuralgia using a murine model of HSV-1 infection. We found that cutaneous HSV-1 infection results in an increase of S100A9 expression in the Dorsal Root Ganglia (DRGs). Infiltrating neutrophils into the DRGs were the main source of S100A9 post HSV-1 infection. Functionally, genetic or pharmacological inhibition of S100A9 impairs the development of HSV-1 infection-induced mechanical pain hypersensitivity. Finally, we found that the pronociceptive role of S100A9 in herpetic neuralgia depends on the TLR4/TNF pathway. These results unraveled previously unknown mechanisms involved in the pathophysiology of herpetic neuralgia and indicate that S100A9 might be an important target for novel therapies aiming acute herpetic neuralgia.

Molecular basis of carrageenan-induced cytokines production in macrophages.

BACKGROUND: Low molecular weight carrageenan (Cg) is a seaweed-derived sulfated polysaccharide widely used as inflammatory stimulus in preclinical studies. However, the molecular mechanisms of Cg-induced inflammation are not fully elucidated. The present study aimed to investigate the molecular basis involved in Cg-induced macrophages activation and cytokines production. METHODS: Primary culture of mouse peritoneal macrophages were stimulated with Kappa Cg. The supernatant and cell lysate were used for ELISA, western blotting, immunofluorescence. Cg-induced mouse colitis was also developed. RESULTS: Here we show that Cg activates peritoneal macrophages to produce pro-inflammatory cytokines such as TNF and IL-1ß. While Cg-induced TNF production/secretion depends on TLR4/MyD88 signaling, the production of pro-IL-1ß relies on TLR4/TRIF/SYK/reactive oxygen species (ROS) signaling pathway. The maturation of pro-IL1ß into IL-1ß is dependent on canonical NLRP3 inflammasome activation via Pannexin-1/P2X7/K+ efflux signaling. In vivo, Cg-induced colitis was reduced in mice in the absence of NLRP3 inflammasome components. CONCLUSIONS: In conclusion, we unravel a critical role of the NLRP3 inflammasome in Cg-induced pro-inflammatory cytokines production and colitis, which is an important discovery on the pro-inflammatory properties of this sulfated polysaccharide for pre-clinical studies. Video abstract Carrageenan (Cg) is one the most used flogistic stimulus in preclinical studies. Nevertheless, the molecular basis of Cg-induced inflammation is not totally elucidated. Herein, Lopes et al. unraveled the molecular basis for Cg-induced macrophages production of biological active IL-1ß. The Cg-stimulated macrophages produces pro-IL-1ß depends on TLR4/TRIF/Syk/ROS, whereas its processing into mature IL-1ß is dependent on the canonical NLRP3 inflammasome.

The role of neutrophils in neuro-immune modulation.

Neutrophils are peripheral immune cells that represent the first recruited innate immune defense against infections and tissue injury. However, these cells can also induce overzealous responses and cause tissue damage. Although the role of neutrophils activating the immune system is well established, only recently their critical implications in neuro-immune interactions are becoming more relevant. Here, we review several aspects of neutrophils in the bidirectional regulation between the nervous and immune systems. First, the role of neutrophils as a diffuse source of acetylcholine and catecholamines is controversial as well as the effects of these neurotransmitters in neutrophil's functions. Second, neutrophils contribute for the activation and sensitization of sensory neurons, and thereby, in events of nociception and pain. In addition, nociceptor activation promotes an axon reflex triggering a local release of neural mediators and provoking neutrophil activation. Third, the recruitment of neutrophils in inflammatory responses in the nervous system suggests these immune cells as innovative targets in the treatment of central infectious, neurological and neurodegenerative disorders. Multidisciplinary studies involving immunologists and neuroscientists are required to define the role of the neurons-neutrophils communication in the pathophysiology of infectious, inflammatory, and neurological disorders.

Thirty days after anterior cruciate ligament transection is sufficient to induce signs of knee osteoarthritis in rats: pain, functional impairment, and synovial inflammation.

OBJECTIVE: To compare the unilateral signs of knee osteoarthritis (KOA) 30 and 60 days after anterior cruciate ligament transection (ACLT). Pain, gait function, synovial fluid inflammation, and histopathological changes in the synovial membrane were analyzed, as well as the interaction between the variables. MATERIALS AND METHODS: Male Wistar rats (n = 32; 219.2 ± 18.6 g) were randomly distributed into four groups of eight animals each. Two groups were submitted to unilateral ACLT surgery to induce KOA and analyzed after 30 (KOA30) and 60 days (KOA60). Two control groups (without surgery) were also assessed after the same time periods (C30 and C60). All the groups were evaluated before ACLT from the least to most stressful tests (skin temperature, mechanical response threshold, gait test, thermal response threshold, and joint swelling), as well as 30 and 60 days after surgery. After euthanasia, the synovial fluid and synovial membrane were collected. RESULTS: Thirty days after ACLT, KOA30 showed decrease paw print area and mechanical response threshold, higher joint swelling, skin temperature, leukocyte count, cytokine levels, and synovitis score. No differences were found between KOA30 and KOA60. CONCLUSION: Our data showed that 30 days after ACLT is sufficient to induce signs of KOA in rats, such as pain, functional impairment, and synovial inflammation, suggesting that a shorter time period can be used as an experimental model.

IL-33 enhances macrophage release of IL-1ß and promotes pain and inflammation in gouty arthritis.

OBJECTIVE: To investigate the role of IL-33 in gouty arthritis. MATERIAL: 174 Balb/c (wild-type) and 54 ST2-/- mice were used in this study. In vitro experiments were conducted in bone marrow-derived macrophages (BMDMs). Synovial fluid samples from gouty arthritis (n = 7) and osteoarthritis (n = 8) hospital patients were used to measure IL-33 and sST2 levels. METHODS: Gout was induced by injection of monosodium urate (MSU) crystals in the knee joint of mice. Pain was determined using the electronic von Frey and static weight bearing. Neutrophil recruitment was determined by H&E staining, Rosenfeld staining slides, and MPO activity. ELISA was used for cytokine and sST2 measurement. The priming effect of IL-33 was determined in BMDM. RESULTS: Synovial fluid of gout patients showed higher IL-33 levels and neutrophil counts than osteoarthritis patients. In mice, the absence of ST2 prevented mechanical pain, knee joint edema, neutrophil recruitment to the knee joint, and lowered IL-1ß and superoxide anion levels. In macrophages, IL-33 enhanced the release of IL-1ß and TNF-α, and BMDMs from ST2-/- showed reduced levels of these cytokines after stimulus with MSU crystals. CONCLUSION: IL-33 mediates gout pain and inflammation by boosting macrophages production of cytokines upon MSU crystals stimulus.

Contribution of spinal cord glial cells to L. amazonensis experimental infection-induced pain in BALB/c mice.

BACKGROUND: The cellular and molecular pathophysiological mecha\nisms of pain processing in neglected parasitic infections such as leishmaniasis remain unknown. The present study evaluated the participation of spinal cord glial cells in the pathophysiology of pain induced by Leishmania amazonensis infection in BALB/c mice. METHODS: Mice received intra-plantar (i.pl.) injection of L. amazonensis (1 × 105) and hyperalgesia, and paw edema were evaluated bilaterally for 40 days. The levels of TNF-α and IL-1ß, MPO activity, and histopathology were assessed on the 40th day. ATF3 mRNA expression was assessed in DRG cells at the 30th day post-infection. Blood TNF-α and IL-1ß levels and systemic parasite burden were evaluated 5-40 days after the infection. At the 30th day post-infection L. amazonensis, the effects of intrathecal (i.t.) treatments with neutralizing antibody anti-CX3CL1, etanercept (soluble TNFR2 receptor), and interleukin-1 receptor antagonist (IL-1ra) on infection-induced hyperalgesia and paw edema were assessed. In another set of experiments, we performed a time course analysis of spinal cord GFAP and Iba-1 (astrocytes and microglia markers, respectively) and used confocal immunofluorescence and Western blot to confirm the expression at the protein level. Selective astrocyte (α-aminoadipate) and microglia (minocycline) inhibitors were injected i.t. to determine the contribution of these cells to hyperalgesia and paw edema. The effects of i.t. treatments with glial and NFκB (PDTC) inhibitors on spinal glial activation, TNF-α, IL-1ß, CX3CR1 and CX3CL1 mRNA expression, and NFκB activation were also evaluated. Finally, the contribution of TNF-α and IL-1ß to CX3CL1 mRNA expression was investigated. RESULTS: L. amazonensis infection induced chronic mechanical and thermal hyperalgesia and paw edema in the infected paw. Mechanical hyperalgesia was also observed in the contralateral paw. TNF-α, IL-1ß, MPO activity, and epidermal/dermal thickness increased in the infected paw, which confirmed the peripheral inflammation at the primary foci of this infection. ATF3 mRNA expression at the ipsilateral DRG of the infected paw was unaltered 30 days post-infection. TNF-α and IL-1ß blood levels were not changed over the time course of disease, and parasitism increased in a time-dependent manner in the ipsilateral draining lymph node. Treatments targeting CX3CL1, TNF-α, and IL-1ß inhibited L. amazonensis-induced ongoing mechanical and thermal hyperalgesia, but not paw edema. A time course of GFAP, Iba-1, and CX3CR1 mRNA expression indicated spinal activation of astrocytes and microglia, which was confirmed at the GFAP and Iba-1 protein level at the peak of mRNA expression (30th day). Selective astrocyte and microglia inhibition diminished infection-induced ipsilateral mechanical hyperalgesia and thermal hyperalgesia, and contralateral mechanical hyperalgesia, but not ipsilateral paw edema. Targeting astrocytes, microglia and NFκB diminished L. amazonensis-induced GFAP, Iba-1, TNF-α, IL-1ß, CX3CR1 and CX3CL1 mRNA expression, and NFκB activation in the spinal cord at the peak of spinal cord glial cells activation. CX3CL1 mRNA expression was also detected in the ipsilateral DRG of infected mice at the 30th day post-infection, and the i.t. injection of TNF-α or IL-1ß in naïve animals induced CX3CL1 mRNA expression in the spinal cord and ipsilateral DRG. CONCLUSIONS: L. amazonensis skin infection produces chronic pain by central mechanisms involving spinal cord astrocytes and microglia-related production of cytokines and chemokines, and NFκB activation contributes to L. amazonensis infection-induced hyperalgesia and neuroinflammation.

DMH-CBD, a cannabidiol analog with reduced cytotoxicity, inhibits TNF production by targeting NF-kB activity dependent on A2A receptor.

Cannabidiol (CBD) is a natural compound with psychoactive therapeutic properties well described. Conversely, the immunological effects of CBD are still poorly explored. In this study, the potential anti-inflammatory effects and underlying mechanisms of CBD and its analog Dimethyl-Heptyl-Cannabidiol (DMH-CBD) were investigated using RAW 264.7 macrophages. CBD and DMH-CBD suppressed LPS-induced TNF production and NF-kB activity in a concentration-dependent manner. Both compounds reduced the NF-kB activity in a µM concentration range: CBD (IC50 = 15 µM) and DMH-CBD (IC50 = 38 µM). However, the concentrations of CBD that mediated NF-kB inhibition were similar to those that cause cytotoxicity (LC50 = 58 µM). Differently, DMH-CBD inhibited the NF-kB activation without cytotoxic effects at the same concentrations, although it provokes cytotoxicity at long-term exposure. The inhibitory action of the DMH-CBD on NF-kB activity was not related to the reduction in IkBα degradation or either p65 (NF-kB) translocation to the nucleus, although it decreased p38 MAP kinase phosphorylation. Additionally, 8-(3-Chlorostyryl) caffeine (CSC), an A2A antagonist, reversed the effect of DMH-CBD on NF-kB activity in a concentration-dependent manner. Collectively, our results demonstrated that CBD reduces NF-kB activity at concentrations intimately associated with those that cause cell death, whereas DMH-CBD decreases NF-kB activity at non-toxic concentrations in an A2A receptor dependent-manner.

Succinate receptor deficiency attenuates arthritis by reducing dendritic cell traffic and expansion of Th17 cells in the lymph nodes.

Rheumatoid arthritis is a chronic inflammatory disease that leads to significant changes in metabolic activity. Succinate, an intermediate of the tricarboxylic acid cycle, has emerged as a metabolic mediator of the innate immune response. However, the involvement of succinate in the generation of the adaptive immune response and establishment of autoimmune response has not been addressed thus far. Here we demonstrated that the succinate-sensing receptor (Sucnr1/GPR91) plays a critical role in the development of immune-mediated arthritis. We found that Sucnr1 acts as a chemotactic gradient sensor that guides dendritic cells (DCs) into the lymph nodes, orchestrating the expansion of the T helper (Th)17-cell population and the development of experimental antigen-induced arthritis. Sucnr1-/- mice show reduced articular hyperalgesia, neutrophil infiltration and inflammatory cytokines in the joint, and reduced frequency of Th17 cells in draining lymph nodes. Adoptive transfer of wild-type (WT) DCs into Sucnr1-/- mice restored the development of arthritis. Moreover, DC-depleted mice transferred with Sucnr1-/- DCs developed less arthritis than mice transferred with WT DCs. In contrast, succinate given together with the immunization boosted the recruitment of DCs and the frequency of Th17 cells in draining lymph nodes, increasing arthritis severity. Therefore, the blockade of Sucnr1 may represent a novel therapeutic target of arthritis.-Saraiva, A. L., Veras, F. P., Peres, R. S., Talbot, J., de Lima, K. A., Luiz, J. P., Carballido, J. M., Cunha, T. M., Cunha, F. Q., Ryffel, B., Alves-Filho, J. C. Succinate receptor deficiency attenuates arthritis by reducing dendritic cell traffic and expansion of Th17 cells in the lymph nodes.

Repurposing of the Nootropic Drug Vinpocetine as an Analgesic and Anti-Inflammatory Agent: Evidence in a Mouse Model of Superoxide Anion-Triggered Inflammation.

Clinically active drugs for the treatment of acute pain have their prescription limited due to the significant side effects they induce. An increase in reactive oxygen species (ROS) has been linked to several conditions, including inflammation and pain processing. Therefore, new or repurposed drugs with the ability of reducing ROS-triggered responses are promising candidates for analgesic drugs. Vinpocetine is a clinically used nootropic drug with antioxidant, anti-inflammatory, and analgesic properties. However, the effects of vinpocetine have not been investigated in a model with a direct relationship between ROS, inflammation, and pain. Based on that, we aimed to investigate the effects of vinpocetine in a model of superoxide anion-induced pain and inflammation using potassium superoxide (KO2) as a superoxide anion donor to trigger inflammation and pain. In the KO2 model, vinpocetine dose-dependently reduced pain-like behaviors (spontaneous pain and hyperalgesia), paw edema, and neutrophil and mononuclear cell recruitment to the paw skin (assessed by H&E staining, fluorescence, and enzymatic assays) and to the peritoneal cavity. Vinpocetine also restored tissue endogenous antioxidant ability and Nrf2 and Ho-1 mRNA expression and reduced superoxide anion production and gp91phox mRNA expression. We also observed the inhibition of IκBα degradation by vinpocetine, which demonstrates a reduction in the activation of NF-κB explaining the diminished production of IL-33, IL-1ß, and TNF-α. Collectively, our data show that vinpocetine alleviates pain and inflammation induced by KO2, which is a mouse model with a direct role of ROS in triggering pain and other inflammatory phenomena. Thus, the results suggest the repurposing of vinpocetine as an anti-inflammatory and analgesic drug.