Sepsis expands a CD39+ plasmablast population that promotes immunosuppression via adenosine-mediated inhibition of macrophage antimicrobial activity.

Sepsis results in elevated adenosine in circulation. Extracellular adenosine triggers immunosuppressive signaling via the A2a receptor (A2aR). Sepsis survivors develop persistent immunosuppression with increased risk of recurrent infections. We utilized the cecal ligation and puncture (CLP) model of sepsis and subsequent infection to assess the role of adenosine in post-sepsis immune suppression. A2aR-deficient mice showed improved resistance to post-sepsis infections. Sepsis expanded a subset of CD39hi B cells and elevated extracellular adenosine, which was absent in mice lacking CD39-expressing B cells. Sepsis-surviving B cell-deficient mice were more resistant to secondary infections. Mechanistically, metabolic reprogramming of septic B cells increased production of ATP, which was converted into adenosine by CD39 on plasmablasts. Adenosine signaling via A2aR impaired macrophage bactericidal activity and enhanced interleukin-10 production. Septic individuals exhibited expanded CD39hi plasmablasts and adenosine accumulation. Our study reveals CD39hi plasmablasts and adenosine as important drivers of sepsis-induced immunosuppression with relevance in human disease.

Neutrophil extracellular traps in rheumatoid arthritis and periodontitis: Contribution of PADI4 gene polymorphisms.

AIM: We sought to investigate the release of neutrophil extracellular traps (NETs) in neutrophils from individuals with rheumatoid arthritis (RA) and controls and compare the presence of NETs in gingival tissues according to periodontal status. Also, the association between single nucleotide polymorphisms (SNPs) of the peptidyl arginine deaminase type 4 (PADI4) gene and the GTG haplotype with RA, periodontitis and NETs was evaluated in vitro. MATERIALS AND METHODS: Peripheral neutrophils were isolated by density gradient, and NET concentration was determined by the PicoGreen method. Immunofluorescence was studied to identify NETs by co-localization of myeloperoxidase (MPO)-citrullinated histone H3 (H3Cit). Genotyping for SNPs (PADI4_89; PADI4_90; PADI4_92; and PADI4_104) was performed in 87 individuals with RA and 111 controls. RESULTS: The release of NETs in vitro was significantly higher in individuals with RA and periodontitis and when stimulated with Porphyromonas gingivalis. Gingival tissues from subjects with RA and periodontitis revealed increased numbers of MPO-H3Cit-positive cells. Individuals with the GTG haplotype showed a higher release of NETs in vitro and worse periodontal parameters. CONCLUSIONS: The release of NETs by circulating neutrophils is associated with RA and periodontitis and is influenced by the presence of the GTG haplotype.

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.

TLR4 abrogates the Th1 immune response through IRF1 and IFN-ß to prevent immunopathology during L. infantum infection.

A striking feature of human visceral leishmaniasis (VL) is chronic inflammation in the spleen and liver, and VL patients present increased production levels of multiple inflammatory mediators, which contribute to tissue damage and disease severity. Here, we combined an experimental model with the transcriptional profile of human VL to demonstrate that the TLR4-IFN-ß pathway regulates the chronic inflammatory process and is associated with the asymptomatic form of the disease. Tlr4-deficient mice harbored fewer parasites in their spleen and liver than wild-type mice. TLR4 deficiency enhanced the Th1 immune response against the parasite, which was correlated with an increased activation of dendritic cells (DCs). Gene expression analyses demonstrated that IRF1 and IFN-ß were expressed downstream of TLR4 after infection. Accordingly, IRF1- and IFNAR-deficient mice harbored fewer parasites in the target organs than wild-type mice due to having an increased Th1 immune response. However, the absence of TLR4 or IFNAR increased the serum transaminase levels in infected mice, indicating the presence of liver damage in these animals. In addition, IFN-ß limits IFN-γ production by acting directly on Th1 cells. Using RNA sequencing analysis of human samples, we demonstrated that the transcriptional signature for the TLR4 and type I IFN (IFN-I) pathways was positively modulated in asymptomatic subjects compared with VL patients and thus provide direct evidence demonstrating that the TLR4-IFN-I pathway is related to the nondevelopment of the disease. In conclusion, our results demonstrate that the TLR4-IRF1 pathway culminates in IFN-ß production as a mechanism for dampening the chronic inflammatory process and preventing immunopathology development.

Neutrophil extracellular traps (NETs) modulate inflammatory profile in obese humans and mice: adipose tissue role on NETs levels.

BACKGROUND: Neutrophil extracellular traps (NETs) are a recently discovered neutrophil defense mechanism which modulates several inflammatory conditions contributing to metabolic profile alterations. Therefore, the present study aimed to evaluate the production of NETs in obese patients and mice, verifying the possible mechanisms associated with the release of NETs by the adipose tissue. METHODS AND RESULTS: The present study investigated NETs production in human adipose tissue and also showing the neutrophils using intravital microscopy in mouse epididymal adipose tissue. Blood and white adipose tissues were obtained from eutrophic and obese individuals and from mice. Lipid, glycemic and leukocyte profiles were evaluated, as well as the levels of NETs and its markers. Bioinformatics and proteomics analyses were performed and the identified key proteins were measured. The main findings showed that the inflammatory markers interleukin-8 (IL-8), heat shock protein 90 (HSP90) and the E1 heat shock protein family (HSPE1) can be modulated by the NETs levels in obesity. Obesity has also been associated with increased cholesterol, glucose intolerance, ionic calcium and NETs. We also observed an increase in catalase and a decreased superoxide dismutase activity. Bioinformatics and proteomics analyses revealed that IL-8, HSP90 and HSPE1 were associated with obesity, inflammation and NETs release. CONCLUSIONS: In conclusion, the present study shows an increase in NETs production during obesity associated with important inflammatory markers in adipose.

Gasdermin-D activation by SARS-CoV-2 triggers NET and mediate COVID-19 immunopathology.

BACKGROUND: The release of neutrophil extracellular traps (NETs) is associated with inflammation, coagulopathy, and organ damage found in severe cases of COVID-19. However, the molecular mechanisms underlying the release of NETs in COVID-19 remain unclear. OBJECTIVES: We aim to investigate the role of the Gasdermin-D (GSDMD) pathway on NETs release and the development of organ damage during COVID-19. METHODS: We performed a single-cell transcriptome analysis in public data of bronchoalveolar lavage. Then, we enrolled 63 hospitalized patients with moderate and severe COVID-19. We analyze in blood and lung tissue samples the expression of GSDMD, presence of NETs, and signaling pathways upstreaming. Furthermore, we analyzed the treatment with disulfiram in a mouse model of SARS-CoV-2 infection. RESULTS: We found that the SARS-CoV-2 virus directly activates the pore-forming protein GSDMD that triggers NET production and organ damage in COVID-19. Single-cell transcriptome analysis revealed that the expression of GSDMD and inflammasome-related genes were increased in COVID-19 patients. High expression of active GSDMD associated with NETs structures was found in the lung tissue of COVID-19 patients. Furthermore, we showed that activation of GSDMD in neutrophils requires active caspase1/4 and live SARS-CoV-2, which infects neutrophils. In a mouse model of SARS-CoV-2 infection, the treatment with disulfiram inhibited NETs release and reduced organ damage. CONCLUSION: These results demonstrated that GSDMD-dependent NETosis plays a critical role in COVID-19 immunopathology and suggests GSDMD as a novel potential target for improving the COVID-19 therapeutic strategy.

Methotrexate promotes recovery of arthritis-induced alveolar bone loss and modifies the composition of the oral-gut microbiota.

OBJECTIVES: The impact of rheumatoid arthritis (RA) on the shaping of the oral and gut microbiome raises the question of whether and how RA treatment modifies microbial communities. We examined changes in the oral and gut microbiota in a mouse model of antigen-induced arthritis (AIA) treated or not with methotrexate (MTX). METHODS: Maxillae and stools were evaluated by the MiSeq platform of the V4 region of the 16S rRNA gene. Alveolar bone parameters were analysed by micro-computed tomography. Moreover, arthritis-induced changes in hyperalgesia and oedema were assessed, along with the impact on periodontal bone health. RESULTS: Microbial communities in MTX-treated AIA mice revealed distinct clusters compared to the control and AIA groups. Overall, MTX impacted the richness and variability of microorganisms in the oral-gut axis microbiome at the phylum level. Regarding the oral microbiome, while in the control group the most dominant phylum was Firmicutes, in the AIA group there was a shift towards the predominance of Campilobacteriota and Bacteroidetes associated with the disease. MTX treatment led to greater dominance of the health-associated phylum Proteobacteria. In the gut microbiome, AIA induction resulted in increased abundance of the Verrucomicrobiota phylum, and MTX treatment restored its levels compared to control. Importantly, the MTX-treated AIA animals had significantly less periodontal bone loss, as well as decreased hyperalgesia and joint oedema compared to the AIA animals. CONCLUSION: Data suggest the benefit of MTX treatment in protecting alveolar bone, in addition to providing new insights on the drug-microbiome interaction in the course of RA.

Neutrophil extracellular traps mediate joint hyperalgesia induced by immune inflammation.

OBJECTIVE: To evaluate the role of neutrophil extracellular traps (NETs) in the genesis of joint hyperalgesia using an experimental model of arthritis and transpose the findings to clinical investigation. METHODS: C57BL/6 mice were subjected to antigen-induced arthritis (AIA) and treated with Pulmozyme (PLZ) to degrade NETs or Cl-amidine to inhibit NET production. Oedema formation, the histopathological score and mechanical hyperalgesia were evaluated. NETs were injected intra-articularly in wild type (WT), Tlr4-/-, Tlr9-/-, Tnfr1-/- and Il1r-/- mice, and the levels of cytokines and Cox2 expression were quantified. NETs were also quantified from human neutrophils isolated from RA patients and individual controls. RESULTS: AIA mice had increased NET concentration in joints, accompanied by increased Padi4 gene expression in the joint cells. Treatment of AIA mice with a peptidyl arginine deiminase 4 inhibitor or with PLZ inhibited the joint hyperalgesia. Moreover, the injection of NETs into joints of naïve animals generated a dose-dependent reduction of mechanical threshold, an increase of articular oedema, inflammatory cytokine production and cyclooxygenase-2 expression. In mice deficient for Tnfr1, Il1r, Tlr4 and Tlr9, joint hyperalgesia induced by NETs was prevented. Last, we found that neutrophils from RA patients were more likely to release NETs, and the increase in synovial fluid NET concentration correlated with an increase in joint pain. CONCLUSION: The findings indicate that NETs cause hyperalgesia possibly through Toll-like receptor (TLR)-4 and TLR-9. These data support the idea that NETs contribute to articular pain, and this pathway can be an alternative target for the treatment of pain in RA.

NLRP12 controls arthritis severity by acting as a checkpoint inhibitor of Th17 cell differentiation.

Nucleotide oligomerization domain (NOD)-like receptor-12 (NLRP12) has emerged as a negative regulator of inflammation. It is well described that the Th17 cell population increases in patients with early Rheumatoid Arthritis (RA), which correlates with the disease activity. Here, we investigated the role of NLRP12 in the differentiation of Th17 cells and the development of experimental arthritis, using the antigen-induced arthritis (AIA) murine model. We found that Nlrp12-/- mice develop severe arthritis characterized by an exacerbated Th17-mediated inflammatory response with increases in the articular hyperalgesia, knee joint swelling, and neutrophil infiltration. Adoptive transfer of Nlrp12-/- cells into WT mice recapitulated the hyperinflammatory response seen in Nlrp12-/- mice and the treatment with anti-IL-17A neutralizing antibody abrogated arthritis development in Nlrp12-/- mice, suggesting that NLRP12 works as an inhibitor of Th17 cell differentiation. Indeed, Th17 cell differentiation markedly increases in Nlrp12-/- T cells cultured under the Th17-skewing condition. Mechanistically, we found that NLRP12 negatively regulates IL-6-induced phosphorylation of STAT3 in T cells. Finally, pharmacological inhibition of STAT3 reduced Th17 cell differentiation and abrogated hyperinflammatory arthritis observed in Nlrp12-/- mice. Thus, we described a novel role for NLRP12 as a checkpoint inhibitor of Th17 cell differentiation, which controls the severity of experimental arthritis.

Nonsurgical periodontal therapy decreases the severity of rheumatoid arthritis and the plasmatic and salivary levels of RANKL and Survivin: a short-term clinical study.

AIM: To investigate the influence of nonsurgical periodontal treatment (NSPT) on clinical periodontal status, rheumatoid arthritis (RA) activity, and plasmatic and salivary levels of biomarkers through a controlled clinical trial on individuals with RA and periodontitis (PE). METHODS: Sixty-six individuals from a convenience sample were considered eligible and consecutively allocated in 3 groups: (1) individuals without PE and RA (-PE-RA, n = 19); (2) individuals without PE and with RA (-PE+RA, n = 23), and (3) individuals with PE and RA (+PE+RA, n = 24). Full-mouth periodontal clinical examinations, Disease Activity Score (DAS-28) evaluations, and analysis in plasma and saliva of RANKL, OPG, RANKL/OPG, and Survivin were performed at baseline (T1) and 45 days after NSPT (T2). RESULTS: NSPT in the +PE+RA group was very effective to improve periodontal condition. At T2, significant reductions in DAS-28 were observed in +PE+RA (p = 0.011). Significantly higher levels of Survivin and RANKL were observed in saliva and plasma from RA individuals (with and without PE) compared to controls. Additionally, Survivin e RANKL demonstrated positive correlations with DAS-28 and an expressively significant reduction in +PE+RA at T2 (p < 0.001). CONCLUSIONS: NSPT was effective on improving both the periodontal and the RA clinical status and reducing the concentration of Survivin and RANKL in saliva and plasma. PRACTICAL IMPLICATIONS: Nonsurgical periodontal treatment was effective on reducing the concentration of Survivin and RANKL and on improving both the periodontal and the RA clinical status of affected individuals. TRIAL REGISTRATION: Brazilian Registry of Clinical Trials (ReBEC) protocol #RBR-8g2bc8 ( http://www.ensaiosclinicos.gov.br/rg/RBR-8g2bc8/ ).

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.

Taurine supplementation increases irisin levels after high intensity physical training in obese women.

BACKGROUND: Irisin is a myokine/adipokine that under stimulus of physical exercise is able to improve thermogenic capacity in adipose tissue. Likewise, taurine supplementation has demonstrated similar effects on energy metabolism. Therefore, we hypothesized that taurine supplementation combined with physical training may induce an increase in irisin concentrations, optimizing energy metabolism in obese individuals. OBJECTIVE: To evaluate if taurine supplementation associated with a high intensity physical training program increases irisin levels in obese women. METHODS: double-blind study with 22 obese women (BMI 32.4 ±â€¯2.0 kg/m2, 36.6 ±â€¯6.4 years and sedentary) who were randomly divided into two groups, control group (GC, n = 14), exercised and supplemented with placebo (3 g of starch), and taurine group (GTAU, n = 8), exercised and supplemented with taurine (3 g). The subjects performed high intensity physical training, Deep Water Running (DWR), for 8 weeks, 3 times/week, for 50 min per training session, at 70-85% maximum heart rate. Resting metabolic rate (RMR) was evaluated by indirect calorimetry, body composition by deuterium oxide, plasma taurine by HPLC, plasma irisin by Multiplex Kit, and food consumption by food records. The results were analyzed by an ANOVA two way repeated measures mixed model, with the Sidak post hoc (p < 0.05). RESULTS: No changes were observed in body composition. DWR increased RMR independent of supplementation (p < 0.001) and irisin levels (pg/mL) showed a significant difference only in the GTAU in 1 h after exercise (p < 0.001). CONCLUSION: DWR associated with taurine supplementation resulted in increased plasma irisin concentrations after physical training in obese adult women.

Targeting nitric oxide as a key modulator of sepsis, arthritis and pain.

Nitric oxide (NO) is produced by enzymatic activity of neuronal (nNOS), endothelial (eNOS), and inducible nitric oxide synthase (iNOS) and modulates a broad spectrum of physiological and pathophysiological conditions. The iNOS isoform is positively regulated at transcriptional level and produces high levels of NO in response to inflammatory mediators and/or to pattern recognition receptor signaling, such as Toll-like receptors. In this review, we compiled the main contributions of our group for understanding of the role of NO in sepsis and arthritis outcome and the peripheral contributions of NO to inflammatory pain development. Although neutrophil iNOS-derived NO is necessary for bacterial killing, systemic production of high levels of NO impairs neutrophil migration to infections through inhibiting neutrophil adhesion on microcirculation and their locomotion. Moreover, neutrophil-derived NO contributes to multiple organ dysfunction in sepsis. In arthritis, NO is chief for bacterial clearance in staphylococcal-induced arthritis; however, it contributes to articular damage and bone mass degradation. NO produced in inflammatory sites also downmodulates pain. The mechanism involved in analgesic effect and inhibition of neutrophil migration is dependent on the activation of the classical sGC/cGMP/PKG pathway. Despite the increasing number of studies performed after the identification of NO as an endothelium-derived relaxing factor, the underlying mechanisms of NO in inflammatory diseases remain unclear.

Modulation of experimental arthritis by vagal sensory and central brain stimulation.

Articular inflammation is a major clinical burden in multiple inflammatory diseases, especially in rheumatoid arthritis. Biological anti-rheumatic drug therapies are expensive and increase the risk of systemic immunosuppression, infections, and malignancies. Here, we report that vagus nerve stimulation controls arthritic joint inflammation by inducing local regulation of innate immune response. Most of the previous studies of neuromodulation focused on vagal regulation of inflammation via the efferent peripheral pathway toward the viscera. Here, we report that vagal stimulation modulates arthritic joint inflammation through a novel "afferent" pathway mediated by the locus coeruleus (LC) of the central nervous system. Afferent vagal stimulation activates two sympatho-excitatory brain areas: the paraventricular hypothalamic nucleus (PVN) and the LC. The integrity of the LC, but not that of the PVN, is critical for vagal control of arthritic joint inflammation. Afferent vagal stimulation suppresses articular inflammation in the ipsilateral, but not in the contralateral knee to the hemispheric LC lesion. Central stimulation is followed by subsequent activation of joint sympathetic nerve terminals inducing articular norepinephrine release. Selective adrenergic beta-blockers prevent the effects of articular norepinephrine and thereby abrogate vagal control of arthritic joint inflammation. These results reveals a novel neuro-immune brain map with afferent vagal signals controlling side-specific articular inflammation through specific inflammatory-processing brain centers and joint sympathetic innervations.

Cholecystokinin inhibits inducible nitric oxide synthase expression by lipopolysaccharide-stimulated peritoneal macrophages.

Cholecystokinin (CCK) was first described as a gastrointestinal hormone. However, apart from its gastrointestinal effects, studies have described that CCK also plays immunoregulatory roles. Taking in account the involvement of inducible nitric oxide synthase- (iNOS-) derived NO in the sepsis context, the present study was undertaken to investigate the role of CCK on iNOS expression in LPS-activated peritoneal macrophages. Our results revealed that CCK reduces NO production and attenuates the iNOS mRNA expression and protein formation. Furthermore, CCK inhibited the nuclear factor- (NF-) κB pathway reducing IκBα degradation and minor p65-dependent translocation to the nucleus. Moreover, CCK restored the intracellular cAMP content activating the protein kinase A (PKA) pathway, which resulted in a negative modulatory role on iNOS expression. In peritoneal macrophages, the CCK-1R expression, but not CCK-2R, was predominant and upregulated by LPS. The pharmacological studies confirmed that CCK-1R subtype is the major receptor responsible for the biological effects of CCK. These data suggest an anti-inflammatory role for the peptide CCK in modulating iNOS-derived NO synthesis, possibly controlling the macrophage activation through NF-κB, cAMP-PKA, and CCK-1R pathways. Based on these findings, CCK could be used as an adjuvant agent to modulate the inflammatory response and prevent systemic complications commonly found during sepsis.

Neutrophil extracellular traps mediate bone erosion in rheumatoid arthritis by enhancing RANKL-induced osteoclastogenesis.

BACKGROUND AND PURPOSE: Rheumatoid arthritis (RA) is a chronic autoimmune disease that can cause bone erosion due to increased osteoclastogenesis. Neutrophils involvement in osteoclastogenesis remains uncertain. Given that neutrophil extracellular traps (NETs) can act as inflammatory mediators in rheumatoid arthritis, we investigated the role of NETs in stimulating bone loss by potentiating osteoclastogenesis during arthritis. EXPERIMENTAL APPROACH: The level of NETs in synovial fluid from arthritis patients was assessed. Bone loss was evaluated by histology and micro-CT in antigen-induced arthritis (AIA)-induced WT mice treated with DNase or in Padi4-deficient mice (Padi4flox/flox LysMCRE ). The size and function of osteoclasts and the levels of RANKL and osteoprotegerin (OPG) released by osteoblasts that were incubated with NETs were measured. The expression of osteoclastogenic marker genes and protein levels were evaluated by qPCR and western blotting. To assess the participation of TLR4 and TLR9 in osteoclastogenesis, cells from Tlr4-/- and Tlr9-/- mice were cultured with NETs. KEY RESULTS: Rheumatoid arthritis patients had higher levels of NETs in synovial fluid than osteoarthritis patients, which correlated with increased levels of RANKL/OPG. Moreover, patients with bone erosion had higher levels of NETs. Inhibiting NETs with DNase or Padi4 deletion alleviated bone loss in arthritic mice. Consistently, NETs enhanced RANKL-induced osteoclastogenesis that was dependent on TLR4 and TLR9 and increased osteoclast resorptive functions in vitro. In addition, NETs stimulated the release of RANKL and inhibited osteoprotegerin in osteoblasts, favouring osteoclastogenesis. CONCLUSIONS AND IMPLICATIONS: Inhibiting NETs could be an alternative strategy to reduce bone erosion in arthritis patients.

5-Lipoxygenase activity increases susceptibility to experimental Paracoccidioides brasiliensis infection.

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by the thermodimorphic fungus Paracoccidioides brasiliensis. Leukotrienes and lipoxins are lipid mediators produced after 5-lipoxygenase (5-LO) activation that exhibit pro- and anti-inflammatory roles, respectively. Here, we have investigated the contribution of 5-LO enzymatic activity in PCM using an experimental model of P. brasiliensis infection. B6.129 wild-type (B6.129) and 5-LO-deficient (5-LO(-/-)) mice were intravenously inoculated with a virulent strain of P. brasiliensis (Pb18), and the survival rate of the infected mice was investigated on different days after yeast infection. 5-LO(-/-) mice exhibited an increased survival rate associated with a decreased number of CFU. The resistance of 5-LO(-/-) during PCM was associated with augmented nitric oxide (NO) production and the formation of compact granulomas. In addition, the absence of 5-LO was associated with a diminished number of CD4(+) CD25(+) regulatory T cells, higher levels of gamma interferon and interleukin-12, and increased T-bet (a T-box transcription factor that directs Th1 lineage commitment) mRNA levels in the lungs. Taken together, our results show for the first time that 5-LO enzymatic activity increases susceptibility to P. brasiliensis, suggesting that this pathway may be a potential target for therapeutic intervention during PCM.

Essential roles of PKA, iNOS, CD95/CD95L, and terminal caspases in suppression of eosinopoiesis by PGE2 and other cAMP-elevating agents.

Up- and downregulation of eosinopoiesis control pulmonary eosinophilia in human asthma. In mice, eosinopoiesis is suppressed in vitro by prostaglandin E2 (PGE2) and in vivo by diethylcarbamazine, through a proapoptotic mechanism sequentially requiring inducible NO synthase (iNOS) and the ligand for death receptor CD95 (CD95L). We examined the roles of iNOS, cAMP-mediated signaling, caspases, and CD95L/CD95 in suppression of eosinopoiesis by PGE2 and other agents signaling through cAMP. Bone-marrow collected from BALB/c mice, or from iNOS-, CD95-, or CD95L-deficient mutants (and wild-type controls), was cultured with interleukin-5 (IL-5), alone or associated with PGE2, cAMP-inducing/mimetic agents, caspase inhibitor zVAD-fmk, iNOS inhibitor aminoguanidine, or combinations thereof, and eosinopoiesis was evaluated at various times. PGE2, added up to 24 hours of culture, dose-dependently suppressed eosinopoiesis, by inducing apoptosis. This effect was (a) paralleled by induction of iNOS in eosinophils; (b) duplicated by sodium nitroprusside, isoproterenol, and cAMP-inducing/mimetic agents; (c) prevented by protein kinase A inhibition. NO was produced through iNOS by dibutyryl-cAMP-stimulated bone-marrow. Overall, PGE2 and isoproterenol shared a requirement for four effector elements (iNOS, CD95L, CD95, and terminal caspases), which together define a pathway targeted by several soluble up- and downmodulators of eosinopoiesis, including drugs, mediators of inflammation, and cytokines.

A clinically-relevant STING agonist restrains human TH17 cell inflammatory profile.

The STING signaling pathway has gained attention over the last few years due to its ability to incite antimicrobial and antitumoral immunity. Conversely, in mouse models of autoimmunity such as colitis and multiple sclerosis, where TH17 cells are implicated in tissue inflammation, STING activation has been associated with the attenuation of immunogenic responses. In this line, STING was found to limit murine TH17 pro-inflammatory program in vitro. Here we demonstrate that 2'3'-c-di-AM(PS)2(Rp,Rp), a STING agonist that has been undergoing clinical trials for antitumor immunotherapy, activates the STING signalosome in differentiating human TH17 cells. Of particular interest, 2'3'-c-di-AM(PS)2(Rp,Rp) reduces IL-17A production and IL23R expression by human TH17 cells while it favors the generation of regulatory T (Treg) cells. These findings suggest that STING agonists may be promising approaches for treating human TH17-mediated chronic inflammation.

Neutrophil Extracellular Traps Aggravate Apical Periodontitis by Stimulating Osteoclast Formation.

INTRODUCTION: Neutrophil extracellular traps (NETs) have been described as structures composed of DNA and proteins, such as elastase and myeloperoxidase, that are able to kill bacteria extracellularly. The aim of the present study was to evaluate the role of NETs in bone resorption observed in pulp infection-induced apical periodontitis in mice. METHODS: Apical periodontitis was experimentally induced by exposing the dental pulp of the mandibular first molar of mice to the oral microenvironment. The expression of NETs was evaluated by immunofluorescence in mice and biopsies of apical periodontitis. Mice were treated with vehicle or DNase I to degrade NETs, and the samples were collected after 7 days. The size of the apical lesion and the osteoclast number were determined in hematoxylin-eosin- and tartrate-resistant acid phosphatase-stained sections, respectively. Osteoclast differentiation and function markers were evaluated by quantitative polymerase chain reaction. The level of NETs in the serum was determined by the myeloperoxidase-DNA PicoGreen assay. RESULTS: We first confirmed the presence of neutrophils and NETs at the site of the lesion in mice and in biopsies of patients with apical periodontitis. The treatment of mice with DNase I reduced the level of NETs in the serum and led to a reduction in apical lesion size and alveolar bone resorption. This effect was associated with a reduction of local inflammatory infiltrate and a reduced number of osteoclasts. We found that the increased expression of Acp5, Ctsk, and Rankl genes associated with osteoclast formation and function were abrogated by the absence of NETs. CONCLUSIONS: Our data highlight NETs as an important player in the pathogenesis of apical periodontitis with regard to the local inflammation and consequent bone resorption after pulp infection.