Kinetics of neutrophil extracellular traps and cytokines in oral mucositis and Candida infection.

OBJECTIVE: This study investigated the concentrations of neutrophil extracellular traps (NET) and salivary cytokines (IL-1ß, IL-6, IL-8/CXCL8, TNF, and TGF-ß1) in patients undergoing chemotherapy and their associations with oral mucositis (OM) and Candida infection. MATERIALS AND METHODS: This prospective longitudinal study performed at a Brazilian service included 60 adults diagnosed with hematolymphoid diseases. Saliva samples were collected on days D0, D3, D10, and D15. Cytokines were analyzed by ELISA and NET formation by identification of the myeloperoxidase-DNA complex. Oral Candida spp. was cultured. RESULTS: OM occurred in 43.3% of patients and oral candidiasis in 20%. However, 66% of individuals had positive cultures for C. albicans. Higher concentrations of IL-6, IL-8/CXCL8, and TNF and lower concentrations of TGF-ß1 were observed in patients with OM. C. albicans infection contributed to the increase in IL-8/CXCL8, TGF-ß1, and TNF. Individuals with OM or with oral candidiasis had significant reductions in NET formation. In contrast, individuals with C. albicans and with concomitant C. albicans and OM exhibited higher NET formation. CONCLUSION: The kinetics of cytokine levels and NET formation in chemotherapy-induced OM appears to be altered by Candida infection, even in the absence of clinical signs of oral candidiasis.

Clinical, immunological, and microbiological analysis of the association between periodontitis and COVID-19: a case-control study.

PURPOSE: Periodontitis and coronavirus disease (COVID-19) share risk factors and activate similar immunopathological pathways, intensifying systemic inflammation. This study investigated the clinical, immunological and microbiological parameters in individuals with COVID-19 and controls, exploring whether periodontitis-driven inflammation contributes to worsening COVID-19 endpoints. METHODS: Case (positive RT-PCR for SARS-CoV-2) and control (negative RT-PCR) individuals underwent clinical and periodontal assessments. Salivary levels of TNF-α, IL-6, IL-1ß, IL-10, OPG, RANKL, neutrophil extracellular traps, and subgingival biofilm were analyzed at two timepoints. Data on COVID-19-related outcomes and comorbidity information were evaluated from medical records. RESULTS: Ninety-nine cases of COVID-19 and 182 controls were included for analysis. Periodontitis was associated with more hospitalization (p = 0.009), more days in the intensive care unit (ICU) (p = 0.042), admission to the semi-ICU (p = 0.047), and greater need for oxygen therapy (p = 0.042). After adjustment for confounders, periodontitis resulted in a 1.13-fold increase in the chance of hospitalization. Salivary IL-6 levels (p = 0.010) were increased in individuals with COVID-19 and periodontitis. Periodontitis was associated with increased RANKL and IL-1ß after COVID-19. No significant changes were observed in the bacterial loads of the periodontopathogens Porphyromona gingivalis, Aggregatibacter actinomycetemcomitans, Tanerella forsythia, and Treponema denticola. CONCLUSIONS: Periodontitis was associated with worse COVID-19 outcomes, suggesting the relevance of periodontal care to reduce the burden of overall inflammation. Understanding the crosstalk between SARS-CoV-2 infection and chronic conditions such as periodontitis that can influence disease outcome is important to potentially prevent complications of COVID-19.

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.

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.

Neutrophil virucidal activity against SARS-CoV-2 is mediated by NETs.

BACKGROUND: The inflammation in the lungs and other vital organs in COVID-19 are characterized by the presence of neutrophils and high concentration of neutrophil extracellular traps (NETs), which also seems to mediate host tissue damage. However, it is not known whether NETs could have virucidal activity against SARS-CoV-2. METHODS: We investigated whether NETs could prevent SARS-CoV-2 replication in neutrophils and epithelial cells, and what the consequence of NETs degradation in K18-humanized ACE2 transgenic mice infected with SARS-CoV-2. RESULTS: Here, by immunofluorescence microscopy we observed that viral particles co-localize with NETs in neutrophils isolated from COVID-19 patients or from healthy individuals and infected in vitro. The inhibition of NETs production increased virus replication in neutrophils. In parallel, we observed that NETs inhibited virus abilities to infect and replicate in epithelial cells after 24 h of infection. Degradation of NETs with DNase I prevented their virucidal effect in vitro. Using K18-humanized ACE2 transgenic mice we observed a higher viral load in animals treated with DNase I. On the other hand, the virucidal effect of NETs was not dependent on neutrophil elastase or myeloperoxidase activity. CONCLUSION: Our results provide evidence of the role of NETosis as a mechanism of SARS-CoV-2 viral capture and inhibition.

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.

Plasmin and plasminogen prevent sepsis severity by reducing neutrophil extracellular traps and systemic inflammation.

Sepsis is a lethal syndrome characterized by systemic inflammation and abnormal coagulation. Despite therapeutic advances, sepsis mortality remains substantially high. Herein, we investigated the role of the plasminogen/plasmin (Plg/Pla) system during sepsis. Plasma levels of Plg were significantly lower in mice subjected to severe compared with nonsevere sepsis, whereas systemic levels of IL-6, a marker of sepsis severity, were higher in severe sepsis. Plg levels correlated negatively with IL-6 in both septic mice and patients, whereas plasminogen activator inhibitor-1 levels correlated positively with IL-6. Plg deficiency render mice susceptible to nonsevere sepsis induced by cecal ligation and puncture (CLP), resulting in greater numbers of neutrophils and M1 macrophages, liver fibrin(ogen) deposition, lower efferocytosis, and increased IL-6 and neutrophil extracellular trap (NET) release associated with organ damage. Conversely, inflammatory features, fibrin(ogen), and organ damage were substantially reduced, and efferocytosis was increased by exogenous Pla given during CLP- and LPS-induced endotoxemia. Plg or Pla protected mice from sepsis-induced lethality and enhanced the protective effect of antibiotics. Mechanistically, Plg/Pla-afforded protection was associated with regulation of NET release, requiring Pla-protease activity and lysine binding sites. Plg/Pla are important host-protective players during sepsis, controlling local and systemic inflammation and collateral organ damage.

FK506 impairs neutrophil migration that results in increased polymicrobial sepsis susceptibility.

OBJECTIVE: This study aimed to investigate the effects of FK506 on experimental sepsis immunopathology. It investigated the effect of FK506 on leukocyte recruitment to the site of infection, systemic cytokine production, and organ injury in mice with sepsis. METHODS: Using a murine cecal ligation and puncture (CLP) peritonitis model, the experiments were performed with wild-type (WT) mice and mice deficient in the gene Nfat1 (Nfat1-/-) in the C57BL/6 background. Animals were treated with 2.0 mg/kg of FK506, subcutaneously, 1 h before the sepsis model, twice a day (12 h/12 h). The number of bacteria colony forming units (CFU) was manually counted. The number of neutrophils in the lungs was estimated by the myeloperoxidase (MPO) assay. The expression of CXCR2 in neutrophils was determined using flow cytometry analysis. The expression of inflammatory cytokines in macrophage was determined using ELISA. The direct effect of FK506 on CXCR2 internalization was evaluated using HEK-293T cells after CXCL2 stimulation by the BRET method. RESULTS: FK506 treatment potentiated the failure of neutrophil migration into the peritoneal cavity, resulting in bacteremia and an exacerbated systemic inflammatory response, which led to higher organ damage and mortality rates. Failed neutrophil migration was associated with elevated CXCL2 chemokine plasma levels and lower expression of the CXCR2 receptor on circulating neutrophils compared with non-treated CLP-induced septic mice. FK506 did not directly affect CXCL2-induced CXCR2 internalization by transfected HEK-293 cells or mice neutrophils, despite increasing CXCL2 release by LPS-treated macrophages. Finally, the CLP-induced response of Nfat1-/- mice was similar to those observed in the Nfat1+/+ genotype, suggesting that the FK506 effect is not dependent on the NFAT1 pathway. CONCLUSION: Our data indicate that the increased susceptibility to infection of FK506-treated mice is associated with failed neutrophil migration due to the reduced membrane availability of CXCR2 receptors in response to exacerbated levels of circulating CXCL2.

Methotrexate and Non-Surgical Periodontal Treatment Change the Oral-Gut Microbiota in Rheumatoid Arthritis: A Prospective Cohort Study.

This study evaluated the changes in the composition of oral-gut microbiota in patients with rheumatoid arthritis (RA) caused by methotrexate (MTX) and non-surgical periodontal treatment (NSPT). Assessments were performed at baseline (T0), 6 months after MTX treatment (T1), and 45 days after NSPT (T2). The composition of the oral and gut microbiota was assessed by amplifying the V4 region of the 16S gene from subgingival plaques and stools. The results of the analysis of continuous variables were presented descriptively and non-parametric tests and Spearman's correlation were adopted. A total of 37 patients (27 with periodontitis) were evaluated at T0; 32 patients (24 with periodontitis) at T1; and 28 patients (17 with periodontitis) at T2. MTX tended to reduce the alpha diversity of the oral-gut microbiota, while NSPT appeared to increase the number of different species of oral microbiota. MTX and NSPT influenced beta diversity in the oral microbiota. The relative abundance of oral microbiota was directly influenced by periodontal status. MTX did not affect the periodontal condition but modified the correlations that varied from weak to moderate (p < 0.05) between clinical parameters and the microbiota. MTX and NSPT directly affected the composition and richness of the oral-gut microbiota. However, MTX did not influence periodontal parameters.

Increased blood neutrophil extracellular traps (NETs) associated with early life stress: translational findings in recent-onset schizophrenia and rodent model.

Higher levels of interleukin (IL)-6 and elevated neutrophil counts are consistently reported in the blood of patients with schizophrenia. Stressors during childhood and/or adolescence are major socioenvironmental risk factors for schizophrenia and may contribute to immune dysregulation. Previous studies using blood cytokines to stratify patients with schizophrenia suggest that only a subset presents a low-grade inflammatory state. However, these studies have not addressed whether environmental factors such as childhood maltreatment contributed to identifying inflammatory clusters. Moreover, a neutrophil-related mechanism (Neutrophil Extracellular Traps; NETs) central to both the initiation and chronicity of autoimmune and inflammatory diseases has never been investigated in psychiatry. Elevated NETs in schizophrenia may predispose patients to inflammatory and autoimmune diseases resulting in reduced life expectancy. We, therefore, investigated NETs as a novel mechanism and biological target in early schizophrenia and their role together with IL-6 and childhood maltreatment in identifying cluster subgroups. We found increased NETs in the plasma of patients with early schizophrenia (n = 78) compared to both their unaffected siblings (n = 25) and community controls (n = 78), irrespective of sex, body mass index, psychoactive drug use, or tobacco smoking. Increased NETs in patients were unrelated to antipsychotic treatment, which was further tested in vitro using fresh neutrophils. By applying unsupervised two-step clustering analysis, we integrated values of NETs, IL-6, and childhood maltreatment scores. We identified two main clusters; childhood maltreatment scores and NETs were the most important variables contributing to cluster separation (high-CL1 and low-CL2), while IL-6 was the least contributor. Patients allocated in the high-CL1 (61.5%) had significantly higher childhood maltreatment scores, NETs, and IL-6 levels than the remaining groups (patients low-CL2, siblings, and controls high-CL1 and low-CL2). We complemented these findings with a rat model based on stress exposure during adolescence that results in several schizophrenia-like changes in adulthood. We found that adolescent stressed rats had higher NETs and IL-6 levels in serum compared to non-stressed rats with a tendency to produce more NETs from the bone marrow. Altogether, this study brings a novel cellular-based mechanism in schizophrenia that, combined with early-stress, could be useful to identify subgroups for more personalised treatments.

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.

Toxic mechanisms of cigarette smoke and heat-not-burn tobacco vapor inhalation on rheumatoid arthritis.

Tobacco combustion exposure worsens rheumatoid arthritis (RA). Non-combustible tobacco devices, as heat-not-burn tobacco (HNBT), are emerging as harm reduction to smokers by releasing nicotine and lower combustible tobacco products. Nevertheless, HNBT toxicity remains unclear. Hence, here we investigated the impacts of the tobacco combustible product (cigarette smoke; CS) or HNBT vapor exposures on antigen-induced arthritis (AIA) in C57BL/6 mice. Animals were exposed to airflow, HNBT vapor, or CS during 1 h/twice a day, under the Health Canada Intense (HCI) smoking regime, between days 14 to 20 after the first immunization. At day 21, 16 h after the last exposures, mice were i.a. challenged and the AIA effects were evaluated 24 h later. CS- or HNBT-exposed mice presented equivalent blood nicotine levels. CS exposure worsened articular symptoms, pulmonary inflammation, and expression of lung metallothioneins. Nevertheless, CS or HNBT exposures reduced lymphoid organs' cellularity, splenocyte proliferation and IL-2 secretion. Additional in vitro CS or HNBT exposures confirmed the harmful effects on splenocytes, which were partially mediated by the activation of nicotine/α7nAchR pathway. Associated, data demonstrate the toxic mechanisms of CS or HNBT inhalation at HCI regime on RA, and highlight that further investigations are fundamental to assure the toxicity of emerging tobacco products on the immune system during specific challenges.

Hydroquinone Exposure Worsens Rheumatoid Arthritis through the Activation of the Aryl Hydrocarbon Receptor and Interleukin-17 Pathways.

Rheumatoid arthritis (RA) development is strongly associated with cigarette smoke exposure, which activates the aryl hydrocarbon receptor (AhR) as a trigger for Th17 inflammatory pathways. We previously demonstrated that the exposure to hydroquinone (HQ), one of the major compounds of cigarette tar, aggravates the arthritis symptomatology in rats. However, the mechanisms related to the HQ-related RA still remain elusive. Cell viability, cytokine secretion, and gene expression were measured in RA human fibroblast-like synoviocytes (RAHFLS) treated with HQ and stimulated or not with TNF-α. Antigen-induced arthritis (AIA) was also elicited in wild type (WT), AhR -/- or IL-17R -/- C57BL/6 mice upon daily exposure to nebulized HQ (25ppm) between days 15 to 21. At day 21, mice were challenged with mBSA and inflammatory parameters were assessed. The in vitro HQ treatment up-regulated TNFR1, TNFR2 expression, and increased ROS production. The co-treatment of HQ and TNF-α enhanced the IL-6 and IL-8 secretion. However, the pre-incubation of RAHFLS with an AhR antagonist inhibited the HQ-mediated cell proliferation and gene expression profile. About the in vivo approach, the HQ exposure worsened the AIA symptoms (edema, pain, cytokines secretion and NETs formation) in WT mice. These AIA effects were abolished in HQ-exposed AhR -/- and IL-17R -/- animals though. Our data demonstrated the harmful HQ influence over the onset of arthritis through the activation and proliferation of synoviocytes. The HQ-related RA severity was also associated with the activation of AhR and IL-17 pathways, highlighting how cigarette smoke compounds can contribute to the RA progression.

Are neutrophil extracellular traps the link for the cross-talk between periodontitis and rheumatoid arthritis physiopathology?

OBJECTIVES: Neutrophil extracellular traps (NETs) play a role in the pathogenesis of periodontitis and rheumatoid arthritis (RA). However, it remains poorly understood whether NETs participate in the cross-talk between periodontitis and RA. Herein, we investigated the production of NETs in individuals with periodontitis and RA and its association with clinical parameters. The impact of periodontal therapy on RA and NET release was also assessed. METHODS: The concentration of NETs and cytokines was determined in the saliva and plasma of individuals with early RA (n = 24), established RA (n = 64) and individuals without RA (n = 76). The influence of periodontitis on the production of NETs and cytokines was also evaluated. RESULTS: Individuals with early RA had a higher concentration of NETs in saliva and plasma than individuals with established RA or without RA. Periodontitis resulted in an increase in the concentration of NETs of groups of individuals without RA and with early RA. The proportion of individuals with high concentrations of IL-6, IL-10 and GM-CSF was higher among individuals with periodontitis than among individuals without periodontitis. The concentrations of TNF-α, IL-6, IL-17/IL-25 and IL-28A were particularly high in individuals with early RA. Worse periodontal clinical parameters, RA onset and RA activity were significantly associated with circulating NETs. Periodontal therapy was associated with a reduction in the concentration of NETs and inflammatory cytokines and amelioration in periodontitis and RA. CONCLUSION: This study reveals that NETs are a possible link between periodontitis and RA, with periodontal therapy resulting in a dramatic switch in circulating NET levels.

Antinociceptive and anti-inflammatory activities of Copaifera pubiflora Benth oleoresin and its major metabolite ent-hardwickiic acid.

ETHNOPHARMACOLOGICAL RELEVANCE: Copaifera species folkloric names are "copaíbas, copaibeiras, copaívas or oil stick", which are widely used in Brazilian folk medicine. Among all ethnopharmacological applications described for Copaifera spp oleoresins, their anti-inflammatory effect stands out. However, the knowledge of anti-inflammatory and antinociceptive properties of Copaifera pubiflora Benth is scarce. AIM OF THE STUDY: To investigate the cytotoxic, anti-inflammatory, and antinociceptive activities of C. pubiflora oleoresin (CPO), and its major compound ent-hardwickiic acid (HA). MATERIAL AND METHODS: The phosphatase assay was used to evaluate the cytotoxicity of CPO and HA in three different cell lines. CPO and HA doses of 1, 3, and 10 mg/kg were employed in the biological assays. The assessment of motor activity was performed using open-field and rotarod tests. Anti-inflammatory activity of CPO and HA was assessed through luciferase assay, measurement of INF-γ, IL-1ß, IL-6, IL-10, and TNF-α in a multi-spot system with the immortalized cell line THP-1, zymosan-induced arthritis, and carrageenan-induced paw edema. Acetic acid-induced abdominal writhing and formalin tests were undertaken to evaluate the antinociceptive potential of CPO and HA. In addition, the evaluation using carrageenan was performed to investigate the effect of CPO in pain intensity to a mechanical stimulus (mechanical hyperalgesia), using the von Frey filaments. A tail-flick test was used to evaluate possible central CPO and HA actions. RESULTS: In the cytotoxicity evaluation, CPO and HA were not cytotoxic to the cell lines tested. CPO and HA (10 mg/kg) did not affect animals' locomotor capacity in both open-field and rotarod tests. In the luciferase assay, CPO and HA significantly reduced luciferase activity (p < 0.05). This reduction indicates a decrease in NF-κB activity. HA and CPO decreased INF-γ, IL-1ß, IL-6, IL-10, and TNF-α at 24 and 72 h in the multi-spot system. In zymosan-induced arthritis, CPO and HA decreased the number of neutrophils in the joint of arthritic mice and the number of total leukocytes (p < 0.05). In experimental arthritis HA significantly decreased joint swelling (p < 0.05). CPO and HA also increased the mechanical threshold during experimental arthritis. HA and CPO significantly inhibited the carrageenan-induced paw edema, being the doses of 10 mg/kg the most effective, registering maximum inhibitions of 58 ± 8% and 76 ± 6% respectively, p < 0.05. CPO and HA reduced the nociceptive behavior in both phases of formalin at all tested doses. The highest doses tested displayed inhibitions of 87 ± 1% and 72 ± 4%, respectively, p < 0.001, in the first phase, and 87 ± 1% and 81 ± 2%, respectively, p < 0.001, in the second phase. Oral treatment of CPO and HA (1, 3, 10 mg/kg) significantly reduced the nociceptive response in acetic acid-induced abdominal writhings, and the 10 mg/kg dose was the most effective with maximum inhibitions of 86 ± 2% and 82 ± 1%, respectively, p < 0.001. Both HA and CPO significantly decreased the intensity of mechanical inflammatory hyper-nociception on carrageenan-induced hyperalgesia at all tested doses, and 10 mg/kg was the most effective dose with maximum inhibitions of 73 ± 5% and 74 ± 7%, respectively, p < 0.05.CPO increased the tail-flick latencies in mice, and concomitant administration of naloxone partially reduced its effect. CONCLUSIONS: CPO and HA may inhibit the production of inflammatory cytokines by suppressing the NF-κB signaling pathway, resulting in anti-inflammatory and antinociceptive activities.

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.

Hydroquinone exposure worsens rheumatoid arthritis through the activation of the aryl hydrocarbon receptor and interleukin-17 pathways

Rheumatoid arthritis (RA) development is strongly associated with cigarette smoke exposure, which activates the aryl hydrocarbon receptor (AhR) as a trigger for Th17 inflammatory pathways. We previously demonstrated that the exposure to hydroquinone (HQ), one of the major compounds of cigarette tar, aggravates the arthritis symptomatology in rats. However, the mechanisms related to the HQ-related RA still remain elusive. Cell viability, cytokine secretion, and gene expression were measured in RA human fibroblast-like synoviocytes (RAHFLS) treated with HQ and stimulated or not with TNF-α. Antigen-induced arthritis (AIA) was also elicited in wild type (WT), AhR −/− or IL-17R −/− C57BL/6 mice upon daily exposure to nebulized HQ (25ppm) between days 15 to 21. At day 21, mice were challenged with mBSA and inflammatory parameters were assessed. The in vitro HQ treatment up-regulated TNFR1, TNFR2 expression, and increased ROS production. The co-treatment of HQ and TNF-α enhanced the IL-6 and IL-8 secretion. However, the pre-incubation of RAHFLS with an AhR antagonist inhibited the HQ-mediated cell proliferation and gene expression profile. About the in vivo approach, the HQ exposure worsened the AIA symptoms (edema, pain, cytokines secretion and NETs formation) in WT mice. These AIA effects were abolished in HQ-exposed AhR −/− and IL-17R −/− animals though. Our data demonstrated the harmful HQ influence over the onset of arthritis through the activation and proliferation of synoviocytes. The HQ-related RA severity was also associated with the activation of AhR and IL-17 pathways, highlighting how cigarette smoke compounds can contribute to the RA progression.

SARS-CoV-2-triggered neutrophil extracellular traps mediate COVID-19 pathology.

Severe COVID-19 patients develop acute respiratory distress syndrome that may progress to cytokine storm syndrome, organ dysfunction, and death. Considering that neutrophil extracellular traps (NETs) have been described as important mediators of tissue damage in inflammatory diseases, we investigated whether NETs would be involved in COVID-19 pathophysiology. A cohort of 32 hospitalized patients with a confirmed diagnosis of COVID-19 and healthy controls were enrolled. The concentration of NETs was augmented in plasma, tracheal aspirate, and lung autopsies tissues from COVID-19 patients, and their neutrophils released higher levels of NETs. Notably, we found that viable SARS-CoV-2 can directly induce the release of NETs by healthy neutrophils. Mechanistically, NETs triggered by SARS-CoV-2 depend on angiotensin-converting enzyme 2, serine protease, virus replication, and PAD-4. Finally, NETs released by SARS-CoV-2-activated neutrophils promote lung epithelial cell death in vitro. These results unravel a possible detrimental role of NETs in the pathophysiology of COVID-19. Therefore, the inhibition of NETs represents a potential therapeutic target for COVID-19.

Regulation of murine arthritis by systemic, spinal, and intra-articular adrenoceptors.

BACKGROUND: The regulation of the immune system by the sympathetic nervous system is allowing the design of novel treatments for inflammatory disorders such as arthritis. In this study, we have analyzed the effects of α- and ß-adrenoceptor agonists injected subcutaneously, intrathecally, or intra-articularly in zymosan-induced arthritis. METHODS: Murine arthritis was induced by intra-articular (knee joint) injection of zymosan. α1 (phenylephrine), α2 (clonidine), ß1 (dobutamine), or ß2 (salbutamol)-adrenoceptor agonists were injected subcutaneously (sc), intrathecally (it), or intra-articularly (ia) to activate peripheral, spinal, or intra-articular adrenoceptors and to study their effects on articular edema formation and neutrophil migration into the synovial cavity. RESULTS: Treatments with phenylephrine did not affect the edema formation, but it increased neutrophil migration when injected subcutaneously (155.3%) or intra-articularly (187.7%). Treatments with clonidine inhibited neutrophil migration (59.9% sc, 68.7% it, 42.8% ia) regardless of the route of administration, but it inhibited edema formation only when injected intrathecally (66.7%) or intra-articularly (36%) but not subcutaneously. Treatments with dobutamine inhibited both edema (42.0% sc, 69.5% it, 61.6% ia) and neutrophil migration (28.4% sc, 70.3% it, 82.4% ia) in a concentration dependent manner. Likewise, all the treatments with salbutamol also inhibited edema formation (89.9% sc, 62.4% it, 69.8% ia) and neutrophil migration (76.6% sc, 39.1% it, 71.7% ia). CONCLUSION: Whereas the ß-adrenoceptor agonists induced anti-inflammatory effects regardless of their route of administration, α1- and α2-adrenoceptor agonists induced either pro- and anti-inflammatory effects, respectively.