CdSe magic-sized quantum dots attenuate reactive oxygen species generated by neutrophils and macrophages with implications in experimental arthritis.

The biological applicability of nanomaterials has been limited due to cytotoxicity. Studies have described the effects of nanomaterials on different tissues and cell types, but their actions on immune cells are less elucidated. This study describes unprecedented in vitro and in vivo antioxidant activities of cadmium selenide magic-sized quantum dots (CdSe MSQDs) with implications on rheumatoid arthritis. While the generation of ROS induced by nanomaterials is linked to cytotoxicity, we found that CdSe MSQDs reduced ROS production by neutrophils and macrophages following opsonized-zymosan stimuli, and we did not find cytotoxic effects. Interestingly, inherent antioxidant properties of CdSe MSQDs were confirmed through DPPH, FRAP, and ORAC assays. Furthermore, CdSe MSQDs reduced ROS levels generated by infiltrating leukocytes into joints in experimental model of rheumatoid arthritis. Briefly, we describe a novel application of CdSe MSQDs in modulating the inflammatory response in experimental rheumatoid arthritis through an unexpected antioxidant activity.

Angiotensin type 2 receptor antagonism as a new target to manage gout.

BACKGROUND: There is a growing search for therapeutic targets in the treatment of gout. The present study aimed to evaluate the analgesic and anti-inflammatory potential of angiotensin type 2 receptor (AT2R) antagonism in an acute gout attack mouse model. METHODS: Male wild-type (WT) C57BL/6 mice either with the AT2R antagonist, PD123319 (10 pmol/joint), or with vehicle injections, or AT2R KO mice, received intra-articular (IA) injection of monosodium urate (MSU) crystals (100 µg/joint), that induce the acute gout attack, and were tested for mechanical allodynia, thermal hyperalgesia, spontaneous nociception and ankle edema development at several times after the injections. To test an involvement of AT2R in joint pain, mice received an IA administration of angiotensin II (0.05-5 nmol/joint) with or without PD123319, and were also evaluated for pain and edema development. Ankle joint tissue samples from mice undergoing the above treatments were assessed for myeloperoxidase activity, IL-1ß release, mRNA expression analyses and nitrite/nitrate levels, 4 h after injections. RESULTS: AT2R antagonism has robust antinociceptive effects on mechanical allodynia (44% reduction) and spontaneous nociception (56%), as well as anti-inflammatory effects preventing edema formation (45%), reducing myeloperoxidase activity (54%) and IL-1ß levels (32%). Additionally, Agtr2tm1a mutant mice have largely reduced painful signs of gout. Angiotensin II administration causes pain and inflammation, which was prevented by AT2R antagonism, as observed in mechanical allodynia 4 h (100%), spontaneous nociception (46%), cold nociceptive response (54%), edema formation (83%), myeloperoxidase activity (48%), and IL-1ß levels (89%). PD123319 treatment also reduces NO concentrations (74%) and AT2R mRNA levels in comparison with MSU untreated mice. CONCLUSION: Our findings show that AT2R activation contributes to acute pain in experimental mouse models of gout. Therefore, the antagonism of AT2R may be a potential therapeutic option to manage gout arthritis.

CCR2 Plays a Protective Role in Rocio Virus-Induced Encephalitis by Promoting Macrophage Infiltration Into the Brain.

Rocio virus (ROCV) is a highly neuropathogenic mosquito-transmitted flavivirus responsible for an unprecedented outbreak of human encephalitis during 1975-1976 in Sao Paulo State, Brazil. Previous studies have shown an increased number of inflammatory macrophages in the central nervous system (CNS) of ROCV-infected mice, implying a role for macrophages in the pathogenesis of ROCV. Here, we show that ROCV infection results in increased expression of CCL2 in the blood and in infiltration of macrophages into the brain. Moreover, we show, using CCR2 knockout mice, that CCR2 expression is essential for macrophage infiltration in the brain during ROCV infection and that the lack of CCR2 results in increased disease severity and mortality. Thus, our findings show the protective role of CCR2-mediated infiltration of macrophages in the brain during ROCV infection.

Diagnosis of canine leptospirosis using an immunomagnetic separation-PCR method.

Diagnosis of leptospirosis by PCR is hampered due to the presence of substances on biological fluids. Here, we report an immunomagnetic separation step prior to PCR which improved the detection of Leptospira spp. in blood and urine samples from dogs. It resulted in a significant improvement on sensitivity for diagnosis of canine leptospirosis.

Chromomycin A5 induces bona fide immunogenic cell death in melanoma.

Purpose: Some first-line cytotoxic chemotherapics, e.g. doxorubicin, paclitaxel and oxaliplatin, induce activation of the immune system through immunogenic cell death (ICD). Tumor cells undergoing ICD function as a vaccine, releasing damage-associated molecular patterns (DAMPs), which act as adjuvants, and neoantigens of the tumor are recognized as antigens. ICD induction is rare, however it yields better and long-lasting antitumor responses to chemotherapy. Advanced metastatic melanoma (AMM) is incurable for more than half of patients. The discovery of ICD inducers against AMM is an interesting drug discovery strategy with high translational potential. Here we evaluated ICD induction of four highly cytotoxic chromomycins A (CA5-8). Methods: ICD features and DAMPs were evaluated using several in vitro techniques with metastatic melanoma cell line (B16-F10) exposed to chromomcins A5-8 such as flow cytometry, western blot, RT-PCR and luminescence. Additionally in vivo vaccination assays with CA5-treated cells in a syngeneic murine model (C57Bl/6) were performed to confirm ICD evaluating the immune cells activation and their antitumor activity. Results: B16-F10 treated with CA5-8 and doxorubicin exhibited ICD features such as autophagy and apoptosis, externalization of calreticulin, and releasing of HMGB1. However, CA5-treated cells had the best profile, also inducing ATP release, ERp57 externalization, phosphorylation of eIF2α and altering expression of transcription of genes related to autophagy, endoplasmic reticulum stress, and apoptosis. Bona fide ICD induction by CA5 was confirmed by vaccination of C57BL/6 mice with CA5-treated cells which activated antigen-presenting cells and T lymphocytes and stimulated antitumor activity. Conclusion: CA5 induces bona fide immunogenic cell death on melanoma.

PD-1/PD-L1 Inhibition Enhances Chemotherapy-Induced Neuropathic Pain by Suppressing Neuroimmune Antinociceptive Signaling.

Cytotoxic agents synergize with immune checkpoint inhibitors and improve outcomes for patients with several cancer types. Nonetheless, a parallel increase in the incidence of dose-limiting side effects, such as peripheral neuropathy, is often observed. Here, we investigated the role of the programmed cell death-1 (PD-1)/programmed death-ligand 1 (PD-L1) axis in the modulation of paclitaxel-induced neuropathic pain. We found that human and mouse neural tissues, including the dorsal root ganglion (DRG), expressed basal levels of PD-1 and PD-L1. During the development of paclitaxel-induced neuropathy, an increase in PD-L1 expression was observed in macrophages from the DRG. This effect depended on Toll-like receptor 4 activation by paclitaxel. Furthermore, PD-L1 inhibited pain behavior triggered by paclitaxel or formalin in mice, suggesting that PD-1/PD-L1 signaling attenuates peripheral neuropathy development. Consistent with this, we observed that the combined use of anti-PD-L1 plus paclitaxel increased mechanical allodynia and chronic neuropathy development induced by single agents. This effect was associated with higher expression of inflammatory markers (Tnf, Il6, and Cx3cr1) in peripheral nervous tissue. Together, these results suggest that PD-1/PD-L1 inhibitors enhance paclitaxel-induced neuropathic pain by suppressing PD-1/PD-L1 antinociceptive signaling.

Citrullinated human fibrinogen triggers arthritis through an inflammatory response mediated by IL-23/IL-17 immune axis.

Rheumatoid arthritis (RA) is an autoimmune disease that causes joint destruction. Although its etiology remains unknown, citrullinated proteins have been considered as an auto-antigen able to trigger an inflammatory response in RA. Herein, we modified the classical antigen-induced arthritis (AIA) model by using citrullinated human plasma fibrinogen (hFIB) as an immunogen to investigate the mechanism of inflammation-driven joint damage by citrullinated hFIB in C57BL/6 mice. We found that hFIB-immunized mice showed high serum levels of anti-citrullinated peptides antibodies (ACPAs). Moreover, hFIB immunized mice showed increased mechanical hyperalgesia, massive leukocyte infiltration, high levels of inflammatory mediators, and progressive joint damage after the intra-articular challenge with citrullinated hFIB. Interestingly, hFIB-induced arthritis was dependent on IL-23/IL-17 immune axis-mediated inflammatory responses since leukocyte infiltration and mechanical hyperalgesia were abrogated in Il17ra-/- and Il23a-/- mice. Thus, we have characterized a novel model of experimental arthritis suitable to investigate the contribution of ACPAs and Th17 cell-mediated immune response in the pathogenesis of RA.

Cardiovascular and Autonomic Dysfunction in Murine Ligature-Induced Periodontitis.

The present study examined the hemodynamics [arterial pressure (AP), AP variability (APV), heart rate (HR), and heart rate variability (HRV)], cardiac function (echocardiographycally), and myocardial inflammation in Balb/c mice submitted to Periodontitis, through the ligation of the left first molar, or Sham surgical procedure. The first protocol indicated that the AP was similar (136 ± 2 vs. 132 ± 3 mmHg in Sham), while the HR was higher in mice with Periodontitis (475 ± 20 vs. 412 ± 18 bpm in Sham), compared to their Sham counterparts. The APV was higher in mice with Periodontitis when evaluated in the time domain (4.5 ± 0.3 vs. 3.4 ± 0.2 mmHg in Sham), frequency domain (power of the LF band of systolic AP), or through symbolic analysis (patterns 0V + 1V), indicating a sympathetic overactivity. The HRV was similar in the mice with Periodontitis, as compared to their Sham counterparts. In the second protocol, the mice with Periodontitis showed decreased cardiac output (10 ± 0.8 vs. 15 ± 1.4 mL/min in Sham) and ejection fraction (37 ± 3 vs. 47 ± 2% in Sham) associated with increased myocardial cytokines (Interleukin-17, Interleukin-6, and Interleukin-4). This study shows that experimental Periodontitis caused cardiac dysfunction, increased heart cytokines, and sympathetic overactivity, in line with epidemiological studies indicating an increased risk of cardiovascular events in clinical Periodontitis.

Acute Increase in O-GlcNAc Improves Survival in Mice With LPS-Induced Systemic Inflammatory Response Syndrome.

Sepsis is a systemic inflammatory response syndrome (SIRS) resulting from a severe infection that is characterized by immune dysregulation, cardiovascular derangements, and end-organ dysfunction. The modification of proteins by O-linked N-acetylglucosamine (O-GlcNAcylation) influences many of the key processes that are altered during sepsis, including the production of inflammatory mediators and vascular contractility. Here, we investigated whether O-GlcNAc affects the inflammatory response and cardiovascular dysfunction associated with sepsis. Mice received an intraperitoneal injection of lipopolysaccharide (LPS, 20 mg/Kg) to induce endotoxic shock and systemic inflammation, resembling sepsis-induced SIRS. The effects of an acute increase in O-GlcNAcylation, by treatment of mice with glucosamine (GlcN, 300 mg/Kg, i.v.) or thiamet-G (ThG, 150 µg/Kg, i.v.), on LPS-associated mortality, production and release of cytokines by macrophages and vascular cells, vascular responsiveness to constrictors and blood pressure were then determined. Mice under LPS-induced SIRS exhibited a systemic and local inflammatory response with increased levels of interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and tumor necrosis factor (TNF-α), as well as severe hypotension and vascular hyporesponsiveness, characterized by reduced vasoconstriction to phenylephrine. In addition, LPS increased neutrophil infiltration in lungs and produced significant lethality. Treatment with GlcN and ThG reduced systemic inflammation and attenuated hypotension and the vascular refractoriness to phenylephrine, improving survival. GlcN and ThG also decreased LPS-induced production of inflammatory cytokines by bone marrow-derived macrophages and nuclear transcription factor-kappa B (NF-κB) activation in RAW 264.7 NF-κB promoter macrophages. Treatment of mice with ThG increased O-glycosylation of NF-κB p65 subunit in mesenteric arteries, which was associated with reduced Ser536 phosphorylation of NF-κB p65. Finally, GlcN also increased survival rates in mice submitted to cecal ligation and puncture (CLP), a sepsis model. In conclusion, increased O-GlcNAc reduces systemic inflammation and cardiovascular disfunction in experimental sepsis models, pointing this pathway as a potential target for therapeutic intervention.

Inflammatory effect of Bothropstoxin-I from the Bothrops jararacussu venom mediated by NLRP3 inflammasome involves ATP and P2X7 receptor

Muscle tissue damage is one of the local effects described in bothropic envenomations. Bothropstoxin-I (BthTX-I), from B. jararacussu venom, is a K49-phospholipase A2 that induces a massive muscle tissue injury, and, consequently, local inflammatory reaction. The NLRP3 inflammasome is a sensor that triggers inflammation by activating caspase 1 and releasing IL-1b and/or inducing pyroptotic cell death in response to tissue damage. We, therefore, aimed to address activation of NLRP3 inflammasome by BthTX-I-associated injury and the mechanism involved in this process. Intramuscular injection of BthTX-I results in infiltration of neutrophils and macrophages in gastrocnemius muscle, which is reduced in NLRP3- and Caspase-1-deficient mice. The in vitro IL-1β production induced by BthTX-I- inperitoneal macrophages requires caspase 1/11, ASC and NLRP3 and is dependent of ATP-induced K+ efflux and P2X7R. BthTX-I induces a dramatic release of ATP from C2C12 myotubes, therefore representing the major mechanism for P2X7R-dependent inflammasome activation in macrophages. A similar result was obtained when human monocyte-derived macrophages were treated with BthTX-I. These findings demonstrated the inflammatory effect of BthTX-I on muscle tissue, pointing out a role for the ATP released by damaged cells for the NLRP3 activation on macrophages, contributing to the understanding of the microenvironment of the tissue damage of the Bothrops envenomation.

Diagnosis of canine leptospirosis using an immunomagnetic separation-PCR method

Diagnosis of leptospirosis by PCR is hampered due to the presence of substances on biological fluids. Here, we report an immunomagnetic separation step prior to PCR which improved the detection of Leptospira spp. in blood and urine samples from dogs. It resulted in a significant improvement on sensitivity for diagnosis of canine leptospirosis.