Trained immunity of synovial macrophages is associated with exacerbated joint inflammation and damage after Staphylococcus aureus infection.

OBJECTIVES: Investigate whether and which synoviocytes would acquire trained immunity characteristics that could exacerbate joint inflammation following a secondary Staphylococcus aureus infection. METHODS: Lipopolysaccharide (LPS) and S. aureus were separately or double injected (21 days of interval) into the tibiofemoral joint cavity of male C57BL/6 mice. At different time points after these stimulations, mechanical nociception was analyzed followed by the analysis of signs of inflammation and damage in the affected joints. The trained immunity markers, including the glycolytic and mTOR pathway, were analyzed in whole tissue or isolated synoviocytes. A group of mice was treated with Rapamycin, an mTOR inhibitor before LPS or S. aureus stimulation. RESULTS: The double LPS - S. aureus hit promoted intense joint inflammation and damage compared to single joint stimulation, including markers in synoviocyte activation, production of proinflammatory cytokines, persistent nociception, and bone damage, despite not reducing the bacterial clearance. The double LPS - S. aureus hit joints increased the synovial macrophage population expressing CX3CR1 alongside triggering established epigenetic modifications associated with trained immunity events in these cells, such as the upregulation of the mTOR signaling pathway (p-mTOR and HIF1α) and the trimethylation of histone H3. Mice treated with Rapamycin presented reduced CX3CR1+ macrophage activation, joint inflammation, and bone damage. CONCLUSIONS: There is a trained immunity phenotype in CX3CR1+ synovial macrophages that contributes to the exacerbation of joint inflammation and damage during septic arthritis caused by S. aureus.

Safety and immunogenicity of a live-attenuated chikungunya virus vaccine in endemic areas of Brazil: interim results of a double-blind, randomised, placebo-controlled phase 3 trial in adolescents.

BACKGROUND: Chikungunya outbreaks have been reported in Brazil since 2014. Adolescents are a sensitive population who would benefit from a prophylactic vaccine. This study assessed the immunogenicity and safety of the vaccine VLA1553 in adolescents in Brazil. With an overall trial duration of 12 months, we now report data on safety and immunogenicity over a period of 28 days after vaccination. METHODS: In this double-blind, randomised, placebo-controlled phase 3 trial, adolescents aged 12 to <18 years were recruited. The trial was performed at ten trial sites across Brazil. Eligible participants were generally healthy. The main exclusion criteria comprised immune-mediated or chronic arthritis or arthralgia, a known or suspected defect of the immune system, or any live vaccine received within the 4 weeks before trial vaccination. Randomisation was stratified by baseline serostatus in a 2:1 ratio to receive VLA1553 (at a dose of 1 × 104 TCID50 per 0·5 mL [ie, 50% tissue culture infectious dose]) or placebo. VLA1553 or placebo was administered intramuscularly as a single-dose immunisation on day 1. The primary endpoint was the proportion of baseline seronegative participants with chikungunya virus neutralising antibody levels of 150 or more in µPRNT50 (a micro plaque reduction neutralisation test), which was considered a surrogate of protection. The safety analysis included all participants receiving a trial vaccination. Immunogenicity analyses were performed in a subset. The trial is registered with ClinicalTrials.gov, NCT04650399. FINDINGS: Between Feb 14, 2022, and March 14, 2023, 754 participants received a trial vaccination (502 received VLA1553 and 252 received placebo) with a per-protocol population of 351 participants for immunogenicity analyses (303 in the VLA1553 group and 48 in the placebo group). In participants who were seronegative at baseline, VLA1553 induced seroprotective chikungunya virus neutralising antibody levels in 247 of 250 (98·8%, 95% CI 96·5-99·8) participants 28 days after vaccination. In seropositive participants, the baseline seroprotection rate of 96·2% increased to 100% after vaccination with VLA1553. Most (365 [93%] of 393) adverse events were of mild or moderate intensity, VLA1553 was generally well tolerated. When compared with placebo, participants exposed to VLA1553 had a significantly higher frequency of related adverse events (351 [69·9%] of 502 vs 121 [48·0%] of 252; p<0·0001), mostly headache, myalgia, fatigue, and fever. Among four reported serious adverse events (three in the VLA1553 group and one in the placebo group), one was classified as possibly related to VLA1553: a high-grade fever. Among 20 adverse events of special interest (ie, symptoms suggesting chikungunya-like disease), 16 were classified as related to trial vaccination (15 in the VLA1553 group and one in the placebo group), with severe symptoms reported in four participants (fever, headache, or arthralgia). 17 adverse events of special interest resolved within 1 week. Among 85 participants with arthralgia (68 in the VLA1553 group and 17 in the placebo group), eight adolescents had short-lived (range 1-5 days), mostly mild recurring episodes (seven in the VLA1553 group and one in the placebo group). The median duration of arthralgia was 1 day (range 1-5 days). The frequency of injection site adverse events for VLA1553 was higher than in the placebo group (161 [32%] vs 62 [25%]), but rarely severe (two [<1%] in the VLA1553 group and one [<1%] in the placebo group). After administration of VLA1553, there was a significantly lower frequency of solicited adverse events in participants who were seropositive at baseline compared with those who were seronegative (53% vs 74%; p<0·0001) including headache, fatigue, fever, and arthralgia. INTERPRETATION: VLA1553 was generally safe and induced seroprotective titres in almost all vaccinated adolescents with favourable safety data in adolescents who were seropositive at baseline. The data support the use of VLA1553 for the prevention of disease caused by the chikungunya virus among adolescents and in endemic areas. FUNDING: Coalition for Epidemic Preparedness Innovation and EU Horizon 2020. TRANSLATION: For the Portuguese translation of the abstract see Supplementary Materials section.

Blunted inflammatory response is associated with a lower response to a weight loss dietary intervention in liver recipients.

BACKGROUND & AIMS: Obesity is associated with chronic low-grade inflammation, and adipose tissue inflammation is required for fatty tissue remodeling. Interestingly, immunosuppressed patients, as liver transplant recipients, often experience excessive weight gain. We investigated how liver recipients' inflammatory response affects body weight loss induced by dietary treatment. METHODS: Overweight liver recipients were paired with non-transplanted subjects to compare their peripheral immune profiles. RESULTS: Transplanted patients had similar profiles of peripheral blood mononuclear cells compared to controls but lower CD8lowCD56+CD16+NK cells and higher B lymphocytes. Patients showed lower serum concentrations of IFN-γ, TNF, IL-4, IL-2, and IL-10 and lower inflammatory responsiveness of peripheral blood mononuclear cells under inflammatory stimuli. Liver recipients paired with non-transplanted subjects followed a weight loss dietary plan for 6 months to verify body composition changes. After 3 and 6 months of nutritional follow-up, the control group lost more body weight than the liver recipient group. The control group decreased fat mass and waist circumference, which was not observed in transplanted patients. CONCLUSION: Therefore, liver recipients under immunosuppressant treatment responded less to different inflammatory stimuli. This impaired inflammatory milieu might be implicated in the lack of response to weight loss dietary intervention. Inflammation may be essential to trigger the weight loss induced by dietary prescription. CLINICAL TRIAL REGISTRY: ClinicalTrials.gov identification number: NCT03103984.

Chronic ethanol exposure decreases H3K27me3 in the Il6 promoter region of macrophages and generates persistent dysfunction on neutrophils during fungal infection.

OBJECTIVE AND DESIGN: The aim of this study was to investigate the effects of ethanol exposure on epigenetic markers in bone marrow (BM) and their impact on inflammatory response during Aspergillus fumigatus infection. RESULTS: Chronic ethanol exposure decreased H3K27me3 enrichment in the Il6 promoter region while increased H3K4me3 enrichment in Tnf. Chimeric mice were generated by transplanting BM from mice exposed to ethanol or water. Infection of ethanol-chimeric mice culminated in higher clinical scores, although there was no effect on mortality. However, previous chronic exposure to ethanol affects persistently the inflammatory response in lung tissue, demonstrated by increased lung damage, neutrophil accumulation and IL-6, TNF and CXCL2 production in ethanol-chimeric mice, resulting in a decreased neutrophil infiltration into the alveolar space. Neutrophil killing and phagocytosis were also significantly lower. Moreover, BM derived macrophages (BMDM) from ethanol-chimeric mice stimulated with A. fumigatus conidia exhibited higher levels of TNF, CXCL2 and IL-6 release and a higher killing activity. The Il6 promoter of BMDM from ethanol-chimeric mice exhibited a reduction in H3K27me3 enrichment, a finding also observed in BM donors exposed to ethanol. CONCLUSIONS: These evidences demonstrate that prior chronic alcohol exposure of bone-marrow modify immune effector cells functions impairing the inflammatory response during A. fumigatus infection. These findings highlight the persistent impact of chronic ethanol exposure on infectious disease outcomes.

Efficacy and safety of Butantan-DV in participants aged 2-59 years through an extended follow-up: results from a double-blind, randomised, placebo-controlled, phase 3, multicentre trial in Brazil.

BACKGROUND: A single-dose dengue vaccine that protects individuals across a wide age range and regardless of dengue serostatus is an unmet need. We assessed the safety and efficacy of the live, attenuated, tetravalent Butantan-dengue vaccine (Butantan-DV) in adults, adolescents, and children. We previously reported the primary and secondary efficacy and safety endpoints in the initial 2 years of follow-up. Here we report the results through an extended follow-up period, with an average of 3·7 years of follow-up. METHODS: In this double-blind, randomised, placebo-controlled, phase 3, multicentre trial in Brazil, healthy participants (aged 2-59 years) who had not previously received a dengue vaccine were enrolled and randomly assigned 2:1 (stratified by age 18-59 years, 7-17 years, and 2-6 years) using a central electronic randomisation system to receive 0·5 mL of Butantan-DV (containing approximately 103 plaque-forming units of each of the four vaccine virus strains) or placebo, administered subcutaneously. Syringes containing vaccine or placebo were prepared by an unmasked trial pharmacist who was not involved in any subsequent participant assessments; other site staff and the participants remained unaware of the group allocations. Vaccine efficacy was calculated with the accrual of virologically confirmed dengue (VCD) cases (by RT-PCR) at least 28 days after vaccination up until the cutoff (at least 2 years of follow-up from the last participant enrolled). The primary endpoint was vaccine efficacy against VCD after day 28 by any dengue virus (DENV) serotype regardless of dengue serostatus at baseline in the per-protocol population. The primary and secondary safety endpoints up until day 21 were previously reported; secondary safety endpoints include the frequency of unsolicited vaccine-related adverse events after day 22. Safety analyses were done on all participants as treated. This trial is registered with ClinicalTrials.gov (NCT02406729) and is ongoing. FINDINGS: Of 16 363 participants assessed for eligibility, 16 235 were randomly assigned between Feb 22, 2016, and July 5, 2019, and received single-dose Butantan-DV (10 259 participants) or placebo (5976 participants). 16 162 participants (Butantan-DV n=10 215; placebo n=5947) were included in the per-protocol population and 16 235 (Butantan-DV n=10 259; placebo n=5976) in the safety population. At the data cutoff (July 13, 2021), participants had 2-5 years of follow-up (mean 3·7 years [SD 1·0], median 4·0 years [IQR 3·2-4·5]). 356 VCD cases were captured through the follow-up (128 in the vaccine group and 228 in the placebo group). Vaccine efficacy against VCD caused by any DENV serotype was 67·3% (95% CI 59·4-73·9); cases caused by DENV-3 or DENV-4 were not observed. The proportions of participants who had serious adverse events were similar between treatment groups (637 [6·2%] in the vaccine group and 395 [6·6%] in the placebo group) up until the cutoff. INTERPRETATION: A single dose of Butantan-DV was generally well tolerated and efficacious against symptomatic VCD (caused by DENV-1 and DENV-2) for a mean of 3·7 years. These findings support the continued development of Butantan-DV to prevent dengue disease in children, adolescents, and adults regardless of dengue serostatus. FUNDING: Instituto Butantan and Merck Sharp & Dohme LLC, a subsidiary of Merck & Co. TRANSLATIONS: For the Spanish and Portuguese translations of the abstract see Supplementary Materials section.

Treatment with lipoxin A4 improves influenza A infection outcome, induces macrophage reprogramming, anti-inflammatory and pro-resolutive responses.

INTRODUCTION: Influenza A is a virus from the Orthomixoviridae family responsible for high lethality rates and morbidity, despite clinically proven vaccination strategies and some anti-viral therapies. The eicosanoid Lipoxin A4 (LXA4) promotes the resolution of inflammation by decreasing cell recruitment and pro-inflammatory cytokines release, but also for inducing activation of apoptosis, efferocytosis, and macrophage reprogramming. OBJECTIVE: Here, we evaluated whether a synthetic lipoxin mimetic, designated AT-01-KG, would improve the course of influenza A infection in a murine model. METHOD: Mice were infected with influenza A/H1N1 and treated with AT-01-KG (1.7 µg/kg/day, i.p.) at day 3 post-infection. RESULTS: AT-01-KG attenuated mortality, reducing leukocyte infiltration and lung damage at day 5 and day 7 post-infection. AT-01-KG is a Formyl Peptide Receptor 2 (designated FPR2/3 in mice) agonist, and the protective responses were not observed in fpr2/3 -/- animals. In mice treated with LXA4 (50 µg/kg/day, i.p., days 3-6 post-infection), at day 7, macrophage reprogramming was observed, as seen by a decrease in classically activated macrophages and an increase in alternatively activated macrophages in the lungs. Furthermore, the number of apoptotic cells and cells undergoing efferocytosis was increased in the lavage of treated mice. Treatment also modulated the adaptive immune response, increasing the number of T helper 2 cells (Th2) and regulatory T (Tregs) cells in the lungs of the treated mice. CONCLUSION: Therefore, treatment with a lipoxin A4 analog was beneficial in a model of influenza A infection in mice. The drug decreased inflammation and promoted resolution and beneficial immune responses, suggesting it may be useful in patients with severe influenza.

Treatment with lipoxin A 4 improves influenza A infection outcome through macrophage reprogramming, anti-inflammatory and pro-resolutive responses.

Objective and design: Here, we evaluated whether a synthetic lipoxin mimetic, designated AT-01-KG, would improve the course of influenza A infection in a murine model. Treatment: Mice were infected with influenza A/H1N1 and treated with AT-01-KG (1.7 mg/kg/day, i.p.) at day 3 post-infection. Methods: Mortality rate was assessed up to day 21 and inflammatory parameters were assessed at days 5 and 7. Results: AT-01-KG attenuated mortality, reducing leukocyte infiltration and lung damage at day 5 and day 7 post-infection. AT-01-KG is a Formyl Peptide Receptor 2 (designated FPR2/3 in mice) agonist, and the protective responses were not observed in FPR2/3 -/- animals. In mice treated with LXA4 (50mg/kg/day, i.p., days 3-6 post-infection), at day 7, macrophage reprogramming was observed, as seen by a decrease in classically activated macrophages and an increase in alternatively activated macrophages in the lungs. Furthermore, the number of apoptotic cells and cells undergoing efferocytosis was increased in the lavage of treated mice. Treatment also modulated the adaptive immune response, increasing the number of anti-inflammatory T cells (Th2) and regulatory T (Tregs) cells in the lungs of the treated mice. Conclusions: Therefore, treatment with a lipoxin A4 analog was beneficial in a model of influenza A infection in mice. The drug decreased inflammation and promoted resolution and beneficial immune responses, suggesting it may be useful in patients with severe influenza.

The glycosaminoglycan-binding chemokine fragment CXCL9(74-103) reduces inflammation and tissue damage in mouse models of coronavirus infection.

Introduction: Pulmonary diseases represent a significant burden to patients and the healthcare system and are one of the leading causes of mortality worldwide. Particularly, the COVID-19 pandemic has had a profound global impact, affecting public health, economies, and daily life. While the peak of the crisis has subsided, the global number of reported COVID-19 cases remains significantly high, according to medical agencies around the world. Furthermore, despite the success of vaccines in reducing the number of deaths caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there remains a gap in the treatment of the disease, especially in addressing uncontrolled inflammation. The massive recruitment of leukocytes to lung tissue and alveoli is a hallmark factor in COVID-19, being essential for effectively responding to the pulmonary insult but also linked to inflammation and lung damage. In this context, mice models are a crucial tool, offering valuable insights into both the pathogenesis of the disease and potential therapeutic approaches. Methods: Here, we investigated the anti-inflammatory effect of the glycosaminoglycan (GAG)-binding chemokine fragment CXCL9(74-103), a molecule that potentially decreases neutrophil transmigration by competing with chemokines for GAG-binding sites, in two models of pneumonia caused by coronavirus infection. Results: In a murine model of betacoronavirus MHV-3 infection, the treatment with CXCL9(74-103) decreased the accumulation of total leukocytes, mainly neutrophils, to the alveolar space and improved several parameters of lung dysfunction 3 days after infection. Additionally, this treatment also reduced the lung damage. In the SARS-CoV-2 model in K18-hACE2-mice, CXCL9(74-103) significantly improved the clinical manifestations of the disease, reducing pulmonary damage and decreasing viral titers in the lungs. Discussion: These findings indicate that CXCL9(74-103) resulted in highly favorable outcomes in controlling pneumonia caused by coronavirus, as it effectively diminishes the clinical consequences of the infections and reduces both local and systemic inflammation.

A single dose of angiotensin-(1-7) resolves eosinophilic inflammation and protects the lungs from a secondary inflammatory challenge.

OBJECTIVE: Angiotensin-(1-7) [Ang-(1-7)] is a pro-resolving mediator. It is not known whether the pro-resolving effects of Ang-(1-7) are sustained and protect the lung from a subsequent inflammatory challenge. This study sought to investigate the impact of treatment in face of a second allergic or lipopolysaccharide (LPS) challenge. METHODS: Mice, sensitized and challenged with ovalbumin (OVA), received a single Ang-(1-7) dose at the peak of eosinophilic inflammation, 24 h after the final OVA challenge. Subsequently, mice were euthanized at 48, 72, 96, and 120 h following the OVA challenge, and cellular infiltrate, inflammatory mediators, lung histopathology, and macrophage-mediated efferocytic activity were evaluated. The secondary inflammatory stimulus (OVA or LPS) was administered 120 h after the last OVA challenge, and subsequent inflammatory analyses were performed. RESULTS: Treatment with Ang-(1-7) resulted in elevated levels of IL-10, CD4+Foxp3+, Mres in the lungs and enhanced macrophage-mediated efferocytic capacity. Moreover, in allergic mice treated with Ang-(1-7) and then subjected to a secondary OVA challenge, inflammation was also reduced. Similarly, in mice exposed to LPS, Ang-(1-7) effectively prevented the lung inflammation. CONCLUSION: A single dose of Ang-(1-7) resolves lung inflammation and protect the lung from a subsequent inflammatory challenge highlighting its potential therapeutic for individuals with asthma.

Congenital Zika virus infection impacts on male mouse offspring's reproductive biology.

In brief: Congenital ZIKV infection promotes alarming effects on male offspring's reproductive biology. This study showed the presence of the ZIKV antigen in the testis parenchyma, decreased testosterone levels, and sperm abnormalities in male offspring born to infected mothers. Abstract: Infection with ZIKV during pregnancy is associated with fetal developmental problems. Although neurological issues are being explored more in experimental studies, limited research has focused on the reproductive health consequences for offspring born to infected mothers. In this context, this study aimed to assess the impact of ZIKV infection during pregnancy on the testes and sperm of adult male offspring. Female mice were intraperitoneally inoculated with a Brazil strain of ZIKV during the 5.5th day of embryonic gestation. The offspring were evaluated 12 weeks after birth to analyze cellular and molecular changes in the testes and sperm. A novel approach combining variable-angle spectroscopic ellipsometry and machine learning modeling was also introduced for sperm sample analysis. The study revealed the presence of ZIKV protein in the testis parenchyma of adult male offspring born to infected mothers. It was shown that the testes exhibited altered steroidogenesis and inflammatory mediators, in addition to significant issues with spermiogenesis that resulted in sperm with DNA fragmentation, head defects, and protamination failure. Additionally, sperm dielectric properties and artificial intelligence showed potential for rapid identification and classification of sperm samples from infected mice. These findings provide crucial insights into the reproductive risks for men born from ZIKV-infected pregnant women.

Role of IL-18 in adipose tissue remodeling and metabolic dysfunction.

BACKGROUND/OBJECTIVES: Proinflammatory cytokines are increased in obese adipose tissue, including inflammasome key masters. Conversely, IL-18 protects against obesity and metabolic dysfunction. We focused on the IL-18 effect in controlling adipose tissue remodeling and metabolism. MATERIALS/SUBJECTS AND METHODS: We used C57BL/6 wild-type (WT) and interleukine-18 deficient (IL-18-/-) male mice fed a chow diet and samples from bariatric surgery patients. RESULTS: IL-18-/- mice showed increased adiposity and proinflammatory cytokine levels in adipose tissue, leading to glucose intolerance. IL-18 was widely secreted by stromal vascular fraction but not adipocytes from mice's fatty tissue. Chimeric model experiments indicated that IL-18 controls adipose tissue expansion through its presence in tissues other than bone marrow. However, IL-18 maintains glucose homeostasis when present in bone marrow cells. In humans with obesity, IL-18 expression in omental tissue was not correlated with BMI or body fat mass but negatively correlated with IRS1, GLUT-4, adiponectin, and PPARy expression. Also, the IL-18RAP receptor was negatively correlated with IL-18 expression. CONCLUSIONS: IL-18 signaling may control adipose tissue expansion and glucose metabolism, as its absence leads to spontaneous obesity and glucose intolerance in mice. We suggest that resistance to IL-18 signaling may be linked with worse glucose metabolism in humans with obesity.

Ionizable Lipid Nanoparticle-Mediated TRAIL mRNA Delivery in the Tumor Microenvironment to Inhibit Colon Cancer Progression.

Introduction: Immunotherapy has revolutionized cancer treatment by harnessing the immune system to enhance antitumor responses while minimizing off-target effects. Among the promising cancer-specific therapies, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has attracted significant attention. Methods: Here, we developed an ionizable lipid nanoparticle (LNP) platform to deliver TRAIL mRNA (LNP-TRAIL) directly to the tumor microenvironment (TME) to induce tumor cell death. Our LNP-TRAIL was formulated via microfluidic mixing and the induction of tumor cell death was assessed in vitro. Next, we investigated the ability of LNP-TRAIL to inhibit colon cancer progression in vivo in combination with a TME normalization approach using Losartan (Los) or angiotensin 1-7 (Ang(1-7)) to reduce vascular compression and deposition of extracellular matrix in mice. Results: Our results demonstrated that LNP-TRAIL induced tumor cell death in vitro and effectively inhibited colon cancer progression in vivo, particularly when combined with TME normalization induced by treatment Los or Ang(1-7). In addition, potent tumor cell death as well as enhanced apoptosis and necrosis was found in the tumor tissue of a group treated with LNP-TRAIL combined with TME normalization. Discussion: Together, our data demonstrate the potential of the LNP to deliver TRAIL mRNA to the TME and to induce tumor cell death, especially when combined with TME normalization. Therefore, these findings provide important insights for the development of novel therapeutic strategies for the immunotherapy of solid tumors.

Resolution pharmacology and the treatment of infectious diseases.

Inflammation is elicited by the host in response to microbes, and is believed to be essential for protection against infection. However, we have previously hypothesized that excessive or misplaced inflammation may be a major contributor to tissue dysfunction and death associated with viral and bacterial infections. The resolutive phase of inflammation is a necessary condition to achieve homeostasis after acute inflammation. It is possible that targeting inflammation resolution may be beneficial for the host during infection. In this review, we summarize the evidence demonstrating the expression, roles and effects of the best described pro-resolving molecules in the context of bacterial and viral infections. Pro-resolving molecules play a pivotal role in modulating a spectrum of pathways associated with tissue inflammation and damage during both viral and bacterial infections. These molecules offer a blend of anti-inflammatory, pro-resolving and sometimes anti-infective benefits, all the while circumventing the undesired and immune-suppressive unwanted effects associated with glucocorticoids. Whether these beneficial effects will translate into benefits to patients clearly deserve further investigation.

Essential role of the CCL2-CCR2 axis in Mayaro virus-induced disease.

Mayaro virus (MAYV) is an emerging arbovirus member of the Togaviridae family and Alphavirus genus. MAYV infection causes an acute febrile illness accompanied by persistent polyarthralgia and myalgia. Understanding the mechanisms involved in arthritis caused by alphaviruses is necessary to develop specific therapies. In this work, we investigated the role of the CCL2/CCR2 axis in the pathogenesis of MAYV-induced disease. For this, wild-type (WT) C57BL/6J and CCR2-/- mice were infected with MAYV subcutaneously and evaluated for disease development. MAYV infection induced an acute inflammatory disease in WT mice. The immune response profile was characterized by an increase in the production of inflammatory mediators, such as IL-6, TNF, and CCL2. Higher levels of CCL2 at the local and systemic levels were followed by the significant recruitment of CCR2+ macrophages and a cellular response orchestrated by these cells. CCR2-/- mice showed an increase in CXCL-1 levels, followed by a replacement of the macrophage inflammatory infiltrate by neutrophils. Additionally, the absence of the CCR2 receptor protected mice from bone loss induced by MAYV. Accordingly, the silencing of CCL2 chemokine expression in vivo and the pharmacological blockade of CCR2 promoted a partial improvement in disease. Cell culture data support the mechanism underlying the bone pathology of MAYV, in which MAYV infection promotes a pro-osteoclastogenic microenvironment mediated by CCL2, IL-6, and TNF, which induces the migration and differentiation of osteoclast precursor cells. Overall, these data contribute to the understanding of the pathophysiology of MAYV infection and the identification future of specific therapeutic targets in MAYV-induced disease.IMPORTANCEThis work demonstrates the role of the CCL2/CCR2 axis in MAYV-induced disease. The infection of wild-type (WT) C57BL/6J and CCR2-/- mice was associated with high levels of CCL2, an important chemoattractant involved in the recruitment of macrophages, the main precursor of osteoclasts. In the absence of the CCR2 receptor, there is a mitigation of macrophage migration to the target organs of infection and protection of these mice against bone loss induced by MAYV infection. Much evidence has shown that host immune response factors contribute significantly to the tissue damage associated with alphavirus infections. Thus, this work highlights molecular and cellular targets involved in the pathogenesis of arthritis triggered by MAYV and identifies novel therapeutic possibilities directed to the host inflammatory response unleashed by MAYV.

Live, Attenuated, Tetravalent Butantan-Dengue Vaccine in Children and Adults.

BACKGROUND: Butantan-Dengue Vaccine (Butantan-DV) is an investigational, single-dose, live, attenuated, tetravalent vaccine against dengue disease, but data on its overall efficacy are needed. METHODS: In an ongoing phase 3, double-blind trial in Brazil, we randomly assigned participants to receive Butantan-DV or placebo, with stratification according to age (2 to 6 years, 7 to 17 years, and 18 to 59 years); 5 years of follow-up is planned. The objectives of the trial were to evaluate overall vaccine efficacy against symptomatic, virologically confirmed dengue of any serotype occurring more than 28 days after vaccination (the primary efficacy end point), regardless of serostatus at baseline, and to describe safety up to day 21 (the primary safety end point). Here, vaccine efficacy was assessed on the basis of 2 years of follow-up for each participant, and safety as solicited vaccine-related adverse events reported up to day 21 after injection. Key secondary objectives were to assess vaccine efficacy among participants according to dengue serostatus at baseline and according to the dengue viral serotype; efficacy according to age was also assessed. RESULTS: Over a 3-year enrollment period, 16,235 participants received either Butantan-DV (10,259 participants) or placebo (5976 participants). The overall 2-year vaccine efficacy was 79.6% (95% confidence interval [CI], 70.0 to 86.3) - 73.6% (95% CI, 57.6 to 83.7) among participants with no evidence of previous dengue exposure and 89.2% (95% CI, 77.6 to 95.6) among those with a history of exposure. Vaccine efficacy was 80.1% (95% CI, 66.0 to 88.4) among participants 2 to 6 years of age, 77.8% (95% CI, 55.6 to 89.6) among those 7 to 17 years of age, and 90.0% (95% CI, 68.2 to 97.5) among those 18 to 59 years of age. Efficacy against DENV-1 was 89.5% (95% CI, 78.7 to 95.0) and against DENV-2 was 69.6% (95% CI, 50.8 to 81.5). DENV-3 and DENV-4 were not detected during the follow-up period. Solicited systemic vaccine- or placebo-related adverse events within 21 days after injection were more common with Butantan-DV than with placebo (58.3% of participants, vs. 45.6%). CONCLUSIONS: A single dose of Butantan-DV prevented symptomatic DENV-1 and DENV-2, regardless of dengue serostatus at baseline, through 2 years of follow-up. (Funded by Instituto Butantan and others; DEN-03-IB ClinicalTrials.gov number, NCT02406729, and WHO ICTRP number, U1111-1168-8679.).

Nanoparticle-based DNA vaccine protects against SARS-CoV-2 variants in female preclinical models.

A safe and effective vaccine with long-term protection against SARS-CoV-2 variants of concern (VOCs) is a global health priority. Here, we develop lipid nanoparticles (LNPs) to provide safe and effective delivery of plasmid DNA (pDNA) and show protection against VOCs in female small animal models. Using a library of LNPs encapsulating unique barcoded DNA (b-DNA), we screen for b-DNA delivery after intramuscular administration. The top-performing LNPs are further tested for their capacity of pDNA uptake in antigen-presenting cells in vitro. The lead LNP is used to encapsulate pDNA encoding the HexaPro version of SARS-CoV-2 spike (LNP-HPS) and immunogenicity and protection is tested in vivo. LNP-HPS elicit a robust protective effect against SARS-CoV-2 Gamma (P.1), correlating with reduced lethality, decreased viral load in the lungs and reduced lung damage. LNP-HPS induce potent humoral and T cell responses against P.1, and generate high levels of neutralizing antibodies against P.1 and Omicron (B.1.1.529). Our findings indicate that the protective efficacy and immunogenicity elicited by LNP-HPS are comparable to those achieved by the approved COVID-19 vaccine from Biontech/Pfizer in animal models. Together, these findings suggest that LNP-HPS hold great promise as a vaccine candidate against VOCs.

The role of neurotrophin polymorphisms and susceptibility to neural damage in leprosy.

OBJECTIVES: Mycobacterium leprae is able to infect Schwann cells leading to neural damage. Neurotrophins are involved in nervous system plasticity and impact neural integrity during diseases. Investigate the association between single nucleotide polymorphisms in neurotrophin genes and leprosy phenotypes, especially neural damage. DESIGN: We selected single nucleotide polymorphisms in neurotrophins or their receptors genes associated with neural disorders: rs6265 and rs11030099 of brain-derived neurotrophic factor (BDNF), rs6330 of BDNF, rs6332 in NT3 and rs2072446 of P75NTR. The association of genetic frequencies with leprosy phenotypes was investigated in a case-control study. RESULTS: An association of the BDNF single nucleotide polymorphism rs11030099 with the number of affected nerves was demonstrated. The "AA+AC" genotypes were demonstrated to be protective against nerve impairment. However, this variation does not affect BDNF serum levels. BDNF is an important factor for myelination of Schwann cells and polymorphisms in this gene can be associated with leprosy outcome. Moreover, rs11030099 is located in the binding region for micro-RNA (miRNA) 26a that could be involved in control of BDNF expression. We demonstrated different expression levels of this miRNA in polar forms of leprosy. CONCLUSION: Our findings demonstrate for the first time an association between the polymorphism rs11030099 in the BDNF gene and neural commitment in leprosy and may indicate a possible role of miRNA-26a acting synergistically to these genetic variants in neural damage development.

siRNA lipid nanoparticles for CXCL12 silencing modulate brain immune response during Zika infection.

CXCL12 is a key chemokine implicated in neuroinflammation, particularly during Zika virus (ZIKV) infection. Specifically, CXCL12 is upregulated in circulating cells of ZIKV infected patients. Here, we developed a lipid nanoparticle (LNP) to deliver siRNA in vivo to assess the impact of CXCL12 silencing in the context of ZIKV infection. The biodistribution of the LNP was assessed in vivo after intravenous injection using fluorescently tagged siRNA. Next, we investigated the ability of the developed LNP to silence CXCL12 in vivo and assessed the resulting effects in a murine model of ZIKV infection. The LNP encapsulating siRNA significantly inhibited CXCL12 levels in the spleen and induced microglial activation in the brain during ZIKV infection. This activation was evidenced by the enhanced expression of iNOS, TNF-α, and CD206 within microglial cells. Moreover, T cell subsets exhibited reduced secretion of IFN-É£ and IL-17 following LNP treatment. Despite no observable alteration in viral load, CXCL12 silencing led to a significant reduction in type-I interferon production compared to both ZIKV-infected and uninfected groups. Furthermore, we found grip strength deficits in the group treated with siRNA-LNP compared to the other groups. Our data suggest a correlation between the upregulated pro-inflammatory cytokines and the observed decrease in strength. Collectively, our results provide evidence that CXCL12 silencing exerts a regulatory influence on the immune response in the brain during ZIKV infection. In addition, the modulation of T-cell activation following CXCL12 silencing provides valuable insights into potential protective mechanisms against ZIKV, offering novel perspectives for combating this infection.

Dietary Vitamin D Mitigates Coronavirus-Induced Lung Inflammation and Damage in Mice.

The COVID-19 pandemic caused by the SARS-CoV-2 (ß-CoV) betacoronavirus has posed a significant threat to global health. Despite the availability of vaccines, the virus continues to spread, and there is a need for alternative strategies to alleviate its impact. Vitamin D, a secosteroid hormone best known for its role in bone health, exhibits immunomodulatory effects in certain viral infections. Here, we have shown that bioactive vitamin D (calcitriol) limits in vitro replication of SARS-CoV-2 and murine coronaviruses MHV-3 and MHV-A59. Comparative studies involving wild-type mice intranasally infected with MHV-3, a model for studying ß-CoV respiratory infections, confirmed the protective effect of vitamin D in vivo. Accordingly, mice fed a standard diet rapidly succumbed to MHV-3 infection, whereas those on a vitamin D-rich diet (10,000 IU of Vitamin D3/kg) displayed increased resistance to acute respiratory damage and systemic complications. Consistent with these findings, the vitamin D-supplemented group exhibited lower viral titers in their lungs and reduced levels of TNF, IL-6, IL-1ß, and IFN-γ, alongside an enhanced type I interferon response. Altogether, our findings suggest vitamin D supplementation ameliorates ß-CoV-triggered respiratory illness and systemic complications in mice, likely via modulation of the host's immune response to the virus.

The effects of Phycocyanobilin on experimental arthritis involve the reduction in nociception and synovial neutrophil infiltration, inhibition of cytokine production, and modulation of the neuronal proteome.

Introduction: The antinociceptive and pharmacological activities of C-Phycocyanin (C-PC) and Phycocyanobilin (PCB) in the context of inflammatory arthritis remain unexplored so far. In the present study, we aimed to assess the protective actions of these compounds in an experimental mice model that replicates key aspects of human rheumatoid arthritis. Methods: Antigen-induced arthritis (AIA) was established by intradermal injection of methylated bovine serum albumin in C57BL/6 mice, and one hour before the antigen challenge, either C-PC (2, 4, or 8 mg/kg) or PCB (0.1 or 1 mg/kg) were administered intraperitoneally. Proteome profiling was also conducted on glutamate-exposed SH-SY5Y neuronal cells to evaluate the PCB impact on this key signaling pathway associated with nociceptive neuronal sensitization. Results and discussion: C-PC and PCB notably ameliorated hypernociception, synovial neutrophil infiltration, myeloperoxidase activity, and the periarticular cytokine concentration of IFN-γ, TNF-α, IL-17A, and IL-4 dose-dependently in AIA mice. In addition, 1 mg/kg PCB downregulated the gene expression for T-bet, RORγ, and IFN-γ in the popliteal lymph nodes, accompanied by a significant reduction in the pathological arthritic index of AIA mice. Noteworthy, neuronal proteome analysis revealed that PCB modulated biological processes such as pain, inflammation, and glutamatergic transmission, all of which are involved in arthritic pathology. Conclusions: These findings demonstrate the remarkable efficacy of PCB in alleviating the nociception and inflammation in the AIA mice model and shed new light on mechanisms underlying the PCB modulation of the neuronal proteome. This research work opens a new avenue to explore the translational potential of PCB in developing a therapeutic strategy for inflammation and pain in rheumatoid arthritis.