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The E3 ubiquitin ligase Smurf1 catalyzes the ubiquitination and proteasomal degradation of several protein substrates related to inflammatory responses and antiviral signaling. This study investigated the role of Smurf1 in modulating inflammation induced by Betacoronavirus infection. Bone marrow-derived macrophages (BMDMs) from C57BL/6 (wild-type) or Smurf1-deficient (Smurf1−/−) mice were infected with MHV-A59 to evaluate the inflammatory response in vitro. Smurf1 was found to be required to downregulate the macrophage production of pro-inflammatory mediators, including TNF, and CXCL1; to control viral release from infected cells; and to increase cell viability. To assess the impact of Smurf 1 in vivo, we evaluated the infection of mice with MHV-A59 through the intranasal route. Smurf1−/− mice infected with a lethal inoculum of MHV-A59 succumbed earlier to infection. Intranasal inoculation with a 10-fold lower dose of MHV-A59 resulted in hematological parameter alterations in Smurf1−/− mice suggestive of exacerbated systemic inflammation. In the lung parenchyma, Smurf1 expression was essential to promote viral clearance, downregulating IFN-β mRNA and controlling the inflammatory profile of macrophages and neutrophils. Conversely, Smurf1 did not affect IFN-β mRNA regulation in the liver, but it was required to increase TNF and iNOS expression in neutrophils and decrease TNF expression in macrophages. In addition, Smurf1−/− mice exhibited augmented liver injuries, accompanied by high serum levels of alanine aminotransferase (ALT). These findings suggest that Smurf1 plays a critical role in regulating the inflammatory response in macrophages and attenuating systemic inflammation during Betacoronavirus infection.

Cutaneous leishmaniasis (CL) is a neglected tropical disease. The treatment is restricted to drugs, such as meglumine antimoniate and amphotericin B, that exhibit toxic effects, high cost, long-term treatment, and limited efficacy. The development of new alternative therapies, including the identification of effective drugs for the topical and oral treatment of CL, is of great interest. In this sense, a combination of topical photodynamic therapy (PDT) with chloroaluminum phthalocyanine liposomes (Lip-ClAlPc) and the oral administration of a self-emulsifying drug delivery system containing fexinidazole (SEDDS-FEX) emerges as a new strategy. The aim of the present study was to prepare, characterize, and evaluate the efficacy of combined therapy with Lip-ClAlPc and SEDDS-FEX in the experimental treatment of Leishmania (Leishmania) major. Lip-ClAlPc and SEDDS-FEX were prepared, and the antileishmanial efficacy study was conducted with the following groups: 1. Lip-ClAlPc (0.05 mL); 2. SEDDS-FEX (50 mg/kg/day); 3. Lip-ClAlPc (0.05 mL)+SEDDS-FEX (50 mg/kg/day) combination; 4. FEX suspension (50 mg/kg/day); and 5. control (untreated). BALB/c mice received 10 sessions of topical Lip-ClAlPc on alternate days and 20 consecutive days of SEDDS-FEX or FEX oral suspension. Therapeutical efficacy was evaluated via the parasite burden (limiting-dilution assay), lesion size (mm), healing of the lesion, and histological analyses. Lip-ClAlPc and SEDDS-FEX presented physicochemical characteristics that are compatible with the administration routes used in the treatments. Lip-ClAlPc+SEDDS-FEX led to a significant reduction in the parasitic burden in the lesion and spleen when compared to the control group (p < 0.05) and the complete healing of the lesion in 43% of animals. The Lip-ClAlPc+SEDDS-FEX combination may be promising for the treatment of CL caused by L. major.

The COVID-19 pandemic caused by the SARS-CoV-2 (β-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 β-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-1β, and IFN-γ, alongside an enhanced type I interferon response. Altogether, our findings suggest vitamin D supplementation ameliorates β-CoV-triggered respiratory illness and systemic complications in mice, likely via modulation of the host’s immune response to the virus.

Exacerbated inflammatory responses are a hallmark of severe coronavirus disease 2019 (COVID-19). Zileuton (Zi) is a selective inhibitor of 5-lipoxygenase, an enzyme involved in the production of several inflammatory/pro-resolving lipid mediators. Herein, we investigated the effect of Zi treatment in a severe acute respiratory syndrome (SARS) model. Mouse hepatitis virus (MHV)3-infected mice treated with Zi significantly improved the clinical score, weight loss, cardiopulmonary function, and survival rates compared with infected untreated animals. The protection observed in Zi-treated mice was associated with a lower inflammatory score, reduced dendritic cell-producing tumor necrosis factor (TNF), and increased neutrophil-producing interleukin (IL)-10 in the lungs three days after infection (dpi). At 5 dpi, the lungs of treated mice showed an increase in Th2-, Treg CD4+-, and Treg CD8+-producing IL-10 and reduced Th1 infiltrating cells. Furthermore, similar results were found upon Zi treatment after SARS-CoV-2 infection in transgenic mice expressing the human angiotensin I-converting enzyme 2 (ACE2) receptor driven by the cytokeratin-18 (K18) gene promoter (K18-hACE2), significantly improving the clinical score, weight loss, and lung inflammatory score compared with untreated animals. Our data suggest that Zi protects against developing severe lung disease during SARS induced by betacoronavirus without affecting the host’s capacity to deal with infection.

Background: Zika virus (ZIKV) infection during pregnancy may cause major congenital defects, including microcephaly, ocular, articular and muscle abnormalities, which are collectively defined as Congenital Zika Syndrome. Here, we performed an in-depth characterization of the effects of congenital ZIKV infection (CZI) in immunocompetent mice. Methods: Pregnant dams were inoculated with ZIKV on embryonic day 5.5 in the presence or absence of a sub-neutralizing dose of a pan-flavivirus monoclonal antibody (4G2) to evaluate the potential role of antibody-dependent enhancement phenomenon (ADE) during short and long outcomes of CZI. Findings: ZIKV infection induced maternal immune activation (MIA), which was associated with occurrence of foetal abnormalities and death. Therapeutic administration of AH-D antiviral peptide during the early stages of pregnancy prevented ZIKV replication and death of offspring. In the post-natal period, CZI was associated with a decrease in whole brain volume, ophthalmologic abnormalities, changes in testicular morphology, and disruption in bone microarchitecture. Some alterations were enhanced in the presence of 4G2 antibody. Interpretation: Our results reveal that early maternal ZIKV infection causes several birth defects in immunocompetent mice, which can be potentiated by ADE phenomenon and are associated with MIA. Additionally, antiviral treatment with AH-D peptide may be beneficial during early maternal ZIKV infection. Fund: This work was supported by the Brazilian National Science Council (CNPq, Brazil), Minas Gerais Foundation for Science (FAPEMIG), Funding Authority for Studies and Projects (FINEP), Coordination of Superior Level Staff Improvement (CAPES), National Research Foundation of Singapore and Centre for Precision Biology at Nanyang Technological University. Keywords: Congenital Zika virus infection (CZI), Congenital Zika Syndrome (CZS), Maternal immune activation (MIA), Antibody-dependent enhancement (ADE), Short and long-term outcomes

Chikungunya (CHIKV) is an arthritogenic alphavirus that causes a self-limiting disease usually accompanied by joint pain and/or polyarthralgia with disabling characteristics. Immune responses developed during the acute phase of CHIKV infection determine the rate of disease progression and resolution. Annexin A1 (AnxA1) is involved in both initiating inflammation and preventing over-response, being essential for a balanced end of inflammation. In this study, we investigated the role of the AnxA1-FPR2/ALX pathway during CHIKV infection. Genetic deletion of AnxA1 or its receptor enhanced inflammatory responses driven by CHIKV. These knockout mice showed increased neutrophil accumulation and augmented tissue damage at the site of infection compared with control mice. Conversely, treatment of wild-type animals with the AnxA1 mimetic peptide (Ac2–26) reduced neutrophil accumulation, decreased local concentration of inflammatory mediators and diminished mechanical hypernociception and paw edema induced by CHIKV-infection. Alterations in viral load were mild both in genetic deletion or with treatment. Combined, our data suggest that the AnxA1-FPR2/ALX pathway is a potential therapeutic strategy to control CHIKV-induced acute inflammation and polyarthralgia.

Acetaminophen (APAP) is usually safe when administrated in therapeutic doses; however, APAP overdose can lead to severe liver injury. APAP can cause direct hepatocyte damage, and stimulates an inflammatory response leading to oxidative stress. Supressor of Cytokine Signaling (SOCS) 2 modulates cytokine and growth factor signaling, and plays a role in the regulation of hepatic cellular processes. Our study evaluated the role of SOCS2 in APAP liver injury. The administration of a toxic dose (600 mg/kg) of APAP caused significant liver necrosis in WT mice. In SOCS2−/− mice, there was significantly more necrosis, neutrophil recruitment, and expression of the neutrophil-active chemokine CXCL-1. Expression of proinflammatory cytokines, such as TNF-α and IL-6, was elevated, while expression of anti-inflammatory cytokines, IL-10 and TGF-β, was diminished. In vitro, SOCS2−/− hepatocytes expressed more p-NF-kB and produced more ROS than WT hepatocytes when exposed to APAP. SOCS2−/− hepatocytes were more sensitive to cell death in the presence of IL-6 and hydrogen peroxide. The administration of catalase in vitro and in vivo resulted in a pronounced reduction of cells/mice death and necrosis in the SOCS2−/− group. We have demonstrated that SOCS2 has a protective role in the liver by controlling pro-oxidative and inflammatory mechanisms induced by APAP.

Influenza A virus (IAV) infection causes severe pulmonary disease characterized by intense leukocyte infiltration. Phosphoinositide-3 kinases (PI3Ks) are central signaling enzymes, involved in cell growth, survival, and migration. Class IB PI3K or phosphatidyl inositol 3 kinase-gamma (PI3Kγ), mainly expressed by leukocytes, is involved in cell migration during inflammation. Here, we investigated the contribution of PI3Kγ for the inflammatory and antiviral responses to IAV. PI3Kγ knockout (KO) mice were highly susceptible to lethality following infection with influenza A/WSN/33 H1N1. In the early time points of infection, infiltration of neutrophils was higher than WT mice whereas type-I and type-III IFN expression and p38 activation were reduced in PI3Kγ KO mice resulting in higher viral loads when compared with WT mice. Blockade of p38 in WT macrophages infected with IAV reduced levels of interferon-stimulated gene 15 protein to those induced in PI3Kγ KO macrophages, suggesting that p38 is downstream of antiviral responses mediated by PI3Kγ. PI3Kγ KO-derived fibroblasts or macrophages showed reduced type-I IFN transcription and altered pro-inflammatory cytokines suggesting a cell autonomous imbalance between inflammatory and antiviral responses. Seven days after IAV infection, there were reduced infiltration of natural killer cells and CD8+ T lymphocytes, increased concentration of inflammatory cytokines in bronchoalveolar fluid, reduced numbers of resolving macrophages, and IL-10 levels in PI3Kγ KO. This imbalanced environment in PI3Kγ KO-infected mice culminated in enhanced lung neutrophil infiltration, reactive oxygen species release, and lung damage that together with the increased viral loads, contributed to higher mortality in PI3Kγ KO mice compared with WT mice. In humans, we tested the genetic association of disease severity in influenza A/H1N1pdm09-infected patients with three potentially functional PIK3CG single-nucleotide polymorphisms (SNPs), rs1129293, rs17847825, and rs2230460. We observed that SNPs rs17847825 and rs2230460 (A and T alleles, respectively) were significantly associated with protection from severe disease using the recessive model in patients infected with influenza A(H1N1)pdm09. Altogether, our results suggest that PI3Kγ is crucial in balancing antiviral and inflammatory responses to IAV infection.

RationaleInfluenza A infections are a leading cause of morbidity and mortality worldwide especially when associated with secondary pneumococcal infections. Inflammation is important to control pathogen proliferation but may also cause tissue injury and death. CXCR1/2 are chemokine receptors relevant for the recruitment of neutrophils. We investigated the role of CXCR1/2 during influenza, pneumococcal, and post-influenza pneumococcal infections.MethodsMice were infected with influenza A virus (IAV) or Streptococcus pneumoniae and then treated daily with the CXCR1/2 antagonist DF2162. To study secondary pneumococcal infection, mice were infected with a sublethal inoculum of IAV then infected with S. pneumoniae 14 days later. DF2162 was given in a therapeutic schedule from days 3 to 6 after influenza infection. Lethality, weight loss, inflammation, virus/bacteria counts, and lung injury were assessed.ResultsCXCL1 and CXCL2 were produced at high levels during IAV infection. DF2162 treatment decreased morbidity and this was associated with decreased infiltration of neutrophils in the lungs and reduced pulmonary damage and viral titers. During S. pneumoniae infection, DF2162 treatment decreased neutrophil recruitment, pulmonary damage, and lethality rates, without affecting bacteria burden. Therapeutic treatment with DF2162 during sublethal IAV infection reduced the morbidity associated with virus infection and also decreased the magnitude of inflammation, lung damage, and number of bacteria in the blood of mice subsequently infected with S. pneumoniae.ConclusionModulation of the inflammatory response by blocking CXCR1/2 improves disease outcome during respiratory influenza and pneumococcal infections, without compromising the ability of the murine host to deal with infection. Altogether, inhibition of CXCR1/2 may be a valid therapeutic strategy for treating lung infections caused by these pathogens, especially controlling secondary bacterial infection after influenza.

ABSTRACT Zika virus (ZIKV) infection is a global health emergency that causes significant neurodegeneration. Neurodegenerative processes may be exacerbated by N-methyl-d-aspartate receptor (NMDAR)-dependent neuronal excitoxicity. Here, we have exploited the hypothesis that ZIKV-induced neurodegeneration can be rescued by blocking NMDA overstimulation with memantine. Our results show that ZIKV actively replicates in primary neurons and that virus replication is directly associated with massive neuronal cell death. Interestingly, treatment with memantine or other NMDAR blockers, including dizocilpine (MK-801), agmatine sulfate, or ifenprodil, prevents neuronal death without interfering with the ability of ZIKV to replicate in these cells. Moreover, in vivo experiments demonstrate that therapeutic memantine treatment prevents the increase of intraocular pressure (IOP) induced by infection and massively reduces neurodegeneration and microgliosis in the brain of infected mice. Our results indicate that the blockade of NMDARs by memantine provides potent neuroprotective effects against ZIKV-induced neuronal damage, suggesting it could be a viable treatment for patients at risk for ZIKV infection-induced neurodegeneration. IMPORTANCE Zika virus (ZIKV) infection is a global health emergency associated with serious neurological complications, including microcephaly and Guillain-Barré syndrome. Infection of experimental animals with ZIKV causes significant neuronal damage and microgliosis. Treatment with drugs that block NMDARs prevented neuronal damage both in vitro and in vivo. These results suggest that overactivation of NMDARs contributes significantly to the neuronal damage induced by ZIKV infection, and this is amenable to inhibition by drug treatment.

Metabolic alterations are associated with arthritis apart from obesity. However, it is still unclear which is the underlying process behind these metabolic changes. Here, we investigate the role of tumor necrosis factor (TNF) in this process in an acute model of antigen-induced arthritis (AIA). Immunized male BALB/c mice received an intra-articular injection of PBS (control) or methylated bovine serum albumin (mBSA) into their knees, and were also pre-treated with different drugs: Etanercept, an anti-TNF drug, DF2156A, a CXCR1/2 receptor antagonist, or a monoclonal antibody RB6-8C5 to deplete neutrophils. Local challenge with mBSA evoked an acute neutrophil influx into the knee joint, and enhanced the joint nociception, along with a transient systemic metabolic alteration (higher levels of glucose and lipids, and altered adipocytokines). Pre-treatment with the conventional biological Etanercept, an inhibitor of TNF action, ameliorated the nociception and the acute joint inflammation dominated by neutrophils, and markedly improved many of the altered systemic metabolites (glucose and lipids), adipocytokines and PTX3. However, the lessening of metabolic changes was not due to diminished accumulation of neutrophils in the joint by Etanercept. Reduction of neutrophil recruitment by pre-treating AIA mice with DF2156A, or even the depletion of these cells by using RB6-8C5 reduced all of the inflammatory parameters and hypernociception developed after AIA challenge, but could not prevent the metabolic changes. Therefore, the induction of joint inflammation provoked acute metabolic alterations which were involved with TNF. We suggest that the role of TNF in arthritis-associated metabolic changes is not due to local neutrophils, which are the major cells present in this model, but rather due to cytokines.

Rheumatoid Arthritis (RA) is a chronic disease characterized by persistent inflammation and pain. Alternative therapies to reduce these symptoms are needed. Marine algae are valuable sources of diverse bioactive compounds. Lithothamnion muelleri (Hapalidiaceae) is a marine algae with anti-inflammatory, antitumor, and immunomodulatory properties. Here, we investigated the potential anti-inflammatory and analgesic activities of L. muelleri in a murine model of antigen-induced arthritis (AIA) in mice. Our results demonstrate that treatment with L. muelleri prevented inflammation and hypernociception in arthritic mice. Mechanistically, the crude extract and the polysaccharide-rich fractions of L. muelleri may act impairing the production of the chemokines CXCL1 and CXCL2, and consequently inhibit neutrophil influx to the knee joint by dampening the adhesion step of leukocyte recruitment in the knee microvessels. Altogether our results suggest that treatment with L.muelleri has a potential therapeutic application in arthritis treatment.