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Highlights•The effect of NSAIDs on equine TNF-α during endotoxemia remains poorly understood.•Effects of flunixin meglumine (FM) and meloxicam on TNF-α were compared in vitro.•FM and meloxicam did not inhibit TNF-α production in LPS-stimulated PBMCs.•FM significantly inhibited TNF-α levels in LPS-stimulated neutrophils.•FM may play a key role in early endotoxemia when neutrophils are mobilized. Systemic inflammatory response syndrome (SIRS) induced by endotoxemia is usually secondary to colitis and is a major cause of high morbidity and mortality in horses. Non-steroidal anti-inflammatory drugs (NSAIDs), such as flunixin meglumine (FM) and meloxicam (MX), are used to improve clinical outcomes in SIRS/endotoxemia. These NSAIDs suppress tumor necrosis factor-alpha (TNF-α) levels; however, the underlying mechanisms remain unclear. The aim of this study was to investigate the inhibitory effects of FM and MX on TNF-α in lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMCs) and neutrophils in vitro. Blood samples were collected from three healthy thoroughbred horses, and PBMCs and neutrophils were isolated using density gradient centrifugation. Cells were cultured with LPS (0.3 ng/mL) and FM or MX (5, 10, or 50 μM). TNF-α gene expression was analyzed using quantitative real-time PCR, and protein levels were measured using ELISA. No significant inhibitory effects of FM or MX on TNF-α gene or protein expression were observed in LPS-stimulated PBMCs. However, FM significantly inhibited the increase in TNF-α protein levels in LPS-stimulated neutrophils in a concentration-dependent manner (p < 0.05). MX showed a similar tendency in LPS-stimulated neutrophils, but the differences were not significant. The regulation of neutrophil-derived TNF-α by FM administration could be a promising therapeutic strategy for equine SIRS/endotoxemia, providing mechanistic insight for optimizing anti-inflammatory therapy.

The clinical outcome of SARS-CoV-2 infection spans from asymptomatic viral elimination to lethal COVID-19 pneumonia, which is due to type I interferon (IFN) deficiency in at least 15–20% of cases. We report two unrelated male patients with critical COVID-19 who are heterozygous for rare deleterious variants in RB1CC1, encoding the autophagy-related FIP200 protein. Airway epithelial cells genetically deprived of FIP200 or cell lines expressing the RB1CC1/FIP200 patient variants exhibit elevated SARS-CoV-2 replication and impaired autophagic flux. The antiviral function of FIP200 is independent of canonical autophagy and type I IFN, but involves the selective autophagy receptor NDP52. We identify a non-canonical function of FIP200 in a novel lysosomal degradation pathway, in which SARS-CoV-2 virions are targeted to single-membrane compartments for degradation of viral RNA in LC3B-positive acidified vesicles. This pathway is impaired in FIP200-deficient cells and in cells expressing FIP200 patient haplotypes. Collectively, we describe a cell-autonomous anti-SARS-CoV-2 restriction pathway, dependent on FIP200 and NDP52, and independent of canonical autophagy and type I IFN, which can underlie critical COVID-19 pneumonia. The variability in clinical outcomes of SARS-CoV-2 infection is partly due to deficiencies in production or response to type I interferons (IFN). Here, the authors describe a FIP200-dependent lysosomal degradation pathway, independent of canonical autophagy and type I IFN, that restricts SARS-CoV-2 replication, offering insights into critical COVID-19 pneumonia mechanisms.

Highlights•Convalescent plasma is an alternative COVID-19 therapy when no other is available.•We evaluated the long-term immune response in treated and untreated individuals.•A higher seroconversion rate early post infusion is observed compared to untreated.•However, long-term humoral and cellular immune responses are similar between groups. During the SARS-CoV-2 pandemic, the use of convalescent plasma (CP) in high-risk patients was proposed and widely implemented in several countries as a potential COVID-19 therapy. Nonetheless, CP therapy’s impact on immune response is nowadays poorly understood, including the correlation between IgG levels, neutralization capacity, and cellular immune response against SARS-CoV-2. Here we evaluated, in a cohort of patients with COVID-19 requiring hospitalization and having received or not CP, as well as in CP donors (recovered from mild disease), the humoral and cellular immune response assessed by titers of SARS-CoV-2 IgG, neutralizing antibodies, and IFN-γ+/IL-2+ ELISpot during the first month (early) and up to nine months (long-term) after symptom onset. Results showed higher seropositivity and seroconversion rates between 7–12 days after plasma infusion in CP-recipients. However, similar IgG and neutralizing immune response kinetics between CP-recipients and non-recipients was observed during the first and until the ninth month of analysis. A positive correlation between IgG and neutralizing levels was detected. Compared to outpatient donors, hospitalized individuals showed a higher response at 3 and 6 months after symptoms onset. A sustained SARS-CoV-2-specific CD4+ and CD8+ T cell response was observed in outpatients and hospitalized patients, regardless of the CP treatment. We concluded that the CP infusion did not affect the long-term SARS-CoV-2 specific humoral and cellular immune responses. Nonetheless, CP may provide a therapeutic window by promoting a higher humoral response during the acute phase of COVID-19. Graphical abstractImage, graphical abstract