Macrophage-derived human resistin promotes perivascular adipose tissue dysfunction in experimental inflammatory arthritis.

Cardiovascular disease (CVD) is the leading cause of death in rheumatoid arthritis (RA). Resistin is an adipokine that induces adipose tissue inflammation and activation of monocytes/macrophages via adenylate cyclase-associated protein-1 (CAP1). Resistin levels are increased in RA and might cause perivascular adipose tissue (PVAT) dysfunction, leading to vascular damage and CVD. This study aimed to investigate the role of resistin in promoting PVAT dysfunction by increasing local macrophage and inflammatory cytokines content in antigen-induced arthritis (AIA). Resistin pharmacological effects were assessed by using C57Bl/6J wild-type (WT) mice, humanized resistin mice expressing human resistin in monocytes-macrophages (hRTN+/-/-), and resistin knockout mice (RTN-/-) with AIA and respective controls. We investigated AIA disease activity and functional, cellular, and molecular parameters of the PVAT. Resistin did not contribute to AIA disease activity and its concentrations were augmented in the PVAT and plasma of WT AIA and hRTN+/-/- AIA animals. In vitro exposure of murine arteries to resistin impaired vascular function by decreasing the anti-contractile effect of PVAT. WT AIA mice and hRTN+/-/- AIA mice exhibited PVAT dysfunction and knockdown of resistin prevented it. Macrophage-derived cytokines, markers of types 1 and 2 macrophages, and CAP1 expression were increased in the PVAT of resistin humanized mice with AIA, but not in knockout mice for resistin. This study reveals that macrophage-derived resistin promotes PVAT inflammation and dysfunction regardless of AIA disease activity. Resistin might represent a translational target to reduce RA-driven vascular dysfunction and CVD.

Carvacrol nanocapsules as a new antifungal strategy: Characterization and evaluation against fungi important for grape quality and to control the synthesis of ochratoxins.

Fungi are a problem for viticulture as they can lead to deterioration of grapes and mycotoxins production. Despite the widespread use of synthetic fungicides to control fungi, their impact on the agricultural ecosystem and human health demand safer and eco-friendly alternatives. This study aimed to produce, characterize and assess the antifungal activity of carvacrol loaded in nanocapsules of Eudragit® and chia mucilage as strategy for controlling Botrytis cinerea, Aspergillus flavus, Aspergillus carbonarius, and Aspergillus niger. Eudragit® and chia mucilage were suitable wall materials, as both favored the encapsulation of carvacrol into nanometric diameter particles. Fourier Transform Infrared Spectroscopy (FTIR) analysis suggested a successful incorporation of carvacrol into both nanocapsules, which was confirmed by presenting a good encapsulation efficiency and loading capacity. Thermogravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC) analyses revealed adequate thermal resistance. All fungi were sensible to carvacrol treatments and B. cinerea was the most sensitive compared to the Aspergillus species. Lower concentrations of encapsulated carvacrol than the unencapsulated form were required to inhibit fungi in the in vitro and grape assays. Additionally, lower levels of carvacrol (unencapsulated or encapsulated) were used to inhibit fungal growth and ochratoxin synthesis on undamaged grapes in comparison to those superficially damaged, highlighting the importance of management practices designed to preserve berry integrity during cultivation, storage or commercialization. When sublethal doses of carvacrol were used, the growth of A. niger and A. carbonarius was suppressed by at least 45 %, and ochratoxins were not found. The nanoencapsulation of carvacrol using Eudragit® and chia mucilage has proven to be an alternative to mitigate the problems with fungi and mycotoxins faced by the grape and wine sector.

Genomic analysis reveals genes that encode the synthesis of volatile compounds by a Bacillus velezensis-based biofungicide used in the treatment of grapes to control Aspergillus carbonarius.

Fungal control strategies based on the use of Bacillus have emerged in agriculture as eco-friendly alternatives to replace/reduce the use of synthetic pesticides. Bacillus sp. P1 was reported as a new promising strain for control of Aspergillus carbonarius, a known producer of ochratoxin A, categorized as possible human carcinogen with high nephrotoxic potential. Grape quality can be influenced by vineyard management practices, including the use of fungal control agents. The aim of this study was to evaluate, for the first time, the quality parameters of Chardonnay grapes exposed to an antifungal Bacillus-based strategy for control of A. carbonarius, supporting findings by genomic investigations. Furthermore, genomic tools were used to confirm that the strain P1 belongs to the non-pathogenic species Bacillus velezensis and also to certify its biosafety. The genome of B. velezensis P1 harbors genes that are putatively involved in the production of volatiles and hydrolytic enzymes, which are responsible for releasing the free form of aroma compounds. In addition to promote biocontrol of phytopathogenic fungi and ochratoxins, the treatment with B. velezensis P1 did not change the texture (hardness and firmness), color and pH of the grapes. Heat map and hierarchical clustering analysis (HCA) of volatiles evaluated by GC/MS revealed that Bacillus-treated grapes showed higher levels of compounds with a pleasant odor descriptions such as 3-hydroxy-2-butanone, 2,3-butanediol, 3-methyl-1-butanol, 3,4-dihydro-ß-ionone, ß-ionone, dihydroactinidiolide, linalool oxide, and ß-terpineol. The results of this study indicate that B. velezensis P1 presents desirable properties to be used as a biocontrol agent.

Spinal HMGB1 participates in the early stages of paclitaxel-induced neuropathic pain via microglial TLR4 and RAGE activation.

Introduction: Chemotherapy-induced neuropathic pain (CINP) is one of the main adverse effects of chemotherapy treatment. At the spinal level, CINP modulation involves glial cells that upregulate Toll-like receptor 4 (TLR4) and signaling pathways, which can be activated by pro-inflammatory mediators as the high mobility group box-1 (HMGB1). Objective: To evaluate the spinal role of HMGB1 in the paclitaxel-induced neuropathic pain via receptor for advanced glycation end products (RAGE) and TLR4 activation expressed in glial cells. Methods: Male C57BL/6 Wild type and TLR4 deficient mice were used in the paclitaxel-induced neuropathic pain model. The nociceptive threshold was measured using the von Frey filament test. In addition, recombinant HMGB1 was intrathecally (i.t.) injected to confirm its nociceptive potential. To evaluate the spinal participation of RAGE, TLR4, NF-kB, microglia, astrocytes, and MAPK p38 in HMGB1-mediated nociceptive effect during neuropathic pain and recombinant HMGB1-induced nociception, the drugs FPS-ZM1, LPS-RS, PDTC, minocycline, fluorocitrate, and SML0543 were respectively administrated by i.t. rout. Microglia, astrocytes, glial cells, RAGE, and TLR4 protein expression were analyzed by Western blot. ELISA immunoassay was also used to assess HMGB1, IL-1ß, and TNF-α spinal levels. Results: The pharmacological experiments demonstrated that spinal RAGE, TLR4, microglia, astrocytes, as well as MAPK p38 and NF-kB signaling are involved with HMGB1-induced nociception and paclitaxel-induced neuropathic pain. Furthermore, HMGB1 spinal levels were increased during the early stages of neuropathic pain and associated with RAGE, TLR4 and microglial activation. RAGE and TLR4 blockade decreased spinal levels of pro-inflammatory cytokines during neuropathic pain. Conclusion: Taken together, our findings indicate that HMGB1 may be released during the early stages of paclitaxel-induced neuropathic pain. This molecule activates RAGE and TLR4 receptors in spinal microglia, upregulating pro-inflammatory cytokines that may contribute to neuropathic pain.

Genome analysis of Pseudomonas strain 4B with broad antagonistic activity against toxigenic fungi.

Pseudomonas sp. 4B isolated from the effluent pond of a bovine abattoir was investigated as antifungal against toxigenic fungi. The complete genome of Pseudomonas 4B was sequenced using the Illumina MiSeq platform. Phylogenetic analysis and genome comparisons indicated that the strain belongs to the Pseudomonas aeruginosa group. In silico investigation revealed gene clusters associated with the biosynthesis of several antifungals, including pyocyanin, rhizomide, thanamycin, and pyochelin. This bacterium was investigated through antifungal assays, showing an inhibitory effect against all toxigenic fungi tested. Bacterial cells reduced the diameter of fungal colonies, colony growth rate, and sporulation of each indicator fungi in 10-day simultaneous growing tests. The co-incubation of bacterial suspension and fungal spores in yeast extract-sucrose broth for 48 h resulted in reduced spore germination. During simultaneous growth, decreased production of aflatoxin B1 and ochratoxin A by Aspergillus flavus and Aspergillus carbonarius, respectively, was observed. Genome analysis and in vitro studies showed the ability of P. aeruginosa 4B to reduce fungal growth parameters and mycotoxin levels, indicating the potential of this bacterium to control toxigenic fungi. The broad antifungal activity of this strain may represent a sustainable alternative for the exploration and subsequent use of its possible metabolites in order to control mycotoxin-producing fungi.

Measurement of Neutrophil Extracellular Traps as a Biomarker for the Differential Diagnosis Between Antineutrophil Cytoplasmic Antibody-Positive Individuals With Antineutrophil Cytoplasmic Antibody-Associated Vasculitis and Nonautoimmune Diseases.

BACKGROUND/OBJECTIVE: Neutrophil extracellular traps (NETs) have a correlation with disease activity in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). However, it is not known whether there is an association between NETs and the presence of ANCA in other diseases. This study aimed to assess the occurrence of NETs in individuals with ANCA and whether serum NET quantitation is capable of distinguishing them with regard to the diagnosis. METHODS: This was a cross-sectional, observational study. From the positive ANCA by indirect immunofluorescence, 94 individuals were divided into groups: AAV, infectious diseases, and neoplastic diseases. Healthy controls served for comparisons. Neutrophil extracellular traps were evaluated through the investigation of NET remnants, by detecting cell-free DNA bound to proteins such as histone, myeloperoxidase, and neutrophil elastase (NE). RESULTS: In patients with perinuclear ANCA (p-ANCA) the detection of NETs by NE was able to distinguish AAV from infection/neoplasia and healthy controls. Receiver operating characteristic curves for serum NETs by NE in patients with p-ANCA were drawn in 2 situations: AAV versus infection/neoplasia, showing a sensitivity of 0.65 and specificity of 0.84, with an area under the curve of 65%; and AAV versus controls, showing a sensitivity of 0.84 and a specificity of 0.88, with an area under the curve of 96%. CONCLUSIONS: For p-ANCA-positive individuals, we found higher serum NETs detected by NE-DNA in those with chronic infectious and neoplastic diseases than in AAV individuals and healthy controls. This allows us to infer that the evaluation of serum NETs may be of value as a biomarker for differential diagnosis.

Bio-functional and prebiotics properties of products based on whole grain sorghum fermented with lactic acid bacteria.

BACKGROUND: Products fermented with lactic acid bacteria based on whole grain flours of red or white sorghum (Sorghum bicolor (L.) Moench) added with malted sorghum flour, or with skim milk (SM) were developed. Composition, protein amino acid profile, total acidity, pH, prebiotic potential, and bio-functional properties after simulation of gastrointestinal digestion were evaluated. RESULTS: In all cases, a pH of 4.5 was obtained in approximately 4.5 h. The products added with SM presented higher acidity. Products made only with sorghum presented higher total dietary fiber, but lower protein content than products with added SM, the last ones having higher lysine content. All products exhibited prebiotic potential, white sorghum being a better ingredient to promote the growth of probiotic bacteria. The addition of malted sorghum or SM significantly increased the bio-functional properties of the products: the sorghum fermented products added with SM presented the highest antioxidant (ABTS•+ inhibition, 4.7 ± 0.2 mM Trolox), antihypertensive (Angiotensin converting enzyme-I inhibition, 57.3 ± 0.5%) and antidiabetogenic (dipeptidyl-peptidase IV inhibition, 31.3 ± 2.1%) activities, while the products added with malted sorghum presented the highest antioxidant (reducing power, 1.6 ± 0.1 mg ascorbic acid equivalent/mL) and antidiabetogenic (α-amylase inhibition, 38.1 ± 0.9%) activities. CONCLUSION: The fermented whole grain sorghum-based products could be commercially exploited by the food industry to expand the offer of the three high-growth markets: gluten-free products, plant-based products (products without SM), and functional foods. © 2023 Society of Chemical Industry.

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.

Enoxaparin improves COVID-19 by reducing Neutrophils Extracellular Traps (NETs) production.

BACKGROUND: COVID-19 causes consequences such as imbalance of the immune system and thrombotic events. During the infection process, NETs in excess induce a pro-inflammatory response and disseminated intravascular coagulation. We evaluated the role of enoxaparin as a potential inhibitor of NETs. METHODS: K18-hACE2 animals infected with the SARS-CoV-2 virus and a group of 23 individuals admitted to the hospital with COVID-19 treated with enoxaparin or without treatment and controls without the disease were included. RESULTS: Enoxaparin decreased the levels of NETs, reduced the signs of the disease and mitigated lung damage in the animals infected with SARS-CoV-2. These effects were partially associated with prevention of SARS-CoV-2 entry and NETs synthesis. Clinical data revealed that treatment with enoxaparin decreased the levels of inflammatory markers, the levels of NETs in isolated neutrophils and the organ dysfunction. CONCLUSION: This study provides evidence for the beneficial effects of enoxaparin in COVID-19 in addition to its anticoagulant role.

Tonsils are major sites of persistence of SARS-CoV-2 in children.

In the present study, we show that SARS-CoV-2 can infect palatine tonsils, adenoids, and secretions in children without symptoms of COVID-19, with no history of recent upper airway infection. We studied 48 children undergoing tonsillectomy due to snoring/OSA or recurrent tonsillitis between October 2020 and September 2021. Nasal cytobrushes, nasal washes, and tonsillar tissue fragments obtained at surgery were tested by RT-qPCR, immunohistochemistry (IHC), flow cytometry, and neutralization assay. We detected the presence of SARS-CoV-2 in at least one specimen tested in 27% of patients. IHC revealed the presence of the viral nucleoprotein in epithelial surface and in lymphoid cells in both extrafollicular and follicular regions, in adenoids and palatine tonsils. Also, IHC for the SARS-CoV-2 non-structural protein NSP-16 indicated the presence of viral replication in 53.8% of the SARS-CoV-2-infected tissues. Flow cytometry showed that CD20+ B lymphocytes were the most infected phenotypes, followed by CD4+ lymphocytes and CD123 dendritic cells, CD8+ T lymphocytes, and CD14+ macrophages. Additionally, IF indicated that infected tonsillar tissues had increased expression of ACE2 and TMPRSS2. NGS sequencing demonstrated the presence of different SARS-CoV-2 variants in tonsils from different tissues. SARS-CoV-2 antigen detection was not restricted to tonsils but was also detected in nasal cells from the olfactory region. Palatine tonsils and adenoids are sites of prolonged RNA presence by SARS-CoV-2 in children, even without COVID-19 symptoms. IMPORTANCE This study shows that SRS-CoV-2 of different lineages can infect tonsils and adenoids in one quarter of children undergoing tonsillectomy. These findings bring advancement to the area of SARS-CoV-2 pathogenesis, by showing that tonsils may be sites of prolonged infection, even without evidence of recent COVID-19 symptoms. SARS-CoV-2 infection of B and T lymphocytes, macrophages, and dendritic cells may interfere with the mounting of immune responses in these secondary lymphoid organs. Moreover, the shedding of SARS-CoV-2 RNA in respiratory secretions from silently infected children raises concern about possible diagnostic confusion in the presence of symptoms of acute respiratory infections caused by other etiologies.

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.

The TIGIT+ T regulatory cells subset associates with nosocomial infection and fatal outcome in COVID-19 patients under mechanical ventilation.

The TIGIT+FOXP3+Treg subset (TIGIT+Tregs) exerts robust suppressive activity on cellular immunity and predisposes septic individuals to opportunistic infection. We hypothesized that TIGIT+Tregs could play an important role in intensifying the COVID-19 severity and hampering the defense against nosocomial infections during hospitalization. Herein we aimed to verify the association between the levels of the TIGIT+Tregs with the mechanical ventilation requirement, fatal outcome, and bacteremia during hospitalization. TIGIT+Tregs were immunophenotyped by flow cytometry from the peripheral blood of 72 unvaccinated hospitalized COVID-19 patients at admission from May 29th to August 6th, 2020. The patients were stratified during hospitalization according to their mechanical ventilation requirement and fatal outcome. COVID-19 resulted in a high prevalence of the TIGIT+Tregs at admission, which progressively increased in patients with mechanical ventilation needs and fatal outcomes. The prevalence of TIGIT+Tregs positively correlated with poor pulmonary function and higher plasma levels of LDH, HMGB1, FGL2, and TNF. The non-survivors presented higher plasma levels of IL-33, HMGB1, FGL2, IL-10, IL-6, and 5.54 times more bacteremia than survivors. Conclusions: The expansion of the TIGIT+Tregs in COVID-19 patients was associated with inflammation, lung dysfunction, bacteremia, and fatal outcome.

NET-targeted therapy: effects, limitations, and potential strategies to enhance treatment efficacy.

Neutrophil extracellular traps (NETs) are complex structures released by activated neutrophils during inflammatory responses. Due to their unique potential for causing tissue damage and modulating immune responses, there is increasing interest in studying these structures as potential targets for the treatment of infectious diseases, autoimmune diseases, and cancer. However, therapeutic targeting of NETs might trigger deleterious effects that may limit treatment efficacy. NET disruption may increase the microbial load in infection; in autoimmunity, NET targeting might impair peripheral tolerance, but it might reduce adaptive immune responses in cancer. In this review, we explore the therapeutic and deleterious effects of NET-targeted therapy while shedding light on novel strategies to overcome treatment-related limitations and enhance treatment efficacy.

The metabolic function of pyruvate kinase M2 regulates reactive oxygen species production and microbial killing by neutrophils.

Neutrophils rely predominantly on glycolytic metabolism for their biological functions, including reactive oxygen species (ROS) production. Although pyruvate kinase M2 (PKM2) is a glycolytic enzyme known to be involved in metabolic reprogramming and gene transcription in many immune cell types, its role in neutrophils remains poorly understood. Here, we report that PKM2 regulates ROS production and microbial killing by neutrophils. Zymosan-activated neutrophils showed increased cytoplasmic expression of PKM2. Pharmacological inhibition or genetic deficiency of PKM2 in neutrophils reduced ROS production and Staphylococcus aureus killing in vitro. In addition, this also resulted in phosphoenolpyruvate (PEP) accumulation and decreased dihydroxyacetone phosphate (DHAP) production, which is required for de novo synthesis of diacylglycerol (DAG) from glycolysis. In vivo, PKM2 deficiency in myeloid cells impaired the control of infection with Staphylococcus aureus. Our results fill the gap in the current knowledge of the importance of lower glycolysis for ROS production in neutrophils, highlighting the role of PKM2 in regulating the DHAP and DAG synthesis to promote ROS production in neutrophils.

SARS-CoV-2 uses CD4 to infect T helper lymphocytes.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the agent of a major global outbreak of respiratory tract disease known as Coronavirus Disease 2019 (COVID-19). SARS-CoV-2 infects mainly lungs and may cause several immune-related complications, such as lymphocytopenia and cytokine storm, which are associated with the severity of the disease and predict mortality. The mechanism by which SARS-CoV-2 infection may result in immune system dysfunction is still not fully understood. Here, we show that SARS-CoV-2 infects human CD4+ T helper cells, but not CD8+ T cells, and is present in blood and bronchoalveolar lavage T helper cells of severe COVID-19 patients. We demonstrated that SARS-CoV-2 spike glycoprotein (S) directly binds to the CD4 molecule, which in turn mediates the entry of SARS- CoV-2 in T helper cells. This leads to impaired CD4 T cell function and may cause cell death. SARS-CoV-2-infected T helper cells express higher levels of IL-10, which is associated with viral persistence and disease severity. Thus, CD4-mediated SARS-CoV-2 infection of T helper cells may contribute to a poor immune response in COVID-19 patients.

A novel insight on SARS-CoV-2 S-derived fragments in the control of the host immunity.

Despite all efforts to combat the pandemic of COVID-19, we are still living with high numbers of infected persons, an overburdened health care system, and the lack of an effective and definitive treatment. Understanding the pathophysiology of the disease is crucial for the development of new technologies and therapies for the best clinical management of patients. Since the manipulation of the whole virus requires a structure with an adequate level of biosafety, the development of alternative technologies, such as the synthesis of peptides from viral proteins, is a possible solution to circumvent this problem. In addition, the use and validation of animal models is of extreme importance to screen new drugs and to compress the organism's response to the disease. Peptides derived from recombinant S protein from SARS-CoV-2 were synthesized and validated by in silico, in vitro and in vivo methodologies. Macrophages and neutrophils were challenged with the peptides and the production of inflammatory mediators and activation profile were evaluated. These peptides were also inoculated into the swim bladder of transgenic zebrafish larvae at 6 days post fertilization (dpf) to mimic the inflammatory process triggered by the virus, which was evaluated by confocal microscopy. In addition, toxicity and oxidative stress assays were also developed. In silico and molecular dynamics assays revealed that the peptides bind to the ACE2 receptor stably and interact with receptors and adhesion molecules, such as MHC and TCR, from humans and zebrafish. Macrophages stimulated with one of the peptides showed increased production of NO, TNF-α and CXCL2. Inoculation of the peptides in zebrafish larvae triggered an inflammatory process marked by macrophage recruitment and increased mortality, as well as histopathological changes, similarly to what is observed in individuals with COVID-19. The use of peptides is a valuable alternative for the study of host immune response in the context of COVID-19. The use of zebrafish as an animal model also proved to be appropriate and effective in evaluating the inflammatory process, comparable to humans.

COVID-19-related hyperglycemia is associated with infection of hepatocytes and stimulation of gluconeogenesis.

Occurrence of hyperglycemia upon infection is associated with worse clinical outcome in COVID-19 patients. However, it is still unknown whether SARS-CoV-2 directly triggers hyperglycemia. Herein, we interrogated whether and how SARS-CoV-2 causes hyperglycemia by infecting hepatocytes and increasing glucose production. We performed a retrospective cohort study including patients that were admitted at a hospital with suspicion of COVID-19. Clinical and laboratory data were collected from the chart records and daily blood glucose values were analyzed to test the hypothesis on whether COVID-19 was independently associated with hyperglycemia. Blood glucose was collected from a subgroup of nondiabetic patients to assess pancreatic hormones. Postmortem liver biopsies were collected to assess the presence of SARS-CoV-2 and its transporters in hepatocytes. In human hepatocytes, we studied the mechanistic bases of SARS-CoV-2 entrance and its gluconeogenic effect. SARS-CoV-2 infection was independently associated with hyperglycemia, regardless of diabetic history and beta cell function. We detected replicating viruses in human hepatocytes from postmortem liver biopsies and in primary hepatocytes. We found that SARS-CoV-2 variants infected human hepatocytes in vitro with different susceptibility. SARS-CoV-2 infection in hepatocytes yields the release of new infectious viral particles, though not causing cell damage. We showed that infected hepatocytes increase glucose production and this is associated with induction of PEPCK activity. Furthermore, our results demonstrate that SARS-CoV-2 entry in hepatocytes occurs partially through ACE2- and GRP78-dependent mechanisms. SARS-CoV-2 infects and replicates in hepatocytes and exerts a PEPCK-dependent gluconeogenic effect in these cells that potentially is a key cause of hyperglycemia in infected patients.

C5aR1 signaling triggers lung immunopathology in COVID-19 through neutrophil extracellular traps.

Patients with severe COVID-19 develop acute respiratory distress syndrome (ARDS) that may progress to cytokine storm syndrome, organ dysfunction, and death. Considering that complement component 5a (C5a), through its cellular receptor C5aR1, has potent proinflammatory actions and plays immunopathological roles in inflammatory diseases, we investigated whether the C5a/C5aR1 pathway could be involved in COVID-19 pathophysiology. C5a/C5aR1 signaling increased locally in the lung, especially in neutrophils of critically ill patients with COVID-19 compared with patients with influenza infection, as well as in the lung tissue of K18-hACE2 Tg mice (Tg mice) infected with SARS-CoV-2. Genetic and pharmacological inhibition of C5aR1 signaling ameliorated lung immunopathology in Tg-infected mice. Mechanistically, we found that C5aR1 signaling drives neutrophil extracellular traps-dependent (NETs-dependent) immunopathology. These data confirm the immunopathological role of C5a/C5aR1 signaling in COVID-19 and indicate that antagonists of C5aR1 could be useful for COVID-19 treatment.