key: cord-0700799-tqhwzf9n authors: Teixeira, Paula C.; Dorneles, Gilson P.; Santana Filho, Paulo C.; da Silva, Igor M.; Schipper, Lucas L.; Al Postiga, Isabelle; Andretta Moreira Neves, Carla; Carlos Rodrigues Júnior, Luiz; Peres, Alessandra; Trindade de Souto, Janeusa; Gonçalves da Fonseca, Simone; Eller, Sarah; Oliveira, Tiago F.; Rotta, Liane N.; Elizabeth Thompson, Claudia; Romão, Pedro R.T. title: Increased LPS levels coexist with systemic inflammation and result in monocyte activation in severe COVID-19 patients date: 2021-09-09 journal: Int Immunopharmacol DOI: 10.1016/j.intimp.2021.108125 sha: 8cf4aa544986e84c8285eca1d916a12b9e320728 doc_id: 700799 cord_uid: tqhwzf9n Mucosal barrier alterations may play a role in the pathogenesis of several diseases, including COVID-19. In this study we evaluate the association between bacterial translocation markers and systemic inflammation at the earliest time-point after hospitalization and at the last 72 h of hospitalization in survivors and non-survivors COVID-19 patients. Sixty-six SARS-CoV-2 RT-PCR positive patients and nine non-COVID-19 pneumonia controls were admitted in this study. Blood samples were collected at hospital admission (T1) (Controls and COVID-19 patients) and 0-72 h before hospital discharge (T2, alive or dead) to analyze systemic cytokines and chemokines, lipopolysaccharide (LPS) concentrations and soluble CD14 (sCD14) levels. THP-1 human monocytic cell line was incubated with plasma from survivors and non-survivors COVID-19 patients and their phenotype, activation status, TLR4, and chemokine receptors were analyzed by flow cytometry. COVID-19 patients presented higher IL-6, IFN-γ, TNF-α, TGF-β1, CCL2/MCP-1, CCL4/MIP-1β, and CCL5/RANTES levels than controls. Moreover, LPS and sCD14 were higher at hospital admission in SARS-CoV-2-infected patients. Non-survivors COVID-19 patients had increased LPS levels concomitant with higher IL-6, TNF-α, CCL2/MCP-1, and CCL5/RANTES levels at T2. Increased expression of CD16 and CCR5 were identified in THP-1 cells incubated with the plasma of survivor patients obtained at T2. The incubation of THP-1 with T2 plasma of non-survivors COVID-19 leads to higher TLR4, CCR2, CCR5, CCR7, and CD69 expression. In conclusion, the coexistence of increased microbial translocation and hyperinflammation in patients with severe COVID-19 may lead to higher monocyte activation, which may be associated with worsening outcomes, such as death. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes Coronavirus disease 2019 (COVID-19), a disease with diverse clinical manifestations (1) . While most COVID-19 cases are asymptomatic or mild, the severe form of COVID-19 can occur with detrimental manifestations such as acute respiratory distress syndrome (ARDS), multi-organ failure, and death (2) . The COVID-19 immunopathology is mainly characterized by a hyperinflammatory state, strong innate cells response, and lymphocyte activation with an exhausted profile (3, 4) The mechanism underlying the inflammatory state of COVID-19 is not fully understood at this time. Although the respiratory tract is the main site of infection for COVID-19, the pathogenesis of the disease can also involve different organs and systems, such as the 4 heart, kidneys, intestines, vasculature, and liver (3) . In the gastrointestinal (GI) tract, symptoms such as diarrhea and abdominal distention have been reported in several patients (5) and enterocytes in the ileum and colon express the angiotensin-converting enzyme 2 (ACE-2) receptor and may act as a site for SARS-CoV-2 entrance and predispose to enteric infection (6) . Furthermore, the transmembrane serine protease 2 (TMPRSS2), also highly expressed in enterocytes, is able to prime the spike protein of SARS-CoV-2 and mediate the virus entry into cells (7) . Additionally, RNA viral particles were detected in stool samples, arising the possibility that bacterial translocation and microbial products from the GI tract to the peripheral blood might contribute to the hyperinflammatory state and COVID-19 severity (8) . The gut and lungs are anatomically distinct but the proximal communication and complex pathways involving their respective microbiota indicate the existence of a gut-lung axis. In this sense, airways infections by viruses and bacteria are accompanied by alterations in the composition of intestinal microbiota, increased GI symptoms, gut dysfunction and barrier disruption (8) . Ahlawat and Sharma (9) and others (10) (11) (12) proposed a gut-lung axis during the SARS-CoV-2 infection through the hyperactive host immune system releasing higher levels of inflammatory cytokines with consequent lung hyper-permeability, gut infection, and disruption of the intestinal permeability that leads to the leakage of gut microbes and associated metabolites into circulation. Interestingly, disruption in gut barriers and microbial translocation is more likely to occur in older individuals and those with chronic diseases, which contribute to the exacerbation of systemic inflammation by activating innate immune cells, mainly monocytes and neutrophils (10) . Lipopolysaccharide (LPS) is a product derived from the membrane of gram-negative bacteria that acts as a potent immune-activating stimulus when recognized by innate immune cells expressing toll-like receptor 4 (TLR4) (13) . Then, innate immune cells 5 become activated and produce high amounts of proinflammatory mediators including cytokines, such as interleukin (IL)-6 and tumor necrosis factor-alpha (TNF-) and chemokines (i.e., CCL2/MCP-1 and CCL5/RANTES) (14) . In this sense, peripheral blood LPS is a well-recognized marker of translocation of bacterial products after events that compromise the integrity of the intestinal mucosa (15, 16) . Moreover, soluble CD14 (sCD14) is released after cleavage from the membrane form (mCD14) on the surface of monocytes through exposure to the LPS stimulation and is considered a marker of microbial translocation (16, 17) . There is emerging evidence indicates that severe SARS-CoV-2 infection induces disruption in the gut barrier contributing to the systemic spread of bacteria and microbial products which affect the host's response to the infection (11, 18, 19) . Mechanistically, it was proposed that SARS-CoV-2 infects enterocytes and causes microbial translocation through transcellular permeability and paracellular pathway using the tight junctions. Additionally, viral infection and replication culminate in the activation of innate and adaptive immunity that induces intestinal damage and increase the intestinal permeability precipitating intestinal barrier failure and bacterial translocation, which in turn auto-fuel vicious cycles of systemic inflammation and tissue damage (18, 19) . However, the time course of the presence of microbial product in the peripheral blood of COVID-19 patients during hospitalization remains unknown. Here, we investigated the association between microbial translocation markers and systemic inflammation in the hospital admission and at the discharge time from the hospital in survivors and non-survivors COVID-19 patients. We hypothesized that microbial translocation markers are associated with systemic inflammation and could contribute to the death of hospitalized COVID-19 patients. 6 Hospitalized COVID-19 patients with confirmed SARS-CoV-2 RT-PCR positive test on nasopharyngeal specimens (n= 66) were consecutively recruited after admission in the COVID-19 Unit of Hospital São Camilo (Esteio/RS, Brazil) between June/2020 and December/2020 to a clinical cohort study. The study was approved by the UFCSPA Ethics Committee (CAAE: 38886220.0.0000). Informed consent was obtained from those legally responsible for the patients after the research objectives and procedures were explained. The authors signed an agreement to preserve patient and staff anonymity related to the use of this data. All patients were diagnosed with COVID-19 infection according to the World Health Organization interim guidance and Diagnosis and Treatment Guideline for Novel Coronavirus Pneumonia and were positive for at least two nucleic acid tests for SARS-CoV-2 (20). Clinical and socio-demographic data were collected from the patient's electronic medical records upon admission to the unit. Blood samples from 9 age-and sex-matched SARS-CoV-2 RT-PCR negative controls admitted to hospital with pneumonia were also obtained. 10.000 events were acquired using CELLQuest Pro Software (BD Bioscience, USA) on 9 a FACSCalibur flow cytometer (BD Bioscience, USA) to determine cell phenotype. THP-1 were identified and gated according to each forward scatter (FSC) and side scatter (SSC) profile, and the mean fluorescence intensity (MFI) was evaluated. Normality of data was checked by Kolmogorov-Smirnov, with values presented as mean ± 95%CI for demographic and clinical characteristics and mean ± standard deviation (SD) for inflammatory mediators. Participant's characteristics and inflammatory analysis were between-groups compared through a one-way ANOVA Firstly, we analyzed the baseline characteristics and inflammatory mediators in controls and COVID-19 hospitalized patients. To this, 66 patients admitted at COVID-19 care unit due to COVID-19 complications were enrolled in this study. Additionally, 9 COVID-19 negative controls were analyzed. COVID-19 patients presented higher body mass and body mass index than controls (p<0.01), and 53.8% of patients were classified as obese. The mean length of stay in the hospital were 21.1 (15.6 -26.6) days for COVID-19 patients, 84.8% of them were admitted to the intensive care unit (ICU) due to COVID-19 10 complications, and 34.8% patients died due to complications of SARS-CoV-2 infection. Forty-three (43) patients COVID-19 patients presented a hyperinflammatory profile at hospital admission compared to controls as presented in Table 2 However, only non-survivors had increased IFN-γ (p<0.01) and TNF-α (p<0.05) compared to controls, and higher TNF-α levels (p<0.05) than survivors. Conversely, survivors had higher levels of CCL5/RANTES (p<0.01) and CCL4/MIP-1β (p<0.05) compared to control and non-survivors groups, respectively. Furthermore, reduced TGF-β1 levels were found in both survivors and non-survivors COVID-19 patients compared to controls (p<0.01). Finally, a strong monocytopenia was observed in COVID-19 nonsurvivors compared to COVID-19 survivors (p=0.01). These data indicate that COVID-19 patients have higher levels of inflammatory cytokines/chemokines at the hospital admission and higher TNF-α plasma levels that may be associated with death. activation that may be associated with worse outcomes in severe COVID-19, such as death ( Figure 6 ). Please, Insert the Figure 6 Angiotensin-Converting Enzyme 2 (ACE-2), which is the most recognized receptor that binds to the Spike protein of SARS-CoV-2 and allows the host infection, is widely expressed in epithelial cells along with the gastrointestinal (GI) tract (10, 23) . Thus, barriers, increasing the translocation of microbial products (11, 23) . Increased endotoxemia has been previously described in several pathological conditions associated with hospitalization, becoming a recognized marker of critical ill suffering from sepsis (24) (25) (26) . Furthermore, recent studies described disrupted gut barrier integrity and increased circulating bacteriome in moderate and severe COVID-19 patients (11, 18, 19) . (19) . Corroborating these data, other studies show an important role for endotoxin and sCD14 in heart failure (27, 28) . Microbial translocation to the peripheral blood also contributes to an early activation of monocytic cells. Indeed, we observed higher sCD14 in survivors and non-survivors COVID-19 patients. In line with this, Bowman and coworkers (29) found increased sCD14 levels in COVID-19 patients independent of the severity of disease. Furthermore, the authors revealed that COVID-19 critical ill patients who recovered or deceased had the highest sCD14 and lipopolysaccharide-binding protein (LBP, another marker of endotoxemia) values compared to the other groups (29) . These data highlighted the presence of strong activation of the monocytic lineage on hospital admission due to the SARS-CoV-2 infection in addition to the LPS translocation and inflammatory exacerbation in ICU patients (30) . In accordance with this study, systemic markers of monocyte activation, mainly sCD14 and sCD163, correlated with inflammatory cytokines in a non-intensive care unit COVID-19 cohort patients (31) . Giron and coworkers (32) found strong correlations between markers of tight junction permeability and microbial translocation with systemic inflammatory molecules, which corroborates our data. It was previously postulated that perturbations in gut microbiota and microbial translocation may exacerbate the severity of hyperinflammation in COVID-19 (33) . Moreover, sCD14 tended to be higher at T2 in non-survivors COVID-19 patients. Interestingly Severe COVID-19 patients presented an inflammatory phenotype of monocytes associated with early activation and downregulation of HLA-DR (42, 44) . Monocytes upregulate the CD69 expression, which promotes tissue infiltration and retention (45, 46) . In conclusion, severe COVID-19 is related to increased microbial translocation during hospitalization coexisting with the inflammatory condition of SARS-CoV-2 infection and could lead to higher monocyte activation and worsening clinical outcomes, such as death. The authors declare no conflict of interest. This project was funded, in part, with support from the Fundação de Amparo à Pesquisa SARS-CoV-2 immunity: review and applications to phase 3 vaccine candidates Clinical and immunologic features in severe and moderate forms of Coronavirus Disease Immunology of COVID-19: Current State of the Science Longitudinal immunological analyses reveal inflammatory misfiring in severe COVID-19 patients Don't Overlook Digestive Symptoms in Patients With 2019 Novel Coronavirus Disease (COVID-19) The digestive system is a potential route of 2019-nCov infection: A bioinformatics analysis based on single-cell transcriptomes Intestinal Barrier Function in Health and Disease-Any role of SARS-CoV-2? Microorganisms Prolonged SARS-CoV-2 RNA detection in anal/rectal swabs and stool specimens in COVID-19 patients after negative conversion in nasopharyngeal RT-PCR test Immunological co-ordination between gut and lungs in SARS-CoV-2 infection | Elsevier Enhanced Reader Alterations in Gut Microbiota of Patients With COVID-19 During Time of Hospitalization Endotoxemia and circulating bacteriome in severe COVID-19 patients [Internet]. medRxiv. medRxiv; 2020 Enteric involvement of severe acute respiratory syndrome-associated coronavirus infection Recognition of lipopolysaccharide pattern by TLR4 complexes Experimental and Molecular Medicine Bacterial lipopolysaccharide induces HIF-1 activation in human monocytes via p44/42 MAPK and NF-κB Lipopolysaccharide-induced bacterial translocation is intestine site-specific and associates with intestinal mucosal inflammation Distinct inflammatory profiles in HIV-infected individuals under antiretroviral therapy using cannabis, cocaine or cannabis plus cocaine Tlaskalová-Hogenová H. CD14 is expressed and released as soluble CD14 by human intestinal epithelial cells in vitro: Lipopolysaccharide activation of epithelial cells revisited Severe COVID-19 Is Fueled by Disrupted Gut Barrier Integrity [Internet]. medRxiv. medRxiv; 2020 Elevated markers of gut leakage and inflammasome activation in COVID-19 patients with cardiac involvement Quantitative lipopolysaccharide analysis using HPLC/MS/MS and its combination with the limulus amebocyte lysate assay Systems biological assessment of immunity to mild versus severe COVID-19 infection in humans. Science (80-) BRIEF COMMUNICATIONS Evidence for Gastrointestinal Infection of SARS-CoV-2 Diagnostic and prognostic implications of endotoxemia in critical illness: Results of the MEDIC study Endotoxemia and Sepsis: Important Roles for Lipopolysaccharide Binding Protein and CD14 in Endotoxin Signal Transduction The level of endotoxemia in sepsis varies in relation to the underlying infection: Impact on final outcome Invasive assessment of bacterial endotoxin and inflammatory cytokines in patients with acute heart failure Endotoxin and immune activation in chronic heart failure: A prospective cohort study Levels of Soluble CD14 and Tumor Necrosis Factor Receptors 1 and 2 May Be Predictive of Death in Severe Coronavirus Disease Increased sCD163 and sCD14 Plasmatic Levels and Depletion of Peripheral Blood Pro-Inflammatory Monocytes, Myeloid and Plasmacytoid Dendritic Cells in Patients With Severe COVID-19 Pneumonia. Front Immunol Increased Serum Levels of sCD14 and sCD163 Indicate a Preponderant Role for Monocytes Front Immunol Plasma Markers of Disrupted Gut Permeability in Severe COVID-19 Patients. Front Immunol Could Perturbation of Gut Microbiota Possibly Exacerbate the Severity of COVID-19 via Cytokine Storm? Front Immunol An inflammatory cytokine signature predicts COVID-19 severity and survival The cytokine storm in COVID-19: An overview of the involvement of the chemokine/chemokinereceptor system Chemokines and chemokine receptors during COVID-19 infection Characterization of the inflammatory response to severe COVID-19 Is Gut Microbiota Dysbiosis a Predictor of Increased Susceptibility to Poor Outcome of COVID-19 Patients? An Update Plasma Markers of Disrupted Gut Permeability in Severe COVID-19 Patients 2 COVID-19 patients upregulate toll-like receptor 4-mediated inflammatory signaling that mimics bacterial sepsis Monocyte activation in systemic Covid-19 infection: Assay and rationale A Longitudinal Study of Immune Cells in Severe COVID-19 Patients. Front Immunol Early Phases of COVID-19 Are Characterized by a Reduction in Lymphocyte Populations and the Presence of Atypical Monocytes. Front Immunol COVID-19: immunopathogenesis and Immunotherapeutics CD69: from activation marker to metabolic 28 South African Mixed Ancestry individuals. Sci Rep Severe COVID-19 Is Marked by a Dysregulated Myeloid Cell Compartment Transcriptomic characteristics of bronchoalveolar lavage fluid and peripheral blood mononuclear cells in COVID-19 patients Glycosaminoglycan binding and oligomerization are essential for the in vivo activity of certain chemokines The Role of the CCL2/CCR2 Axis in Mouse Mast Cell Migration In Vitro and In Vivo Recombinant human MCP-1/JE induces chemotaxis, calcium flux, and the respiratory burst in human monocytes CCL5-CCR5 interaction provides antiapoptotic signals for macrophage survival during viral infection Disruption of the CCL5/RANTES-CCR5 pathway restores immune homeostasis and reduces plasma viral load in critical COVID-19 Low CCR7-mediated migration of human monocyte derived dendritic cells in response to human respiratory syncytial virus and human metapneumovirus Signaling Pathways, and Adjuvant Functions in Viral Infection and Prevention