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1.
J Immunol ; 208(4): 979-990, 2022 02 15.
Article in English | MEDLINE | ID: covidwho-1631932

ABSTRACT

Calprotectin is released by activated neutrophils along with myeloperoxidase (MPO) and proteases. It plays numerous roles in inflammation and infection, and is used as an inflammatory biomarker. However, calprotectin is readily oxidized by MPO-derived hypohalous acids to form covalent dimers of its S100A8 and S100A9 subunits. The dimers are susceptible to degradation by proteases. We show that detection of human calprotectin by ELISA declines markedly because of its oxidation by hypochlorous acid and subsequent degradation. Also, proteolysis liberates specific peptides from oxidized calprotectin that is present at inflammatory sites. We identified six calprotectin-derived peptides by mass spectrometry and detected them in the bronchoalveolar lavage fluid of children with cystic fibrosis (CF). We assessed the peptides as biomarkers of neutrophilic inflammation and infection. The content of the calprotectin peptide ILVI was related to calprotectin (r = 0.72, p = 0.01, n = 10). Four of the peptides were correlated with the concentration of MPO (r > 0.7, p ≤ 0.01, n = 21), while three were higher (p < 0.05) in neutrophil elastase-positive (n = 14) than -negative samples (n = 7). Also, five of the peptides were higher (p < 0.05) in the bronchoalveolar lavage fluid from children with CF with infections (n = 21) than from non-CF children without infections (n = 6). The specific peptides liberated from calprotectin will signal uncontrolled activity of proteases and MPO during inflammation. They may prove useful in tracking inflammation in respiratory diseases dominated by neutrophils, including coronavirus disease 2019.


Subject(s)
Bronchoalveolar Lavage Fluid/immunology , Cystic Fibrosis/immunology , Inflammation/immunology , Leukocyte L1 Antigen Complex/metabolism , Neutrophils/immunology , Peptides/metabolism , Respiratory System/metabolism , Child , Child, Preschool , Cystic Fibrosis/diagnosis , Female , Humans , Inflammation/diagnosis , Leukocyte L1 Antigen Complex/genetics , Leukocyte L1 Antigen Complex/immunology , Male , Neutrophil Activation , Oxidation-Reduction , Peptides/genetics , Peptides/immunology , Proteolysis
2.
Viruses ; 13(11)2021 11 03.
Article in English | MEDLINE | ID: covidwho-1542790

ABSTRACT

The detailed characterization of human γδ T lymphocyte differentiation at the single-cell transcriptomic (scRNAseq) level in tumors and patients with coronavirus disease 2019 (COVID-19) requires both a reference differentiation trajectory of γδ T cells and a robust mapping method for additional γδ T lymphocytes. Here, we incepted such a method to characterize thousands of γδ T lymphocytes from (n = 95) patients with cancer or adult and pediatric COVID-19 disease. We found that cancer patients with human papillomavirus-positive head and neck squamous cell carcinoma and Epstein-Barr virus-positive Hodgkin's lymphoma have γδ tumor-infiltrating T lymphocytes that are more prone to recirculate from the tumor and avoid exhaustion. In COVID-19, both TCRVγ9 and TCRVγnon9 subsets of γδ T lymphocytes relocalize from peripheral blood mononuclear cells (PBMC) to the infected lung tissue, where their advanced differentiation, tissue residency, and exhaustion reflect T cell activation. Although severe COVID-19 disease increases both recruitment and exhaustion of γδ T lymphocytes in infected lung lesions but not blood, the anti-IL6R therapy with Tocilizumab promotes γδ T lymphocyte differentiation in patients with COVID-19. PBMC from pediatric patients with acute COVID-19 disease display similar γδ T cell lymphopenia to that seen in adult patients. However, blood γδ T cells from children with the COVID-19-related multisystem inflammatory syndrome are not lymphodepleted, but they are differentiated as in healthy PBMC. These findings suggest that some virus-induced memory γδ T lymphocytes durably persist in the blood of adults and could subsequently infiltrate and recirculate in tumors.


Subject(s)
COVID-19/immunology , Lymphocytes, Tumor-Infiltrating/immunology , Neoplasms/immunology , RNA-Seq , Receptors, Antigen, T-Cell, gamma-delta/immunology , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Adult , Bronchoalveolar Lavage Fluid/immunology , COVID-19/complications , Cell Differentiation , Child , Head and Neck Neoplasms/immunology , Head and Neck Neoplasms/virology , Herpesvirus 4, Human/isolation & purification , Hodgkin Disease/immunology , Hodgkin Disease/virology , Humans , Lung/immunology , Lymphocyte Activation , Lymphocyte Count , Lymphocytes, Tumor-Infiltrating/metabolism , Lymphocytes, Tumor-Infiltrating/physiology , Neoplasms/virology , Papillomaviridae/isolation & purification , Severity of Illness Index , Single-Cell Analysis , Systemic Inflammatory Response Syndrome/immunology , T-Lymphocyte Subsets/physiology
3.
JCI Insight ; 7(1)2022 01 11.
Article in English | MEDLINE | ID: covidwho-1523122

ABSTRACT

Neutrophils are recognized as important circulating effector cells in the pathophysiology of severe coronavirus disease 2019 (COVID-19). However, their role within the inflamed lungs is incompletely understood. Here, we collected bronchoalveolar lavage (BAL) fluids and parallel blood samples of critically ill COVID-19 patients requiring invasive mechanical ventilation and compared BAL fluid parameters with those of mechanically ventilated patients with influenza, as a non-COVID-19 viral pneumonia cohort. Compared with those of patients with influenza, BAL fluids of patients with COVID-19 contained increased numbers of hyperactivated degranulating neutrophils and elevated concentrations of the cytokines IL-1ß, IL-1RA, IL-17A, TNF-α, and G-CSF; the chemokines CCL7, CXCL1, CXCL8, CXCL11, and CXCL12α; and the protease inhibitors elafin, secretory leukocyte protease inhibitor, and tissue inhibitor of metalloproteinases 1. In contrast, α-1 antitrypsin levels and net proteolytic activity were comparable in COVID-19 and influenza BAL fluids. During antibiotic treatment for bacterial coinfections, increased BAL fluid levels of several activating and chemotactic factors for monocytes, lymphocytes, and NK cells were detected in patients with COVID-19 whereas concentrations tended to decrease in patients with influenza, highlighting the persistent immunological response to coinfections in COVID-19. Finally, the high proteolytic activity in COVID-19 lungs suggests considering protease inhibitors as a treatment option.


Subject(s)
Bacterial Infections , Bronchoalveolar Lavage Fluid , COVID-19 , Coinfection , Influenza, Human , Adult , Aged , Bacterial Infections/complications , Bacterial Infections/immunology , Bacterial Infections/metabolism , Bacterial Infections/pathology , Bronchoalveolar Lavage Fluid/chemistry , Bronchoalveolar Lavage Fluid/cytology , Bronchoalveolar Lavage Fluid/immunology , COVID-19/complications , COVID-19/diagnosis , COVID-19/immunology , COVID-19/pathology , Coinfection/immunology , Coinfection/metabolism , Coinfection/pathology , Cytokines/analysis , Female , Humans , Inflammation , Influenza, Human/complications , Influenza, Human/diagnosis , Influenza, Human/immunology , Influenza, Human/pathology , Lung/immunology , Lung/metabolism , Lung/pathology , Male , Middle Aged
4.
J Ethnopharmacol ; 285: 114838, 2022 Mar 01.
Article in English | MEDLINE | ID: covidwho-1509996

ABSTRACT

ETHNOPHARMACOLOGICAL RELEVANCE: Keguan-1, a new traditional Chinese medicine (TCM) prescription contained seven Chinese herbs, is developed to treat coronavirus disease 19 (COVID-19). The first internationally registered COVID-19 randomised clinical trial on integrated therapy demonstrated that Keguan-1 significantly reduced the incidence of ARDS and inhibited the severe progression of COVID-19. AIM OF THE STUDY: To investigate the protective mechanism of Keguan-1 on ARDS, a lipopolysaccharide (LPS)-induced acute lung injury (ALI) model was used to simulate the pathological state of ARDS in patients with COVID-19, focusing on its effect and mechanism on ALI. MATERIALS AND METHODS: Mice were challenged with LPS (2 mg/kg) by intratracheal instillation (i.t.) and were orally administered Keguan-1 (low dose, 1.25 g/kg; medium dose, 2.5 g/kg; high dose, 5 g/kg) after 2 h. Bronchoalveolar lavage fluid (BALF) and lung tissue were collected 6 h and 24 h after i.t. administration of LPS. The levels of inflammatory factors tumour necrosis factor alpha (TNF-α), interleukin (IL)-6, IL-1ß, keratinocyte-derived chemokine (KC or mCXCL1), macrophage inflammatory protein 2 (MIP2 or mCXCL2), angiotensin II (Ang II), and endothelial cell junction-associated proteins were analysed using ELISA or western blotting. RESULTS: Keguan-1 improved the survival rate, respiratory condition, and pathological lung injury; decreased the production of proinflammatory factors (TNF-α, IL-6, IL-1ß, KC, and MIP2) in BALF and the number of neutrophils in the lung tissues; and ameliorated inflammatory injury in the lung tissues of the mice with LPS-induced ALI. Keguan-1 also reduced the expression of Ang II and the adhesion molecule ICAM-1; increased tight junction proteins (JAM-1 and claudin-5) and VE-cadherin expression; and alleviated pulmonary vascular endothelial injury in LPS-induced ALI. CONCLUSION: These results demonstrate that Keguan-1 can improve LPS-induced ALI by reducing inflammation and pulmonary vascular endothelial injury, providing scientific support for the clinical treatment of patients with COVID-19. Moreover, it also provides a theoretical basis and technical support for the scientific use of TCMs in emerging infectious diseases.


Subject(s)
Acute Lung Injury , Antiviral Agents/pharmacology , Bronchoalveolar Lavage Fluid , COVID-19 , Drugs, Chinese Herbal/pharmacology , Lung , Acute Lung Injury/drug therapy , Acute Lung Injury/immunology , Acute Lung Injury/pathology , Animals , Bronchoalveolar Lavage Fluid/immunology , Bronchoalveolar Lavage Fluid/virology , COVID-19/complications , COVID-19/immunology , COVID-19/virology , Capsules , Chemokine CXCL2/analysis , Coix , Forsythia , Interleukin-1beta/analysis , Interleukin-6/analysis , Lonicera , Lung/drug effects , Lung/metabolism , Lung/pathology , Lung/virology , Mice , Mortality , Morus , Peptide Fragments/analysis , Prunus armeniaca , Respiration/drug effects , SARS-CoV-2 , Treatment Outcome , Tumor Necrosis Factor-alpha/analysis
5.
Emerg Microbes Infect ; 10(1): 2194-2198, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1504286

ABSTRACT

Inactivated coronaviruses, including severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) and Middle East respiratory syndrome coronavirus (MERS-CoV), as potential vaccines have been reported to result in enhanced respiratory diseases (ERDs) in murine and nonhuman primate (NHP) pneumonia models after virus challenge, which poses great safety concerns of antibody-dependent enhancement (ADE) for the rapid wide application of inactivated SARS-CoV-2 vaccines in humans, especially when the neutralizing antibody levels induced by vaccination or initial infection quickly wane to nonneutralizing or subneutralizing levels over the time. With passive transfer of diluted postvaccination polyclonal antibodies to mimic the waning antibody responses after vaccination, we found that in the absence of cellular immunity, passive infusion of subneutralizing or nonneutralizing anti-SARS-CoV-2 antibodies could still provide some level of protection against infection upon challenge, and no low-level antibody-enhanced infection was observed. The anti-SARS-CoV-2 IgG-infused group and control group showed similar, mild to moderate pulmonary immunopathology during the acute phase of virus infection, and no evidence of vaccine-related pulmonary immunopathology enhancement was found. Typical immunopathology included elevated MCP-1, IL-8 and IL-33 in bronchoalveolar lavage fluid; alveolar epithelial hyperplasia; and exfoliated cells and mucus in bronchioles. Our results corresponded with the recent observations that no pulmonary immunology was detected in preclinical studies of inactivated SARS-CoV-2 vaccines in either murine or NHP pneumonia models or in large clinical trials and further supported the safety of inactivated SARS-CoV-2 vaccines.


Subject(s)
Antibodies, Viral/immunology , Antibody-Dependent Enhancement , COVID-19 Vaccines/immunology , COVID-19/immunology , Immunogenicity, Vaccine , SARS-CoV-2/immunology , Alveolar Epithelial Cells/pathology , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/toxicity , Bronchioles/chemistry , Bronchioles/pathology , Bronchoalveolar Lavage Fluid/chemistry , Bronchoalveolar Lavage Fluid/immunology , COVID-19/pathology , COVID-19/virology , Cytokines/analysis , Humans , Hyperplasia , Immunoglobulin G/immunology , Immunoglobulin G/toxicity , Lung/pathology , Macaca mulatta , Male , Mice , Mucus , SARS-CoV-2/isolation & purification , SARS-CoV-2/physiology , Vaccines, Inactivated/immunology
6.
Viruses ; 13(11)2021 11 03.
Article in English | MEDLINE | ID: covidwho-1502529

ABSTRACT

The detailed characterization of human γδ T lymphocyte differentiation at the single-cell transcriptomic (scRNAseq) level in tumors and patients with coronavirus disease 2019 (COVID-19) requires both a reference differentiation trajectory of γδ T cells and a robust mapping method for additional γδ T lymphocytes. Here, we incepted such a method to characterize thousands of γδ T lymphocytes from (n = 95) patients with cancer or adult and pediatric COVID-19 disease. We found that cancer patients with human papillomavirus-positive head and neck squamous cell carcinoma and Epstein-Barr virus-positive Hodgkin's lymphoma have γδ tumor-infiltrating T lymphocytes that are more prone to recirculate from the tumor and avoid exhaustion. In COVID-19, both TCRVγ9 and TCRVγnon9 subsets of γδ T lymphocytes relocalize from peripheral blood mononuclear cells (PBMC) to the infected lung tissue, where their advanced differentiation, tissue residency, and exhaustion reflect T cell activation. Although severe COVID-19 disease increases both recruitment and exhaustion of γδ T lymphocytes in infected lung lesions but not blood, the anti-IL6R therapy with Tocilizumab promotes γδ T lymphocyte differentiation in patients with COVID-19. PBMC from pediatric patients with acute COVID-19 disease display similar γδ T cell lymphopenia to that seen in adult patients. However, blood γδ T cells from children with the COVID-19-related multisystem inflammatory syndrome are not lymphodepleted, but they are differentiated as in healthy PBMC. These findings suggest that some virus-induced memory γδ T lymphocytes durably persist in the blood of adults and could subsequently infiltrate and recirculate in tumors.


Subject(s)
COVID-19/immunology , Lymphocytes, Tumor-Infiltrating/immunology , Neoplasms/immunology , RNA-Seq , Receptors, Antigen, T-Cell, gamma-delta/immunology , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Adult , Bronchoalveolar Lavage Fluid/immunology , COVID-19/complications , Cell Differentiation , Child , Head and Neck Neoplasms/immunology , Head and Neck Neoplasms/virology , Herpesvirus 4, Human/isolation & purification , Hodgkin Disease/immunology , Hodgkin Disease/virology , Humans , Lung/immunology , Lymphocyte Activation , Lymphocyte Count , Lymphocytes, Tumor-Infiltrating/metabolism , Lymphocytes, Tumor-Infiltrating/physiology , Neoplasms/virology , Papillomaviridae/isolation & purification , Severity of Illness Index , Single-Cell Analysis , Systemic Inflammatory Response Syndrome/immunology , T-Lymphocyte Subsets/physiology
7.
Physiol Rep ; 9(20): e15075, 2021 10.
Article in English | MEDLINE | ID: covidwho-1485552

ABSTRACT

Exercise has substantial health benefits, but the effects of exercise on immune status and susceptibility to respiratory infections are less clear. Furthermore, there is limited research examining the effects of prolonged exercise on local respiratory immunity and antiviral activity. To assess the upper respiratory tract in response to exercise, we collected nasal lavage fluid (NALF) from human subjects (1) at rest, (2) after 45 min of moderate-intensity exercise, and (3) after 180 min of moderate-intensity exercise. To assess immune responses of the lower respiratory tract, we utilized a murine model to examine the effect of exercise duration on bronchoalveolar lavage (BAL) fluid immune cell content and lung gene expression. NALF cell counts did not change after 45 min of exercise, whereas 180 min significantly increased total cells and leukocytes in NALF. Importantly, fold change in NALF leukocytes correlated with the post-exercise fatigue rating in the 180-min exercise condition. The acellular portion of NALF contained strong antiviral activity against Influenza A in both resting and exercise paradigms. In mice undergoing moderate-intensity exercise, BAL total cells and neutrophils decreased in response to 45 or 90 min of exercise. In lung lobes, increased expression of heat shock proteins suggested that cellular stress occurred in response to exercise. However, a broad upregulation of inflammatory genes was not observed, even at 180 min of exercise. This work demonstrates that exercise duration differentially alters the cellularity of respiratory tract fluids, antiviral activity, and gene expression. These changes in local mucosal immunity may influence resistance to respiratory viruses, including influenza or possibly other pathogens in which nasal mucosa plays a protective role, such as rhinovirus or SARS-CoV-2.


Subject(s)
Exercise/physiology , Influenza A virus/immunology , Leukocytes/immunology , Lung/immunology , Nasal Lavage Fluid/immunology , Neutrophils/immunology , Adolescent , Adult , Animals , Bronchoalveolar Lavage Fluid/cytology , Bronchoalveolar Lavage Fluid/immunology , Female , Gene Expression , Humans , Leukocytes/metabolism , Lung/cytology , Lung/metabolism , Male , Mice , Mice, Inbred BALB C , Nasal Lavage/methods , Nasal Lavage Fluid/cytology , Nasal Mucosa/cytology , Nasal Mucosa/immunology , Nasal Mucosa/metabolism , Neutrophils/metabolism , Time Factors , Young Adult
8.
Science ; 374(6573): 1343-1353, 2021 Dec 10.
Article in English | MEDLINE | ID: covidwho-1483979

ABSTRACT

Neutralizing antibody responses gradually wane against several variants of concern (VOCs) after vaccination with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine messenger RNA-1273 (mRNA-1273). We evaluated the immune responses in nonhuman primates that received a primary vaccination series of mRNA-1273 and were boosted about 6 months later with either homologous mRNA-1273 or heterologous mRNA-1273.ß, which encompasses the spike sequence of the B.1.351 Beta variant. After boost, animals had increased neutralizing antibody responses across all VOCs, which was sustained for at least 8 weeks after boost. Nine weeks after boost, animals were challenged with the SARS-CoV-2 Beta variant. Viral replication was low to undetectable in bronchoalveolar lavage and significantly reduced in nasal swabs in all boosted animals, suggesting that booster vaccinations may be required to sustain immunity and protection.


Subject(s)
/immunology , COVID-19 Vaccines/immunology , COVID-19/prevention & control , Immunogenicity, Vaccine , SARS-CoV-2/immunology , /administration & dosage , Animals , Antibodies, Neutralizing/blood , Antibodies, Viral/analysis , Antibodies, Viral/blood , Bronchoalveolar Lavage Fluid/immunology , Bronchoalveolar Lavage Fluid/virology , COVID-19/immunology , COVID-19/virology , COVID-19 Vaccines/administration & dosage , Immunity, Mucosal , Immunization, Secondary , Macaca mulatta , Nose/immunology , Nose/virology , RNA, Viral/analysis , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , SARS-CoV-2/physiology , T Follicular Helper Cells/immunology , Th1 Cells/immunology , Virus Replication
9.
Front Immunol ; 12: 752612, 2021.
Article in English | MEDLINE | ID: covidwho-1456293

ABSTRACT

Background: Lymphopenia and the neutrophil/lymphocyte ratio may have prognostic value in COVID-19 severity. Objective: We investigated neutrophil subsets and functions in blood and bronchoalveolar lavage (BAL) of COVID-19 patients on the basis of patients' clinical characteristics. Methods: We used a multiparametric cytometry profiling based to mature and immature neutrophil markers in 146 critical or severe COVID-19 patients. Results: The Discovery study (38 patients, first pandemic wave) showed that 80% of Intensive Care Unit (ICU) patients develop strong myelemia with CD10-CD64+ immature neutrophils (ImNs). Cellular profiling revealed three distinct neutrophil subsets expressing either the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), the interleukin-3 receptor alpha (CD123), or programmed death-ligand 1 (PD-L1) overrepresented in ICU patients compared to non-ICU patients. The proportion of LOX-1- or CD123-expressing ImNs is positively correlated with clinical severity, cytokine storm (IL-1ß, IL-6, IL-8, TNFα), acute respiratory distress syndrome (ARDS), and thrombosis. BALs of patients with ARDS were highly enriched in LOX-1-expressing ImN subsets and in antimicrobial neutrophil factors. A validation study (118 patients, second pandemic wave) confirmed and strengthened the association of the proportion of ImN subsets with disease severity, invasive ventilation, and death. Only high proportions of LOX-1-expressing ImNs remained strongly associated with a high risk of severe thrombosis independently of the plasma antimicrobial neutrophil factors, suggesting an independent association of ImN markers with their functions. Conclusion: LOX-1-expressing ImNs may help identifying COVID-19 patients at high risk of severity and thrombosis complications.


Subject(s)
COVID-19/complications , Neutrophils/immunology , Scavenger Receptors, Class E/genetics , Thrombosis/etiology , Adult , Aged , B7-H1 Antigen/genetics , B7-H1 Antigen/immunology , Bronchoalveolar Lavage Fluid/immunology , COVID-19/genetics , COVID-19/immunology , COVID-19/virology , Critical Illness , Female , Humans , Interleukin-3 Receptor alpha Subunit/genetics , Interleukin-3 Receptor alpha Subunit/immunology , Interleukin-8/genetics , Interleukin-8/immunology , Male , Middle Aged , Respiratory Distress Syndrome/etiology , Respiratory Distress Syndrome/genetics , Respiratory Distress Syndrome/immunology , SARS-CoV-2/physiology , Scavenger Receptors, Class E/immunology , Thrombosis/genetics , Thrombosis/immunology
10.
Front Immunol ; 12: 705646, 2021.
Article in English | MEDLINE | ID: covidwho-1450806

ABSTRACT

COVID-19 is a disease with a spectrum of clinical responses ranging from moderate to critical. To study and control its effects, a large number of researchers are focused on two substantial aims. On the one hand, the discovery of diverse biomarkers to classify and potentially anticipate the disease severity of patients. These biomarkers could serve as a medical criterion to prioritize attention to those patients with higher prone to severe responses. On the other hand, understanding how the immune system orchestrates its responses in this spectrum of disease severities is a fundamental issue required to design new and optimized therapeutic strategies. In this work, using single-cell RNAseq of bronchoalveolar lavage fluid of nine patients with COVID-19 and three healthy controls, we contribute to both aspects. First, we presented computational supervised machine-learning models with high accuracy in classifying the disease severity (moderate and severe) in patients with COVID-19 starting from single-cell data from bronchoalveolar lavage fluid. Second, we identified regulatory mechanisms from the heterogeneous cell populations in the lungs microenvironment that correlated with different clinical responses. Given the results, patients with moderate COVID-19 symptoms showed an activation/inactivation profile for their analyzed cells leading to a sequential and innocuous immune response. In comparison, severe patients might be promoting cytotoxic and pro-inflammatory responses in a systemic fashion involving epithelial and immune cells without the possibility to develop viral clearance and immune memory. Consequently, we present an in-depth landscape analysis of how transcriptional factors and pathways from these heterogeneous populations can regulate their expression to promote or restrain an effective immune response directly linked to the patients prognosis.


Subject(s)
Bronchoalveolar Lavage Fluid/cytology , Bronchoalveolar Lavage Fluid/immunology , COVID-19/pathology , Lung/cytology , SARS-CoV-2/immunology , B-Lymphocytes/immunology , Biomarkers , Bronchoalveolar Lavage Fluid/chemistry , Dendritic Cells/immunology , Epithelial Cells/cytology , Epithelial Cells/virology , Humans , Killer Cells, Natural/immunology , Lung/chemistry , Machine Learning , Macrophages/immunology , Monocytes/immunology , Neutrophils/immunology , RNA, Viral/genetics , Sequence Analysis, RNA , Severity of Illness Index , Single-Cell Analysis , T-Lymphocytes/immunology
11.
Nat Microbiol ; 6(10): 1245-1258, 2021 10.
Article in English | MEDLINE | ID: covidwho-1380902

ABSTRACT

Respiratory failure is associated with increased mortality in COVID-19 patients. There are no validated lower airway biomarkers to predict clinical outcome. We investigated whether bacterial respiratory infections were associated with poor clinical outcome of COVID-19 in a prospective, observational cohort of 589 critically ill adults, all of whom required mechanical ventilation. For a subset of 142 patients who underwent bronchoscopy, we quantified SARS-CoV-2 viral load, analysed the lower respiratory tract microbiome using metagenomics and metatranscriptomics and profiled the host immune response. Acquisition of a hospital-acquired respiratory pathogen was not associated with fatal outcome. Poor clinical outcome was associated with lower airway enrichment with an oral commensal (Mycoplasma salivarium). Increased SARS-CoV-2 abundance, low anti-SARS-CoV-2 antibody response and a distinct host transcriptome profile of the lower airways were most predictive of mortality. Our data provide evidence that secondary respiratory infections do not drive mortality in COVID-19 and clinical management strategies should prioritize reducing viral replication and maximizing host responses to SARS-CoV-2.


Subject(s)
Bronchoalveolar Lavage Fluid/microbiology , COVID-19/therapy , Respiration, Artificial , SARS-CoV-2/pathogenicity , Adaptive Immunity , Adult , Aged , Bacteria/classification , Bacteria/genetics , Bacteria/isolation & purification , Bacterial Load , Bronchoalveolar Lavage Fluid/immunology , Bronchoalveolar Lavage Fluid/virology , COVID-19/immunology , COVID-19/microbiology , COVID-19/mortality , Critical Illness , Female , Hospitalization , Humans , Immunity, Innate , Male , Microbiota , Middle Aged , Odds Ratio , Prognosis , Prospective Studies , Respiratory System/immunology , Respiratory System/microbiology , Respiratory System/virology , SARS-CoV-2/immunology , Viral Load
12.
JCI Insight ; 6(13)2021 06 18.
Article in English | MEDLINE | ID: covidwho-1346128

ABSTRACT

We explored the potential link between chronic inflammatory arthritis and COVID-19 pathogenic and resolving macrophage pathways and their role in COVID-19 pathogenesis. We found that bronchoalveolar lavage fluid (BALF) macrophage clusters FCN1+ and FCN1+SPP1+ predominant in severe COVID-19 were transcriptionally related to synovial tissue macrophage (STM) clusters CD48hiS100A12+ and CD48+SPP1+ that drive rheumatoid arthritis (RA) synovitis. BALF macrophage cluster FABP4+ predominant in healthy lung was transcriptionally related to STM cluster TREM2+ that governs resolution of synovitis in RA remission. Plasma concentrations of SPP1 and S100A12 (key products of macrophage clusters shared with active RA) were high in severe COVID-19 and predicted the need for Intensive Care Unit transfer, and they remained high in the post-COVID-19 stage. High plasma levels of SPP1 were unique to severe COVID-19 when compared with other causes of severe pneumonia, and IHC localized SPP1+ macrophages in the alveoli of COVID-19 lung. Investigation into SPP1 mechanisms of action revealed that it drives proinflammatory activation of CD14+ monocytes and development of PD-L1+ neutrophils, both hallmarks of severe COVID-19. In summary, COVID-19 pneumonitis appears driven by similar pathogenic myeloid cell pathways as those in RA, and their mediators such as SPP1 might be an upstream activator of the aberrant innate response in severe COVID-19 and predictive of disease trajectory including post-COVID-19 pathology.


Subject(s)
Arthritis, Rheumatoid/immunology , COVID-19/immunology , Monocytes/immunology , Neutrophils/immunology , Osteopontin/immunology , Arthritis, Rheumatoid/metabolism , B7-H1 Antigen/immunology , Bronchoalveolar Lavage Fluid/immunology , CD48 Antigen/immunology , COVID-19/chemically induced , COVID-19/metabolism , Fatty Acid-Binding Proteins/immunology , Humans , Lectins/immunology , Lipopolysaccharide Receptors/immunology , Lipopolysaccharide Receptors/metabolism , Lung/diagnostic imaging , Lung/immunology , Lung/metabolism , Lung/pathology , Macrophages/immunology , Macrophages/metabolism , Membrane Glycoproteins/immunology , Monocytes/metabolism , Neutrophils/metabolism , Osteopontin/blood , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Immunologic/immunology , S100A12 Protein/immunology , S100A12 Protein/metabolism , Synovial Membrane/immunology , Tomography, X-Ray Computed
13.
J Immunol ; 207(5): 1229-1238, 2021 09 01.
Article in English | MEDLINE | ID: covidwho-1344412

ABSTRACT

Infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) or seasonal influenza may lead to respiratory failure requiring intubation and mechanical ventilation. The pathophysiology of this respiratory failure is attributed to local immune dysregulation, but how the immune response to viral infection in the lower airways of the human lung differs between individuals with respiratory failure and those without is not well understood. We used quantitative multiparameter flow cytometry and multiplex cytokine assays to evaluate matched blood and bronchoalveolar lavage (BAL) samples from control human subjects, subjects with symptomatic seasonal influenza who did not have respiratory failure, and subjects with severe seasonal influenza or SARS-CoV-2 infection with respiratory failure. We find that severe cases are associated with an influx of nonclassical monocytes, activated T cells, and plasmablast B cells into the lower airways. Cytokine concentrations were not elevated in the lower airways of moderate influenza patients compared with controls; however, 28 of 35 measured cytokines were significantly elevated in severe influenza, severe SARS-CoV-2 infection, or both. We noted the largest elevations in IL-6, IP-10, MCP-1, and IL-8. IL-1 family cytokines and RANTES were higher in severe influenza infection than severe SARS-CoV-2 infection. Interestingly, only the concentration of IP-10-correlated between blood and BAL during severe infection. Our results demonstrate inflammatory immune dysregulation in the lower airways during severe viral pneumonia that is distinct from lower airway responses seen in human patients with symptomatic, but not severe, illness and suggest that measurement of blood IP-10 concentration may predict this unique dysregulation.


Subject(s)
COVID-19/immunology , Influenza A virus/physiology , Pneumonia, Viral/immunology , Respiratory System/immunology , SARS-CoV-2/physiology , Adult , Aged , Blood Proteins/metabolism , Bronchoalveolar Lavage Fluid/immunology , COVID-19/diagnosis , Chemokine CXCL10/metabolism , Cohort Studies , Female , Humans , Inflammation Mediators/metabolism , Influenza, Human/immunology , Male , Middle Aged , Prospective Studies , Respiratory Insufficiency , Severity of Illness Index
14.
Cancer Cytopathol ; 129(8): 632-641, 2021 08.
Article in English | MEDLINE | ID: covidwho-1342873

ABSTRACT

BACKGROUND: Bronchoalveolar lavage (BAL) in patients with severe coronavirus disease 2019 (COVID-19) may provide additional and complementary findings for the management of these patients admitted to intensive care units (ICUs). This study addresses the cytological features of the infection and highlights the more influential inflammatory components. The correlation between pathological variables and clinical data is also analyzed. METHODS: The authors performed a retrospective analysis of the cytopathological features of BAL in 20 COVID-19 patients and 20 members of a matched cohort from a critical ICU who had acute respiratory distress syndrome caused by other pulmonary conditions. RESULTS: A comparison of the controls (n = 20) and the COVID-19 patients (n = 20) revealed that the latter had a higher neutrophil count (median, 63.8% of the cell count) with lower percentages of macrophages and lymphocytes. An increase in the expression of CD68-positive, monocytic multinucleated giant cells (MGCs) was reported; megakaryocytes were not detected on CD61 staining. Perls staining showed isolated elements. In situ RNA analysis demonstrated scattered chromogenic signals in type II pneumocytes. An ultrastructural analysis confirmed the presence of intracytoplasmic vacuoles containing rounded structures measuring 140 nm in diameter (putative viral particles). In COVID-19 patients, the clinicopathological correlation revealed a positive correlation between lactate dehydrogenase values and MGCs (r = 0.54). CONCLUSIONS: The analysis of BAL samples might be implemented as a routine practice for the evaluation of COVID-19 patients in ICUs in the appropriate clinical scenario. Additional studies using a larger sample size of patients who developed COVID-19 during the second wave of the epidemic in the autumn of 2020 are needed to further support our findings.


Subject(s)
Bronchoalveolar Lavage Fluid/cytology , Bronchoalveolar Lavage Fluid/immunology , COVID-19/diagnosis , COVID-19/immunology , COVID-19/pathology , Adult , Aged , Female , Humans , Male , Middle Aged , Pilot Projects , Pneumonia, Viral/diagnosis , Pneumonia, Viral/immunology , Pneumonia, Viral/pathology , Retrospective Studies , SARS-CoV-2
15.
Science ; 373(6561): eabj0299, 2021 Sep 17.
Article in English | MEDLINE | ID: covidwho-1334532

ABSTRACT

Immune correlates of protection can be used as surrogate endpoints for vaccine efficacy. Here, nonhuman primates (NHPs) received either no vaccine or doses ranging from 0.3 to 100 µg of the mRNA-1273 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine. mRNA-1273 vaccination elicited circulating and mucosal antibody responses in a dose-dependent manner. Viral replication was significantly reduced in bronchoalveolar lavages and nasal swabs after SARS-CoV-2 challenge in vaccinated animals and most strongly correlated with levels of anti­S antibody and neutralizing activity. Lower antibody levels were needed for reduction of viral replication in the lower airway than in the upper airway. Passive transfer of mRNA-1273­induced immunoglobulin G to naïve hamsters was sufficient to mediate protection. Thus, mRNA-1273 vaccine­induced humoral immune responses are a mechanistic correlate of protection against SARS-CoV-2 in NHPs.


Subject(s)
Antibodies, Neutralizing/blood , Antibodies, Viral/blood , COVID-19 Vaccines/administration & dosage , COVID-19 Vaccines/immunology , COVID-19/prevention & control , Immunogenicity, Vaccine , SARS-CoV-2/immunology , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Antibody Affinity , Bronchoalveolar Lavage Fluid/immunology , Bronchoalveolar Lavage Fluid/virology , CD4-Positive T-Lymphocytes/immunology , COVID-19/immunology , COVID-19/virology , Female , Immunization Schedule , Immunization, Passive , Immunization, Secondary , Immunoglobulin G/immunology , Immunologic Memory , Lung/immunology , Lung/virology , Macaca mulatta , Male , Mesocricetus , Nasal Mucosa/immunology , Nasal Mucosa/virology , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/immunology , Vaccination , Vaccine Potency , Virus Replication
16.
Sci Rep ; 11(1): 14151, 2021 07 08.
Article in English | MEDLINE | ID: covidwho-1303792

ABSTRACT

The Coronavirus disease 2019 (COVID-19) has been spreading worldwide with rapidly increased number of deaths. Hyperinflammation mediated by dysregulated monocyte/macrophage function is considered to be the key factor that triggers severe illness in COVID-19. However, no specific targeting molecule has been identified for detecting or treating hyperinflammation related to dysregulated macrophages in severe COVID-19. In this study, previously published single-cell RNA-sequencing data of bronchoalveolar lavage fluid cells from thirteen COVID-19 patients were analyzed with publicly available databases for surface and imageable targets. Immune cell composition according to the severity was estimated with the clustering of gene expression data. Expression levels of imaging target molecules for inflammation were evaluated in macrophage clusters from single-cell RNA-sequencing data. In addition, candidate targetable molecules enriched in severe COVID-19 associated with hyperinflammation were filtered. We found that expression of SLC2A3, which can be imaged by [18F]fluorodeoxyglucose, was higher in macrophages from severe COVID-19 patients. Furthermore, by integrating the surface target and drug-target binding databases with RNA-sequencing data of severe COVID-19, we identified candidate surface and druggable targets including CCR1 and FPR1 for drug delivery as well as molecular imaging. Our results provide a resource in the development of specific imaging and therapy for COVID-19-related hyperinflammation.


Subject(s)
COVID-19/diagnostic imaging , COVID-19/therapy , Molecular Imaging/methods , Molecular Targeted Therapy , Bronchoalveolar Lavage Fluid/cytology , Bronchoalveolar Lavage Fluid/immunology , COVID-19/genetics , COVID-19/immunology , Databases, Nucleic Acid , Drug Delivery Systems , Gene Expression , Glucose Transporter Type 3/genetics , Glucose Transporter Type 3/metabolism , Humans , Inflammation , Macrophages/immunology , Monocytes/immunology , Receptors, CCR1 , Receptors, Formyl Peptide , Severity of Illness Index
17.
Cell Commun Signal ; 19(1): 73, 2021 07 08.
Article in English | MEDLINE | ID: covidwho-1301855

ABSTRACT

BACKGROUND: The coronavirus disease 2019 (COVID-19) outbreak caused by severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) has become an ongoing pandemic. Understanding the respiratory immune microenvironment which is composed of multiple cell types, together with cell communication based on ligand-receptor interactions is important for developing vaccines, probing COVID-19 pathogenesis, and improving pandemic control measures. METHODS: A total of 102 consecutive hospitalized patients with confirmed COVID-19 were enrolled in this study. Clinical information, routine laboratory tests, and flow cytometry analysis data with different conditions were collected and assessed for predictive value in COVID-19 patients. Next, we analyzed public single-cell RNA-sequencing (scRNA-seq) data from bronchoalveolar lavage fluid, which offers the closest available view of immune cell heterogeneity as encountered in patients with varying severity of COVID-19. A weighting algorithm was used to calculate ligand-receptor interactions, revealing the communication potentially associated with outcomes across cell types. Finally, serum cytokines including IL6, IL1ß, IL10, CXCL10, TNFα, GALECTIN-1, and IGF1 derived from patients were measured. RESULTS: Of the 102 COVID-19 patients, 42 cases (41.2%) were categorized as severe. Multivariate logistic regression analysis demonstrated that AST, D-dimer, BUN, and WBC were considered as independent risk factors for the severity of COVID-19. T cell numbers including total T cells, CD4+ and CD8+ T cells in the severe disease group were significantly lower than those in the moderate disease group. The risk model containing the above mentioned inflammatory damage parameters, and the counts of T cells, with AUROCs ranged from 0.78 to 0.87. To investigate the molecular mechanism at the cellular level, we analyzed the published scRNA-seq data and found that macrophages displayed specific functional diversity after SARS-Cov-2 infection, and the metabolic pathway activities in the identified macrophage subtypes were influenced by hypoxia status. Importantly, we described ligand-receptor interactions that are related to COVID-19 serverity involving macrophages and T cell subsets by communication analysis. CONCLUSIONS: Our study showed that macrophages driving ligand-receptor crosstalk contributed to the reduction and exhaustion of CD8+ T cells. The identified crucial cytokine panel, including IL6, IL1ß, IL10, CXCL10, IGF1, and GALECTIN-1, may offer the selective targets to improve the efficacy of COVID-19 therapy. TRIAL REGISTRATION: This is a retrospective observational study without a trial registration number. Video Abstract.


Subject(s)
COVID-19/immunology , COVID-19/pathology , Cell Communication , Macrophages/immunology , Single-Cell Analysis , Aged , Bronchoalveolar Lavage Fluid/immunology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/pathology , COVID-19/epidemiology , COVID-19/physiopathology , China/epidemiology , Cytokines/blood , Cytokines/immunology , Female , Humans , Macrophages/pathology , Male , Middle Aged , Receptors, Cytokine , Retrospective Studies , Sequence Analysis, RNA , Severity of Illness Index
18.
PLoS Pathog ; 17(7): e1009381, 2021 07.
Article in English | MEDLINE | ID: covidwho-1291654

ABSTRACT

Clearance of viral infections, such as SARS-CoV-2 and influenza A virus (IAV), must be fine-tuned to eliminate the pathogen without causing immunopathology. As such, an aggressive initial innate immune response favors the host in contrast to a detrimental prolonged inflammation. The complement pathway bridges innate and adaptive immune system and contributes to the response by directly clearing pathogens or infected cells, as well as recruiting proinflammatory immune cells and regulating inflammation. However, the impact of modulating complement activation in viral infections is still unclear. In this work, we targeted the complement decay-accelerating factor (DAF/CD55), a surface protein that protects cells from non-specific complement attack, and analyzed its role in IAV infections. We found that DAF modulates IAV infection in vivo, via an interplay with the antigenic viral proteins hemagglutinin (HA) and neuraminidase (NA), in a strain specific manner. Our results reveal that, contrary to what could be expected, DAF potentiates complement activation, increasing the recruitment of neutrophils, monocytes and T cells. We also show that viral NA acts on the heavily sialylated DAF and propose that the NA-dependent DAF removal of sialic acids exacerbates complement activation, leading to lung immunopathology. Remarkably, this mechanism has no impact on viral loads, but rather on the host resilience to infection, and may have direct implications in zoonotic influenza transmissions.


Subject(s)
CD55 Antigens/physiology , Influenza A Virus, H1N1 Subtype/isolation & purification , Lung/immunology , Viremia/immunology , Animals , Bronchoalveolar Lavage Fluid/immunology , CD55 Antigens/chemistry , CD55 Antigens/deficiency , Chemotaxis, Leukocyte , Complement Activation , Hemagglutinin Glycoproteins, Influenza Virus/physiology , Host Adaptation , Host Specificity , Host-Pathogen Interactions , Influenza A Virus, H1N1 Subtype/enzymology , Influenza A Virus, H1N1 Subtype/pathogenicity , Influenza A Virus, H1N1 Subtype/physiology , Interferon-gamma/analysis , Lung/pathology , Lung/virology , Mice , Mice, Inbred C57BL , N-Acetylneuraminic Acid , Neuraminidase/physiology , Orthomyxoviridae Infections/immunology , Orthomyxoviridae Infections/pathology , Viral Load , Viral Proteins/physiology , Virulence , Virus Replication , Weight Loss
19.
J Allergy Clin Immunol ; 148(2): 368-380.e3, 2021 08.
Article in English | MEDLINE | ID: covidwho-1260767

ABSTRACT

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can lead to a variety of clinical outcomes, ranging from the absence of symptoms to severe acute respiratory disease and ultimately death. A feature of patients with severe coronavirus disease 2019 (COVID-19) is the abundance of inflammatory cytokines in the blood. Elevated levels of cytokines are predictive of infection severity and clinical outcome. In contrast, studies aimed at defining the driving forces behind the inflammation in lungs of subjects with severe COVID-19 remain scarce. OBJECTIVE: Our aim was to analyze and compare the plasma and bronchoalveolar lavage (BAL) fluids of patients with severe COVID-19 (n = 45) for the presence of cytokines and lipid mediators of inflammation (LMIs). METHODS: Cytokines were measured by using Luminex multiplex assay, and LMIs were measured by using liquid chromatography-tandem mass spectrometry. RESULTS: We revealed high concentrations of numerous cytokines, chemokines, and LMIs in the BAL fluid of patients with severe COVID-19. Of the 13 most abundant mediators in BAL fluid, 11 were chemokines, with CXCL1 and CXCL8 being 200 times more abundant than IL-6 and TNF-α. Eicosanoid levels were also elevated in the lungs of subjects with severe COVID-19. Consistent with the presence chemotactic molecules, BAL fluid samples were enriched for neutrophils, lymphocytes, and eosinophils. Inflammatory cytokines and LMIs in plasma showed limited correlations with those present in BAL fluid, arguing that circulating inflammatory molecules may not be a reliable proxy of the inflammation occurring in the lungs of patients with severe COVID-19. CONCLUSIONS: Our findings indicate that hyperinflammation of the lungs of patients with severe COVID-19 is fueled by excessive production of chemokines and eicosanoids. Therapeutic strategies to dampen inflammation in patients with COVID-19 should be tailored accordingly.


Subject(s)
COVID-19/immunology , Cytokines/immunology , Eicosanoids/immunology , Inflammation/immunology , Lung/immunology , SARS-CoV-2 , Adult , Aged , Bronchoalveolar Lavage Fluid/cytology , Bronchoalveolar Lavage Fluid/immunology , COVID-19/blood , Cytokines/blood , Female , Humans , Inflammation/blood , Lung/cytology , Lymphocytes/immunology , Male , Middle Aged , Neutrophils/immunology , Severity of Illness Index
20.
Viruses ; 13(6)2021 05 21.
Article in English | MEDLINE | ID: covidwho-1244141

ABSTRACT

Patients with coronavirus disease 2019 (COVID-19) predominantly have a respiratory tract infection with various symptoms and high mortality is associated with respiratory failure second to severe disease. The risk factors leading to severe disease remain unclear. Here, we reanalyzed a published single-cell RNA-Seq (scRNA-Seq) dataset and found that bronchoalveolar lavage fluid (BALF) of patients with severe disease compared to those with mild disease contained decreased TH17-type cells, decreased IFNA1-expressing cells with lower expression of toll-like receptor 7 (TLR7) and TLR8, increased IgA-expressing B cells, and increased hyperactive epithelial cells (and/or macrophages) expressing matrix metalloproteinases (MMPs), hyaluronan synthase 2 (HAS2), and plasminogen activator inhibitor-1 (PAI-1), which may together contribute to the pulmonary pathology in severe COVID-19. We propose IFN-I (and TLR7/TLR8) and PAI-1 as potential biomarkers to predict the susceptibility to severe COVID-19.


Subject(s)
COVID-19/pathology , Lung/pathology , B-Lymphocytes/metabolism , B-Lymphocytes/pathology , Biomarkers/metabolism , Bronchoalveolar Lavage Fluid/immunology , COVID-19/immunology , COVID-19/metabolism , Databases, Genetic , Humans , Hyaluronan Synthases/metabolism , Immunoglobulin A/metabolism , Interferon-alpha/metabolism , Lung/immunology , Lung/metabolism , Matrix Metalloproteinases/metabolism , Mucin-1/metabolism , Plasminogen Activator Inhibitor 1/metabolism , RNA-Seq , SARS-CoV-2 , Th17 Cells/metabolism , Th17 Cells/pathology
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