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1.
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
2.
Nat Immunol ; 23(1): 62-74, 2022 01.
Article in English | MEDLINE | ID: covidwho-1514418

ABSTRACT

The molecular mechanisms governing orderly shutdown and retraction of CD4+ type 1 helper T (TH1) cell responses remain poorly understood. Here we show that complement triggers contraction of TH1 responses by inducing intrinsic expression of the vitamin D (VitD) receptor and the VitD-activating enzyme CYP27B1, permitting T cells to both activate and respond to VitD. VitD then initiated the transition from pro-inflammatory interferon-γ+ TH1 cells to suppressive interleukin-10+ cells. This process was primed by dynamic changes in the epigenetic landscape of CD4+ T cells, generating super-enhancers and recruiting several transcription factors, notably c-JUN, STAT3 and BACH2, which together with VitD receptor shaped the transcriptional response to VitD. Accordingly, VitD did not induce interleukin-10 expression in cells with dysfunctional BACH2 or STAT3. Bronchoalveolar lavage fluid CD4+ T cells of patients with COVID-19 were TH1-skewed and showed de-repression of genes downregulated by VitD, from either lack of substrate (VitD deficiency) and/or abnormal regulation of this system.


Subject(s)
Interferon-gamma/immunology , Interleukin-10/immunology , SARS-CoV-2/immunology , Th1 Cells/immunology , Vitamin D/metabolism , 25-Hydroxyvitamin D3 1-alpha-Hydroxylase/metabolism , Basic-Leucine Zipper Transcription Factors/metabolism , Bronchoalveolar Lavage Fluid/cytology , COVID-19/immunology , COVID-19/pathology , Complement C3a/immunology , Complement C3b/immunology , Humans , JNK Mitogen-Activated Protein Kinases/metabolism , Lymphocyte Activation/immunology , Receptors, Calcitriol/metabolism , Respiratory Distress Syndrome/immunology , Respiratory Distress Syndrome/pathology , Respiratory Distress Syndrome/virology , STAT3 Transcription Factor/metabolism , Signal Transduction/immunology , Transcription, Genetic/genetics
3.
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
4.
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
5.
PLoS One ; 16(10): e0254985, 2021.
Article in English | MEDLINE | ID: covidwho-1448572

ABSTRACT

BACKGROUND: The goal of this study was to determine if IL-22:Fc would Acute Respiratory Distress Syndrome (ARDS). SUMMARY BACKGROUND DATA: No therapies exist for ARDS and treatment is purely supportive. Interleukin-22 (IL-22) plays an integral component in recovery of the lung from infection. IL-22:Fc is a recombinant protein with a human FC immunoglobulin that increases the half-life of IL-22. STUDY DESIGN: ARDS was induced in C57BL/6 mice with intra-tracheal lipopolysaccharide (LPS) at a dose of 33.3 or 100 ug. In the low-dose LPS group (LDG), IL-22:FC was administered via tail vein injection at 30 minutes (n = 9) and compared to sham (n = 9). In the high-dose LPS group (HDG), IL-22:FC was administered (n = 11) then compared to sham (n = 8). Euthanasia occurred after bronchioalveolar lavage (BAL) on post-injury day 4. RESULTS: In the LDG, IL-22:FC resulted in decreased protein leak (0.15 vs. 0.25 ug/uL, p = 0.02). BAL protein in animals receiving IL-22:Fc in the HDG was not different. For the HDG, animals receiving IL-22:Fc had lower BAL cell counts (539,636 vs 3,147,556 cells/uL, p = 0.02). For the HDG, IL-6 (110.6 vs. 527.1 pg/mL, p = 0.04), TNF-α (5.87 vs. 25.41 pg/mL, p = 0.04), and G-CSF (95.14 vs. 659.6, p = 0.01) levels were lower in the BAL fluid of IL-22:Fc treated animals compared to sham. CONCLUSIONS: IL-22:Fc decreases lung inflammation and lung capillary leak in ARDS. IL-22:Fc may be a novel therapy for ARDS.


Subject(s)
Immunoglobulin Fc Fragments/pharmacology , Interleukins/pharmacology , Lung Injury/drug therapy , Pneumonia/drug therapy , Respiratory Distress Syndrome/drug therapy , Animals , Bronchoalveolar Lavage Fluid/chemistry , Bronchoalveolar Lavage Fluid/cytology , Female , Lipopolysaccharides/toxicity , Lung Injury/pathology , Lymphocyte Count , Lymphocytes/immunology , Macrophages/immunology , Male , Mice , Mice, Inbred C57BL , Neutrophils/immunology , Pneumonia/pathology , Receptors, Interleukin/metabolism , Recombinant Proteins/pharmacology , Respiratory Distress Syndrome/pathology , Respiratory Mucosa/pathology
7.
Front Immunol ; 12: 658428, 2021.
Article in English | MEDLINE | ID: covidwho-1389178

ABSTRACT

SARS-CoV-2 virus causes upper and lower respiratory diseases including pneumonia, and in some cases, leads to lethal pulmonary failure. Angiotensin converting enzyme-2 (ACE2), the receptor for cellular entry of SARS-CoV-2 virus, has been shown to protect against severe acute lung failure. Here, we provide evidence that SARS-CoV-2 spike protein S1 reduced the mRNA expression of ACE2 and type I interferons in primary cells of lung bronchoalveolar lavage (BAL) from naïve rhesus macaques. The expression levels of ACE2 and type I interferons were also found to be correlated with each other, consistent with the recent finding that ACE2 is an interferon-inducible gene. Furthermore, induction of ACE2 and type I interferons by poly I:C, an interferon inducer, was suppressed by S1 protein in primary cells of BAL. These observations suggest that the downregulation of ACE2 and type I interferons induced by S1 protein may directly contribute to SARS-CoV-2-associated lung diseases.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19 , Interferon Type I/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Animals , Bronchoalveolar Lavage Fluid/cytology , Macaca mulatta , SARS-CoV-2
8.
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
9.
BMC Complement Med Ther ; 21(1): 192, 2021 Jul 05.
Article in English | MEDLINE | ID: covidwho-1317123

ABSTRACT

BACKGROUND: Lippia javanica (lemon bush) is commonly used in the treatment of respiratory ailments, including asthma in southern African countries but there is no scientific evidence to support this claim. This study investigated the anti-inflammatory, antioxidant and anti-asthmatic effects of L. javanica using a rat model of asthma. METHODS: A 5% w/v L. javanica tea infusion was prepared and characterised by liquid chromatography-mass spectrometer (LC-MS). Animals were intraperitoneally sensitized with ovalbumin (OVA) and subsequently challenged intranasal with OVA on day 15 except the control group. Animals were grouped (n = 5/group) for treatment: unsensitised control, sensitised control, sensitised + prednisolone and sensitised + L. javanica at 50 mg/kg/day and 100 mg/kg/day - equivalent to 1 and 2 cups of tea per day, respectively. After 2 weeks of treatment, bronchoalveolar lavage fluid (BALF) was collected for total and differential white blood cell (WBC) count. Nitric oxide (NO), lipid peroxidation and antioxidants were also assessed in BALF. Ovalbumin specific IgE antibody and inflammatory cytokines: IL-4, IL-5, IL-13 and TNF-alpha were measured in serum. Lung and muscle tissues were histological examined. RESULTS: L. javanica was rich in phenolic compounds. OVA sensitisation resulted in development of allergic asthma in rats. L. javanica treatment resulted in a reduction in total WBC count as well as eosinophils, lymphocytes and neutrophils in BALF. L. javanica inhibited Th2-mediated immune response, which was evident by a decrease in serum IgE and inflammatory cytokines: IL-4, IL-5, IL-13 and TNF-α. L. javanica treatment also reduced malondialdehyde (MDA) and NO, and increased superoxide dismutase, glutathione and total antioxidant capacity. Histology showed significant attenuation of lung infiltration of inflammatory cells, alveolar thickening, and bronchiole smooth muscle thickening. CONCLUSION: L. javanica suppressed allergic airway inflammation by reducing Th2-mediated immune response and oxidative stress in OVA-sensitized rats which may be attributed to the presence of phenolic compound in the plant. This finding validates the traditional use of L. javanica in the treatment of respiratory disorders.


Subject(s)
Asthma/drug therapy , Lippia , Teas, Herbal , Animals , Antioxidants/metabolism , Asthma/pathology , Bronchoalveolar Lavage Fluid/cytology , Cytokines/blood , Disease Models, Animal , Eosinophils/metabolism , Glutathione/metabolism , Immunoglobulin E/blood , Leukocyte Count , Lung/pathology , Lymphocytes/metabolism , Malondialdehyde/metabolism , Neutrophils/metabolism , Nitric Oxide/metabolism , Oxidative Stress/drug effects , Rats, Wistar , Superoxide Dismutase/metabolism , Th2 Cells/drug effects
10.
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
11.
BMC Complement Med Ther ; 21(1): 192, 2021 Jul 05.
Article in English | MEDLINE | ID: covidwho-1296592

ABSTRACT

BACKGROUND: Lippia javanica (lemon bush) is commonly used in the treatment of respiratory ailments, including asthma in southern African countries but there is no scientific evidence to support this claim. This study investigated the anti-inflammatory, antioxidant and anti-asthmatic effects of L. javanica using a rat model of asthma. METHODS: A 5% w/v L. javanica tea infusion was prepared and characterised by liquid chromatography-mass spectrometer (LC-MS). Animals were intraperitoneally sensitized with ovalbumin (OVA) and subsequently challenged intranasal with OVA on day 15 except the control group. Animals were grouped (n = 5/group) for treatment: unsensitised control, sensitised control, sensitised + prednisolone and sensitised + L. javanica at 50 mg/kg/day and 100 mg/kg/day - equivalent to 1 and 2 cups of tea per day, respectively. After 2 weeks of treatment, bronchoalveolar lavage fluid (BALF) was collected for total and differential white blood cell (WBC) count. Nitric oxide (NO), lipid peroxidation and antioxidants were also assessed in BALF. Ovalbumin specific IgE antibody and inflammatory cytokines: IL-4, IL-5, IL-13 and TNF-alpha were measured in serum. Lung and muscle tissues were histological examined. RESULTS: L. javanica was rich in phenolic compounds. OVA sensitisation resulted in development of allergic asthma in rats. L. javanica treatment resulted in a reduction in total WBC count as well as eosinophils, lymphocytes and neutrophils in BALF. L. javanica inhibited Th2-mediated immune response, which was evident by a decrease in serum IgE and inflammatory cytokines: IL-4, IL-5, IL-13 and TNF-α. L. javanica treatment also reduced malondialdehyde (MDA) and NO, and increased superoxide dismutase, glutathione and total antioxidant capacity. Histology showed significant attenuation of lung infiltration of inflammatory cells, alveolar thickening, and bronchiole smooth muscle thickening. CONCLUSION: L. javanica suppressed allergic airway inflammation by reducing Th2-mediated immune response and oxidative stress in OVA-sensitized rats which may be attributed to the presence of phenolic compound in the plant. This finding validates the traditional use of L. javanica in the treatment of respiratory disorders.


Subject(s)
Asthma/drug therapy , Lippia , Teas, Herbal , Animals , Antioxidants/metabolism , Asthma/pathology , Bronchoalveolar Lavage Fluid/cytology , Cytokines/blood , Disease Models, Animal , Eosinophils/metabolism , Glutathione/metabolism , Immunoglobulin E/blood , Leukocyte Count , Lung/pathology , Lymphocytes/metabolism , Malondialdehyde/metabolism , Neutrophils/metabolism , Nitric Oxide/metabolism , Oxidative Stress/drug effects , Rats, Wistar , Superoxide Dismutase/metabolism , Th2 Cells/drug effects
12.
Front Immunol ; 12: 663303, 2021.
Article in English | MEDLINE | ID: covidwho-1291384

ABSTRACT

The release of neutrophil extracellular traps (NETs), a process termed NETosis, avoids pathogen spread but may cause tissue injury. NETs have been found in severe COVID-19 patients, but their role in disease development is still unknown. The aim of this study is to assess the capacity of NETs to drive epithelial-mesenchymal transition (EMT) of lung epithelial cells and to analyze the involvement of NETs in COVID-19. Bronchoalveolar lavage fluid of severe COVID-19 patients showed high concentration of NETs that correlates with neutrophils count; moreover, the analysis of lung tissues of COVID-19 deceased patients showed a subset of alveolar reactive pneumocytes with a co-expression of epithelial marker and a mesenchymal marker, confirming the induction of EMT mechanism after severe SARS-CoV2 infection. By airway in vitro models, cultivating A549 or 16HBE at air-liquid interface, adding alveolar macrophages (AM), neutrophils and SARS-CoV2, we demonstrated that to trigger a complete EMT expression pattern are necessary the induction of NETosis by SARS-CoV2 and the secretion of AM factors (TGF-ß, IL8 and IL1ß). All our results highlight the possible mechanism that can induce lung fibrosis after SARS-CoV2 infection.


Subject(s)
COVID-19/physiopathology , Epithelial-Mesenchymal Transition , Extracellular Traps/metabolism , Neutrophils/metabolism , Adult , Biopsy , Bronchoalveolar Lavage Fluid/cytology , COVID-19/complications , COVID-19/immunology , Cell Line , Epithelial Cells/pathology , Humans , Lung/pathology , Pulmonary Fibrosis/etiology , Pulmonary Fibrosis/metabolism
13.
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
14.
Sci Rep ; 11(1): 11462, 2021 06 01.
Article in English | MEDLINE | ID: covidwho-1253984

ABSTRACT

An excessive immune response known as cytokine storm is the hallmark of severe COVID-19. The cause of this cytokine rampage is yet not known. Based on recent epidemiological evidence, we hypothesized that CD80/86 signaling is essential for this hyperinflammation, and that blocking this proinflammatory axis could be an effective therapeutic approach to protect against severe COVID-19. Here we provide exploratory evidence that abatacept, a drug that blocks CD80/86 co-stimulation, produces changes at the systemic level that are highly antagonistic of the proinflammatory processes elicited by COVID-19. Using RNA-seq from blood samples from a longitudinal cohort of n = 38 rheumatic patients treated with abatacept, we determined the immunological processes that are significantly regulated by this treatment. We then analyzed available blood RNA-seq from two COVID19 patient cohorts, a very early cohort from the epicenter of the pandemic in China (n = 3 COVID-19 cases and n = 3 controls), and a recent and larger cohort from the USA (n = 49 severe and n = 51 mild COVD-19 patients). We found a highly significant antagonism between SARS-CoV-2 infection and COVID-19 severity with the systemic response to abatacept. Analysis of previous single-cell RNA-seq data from bronchoalveolar lavage fluid from mild and severe COVID-19 patients and controls, reinforce the implication of the CD80/86 proinflammatory axis. Our functional results further support abatacept as a candidate therapeutic approach to prevent severe COVID-19.


Subject(s)
Abatacept/pharmacology , COVID-19/drug therapy , Cytokine Release Syndrome/prevention & control , Immunosuppressive Agents/pharmacology , SARS-CoV-2/immunology , Signal Transduction/drug effects , Abatacept/therapeutic use , Aged , Arthritis, Rheumatoid/blood , Arthritis, Rheumatoid/drug therapy , Arthritis, Rheumatoid/immunology , B7-1 Antigen/metabolism , B7-2 Antigen/metabolism , Bronchoalveolar Lavage Fluid/cytology , COVID-19/blood , COVID-19/complications , COVID-19/immunology , China , Cytokine Release Syndrome/immunology , Cytokine Release Syndrome/virology , Female , Humans , Immunosuppressive Agents/therapeutic use , Male , Middle Aged , Observational Studies as Topic , RNA-Seq , Severity of Illness Index , Signal Transduction/immunology , Single-Cell Analysis , Spain , United States , Up-Regulation/drug effects , Up-Regulation/immunology
15.
Hum Pathol ; 113: 92-103, 2021 07.
Article in English | MEDLINE | ID: covidwho-1201239

ABSTRACT

Information on bronchoalveolar lavage (BAL) in patients with COVID-19 is limited, and clinical correlation has not been reported. This study investigated the key features of BAL fluids from COVID-19 patients and assessed their clinical significance. A total of 320 BAL samples from 83 COVID-19 patients and 70 non-COVID-19 patients (27 patients with other respiratory viral infections) were evaluated, including cell count/differential, morphology, flow cytometric immunophenotyping, and immunohistochemistry. The findings were correlated with clinical outcomes. Compared to non-COVID-19 patients, BAL from COVID-19 patients was characterized by significant lymphocytosis (p < 0.001), in contrast to peripheral blood lymphopenia commonly observed in COVID-19 patients and the presence of atypical lymphocytes with plasmacytoid/plasmablastic features (p < 0.001). Flow cytometry and immunohistochemistry demonstrated that BAL lymphocytes, including plasmacytoid and plasmablastic cells, were composed predominantly of T cells with a mixture of CD4+ and CD8+ cells. Both populations had increased expression of T-cell activation markers, suggesting important roles of helper and cytotoxic T-cells in the immune response to SARS-CoV-2 infection in the lung. More importantly, BAL lymphocytosis was significantly associated with longer hospital stay (p < 0.05) and longer requirement for mechanical ventilation (p < 0.05), whereas the median atypical (activated) lymphocyte count was associated with shorter hospital stay (p < 0.05), shorter time on mechanical ventilation (p < 0.05) and improved survival. Our results indicate that BAL cellular analysis and morphologic findings provide additional important information for diagnostic and prognostic work-up, and potential new therapeutic strategies for patients with severe COVID-19.


Subject(s)
Bronchoalveolar Lavage Fluid/immunology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , COVID-19/immunology , Lung/immunology , Adult , Aged , Aged, 80 and over , Bronchoalveolar Lavage Fluid/cytology , Female , Humans , Male , Middle Aged , SARS-CoV-2
16.
J Med Virol ; 93(3): 1520-1525, 2021 03.
Article in English | MEDLINE | ID: covidwho-1196469

ABSTRACT

In Coronavirus disease-2019 (COVID-19) cases, hyper inflammation is associated with the severity of the disease. High levels of circulating cytokines were reported in severe COVID-19 patients. Neopterin produced by macrophages on stimulation with interferon-gamma, which is an important cytokine in the antiviral immune response, hence it can be used to predict the severity of disease in COVID-19 cases. In this study, it was aimed to determine the prognostic value of the neopterin for the prediction of severe disease in patients with COVID-19. This single-center, prospective study was conducted in hospitalized COVID-19 patients and healthy volunteers. Severe and mild COVID-19 cases were compared in terms of clinical and laboratory findings as well as serum neopterin levels on hospital admission. To assess the prognostic utility of neopterin between the severe and mild COVID-19 groups, a receiver-operating characteristic (ROC) curve was generated, and the area under the curve (AUC) was calculated. The median serum neopterin level was four times higher in COVID-19 patients than the healthy controls (46 vs. 12 nmol/L; p < .001). The AUC value of serum neopterin was 0.914 (95% confidence interval, 0.85-0.97). The sensitivity and specificity of serum neopterin for the cut-off value of 90 nmol/L to identify severe COVID-19 cases were 100% and 76%, respectively. Serum neopterin levels on hospitalization were significantly higher in severe COVID-19 disease than mild COVID-19 patients. Neopterin levels can be used as an early prognostic biomarker for COVID-19 on admission.


Subject(s)
COVID-19/diagnosis , Interferon-gamma/immunology , Macrophages/immunology , Neopterin/blood , Adult , Biomarkers/blood , Bronchoalveolar Lavage Fluid/cytology , COVID-19/mortality , COVID-19/pathology , Cytokines/blood , Female , Humans , Male , Middle Aged , Prognosis , SARS-CoV-2/immunology , Severity of Illness Index , Young Adult
17.
Genome Med ; 13(1): 64, 2021 04 20.
Article in English | MEDLINE | ID: covidwho-1195928

ABSTRACT

BACKGROUND: Immunosuppressive and anti-cytokine treatment may have a protective effect for patients with COVID-19. Understanding the immune cell states shared between COVID-19 and other inflammatory diseases with established therapies may help nominate immunomodulatory therapies. METHODS: To identify cellular phenotypes that may be shared across tissues affected by disparate inflammatory diseases, we developed a meta-analysis and integration pipeline that models and removes the effects of technology, tissue of origin, and donor that confound cell-type identification. Using this approach, we integrated > 300,000 single-cell transcriptomic profiles from COVID-19-affected lungs and tissues from healthy subjects and patients with five inflammatory diseases: rheumatoid arthritis (RA), Crohn's disease (CD), ulcerative colitis (UC), systemic lupus erythematosus (SLE), and interstitial lung disease. We tested the association of shared immune states with severe/inflamed status compared to healthy control using mixed-effects modeling. To define environmental factors within these tissues that shape shared macrophage phenotypes, we stimulated human blood-derived macrophages with defined combinations of inflammatory factors, emphasizing in particular antiviral interferons IFN-beta (IFN-ß) and IFN-gamma (IFN-γ), and pro-inflammatory cytokines such as TNF. RESULTS: We built an immune cell reference consisting of > 300,000 single-cell profiles from 125 healthy or disease-affected donors from COVID-19 and five inflammatory diseases. We observed a CXCL10+ CCL2+ inflammatory macrophage state that is shared and strikingly abundant in severe COVID-19 bronchoalveolar lavage samples, inflamed RA synovium, inflamed CD ileum, and UC colon. These cells exhibited a distinct arrangement of pro-inflammatory and interferon response genes, including elevated levels of CXCL10, CXCL9, CCL2, CCL3, GBP1, STAT1, and IL1B. Further, we found this macrophage phenotype is induced upon co-stimulation by IFN-γ and TNF-α. CONCLUSIONS: Our integrative analysis identified immune cell states shared across inflamed tissues affected by inflammatory diseases and COVID-19. Our study supports a key role for IFN-γ together with TNF-α in driving an abundant inflammatory macrophage phenotype in severe COVID-19-affected lungs, as well as inflamed RA synovium, CD ileum, and UC colon, which may be targeted by existing immunomodulatory therapies.


Subject(s)
COVID-19/immunology , Cytokines/immunology , Macrophages/immunology , SARS-CoV-2 , Arthritis, Rheumatoid/genetics , Arthritis, Rheumatoid/immunology , Bronchoalveolar Lavage Fluid/cytology , Bronchoalveolar Lavage Fluid/immunology , COVID-19/genetics , Colitis, Ulcerative/genetics , Colitis, Ulcerative/immunology , Colon/immunology , Crohn Disease/genetics , Crohn Disease/immunology , Humans , Inflammation/genetics , Inflammation/immunology , Lung/immunology , Lung Diseases, Interstitial/genetics , Lung Diseases, Interstitial/immunology , Lupus Erythematosus, Systemic/genetics , Lupus Erythematosus, Systemic/immunology , Phenotype , RNA-Seq
18.
BMC Infect Dis ; 21(1): 353, 2021 Apr 15.
Article in English | MEDLINE | ID: covidwho-1190057

ABSTRACT

BACKGROUND: The primary objective of the study is to describe the cellular characteristics of bronchoalveolar lavage fluid (BALF) of COVID-19 patients requiring invasive mechanical ventilation; the secondary outcome is to describe BALF findings between survivors vs non-survivors. MATERIALS AND METHODS: Patients positive for SARS-CoV-2 RT PCR, admitted to ICU between March and April 2020 were enrolled. At ICU admission, BALF were analyzed by flow cytometry. Univariate, multivariate and Spearman correlation analyses were performed. RESULTS: Sixty-four patients were enrolled, median age of 64 years (IQR 58-69). The majority cells in the BALF were neutrophils (70%, IQR 37.5-90.5) and macrophages (27%, IQR 7-49) while a minority were lymphocytes, 1%, TCD3+ 92% (IQR 82-95). The ICU mortality was 32.8%. Non-survivors had a significantly older age (p = 0.033) and peripheral lymphocytes (p = 0.012) were lower compared to the survivors. At multivariate analysis the percentage of macrophages in the BALF correlated with poor outcome (OR 1.336, CI95% 1.014-1.759, p = 0.039). CONCLUSIONS: In critically ill patients, BALF cellularity is mainly composed of neutrophils and macrophages. The macrophages percentage in the BALF at ICU admittance correlated with higher ICU mortality. The lack of lymphocytes in BALF could partly explain a reduced anti-viral response.


Subject(s)
Bronchoalveolar Lavage Fluid/cytology , Bronchoalveolar Lavage Fluid/immunology , COVID-19/epidemiology , COVID-19/immunology , Leukocyte Count , Pneumonia, Viral/epidemiology , Pneumonia, Viral/immunology , Respiration, Artificial , Adult , Aged , Bronchoalveolar Lavage Fluid/virology , COVID-19/mortality , COVID-19/virology , Critical Illness/epidemiology , Female , Humans , Intensive Care Units , Italy/epidemiology , Lymphocytes/cytology , Macrophages/cytology , Male , Middle Aged , Neutrophils/cytology , Pneumonia, Viral/mortality , Pneumonia, Viral/virology , SARS-CoV-2/immunology , SARS-CoV-2/pathogenicity , Survivors/statistics & numerical data , Treatment Outcome
19.
Thorax ; 76(10): 1010-1019, 2021 10.
Article in English | MEDLINE | ID: covidwho-1180971

ABSTRACT

BACKGROUND: Knowledge of the pathophysiology of COVID-19 is almost exclusively derived from studies that examined the immune response in blood. We here aimed to analyse the pulmonary immune response during severe COVID-19 and to compare this with blood responses. METHODS: This was an observational study in patients with COVID-19 admitted to the intensive care unit (ICU). Mononuclear cells were purified from bronchoalveolar lavage fluid (BALF) and blood, and analysed by spectral flow cytometry; inflammatory mediators were measured in BALF and plasma. FINDINGS: Paired blood and BALF samples were obtained from 17 patients, four of whom died in the ICU. Macrophages and T cells were the most abundant cells in BALF, with a high percentage of T cells expressing the ƴδ T cell receptor. In the lungs, both CD4 and CD8 T cells were predominantly effector memory cells (87·3% and 83·8%, respectively), and these cells expressed higher levels of the exhaustion marker programmad death-1 than in peripheral blood. Prolonged ICU stay (>14 days) was associated with a reduced proportion of activated T cells in peripheral blood and even more so in BALF. T cell activation in blood, but not in BALF, was higher in fatal COVID-19 cases. Increased levels of inflammatory mediators were more pronounced in BALF than in plasma. INTERPRETATION: The bronchoalveolar immune response in COVID-19 has a unique local profile that strongly differs from the immune profile in peripheral blood. Fully elucidating COVID-19 pathophysiology will require investigation of the pulmonary immune response.


Subject(s)
COVID-19/immunology , Immunity, Cellular/physiology , Inflammation Mediators/metabolism , Aged , Bronchoalveolar Lavage Fluid/chemistry , Bronchoalveolar Lavage Fluid/cytology , COVID-19/blood , COVID-19/pathology , Critical Care , Critical Illness , Female , Flow Cytometry , Humans , Macrophages/physiology , Male , Middle Aged , T-Lymphocytes/physiology
20.
Cells ; 10(3)2021 03 10.
Article in English | MEDLINE | ID: covidwho-1125934

ABSTRACT

We herein characterize the immunopathological features of two Italian COVID-19 patients who underwent bilateral lung transplantation (bLTx). Removed lungs underwent histopathological evaluation. Gene expression profiling (GEP) for immune-related signatures was performed on lung specimens and SARS-CoV-2-stimulated peripheral blood mononuclear cells (PBMCs). Cytokine levels were measured on lungs, bronchoalveolar lavage fluids and in culture supernatants. Pathological assessment showed extensive lung damage with the pattern of proliferative to fibrotic phases, with diffuse alveolar damage mimicking usual interstitial pneumonia (UIP). Lungs' GEP revealed overexpression of pathogen recognition receptors, effector cytokines and chemokines, immune activation receptors and of the inflammasome components. Multiplex cytokine analysis confirmed a proinflammatory state, with high levels of monocyte/macrophage chemotactic and activating factors and of IL-6 and TNF-α. A similar profile was observed in SARS-CoV-2-stimulated PBMCs collected 7 days after transplant. The pattern of tissue damage observed in the lungs suggests that this may represent the output of protracted disease, resembling a diffuse UIP-like picture. The molecular immune profiling supports the paradigm of a persistent proinflammatory state and sustained humoral immunity, conditions that are maintained despite the iatrogenic immunosuppression.


Subject(s)
COVID-19/surgery , Chemokines/metabolism , Cytokines/metabolism , Leukocytes, Mononuclear/metabolism , Lung Transplantation , Lung/pathology , Respiratory Distress Syndrome/surgery , Adolescent , Bronchoalveolar Lavage Fluid/cytology , Bronchoalveolar Lavage Fluid/virology , COVID-19/blood , Gene Expression Profiling , Gene Expression Regulation/immunology , Genotype , Humans , Inflammasomes/metabolism , Interleukin-6/metabolism , Lung/immunology , Lung/metabolism , Lung/virology , Lymph Nodes/pathology , Lymph Nodes/virology , Male , Middle Aged , Plasma/virology , Polymorphism, Single Nucleotide , Respiratory Distress Syndrome/immunology , Respiratory Distress Syndrome/pathology , Respiratory Distress Syndrome/virology , Tumor Necrosis Factor-alpha/metabolism
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