ScRNA-Seq study of neutrophils reveals vast heterogeneity and breadth of inflammatory responses in severe COVID-19 patients

AbstractSevere cases of COVID-19 are characterized by dysregulated immune responses, but specific mechanisms contributing to the most severe outcomes remain unclear. Neutrophils are the most abundant leukocyte population in human hosts and reach markedly high numbers during severe COVID-19. However, a detailed examination of their responses has been largely overlooked in the COVID-19 literature to date. Here, we report for the first time a dedicated study of neutrophil responses using single-cell RNA sequencing (scRNA-Seq) of fresh leukocytes from 11 hospitalized adult patients with mild and severe COVID-19 disease and 5 healthy controls. We observed that neutrophils display a pronounced inflammatory profile, with dramatic disruption of predicted cell-cell interactions as the severity of the disease increases. We also identified unique mature and immature neutrophil subpopulations based on transcriptomic profiling, including an antiviral phenotype, and changes in the proportion of each population linked to the severity of the disease. Finally, pathway analysis revealed increased markers of oxidative phosphorylation and ribosomal genes, along with downregulation of many antiviral and host defense pathway genes during severe disease compared to mild infections. Collectively, our findings indicate that neutrophils are capable of mounting effective antiviral defenses but adopt a form of immune dysregulation characterized by excess cellular stress, thereby contributing to the pathogenesis of severe COVID-19.

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Pattern of inflammatory immune response determines the clinical course and outcome of COVID-19: unbiased clustering analysis

Scientific Reports ◽

10.1038/s41598-021-87668-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Eunyoung Emily Lee ◽

Kyoung-Ho Song ◽

Woochang Hwang ◽

Sin Young Ham ◽

Hyeonju Jeong ◽

...

Keyword(s):

Immune Response ◽

Immune Responses ◽

Clustering Analysis ◽

Validation Cohort ◽

Inflammatory Responses ◽

Clinical Course ◽

Severe Disease ◽

Outcome Data ◽

Inflammatory Immune Response ◽

Cluster 2

AbstractThe objective of the study was to identify distinct patterns in inflammatory immune responses of COVID-19 patients and to investigate their association with clinical course and outcome. Data from hospitalized COVID-19 patients were retrieved from electronic medical record. Supervised k-means clustering of serial C-reactive protein levels (CRP), absolute neutrophil counts (ANC), and absolute lymphocyte counts (ALC) was used to assign immune responses to one of three groups. Then, relationships between patterns of inflammatory responses and clinical course and outcome of COVID-19 were assessed in a discovery and validation cohort. Unbiased clustering analysis grouped 105 patients of a discovery cohort into three distinct clusters. Cluster 1 (hyper-inflammatory immune response) was characterized by high CRP levels, high ANC, and low ALC, whereas Cluster 3 (hypo-inflammatory immune response) was associated with low CRP levels and normal ANC and ALC. Cluster 2 showed an intermediate pattern. All patients in Cluster 1 required oxygen support whilst 61% patients in Cluster 2 and no patient in Cluster 3 required supplementary oxygen. Two (13.3%) patients in Cluster 1 died, whereas no patient in Clusters 2 and 3 died. The results were confirmed in an independent validation cohort of 116 patients. We identified three different patterns of inflammatory immune response to COVID-19. Hyper-inflammatory immune responses with elevated CRP, neutrophilia, and lymphopenia are associated with a severe disease and a worse outcome. Therefore, targeting the hyper-inflammatory response might improve the clinical outcome of COVID-19.

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Impact of STING Inflammatory Signaling during Intracellular Bacterial Infections

Cells ◽

10.3390/cells11010074 ◽

2021 ◽

Vol 11 (1) ◽

pp. 74

Author(s):

Erika S. Guimarães ◽

Fabio V. Marinho ◽

Nina M. G. P. de Queiroz ◽

Maísa M. Antunes ◽

Sergio C. Oliveira

Keyword(s):

Immune Responses ◽

Bacterial Infections ◽

Inflammatory Responses ◽

Metabolic Reprogramming ◽

Host Cells ◽

Type I Interferons ◽

Type I ◽

Bacterial Survival ◽

Inflammatory Profile ◽

The Impact

The early detection of bacterial pathogens through immune sensors is an essential step in innate immunity. STING (Stimulator of Interferon Genes) has emerged as a key mediator of inflammation in the setting of infection by connecting pathogen cytosolic recognition with immune responses. STING detects bacteria by directly recognizing cyclic dinucleotides or indirectly by bacterial genomic DNA sensing through the cyclic GMP-AMP synthase (cGAS). Upon activation, STING triggers a plethora of powerful signaling pathways, including the production of type I interferons and proinflammatory cytokines. STING activation has also been associated with the induction of endoplasmic reticulum (ER) stress and the associated inflammatory responses. Recent reports indicate that STING-dependent pathways participate in the metabolic reprogramming of macrophages and contribute to the establishment and maintenance of a robust inflammatory profile. The induction of this inflammatory state is typically antimicrobial and related to pathogen clearance. However, depending on the infection, STING-mediated immune responses can be detrimental to the host, facilitating bacterial survival, indicating an intricate balance between immune signaling and inflammation during bacterial infections. In this paper, we review recent insights regarding the role of STING in inducing an inflammatory profile upon intracellular bacterial entry in host cells and discuss the impact of STING signaling on the outcome of infection. Unraveling the STING-mediated inflammatory responses can enable a better understanding of the pathogenesis of certain bacterial diseases and reveal the potential of new antimicrobial therapy.

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A30 DIFFERENCES BETWEEN HUMAN AND MOUSE INTESTINAL EPITHELIAL CELLS IN THEIR INNATE IMMUNE RESPONSES TO BACTERIAL AND HOST STIMULI.

Journal of the Canadian Association of Gastroenterology ◽

10.1093/jcag/gwab002.029 ◽

2021 ◽

Vol 4 (Supplement_1) ◽

pp. 2-3

Author(s):

J M Allaire ◽

A Poon ◽

S M Crowley ◽

X Han ◽

M Stahl ◽

...

Keyword(s):

Epithelial Cells ◽

Immune Responses ◽

Inflammatory Responses ◽

Intestinal Epithelial Cells ◽

Innate Immune ◽

Negative Regulator ◽

Intestinal Epithelial ◽

Innate Immune Responses ◽

Transformed Cell Lines ◽

First Time

Abstract Background Intestinal epithelial cells (IEC) reside in close contact with the gut microbiota. It is thus important that IEC are hypo-responsive to bacterial products to prevent maladaptive inflammatory responses in the gut, such as those seen in Inflammatory bowel diseases (IBD). This suppression of innate immune signaling in IEC is in part due to their strong expression of Single Ig IL1 related receptor (SIGIRR), a negative regulator of interleukin (IL)-1 and toll-like receptor (TLR) signaling. IL37, a newly recognized anti-inflammatory cytokine has been shown to strongly inhibit innate signaling in cells by binding to, and signaling through SIGIRR, leading to suppression of various forms of inflammation in mice. Few studies have looked at the function of IL-37/SIGIRR in IEC and their potential use to balance inflammatory responses. Notably, while many groups have studied IEC immune response in vitro, using transformed IEC lines, our focus is on primary-derived IEC which more accurately reflect in vivo responses. Aims To characterize IEC intrinsic and species-specific immune responses elicited by bacteria and host products as well as the role of IL37/SIGIRR in regulating this innate signaling. Methods We used organoid to study the innate immune responses of primary IEC derived from human or mouse colon (colonoids). After stimulation with inflammatory stimuli (IL1β, FliC and LPS), qPCR, ELISA, Milliplex Multiplex Assay and Western blot were used to determine modification in signalling pathway and cytokine/chemokine secretion. Results Using colonoids derived from healthy donors, we demonstrated that unlike transformed cell lines or mouse IEC, human IEC respond only to the bacterial product FliC, and not to LPS or IL1β. We further characterized human colonoid innate immune responses and despite significant inter-individual variability upon FliC stimulation, all organoids released several chemokines (IL8, CXCL1, CXCL2, CCL2 and CCL20). We showed for the first time that IL37 attenuated these innate immune responses through inhibition of intracellular signaling pathways (p38 and NFkB). Using colonoids derived from wildtype and Sigirr deficient mice, we found that mice IEC were responsive to IL1b and FliC and that the suppressive effects of IL37 were Sigirr dependent. Conclusions Our results show that human IEC show variability among individuals in the magnitude of their innate immune responses, and these responses differ from those obtained from transformed cells and primary mouse IEC. For the first time, we show that IL37 suppresses IEC innate immune responses, through its ability to signal through Sigirr. Further investigations will assess the ability of IL37 to control inflammation of IEC derived from IBD patients, as a potential therapeutic to promote gut health. Funding Agencies CAG, CIHRMSFHR

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Brucella-Induced Downregulation of lncRNA Gm28309 Triggers Macrophages Inflammatory Response Through the miR-3068-5p/NF-κB Pathway

Frontiers in Immunology ◽

10.3389/fimmu.2020.581517 ◽

2020 ◽

Vol 11 ◽

Author(s):

Xingmei Deng ◽

Jia Guo ◽

Zhihua Sun ◽

Laizhen Liu ◽

Tianyi Zhao ◽

...

Keyword(s):

Inflammatory Response ◽

Immune Responses ◽

Nlrp3 Inflammasome ◽

Inflammatory Responses ◽

Underlying Mechanism ◽

Bioinformatic Analysis ◽

Luciferase Reporter ◽

Non Coding Rnas ◽

First Time

ObjectivesThe underlying mechanism of the inflammatory response against Brucellosis caused by Brucella remains poorly understood. This study aimed to determine the role of long non-coding RNAs (lncRNAs) in regulating of inflammatory and anti-Brucella responses.Materials and methodsMicroarray analysis was performed to detect differentially expressed lncRNAs in THP-1 cells infected with an S2308 Brucella strain. The candidate lncRNAs were screened using bioinformatic analysis and siRNAs; bioinformatic prediction and luciferase reporter assay were also conducted, while inflammatory responses was assessed using RT‐qPCR, western blot, immunofluorescence, ELISA, HE, and immunohistochemistry.ResultsThe lncRNA Gm28309 was identified to be involved in regulating inflammation induced by Brucella. Gm28309, localized in the cytoplasm, was down-expressed in RAW264.7 cells infected with S2308. Overexpression of Gm28309 or inhibition of miR-3068-5p repressed p65 phosphorylation and reduced NLRP3 inflammasome and IL-1β and IL-18 secretion. Mechanistically, Gm28309 acted as a ceRNA of miR-3068-5p to activate NF-κB pathway by targeting κB-Ras2, an inhibitor of NF-κB signaling. Moreover, the number of intracellular Brucella was higher when Gm28309 was overexpressed or when miR-3068-5p or p65 was inhibited. However, these effects were reversed by the miR-3068-5p mimic.ConclusionsOur study demonstrates, for the first time, that LncRNAs are involved in regulating immune responses during Brucella infection, and Gm28309, an lncRNA, plays a crucial role in activating NF-κB/NLRP3 inflammasome signaling pathway.

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Reduced inflammatory responses to SARS-CoV-2 infection in children presenting to hospital with COVID19 in China

10.1101/2020.07.02.20145110 ◽

2020 ◽

Author(s):

Guoqing Qian ◽

Yong Zhang ◽

Yang Xu ◽

Weihua Hu ◽

Ian P Hall ◽

...

Keyword(s):

Inflammatory Response ◽

Immune Responses ◽

Tumour Necrosis Factor ◽

Proinflammatory Cytokines ◽

Tumour Necrosis ◽

Inflammatory Responses ◽

Severe Disease ◽

Hubei Province ◽

Cytokine Storm ◽

Necrosis Factor

SummaryBackgroundInfection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in children is associated with better outcomes than in adults. The inflammatory response to COVID-19 infection in children remains poorly characterised.MethodsWe retrospectively analysed the medical records of 127 laboratory-confirmed COVID-19 patients aged 1 month to 16 years from Wuhan and Jingzhou of Hubei Province. Patients presented between January 25th and March 24th 2020. Information on clinical features, laboratory results, plasma cytokines/chemokines and lymphocyte subsets were analysed.FindingsChildren admitted to hospital with COVID-19 were more likely to be male (67.7%) and the median age was 7.3 [IQR 4.9] years. All but one patient with severe disease was aged under 2 and the majority (5/7) had significant co-morbidities. Despite 53% having viral pneumonia on CT scanning only 2 patients had low lymphocyte counts and no differences were observed in the levels of plasma proinflammatory cytokines, including interleukin (IL)-2, IL-4, IL-6, tumour necrosis factor (TNF)-α, and interferon (IFN)-γ between patients with mild, moderate or severe disease.InterpretationsWe demonstrated that the immune responses of children to COVID-19 infection is significantly different from that seen in adults. Our evidence suggests that SARS-CoV-2 does not trigger a robust inflammatory response or ‘cytokine storm’ in children with COVID-19, and this may underlie the generally better outcomes seen in children with this disease. These data also imply anti-cytokine therapies may not be effective in children with moderate COVID-19.FundingThis study was funded by National Natural Foundation of China (No. 81970653).Research in contextEvidence before this studyWe searched PubMed without language restriction for studies published until June 25, 2020, using the search terms “SARS-CoV-2” or “novel coronavirus” or “COVID-19” and “immune responses” or “innate immunity” or “cytokine” or “subset of lymphocyte” and “children” or “adolescent”. Previously published research describes that severe and fatal cases in children are relatively rare. However, the inflammatory responses to COVID-19 infection in children remains poorly characterised.Added value of this studyWe analysed data from 127 laboratory-confirmed COVID-19 patients aged 1 month to 16 years in Hubei province to explore the immune responses to SARS-CoV-2 infection presenting to hospital with COVID-19. Cell numbers of CD3+, CD4+, CD8+ and natural killer T cells were within mostly normal limits even in more severe cases, and the levels of immunoglobulins, and proinflammatory cytokines, including interleukin (IL)-2, IL-4, IL-6, tumour necrosis factor (TNF)-α, and interferon (IFN)-γ were not generally elevated regardless of disease severity.Implications of all the available evidenceThe immune response to SARS-CoV-2 infection of children is significantly different from that seen in adults. The inflammatory responses seen even in children with viral pneumonia on CT are relatively mild and do not trigger the “cytokine storm” seen in some adults with COVID-19. This implies anti-cytokine therapies may not be effective in children with COVID-19.

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Abstract 17530: Characterization of Cardiac-resident Leukocyte Populations in the Steady-state and During Aging

Circulation ◽

10.1161/circ.130.suppl_2.17530 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Gustavo C Ramos ◽

Anne van den Berg ◽

Johannes Weirather ◽

Ulrich Hofmann ◽

Stefan Frantz

Keyword(s):

Steady State ◽

Serum Levels ◽

Cardiac Physiology ◽

Aged Mice ◽

Leukocyte Population ◽

Genes Expression ◽

Inflammatory Profile ◽

Leukocyte Populations ◽

First Time

Although it is well acknowledged that immunological phenomena participate in several cardiac diseases, it has been deeply neglected the fact that the heart is already populated by resident leukocytes before the onset of any sign of disease. In the present work we comprehensively characterized the cardiac-resident leukocyte populations and addressed for the first time in the literature how these cells take part in cardiac physiology and aging. We compared cardiac function, structure and gene expression of healthy naïve C57BL6/J (WT) mice 10 (young), 30 (mid-aged) and 60 (aged) wk old. Flow cytometrical analysis of perfused-digested murine hearts revealed a cardiac-resident leukocyte population comprised of approx. 103 cells/mg (M2-macrophages > B-cells > T-cells > granulocytes). The frequency of resident leukocytes found in cardiac muscle was 17.4 fold higher than in skeletal muscle, suggesting that they might be relevant for cardiac physiology. Importantly, cardiac-resident leukocyte composition shifted towards a pro-inflammatory profile during aging. Additionally, qPCR analysis indicated a significant up-regulation of pro-inflammatory genes such as TNF, IL-1β and IFN-γ in aged mice (e.g. TNF mRNA expression level: 2.1 .10-4 ± 2.0 10-5 in young vs 1.5 .10-3 ± 2.6 .10-4 in aged mice; P < 0.05). Similar results were found regarding to pro-fibrotic and pro-hypertrophic genes expression (e.g. Coll-III, TGF-β and βMyHC). Furthermore, aged WT mice presented increased serum levels of autoantibodies against myosin and higher frequencies of activated T helper cells (CD4+CD69+) in mediastinal lymph nodes as compared to young mice. Echocardiographic and hemodynamic studies revealed that in parallel with increased inflammation, 60 wk old naïve mice spontaneously develop severe cardiac dysfunction (e.g. ejection fraction: 34.6% ± 5.19%). Altogether, these data demonstrated that the heart comprises an important and yet underappreciated resident leukocyte population in the steady-state and that shifts in its composition occur in parallel with the cardiac deterioration observed in aged mice.

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Streptococcus pneumoniae colonisation associates with impaired adaptive immune responses against SARS-CoV-2

10.1101/2021.07.22.21260837 ◽

2021 ◽

Author(s):

Elena Mitsi ◽

Jesus Reine ◽

Britta C Urban ◽

Carla Solorzano ◽

Elissavet Nikolaou ◽

...

Keyword(s):

Immune Responses ◽

Immune Escape ◽

Inflammatory Responses ◽

Severe Disease ◽

Upper Airway ◽

Epidemiological Data ◽

Asymptomatic Infection ◽

Respiratory Pathogens ◽

Apparent Contradiction ◽

Systematic Screening

Although recent epidemiological data suggest that pneumococci may contribute to the risk of SARS-CoV-2 disease, secondary pneumococcal pneumonia has been reported as infrequent. This apparent contradiction may be explained by interactions of SARS-CoV-2 and pneumococcus in the upper airway, resulting in the escape of SARS-CoV-2 from protective host immune responses. Here, we investigated the relationship of these two respiratory pathogens in two distinct cohorts of a) healthcare workers with asymptomatic or mildly symptomatic SARS-CoV-2 infection identified by systematic screening and b) patients with moderate to severe disease who presented to hospital. We assessed the effect of co-infection on host antibody, cellular and inflammatory responses to the virus. In both cohorts, pneumococcal colonisation was associated with diminished anti-viral immune responses, which affected primarily mucosal IgA levels among individuals with mild or asymptomatic infection and cellular memory responses in infected patients. Our findings suggest that S. pneumoniae modulates host immunity to SARS-CoV-2 and raises the question if pneumococcal carriage also enables immune escape of other respiratory viruses through a similar mechanism and facilitates reinfection occurrence.

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Dysregulation of Blood-Brain Barrier and Exacerbated Inflammatory Response in Cx47-Deficient Mice after Induction of EAE

Pharmaceuticals ◽

10.3390/ph14070621 ◽

2021 ◽

Vol 14 (7) ◽

pp. 621

Author(s):

Filippos Stavropoulos ◽

Elena Georgiou ◽

Irene Sargiannidou ◽

Kleopas A. Kleopa

Keyword(s):

Multiple Sclerosis ◽

Inflammatory Responses ◽

Severe Disease ◽

Disease Onset ◽

Altered Expression ◽

Gm Csf ◽

T Cell Infiltration ◽

Tight Junction Formation ◽

Inflammatory Profile ◽

Blood Spinal Cord Barrier

Induction of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), in connexin 32 (Cx32) or Cx47 knockout (KO) mice with deficiency in oligodendrocyte gap junctions (GJs) results in a more severe disease course. In particular, Cx47 KO EAE mice experience an earlier EAE onset and more pronounced disease severity, accompanied by dysregulated pro-inflammatory responses preceding the disease manifestations. In this study, analysis of relevant pro-inflammatory cytokines in wild type EAE, Cx32 KO EAE, and Cx47 KO EAE mice revealed altered expression of Vcam-1 preceding EAE [7 days post injection (dpi)], of Ccl2 at the onset of EAE (12 dpi), and of Gm-csf at the peak of EAE (24 dpi) in Cx47 KO EAE mice. Moreover, Cx47 KO EAE mice exhibited more severe blood-spinal cord barrier (BSCB) disruption, enhanced astrogliosis with defects in tight junction formation at the glia limitans, and increased T-cell infiltration prior to disease onset. Thus, Cx47 deficiency appears to cause dysregulation of the inflammatory profile and BSCB integrity, promoting early astrocyte responses in Cx47 KO EAE mice that lead to a more severe EAE outcome. Further investigation into the role of oligodendrocytic Cx47 in EAE and multiple sclerosis pathology is warranted.

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Communication Sciences and Disorders PhD Graduates' Perceptions of Their PhD Program

Perspectives of the ASHA Special Interest Groups ◽

10.1044/2020_persp-19-00107 ◽

2020 ◽

Vol 5 (2) ◽

pp. 463-478

Author(s):

Elizabeth Crais ◽

Melody Harrison Savage

Keyword(s):

Detailed Examination ◽

Career Expectations ◽

Doctoral Preparation ◽

Creative Solutions ◽

Phd Education ◽

Doctor Of Philosophy ◽

First Time ◽

Students Success ◽

Phd Program

Purpose The shortage of doctor of philosophy (PhD)–level applicants to fill academic and research positions in communication sciences and disorders (CSD) programs calls for a detailed examination of current CSD PhD educational practices and the generation of creative solutions. The intended purposes of the article are to encourage CSD faculty to examine their own PhD program practices and consider the perspectives of recent CSD PhD graduates in determining the need for possible modifications. Method The article describes the results of a survey of 240 CSD PhD graduates and their perceptions of the challenges and facilitators to completing a PhD degree; the quality of their preparation in research, teaching, and job readiness; and ways to improve PhD education. Results Two primary themes emerged from the data highlighting the need for “matchmaking.” The first time point of needed matchmaking is prior to entry among students, mentors, and expectations as well as between aspects of the program that can lead to students' success and graduation. The second important matchmaking need is between the actual PhD preparation and the realities of the graduates' career expectations, and those placed on graduates by their employers. Conclusions Within both themes, graduate's perspectives and suggestions to help guide future doctoral preparation are highlighted. The graduates' recommendations could be used by CSD PhD program faculty to enhance the quality of their program and the likelihood of student success and completion. Supplemental Material https://doi.org/10.23641/asha.11991480

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Transcriptomic Analysis of Inbred Chicken Lines Reveals Infectious Bursal Disease Severity Is Associated with Greater Bursal Inflammation In Vivo and More Rapid Induction of Pro-Inflammatory Responses in Primary Bursal Cells Stimulated Ex Vivo

Viruses ◽

10.3390/v13050933 ◽

2021 ◽

Vol 13 (5) ◽

pp. 933

Author(s):

Amin S. Asfor ◽

Salik Nazki ◽

Vishwanatha R.A.P. Reddy ◽

Elle Campbell ◽

Katherine L. Dulwich ◽

...

Keyword(s):

Rho Gtpases ◽

Inflammatory Responses ◽

Ex Vivo ◽

Severe Disease ◽

Infectious Bursal Disease ◽

Rna Seq ◽

Rapid Induction ◽

Cytoskeletal Regulation ◽

Bursal Disease

In order to better understand differences in the outcome of infectious bursal disease virus (IBDV) infection, we inoculated a very virulent (vv) strain into White Leghorn chickens of inbred line W that was previously reported to experience over 24% flock mortality, and three inbred lines (15I, C.B4 and 0) that were previously reported to display no mortality. Within each experimental group, some individuals experienced more severe disease than others but line 15I birds experienced milder disease based on average clinical scores, percentage of birds with gross pathology, average bursal lesion scores and average peak bursal virus titre. RNA-Seq analysis revealed that more severe disease in line W was associated with significant up-regulation of pathways involved in inflammation, cytoskeletal regulation by Rho GTPases, nicotinic acetylcholine receptor signaling, and Wnt signaling in the bursa compared to line 15I. Primary bursal cell populations isolated from uninfected line W birds contained a significantly greater percentage of KUL01+ macrophages than cells isolated from line 15I birds (p < 0.01) and, when stimulated ex vivo with LPS, showed more rapid up-regulation of pro-inflammatory gene expression than those from line 15I birds. We hypothesize that a more rapid induction of pro-inflammatory cytokine responses in bursal cells following IBDV infection leads to more severe disease in line W birds than in line 15I.

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