Single-cell RNA sequencing reveals characteristics of myeloid cells in pulmonary post-acute sequelae of SARS-CoV-2.

Background: Although our understanding of the immunopathology and subsequent risk and severity of COVID-19 disease is evolving, a detailed account of immune responses that contribute to the long-term consequences of pulmonary complication in COVID-19 infection remain unclear. Few studies have detailed the immune and cytokine profiles associated with post-acute sequalae of SARS-CoV-2 infection with persistent pulmonary symptoms (PPASC). However, the dysregulation of the immune system that drives pulmonary sequelae in COVID-19 survivors and PASC sufferers remains largely unknown. Results: To characterize the immunological features of pulmonary PASC (PPASC), we performed droplet-based single-cell RNA sequencing to study the transcriptomic profiles of peripheral blood mononuclear cells (PBMCs) from participants naïve to SARS-CoV-2 (Control) and infected with SARS-CoV-2 with chronic pulmonary symptoms (PPASC). We analyzed more than 34,139 PBMCs by integrating our dataset with previously reported control datasets (GSM4509024) cell distribution. In total, 11 distinct cell populations were identified based on the expression of canonical markers. The proportion of myeloid-lineage cells ([MLCs]; CD14 + /CD16 + monocytes and dendritic cells) was increased in PPASC compared to controls. MLCs from PPASC displayed up-regulation of genes associated with pulmonary symptoms/fibrosis, while glycolysis metabolism-related genes were downregulated. Similarly, pathway analysis showed that fibrosis- related ( VEGF , WNT , and SMAD ) and cell death pathways were up-regulated, but immune pathways were down-regulated in PPASC. In PPASC, we observed interactive VEGF ligand- receptor pairs among MLCs, and network modules in CD14 + (cluster 4) and CD16 + (Cluster 5) monocytes displayed a significant enrichment for biological pathways linked to adverse COVID- 19 outcomes, fibrosis, and angiogenesis. Further analysis revealed a distinct metabolic alteration in MLCs with a down-regulation of glycolysis/gluconeogenesis in PPASC compared to SARS- CoV-2 naïve samples. Conclusion: This study offers valuable insights into the immune response and cellular landscape in PPASC. The presence of elevated MLC levels and their corresponding gene signatures associated with fibrosis, immune response suppression, and altered metabolic states suggests their potential role as a driver of PPASC.

Low-density granulocytes display immature cells with enhanced NET formation in people living with HIV.

While the protective role of neutrophil extracellular traps (NETs) in limiting human immunodeficiency virus (HIV) spread to susceptible cells has been documented, there is comparatively little insight into whether NET formation is harmful in people living with HIV (PLWH). To gain insight into neutrophil dysregulation and the pathological role of NETs in HIV, we examined expressions of NET-associated markers [cell-free DNA (cfDNA) and citrullinated histone H3 (CitH3)] in the plasmas from a cohort of the Hawaii Aging with HIV-cardiovascular and HIV-seronegative (HIV-) individuals. In a subset of participants, circulating low-density granulocyte (LDG) levels and their maturation and activation status were analyzed via flow cytometry. We demonstrated higher plasma levels of CitH3 in PLWH compared to HIV- individuals. LDGs from PLWH had heightened CD66b, but reduced CD16 expression. The percentages and counts of CD10+ LDGs were significantly decreased in PLWH. In addition, the CD16Lo LDG subsets were enriched in PLWH, compared to HIV- group, indicating that immature LDGs are increased in PLWH. Moreover, LDGs from PLWH exhibited significantly higher NET forming capacity. In summary, our study presents evidence that LDGs from PLWH on ART display an immature and altered phenotype with increased NET formation. Among PLWH, plasma NET levels as well as LDG parameters correlated with blood markers for inflammation and coagulation, suggesting that neutrophil activation and NETs may exert proinflammatory and coagulation effects. Our data provide insights into the pathologic role of LDGs at least in part mediated through NET formation in PLWH.

Corrigendum: Phenotypic alteration of low-density granulocytes in people with pulmonary post-acute sequelae of SARS-CoV-2 infection.

[This corrects the article DOI: 10.3389/fimmu.2022.1076724.].

Elevated circulating monocytes and monocyte activation in COVID-19 convalescent individuals.

Background: Monocytes and macrophages play a pivotal role in inflammation during acute SARS-CoV-2 infection. However, their contribution to the development of post-acute sequelae of SARS-CoV-2 infection (PASC) are not fully elucidated. Methods: A cross-sectional study was conducted comparing plasma cytokine and monocyte levels among three groups: participants with pulmonary PASC (PPASC) with a reduced predicted diffusing capacity for carbon monoxide [DLCOc, <80%; (PG)]; fully recovered from SARS-CoV-2 with no residual symptoms (recovered group, RG); and negative for SARS-CoV-2 (negative group, NG). The expressions of cytokines were measured in plasma of study cohort by Luminex assay. The percentages and numbers of monocyte subsets (classical, intermediate, and non-classical monocytes) and monocyte activation (defined by CD169 expression) were analyzed using flow cytometry analysis of peripheral blood mononuclear cells. Results: Plasma IL-1Ra levels were elevated but FGF levels were reduced in PG compared to NG. Circulating monocytes and three subsets were significantly higher in PG and RG compared to NG. PG and RG exhibited higher levels of CD169+ monocyte counts and higher CD169 expression was detected in intermediate and non-classical monocytes from RG and PG than that found in NG. Further correlation analysis with CD169+ monocyte subsets revealed that CD169+ intermediate monocytes negatively correlated with DLCOc%, and CD169+ non-classical monocytes positively correlated with IL-1α, IL-1ß, MIP-1α, Eotaxin, and IFN-γ. Conclusion: This study present evidence that COVID convalescents exhibit monocyte alteration beyond the acute COVID-19 infection period even in convalescents with no residual symptoms. Further, the results suggest that monocyte alteration and increased activated monocyte subsets may impact pulmonary function in COVID-19 convalescents. This observation will aid in understanding the immunopathologic feature of pulmonary PASC development, resolution, and subsequent therapeutic interventions.

Single-cell RNA sequencing reveals characteristics of myeloid cells in post-acute sequelae of SARS-CoV-2 patients with persistent respiratory symptoms.

Background: Although our understanding of the immunopathology and subsequent risk and severity of COVID-19 disease is evolving, a detailed account of immune responses that contribute to the long-term consequences of pulmonary complications in COVID-19 infection remains unclear. Few studies have detailed the immune and cytokine profiles associated with post-acute sequelae of SARS-CoV-2 infection (PASC) with persistent pulmonary symptoms. The dysregulation of the immune system that drives pulmonary sequelae in COVID-19 survivors and PASC sufferers remains largely unknown. Results: To characterize the immunological features of pulmonary PASC (PPASC), we performed droplet-based single-cell RNA sequencing (scRNA-seq) to study the transcriptomic profiles of peripheral blood mononuclear cells (PBMCs) from a participant naïve to SARS-CoV-2 (Control) (n=1) and infected with SARS-CoV-2 with chronic pulmonary symptoms (PPASC) (n=2). After integrating scRNA-seq data with a naïve participant from a published dataset, 11 distinct cell populations were identified based on the expression of canonical markers. The proportion of myeloid-lineage cells ([MLCs]; CD14+/CD16+monocytes, and dendritic cells) was increased in PPASC (n=2) compared to controls (n=2). MLCs from PPASC displayed up-regulation of genes associated with pulmonary symptoms/fibrosis, while glycolysis metabolism-related genes were downregulated. Similarly, pathway analysis showed that fibrosis-related (VEGF, WNT, and SMAD) and cell death pathways were up-regulated, but immune pathways were down-regulated in PPASC. Further comparison of PPASC with scRNA-seq data with Severe COVID-19 (n=4) data demonstrated enrichment of fibrotic transcriptional signatures. In PPASC, we observed interactive VEGF ligand-receptor pairs among MLCs, and network modules in CD14+ (cluster 4) and CD16+ (Cluster 5) monocytes displayed a significant enrichment for biological pathways linked to adverse COVID-19 outcomes, fibrosis, and angiogenesis. Further analysis revealed a distinct metabolic alteration in MLCs with a down-regulation of glycolysis/gluconeogenesis in PPASC compared to SARS-CoV-2 naïve samples. Conclusion: Analysis of a small scRNA-seq dataset demonstrated alterations in the immune response and cellular landscape in PPASC. The presence of elevated MLC levels and their corresponding gene signatures associated with fibrosis, immune response suppression, and altered metabolic states suggests a potential role in PPASC development.

Characterization of Circulating Fibrocytes in People Living with HIV on Stable Antiretroviral Therapy.

Highly effective combination antiretroviral therapy has reduced HIV infection to a manageable chronic disease, shifting the clinical landscape toward management of noninfectious comorbidities in people living with HIV (PLWH). These comorbidities are diverse, generally associated with accelerated aging, and present within multiple organ systems. Mechanistically, immune dysregulation and chronic inflammation, both of which persist in PLWH with well-controlled virally suppressive HIV infection, are suggested to create and exacerbate noninfectious comorbidity development. Persistent inflammation often leads to fibrosis, which is the common end point pathologic feature associated with most comorbidities. Fibrocytes are bone marrow-derived fibroblast-like cells, which emerged as key effector cells in tissue repair and pathologic fibrotic diseases. Despite their relevance to fibrosis, the circulating fibrocyte concentration in PLWH remains poorly characterized, and an understanding of their functional role in chronic HIV is limited. In this study, utilizing PBMCs from a cross-sectional adult HIV cohort study with matched uninfected controls (HIV-), we aimed to identify and compare circulating fibrocytes in blood. Both the percentage and number of fibrocytes and α-smooth muscle actin+ fibrocytes in circulation did not differ between the HIV+ and HIV- groups. However, circulating fibrocyte levels were significantly associated with increasing age in both the HIV+ and HIV- groups (the percentage and number; r = 0.575, p ≤ 0.0001 and r = 0.558, p ≤ 0.0001, respectively). Our study demonstrates that circulating fibrocyte levels and their fibroblast-like phenotype defined as collagen I and α-smooth muscle actin+ expression are comparable between, and strongly associated with, age irrespective of HIV status.

Identification and Implications of HIV-1 CRF01_AE Subtype in Hawai'i.

Human Immunodeficiency Virus has a high propensity for genetic variation, demonstrated by its complex phylogeny and multiplicity of subtypes. Subtype B is predominant in North America as well as in Hawai'i while CRF01_AE is found in over 50% of cases in the Philippines and Southeast Asia. In a small collaborative study between the Hawai'i Center for AIDS and Philippines General Hospital, molecular phylogenetic subtyping was conducted on HIV+ participants. Two of 15 (13%) participants from the Hawai'i cohort and 12 of 21 (57%) participants from the Philippines cohort were identified as having CRF01_AE subtype of HIV-1, with remaining participants identified as subtype B. While one individual in Hawai'i with CRF01_AE had emigrated from the Philippines, the other participant from Hawai'i with CRF01_AE subtype was a local individual, born and raised in Hawai'i. The authors report that HIV subtype diversity may be increased in Hawai'i and discuss its potential clinical and public health implications.

Phenotypic alteration of low-density granulocytes in people with pulmonary post-acute sequalae of SARS-CoV-2 infection.

Background: Low-density granulocytes (LDGs) are a distinct subset of neutrophils whose increased abundance is associated with the severity of COVID-19. However, the long-term effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on LDG levels and phenotypic alteration remain unexplored. Methods: Using participants naïve to SARS-CoV-2 (NP), infected with SARS-CoV-2 with no residual symptoms (NRS), and infected with SARS-CoV-2 with chronic pulmonary symptoms (PPASC), we compared LDG levels and their phenotype by measuring the expression of markers for activation, maturation, and neutrophil extracellular trap (NET) formation using flow cytometry. Results: The number of LDGs was elevated in PPASC compared to NP. Individuals infected with SARS-CoV-2 (NRS and PPASC) demonstrated increased CD10+ and CD16hi subset counts of LDGs compared to NP group. Further characterization of LDGs demonstrated that LDGs from COVID-19 convalescents (PPASC and NRS) displayed increased markers of NET forming ability and aggregation with platelets compared to LDGs from NP, but no differences were observed between PPASC and NRS. Conclusions: Our data from a small cohort study demonstrates that mature neutrophils with a heightened activation phenotype remain in circulation long after initial SARS-CoV-2 infection. Persistent elevation of markers for neutrophil activation and NET formation on LDGs, as well as an enhanced proclivity for platelet-neutrophil aggregation (PNA) formation in COVID-19 convalescent individuals may be associated with PPASC prognosis and development.