Skin microbiome profile in people living with HIV/AIDS in Cameroon.

The presence of pathogens and the state of diseases, particularly skin diseases, may alter the composition of human skin microbiome. HIV infection has been reported to impair gut microbiome that leads to severe consequences. However, with cutaneous manifestations, that can be life-threatening, due to the opportunistic pathogens, little is known whether HIV infection might influence the skin microbiome and affect the skin homeostasis. This study catalogued the profile of skin microbiome of healthy Cameroonians, at three different skin sites, and compared them to the HIV-infected individuals. Taking advantage on the use of molecular assay coupled with next-generation sequencing, this study revealed that alpha-diversity of the skin microbiome was higher and beta-diversity was altered significantly in the HIV-infected Cameroonians than in the healthy ones. The relative abundance of skin microbes such as Micrococcus and Kocuria species was higher and Cutibacterium species was significantly lower in HIV-infected people, indicating an early change in the human skin microbiome in response to the HIV infection. This phenotypical shift was not related to the number of CD4 T cell count thus the cause remains to be identified. Overall, these data may offer an important lead on the role of skin microbiome in the determination of cutaneous disease state and the discovery of safe pharmacological preparations to treat microbial-related skin disorders.

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.

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.].

In vitro evidence against productive SARS-CoV-2 infection of human testicular cells: Bystander effects of infection mediate testicular injury.

The hallmark of severe COVID-19 involves systemic cytokine storm and multi-organ injury including testicular inflammation, reduced testosterone, and germ cell depletion. The ACE2 receptor is also expressed in the resident testicular cells, however, SARS-CoV-2 infection and mechanisms of testicular injury are not fully understood. The testicular injury could be initiated by direct virus infection or exposure to systemic inflammatory mediators or viral antigens. We characterized SARS-CoV-2 infection in different human testicular 2D and 3D culture systems including primary Sertoli cells, Leydig cells, mixed seminiferous tubule cells (STC), and 3D human testicular organoids (HTO). Data shows that SARS-CoV-2 does not productively infect any testicular cell type. However, exposure of STC and HTO to inflammatory supernatant from infected airway epithelial cells and COVID-19 plasma decreased cell viability and resulted in the death of undifferentiated spermatogonia. Further, exposure to only SARS-CoV-2 Envelope protein caused inflammatory response and cytopathic effects dependent on TLR2, while Spike 1 or Nucleocapsid proteins did not. A similar trend was observed in the K18-hACE2 transgenic mice which demonstrated a disrupted tissue architecture with no evidence of virus replication in the testis that correlated with peak lung inflammation. Virus antigens including Spike 1 and Envelope proteins were also detected in the serum during the acute stage of the disease. Collectively, these data strongly suggest that testicular injury associated with SARS-CoV-2 infection is likely an indirect effect of exposure to systemic inflammation and/or SARS-CoV-2 antigens. Data also provide novel insights into the mechanism of testicular injury and could explain the clinical manifestation of testicular symptoms associated with severe COVID-19.

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.

In vitro evidence against productive SARS-CoV-2 infection of human testicular cells: Bystander effects of infection mediate testicular injury.

The hallmark of severe COVID-19 involves systemic cytokine storm and multi-organ failure including testicular injury and germ cell depletion. The ACE2 receptor is also expressed in the resident testicular cells however, SARS-CoV-2 infection and mechanisms of testicular injury are not fully understood. The testicular injury can likely result either from direct virus infection of resident cells or by exposure to systemic inflammatory mediators or virus antigens. We here characterized SARS-CoV-2 infection in different human testicular 2D and 3D models including primary Sertoli cells, Leydig cells, mixed seminiferous tubule cells (STC), and 3D human testicular organoids (HTO). Data shows that SARS-CoV-2 does not establish a productive infection in any testicular cell types. However, exposure of STC and HTO to inflammatory supernatant from infected airway epithelial cells and COVID-19 plasma depicted a significant decrease in cell viability and death of undifferentiated spermatogonia. Further, exposure to only SARS-CoV-2 envelope protein, but not Spike or nucleocapsid proteins led to cytopathic effects on testicular cells that was dependent on the TLR2 receptor. A similar trend was observed in the K18h-ACE2 mouse model which revealed gross pathology in the absence of virus replication in the testis. Collectively, data strongly indicates that the testicular injury is not due to direct infection of SARS-CoV-2 but more likely an indirect effect of exposure to systemic inflammation or SARS-CoV-2 antigens. Data also provide novel insights into the mechanism of testicular injury and could explain the clinical manifestation of testicular symptoms associated with severe COVID-19.

Skin microbiome profile of healthy Cameroonians and Japanese.

The commensal microbes of the skin have a significant impact on dermal physiology and pathophysiology. Racial and geographical differences in the skin microbiome are suggested and may play a role in the sensitivity to dermatological disorders, including infectious diseases. However, little is known about the skin microbiome profiles of people living in Central Africa, where severe tropical infectious diseases impose a burden on the inhabitants. This study provided the skin profiles of healthy Cameroonians in different body sites and compared them to healthy Japanese participants. The skin microbiome of Cameroonians was distinguishable from that of Japanese in all skin sites examined in this study. For example, Micrococcus was predominantly found in skin samples of Cameroonians but mostly absent in Japanese skin samples. Instead, the relative abundance of Cutibacterium species was significantly higher in healthy Japanese. Principal coordinate analysis of beta diversity showed that the skin microbiome of Cameroonians formed different clusters from Japanese, suggesting a substantial difference in the microbiome profiles between participants of both countries. In addition, the alpha diversity in skin microbes was higher in Cameroonians than Japanese participants. These data may offer insights into the determinant factors responsible for the distinctness of the skin microbiome of people living in Central Africa and Asia.

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.

RIG-I and MDA5 are modulated by bone morphogenetic protein (BMP6) and are essential for restricting Zika virus infection in human Sertoli cells.

Sexual transmission of Zika virus (ZIKV) is associated with virus persistence in the testes and shedding in the seminal fluid for months after recovery. We previously demonstrated that ZIKV can establish long-term replication without causing cytotoxicity in human Sertoli cells (SC), responsible for maintaining the immune privileged compartment of seminiferous tubules. Functional gene expression analyses also predicted activation of multiple virus sensing pathways including TLR3, RIG-I, and MDA5. Here, we elucidated which of the RNA virus sensing receptors play a decisive role in restricting ZIKV replication. We show that both poly I:C and IFN-ß treatment induced a robust antiviral state and reduced ZIKV replication significantly, suggesting that virus sensing and antiviral signaling are functional in SC. Silencing of TLR3, 7, and 9 did not affect virus replication kinetics; however, both RIG-I and MDA5 played a synergistic role in inducing an anti-ZIKV response. Further, the impact of SC-specific immunosuppressive pathways that collectively regulate SC function, specifically the TGF-ß superfamily members, TGF-ß, Activin A, and BMP6, on ZIKV replication was investigated. While ZIKV did not modulate the expression of TGF-ß and Activin A, BMP6 signaling was suppressed at later stages of infection. Notably, treatment with BMP6 increased IFN-ß, p-IRF3, and p-STAT1 levels, and expression of key interferon-stimulated genes including MDA5, suggesting that BMP6 enhances antiviral response in SC. Collectively, this study further delineates the key role of the RIG-I-like receptors in sensing ZIKV in SC, and reveals a novel role of BMP6 in modulating innate immune and antiviral response in the testes.

Paracrine IFN Response Limits ZIKV Infection in Human Sertoli Cells.

Zika virus (ZIKV) is unique among mosquito-borne flaviviruses in its ability to be sexually transmitted. The testes have been implicated as sites of long-term ZIKV replication, and our previous studies have identified Sertoli cells (SC), the nurse cells of the seminiferous epithelium that govern spermatogenesis, as major targets of ZIKV infection. To improve our understanding of the interaction of ZIKV with human SC, we analyzed ZIKV-induced proteome changes in these cells using high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS). Our data demonstrated that interferon (IFN) signaling was the most significantly enriched pathway and the antiviral proteins MX1 and IFIT1 were among the top upregulated proteins in SC following ZIKV infection. The dynamic between IFN response and ZIKV infection kinetics in SC remains unclear, therefore we further determined whether MX1 and IFIT1 serve as antiviral effectors against ZIKV. We found that increased levels of MX1 at the later time points of infection coincided with diminished ZIKV infection while the silencing of MX1 and IFIT1 enhanced peak ZIKV propagation in SC. Furthermore, although IFN-I exposure was found to significantly hinder ZIKV replication in SC, IFN response was attenuated in these cells as compared to other cell types. The data in this study highlight IFN-I as a driver of the antiviral state that limits ZIKV infection in SC and suggests that MX1 and IFIT1 function as antiviral effectors against ZIKV in SC. Collectively, this study provides important biological insights into the response of SC to ZIKV infection and the ability of the virus to persist in the testes.

Axl Promotes Zika Virus Entry and Modulates the Antiviral State of Human Sertoli Cells.

Zika virus (ZIKV) is unique among mosquito-borne flaviviruses in its ability to be sexually transmitted. Persistent ZIKV infection in the testes, which are immune privileged organs, long after peripheral clearance suggests involvement of immunosuppressive pathways; however, the underlying mechanisms remain undetermined. We recently demonstrated that ZIKV infects human Sertoli cells (SC), the major cell type of the seminiferous epithelium responsible for maintaining the immune privileged compartment of seminiferous tubules. Recent reports have identified the TAM (Tyro3, Axl, Mer) receptor tyrosine kinase Axl as an entry receptor and/or immune modulator for ZIKV in a cell type-specific manner. Interestingly, the seminiferous epithelium exhibits high basal expression of the Axl receptor where it is involved in clearance of apoptotic germ cells and immunosuppression. Here, we show that Axl was highly expressed in SC compared to Leydig cells (LC) that correlated with robust ZIKV infection of SC, but not LC. Further, neutralization of Axl receptor and its ligand Gas6 strongly attenuated virus entry in SC. However, inhibition of Axl kinase did not affect ZIKV entry but instead led to decreased protein levels of suppressor of cytokine signaling 1 (SOCS1) and SOCS3, increased expression of interferon-stimulated genes (ISGs), and reduced ZIKV replication. Similarly, treatment of multicellular human testicular organoids with an Axl kinase inhibitor attenuated ZIKV replication and increased ISG expression. Together, our data demonstrate that Axl promotes ZIKV entry and negatively regulates the antiviral state of SC to augment ZIKV infection of the testes and provides new insights into testis antiviral immunity and ZIKV persistence.IMPORTANCE Recent Zika virus (ZIKV) outbreaks have identified sexual transmission as a new route of disease spread not reported for other flaviviruses. ZIKV crosses the blood-testis barrier and establishes infection in seminiferous tubules, the site for spermatozoa development. Currently, there are no therapies to treat ZIKV infection, and the immune mechanisms underlying testicular persistence are unclear. We found that multiple human testicular cell types, except Leydig cells, support ZIKV infection. Axl receptor, which plays a pivotal role in maintaining the immunosuppressive milieu of the testis, is highly expressed in Sertoli cells and augments ZIKV infection by promoting virus entry and negatively regulating the antiviral state. By using testicular organoids, we further describe the antiviral role of Axl inhibition. The significance of our research lies in defining cross talk between Axl and type I interferon signaling as an essential mechanism of immune control that can inform therapeutic efforts to clear ZIKV from the testis.

Feasibility of microbial sample collection on the skin from people in Yaoundé, Cameroon.

Characterization of microbial communities in the skin in healthy individuals and diseased patients holds valuable information for understanding pathogenesis of skin diseases and as a source for developing novel therapies. Notably, resources regarding skin microbiome are limited in developing countries where skin disorders from infectious diseases are extremely common. A simple method for sample collection and processing for skin microbiome studies in such countries is crucial. The aim of this study is to confirm the feasibility of collecting skin microbiota from individuals in Yaoundé, a capital city of Cameroon, and subsequent extraction of bacterial DNA in a resource limited setting. Skin swabs from several individuals in Yaoundé were successfully obtained, and sufficient amount of bacterial 16S ribosomal RNA-coding DNA was collected, which was confirmed by quantitative PCR. The median copy number of 16S ribosomal RNA gene varied across participants and collection sites, with significantly more copies in samples collected from the forehead compared to the left and right forearm, or back. This study demonstrated that collecting surface skin microbes using our swabbing method is feasible in a developing country. We further showed that even with limited resources, we could collect sufficient amount of skin microbiota from the inhabitants in Yaoundé where no studies of skin microbiome were reported, which can be passed to further metagenomic analysis such as next generation sequencing.