In a recent study posted to the bioRxiv* preprint server, researchers characterized the responses of the peripheral blood mononuclear cells (PBMCs) at the individual cell and cell type levels against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and severe acute respiratory syndrome (SARS-CoV) ex vivo.
The ongoing SARS-CoV-2 pandemic is a societal, medical, and financial crisis of increasing significance worldwide. The cell-intrinsic responses observed in vivo in PBMCs during severe and mild forms of SARS-CoV-2 infection vary qualitatively and quantitatively among individuals. Whether these responses are stimulated by the productively infected epithelial cells from the respiratory tract or somewhat induced by the direct interaction of the PBMCs with the viral particles is not yet understood.
Hence, ex vivo systems that provide in vivo correlates of the cellular immunophenotypes of SARS-CoV-2 infection are necessary to develop immunomodulatory strategies against coronavirus disease 2019 (COVID-19). Additionally, the ex vivo models provide a platform for concurrent comparison of two genetically close yet functionally different viruses.
About the study
In the present study, the researchers investigated the cell-intrinsic nonspecific responses and susceptibility to infection of PBMCs from healthy individuals following exposure to SARS-CoV-2 and SARS-CoV in ex vivo conditions.
The unstimulated PBMCs from healthy subjects were exposed to identical infectious titers of purified stocks of SARS-CoV-2 and SARS-CoV, respectively, on Vero E6 cells. The PBMCs exposed to supernatants of uninfected Vero E6 cells were used as the reference in the study.
The viral strains used in the study were SARS-CoV isolate HKU-39849 and SARS-CoV-2 B.1 lineage. The amount of infectious viral particles was quantified using the plaque titration assay. Both the control and study groups were treated with 10µM of ruxolitinib and 20µM of remdesivir 30 minutes before the viral exposure.
The results indicate that human PBMCs were obstinate to productive SARS-CoV-2 and SARS-CoV infection, similar to the lack of detectable expression of angiotensin-converting enzyme 2 (ACE2) receptor ex vivo. Thus, SARS-CoV-2 likely attaches or internalizes to the PBMCs in an ACE2-independent mode, resulting in the association or internalization of viral ribonucleic acid (RNA) to the cells.
Further, the continuous presence of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) inhibitor drug ruxolitinib failed to enable the secretion of viral RNA or infectious particles, suggesting that the JAK/STAT-dependent cell-intrinsic nonspecific immunity is not the reason for the undetectable level of viral particles.
Single-cell RNA sequencing (scRNA-seq) and bulk RNA sequencing indicated that during SARS-CoV-2 infection, interferon (IFN)-stimulated genes (ISGs) exhibited a JAK/STAT-dependent stimulation in many cell types present in PBMCs, significantly in the monocytic cells. In contrast, the upregulation of expression was not observed in the pro-inflammatory cytokines such as tumor necrosis factor α (TNFα), interleukin-1 (IL-1), and interleukin-6 (IL-6) in PBMCs during SARS-CoV-2 infection.
The monocytes positive for RNA of SARS-CoV-2 exhibited an over-expression of profibrotic genes and a lower ISG signature relative to the bystander cells from identical cultures but with undetectable viral RNA. These findings indicate the probability of the preference of SARS-CoV-2 for cells with a low ISG baseline profile or the release of a SARS-CoV-2-specific sensing antagonist after particle internalization during the infection. Further, the SARS-CoV-2 RNA associated with the PBMC in an ACE2 independent manner remained for several days.
According to the authors, this is the first investigation that sheds light on the response of the refractory PBMCs during exposure to SARS-CoV-2 and SARS-CoV particles in the absence of co-stimulating epithelial cell types infected with the same viruses.
The study findings indicate that a direct interaction between PBMCs and SARS-CoV-2 particles in a non-productive infection triggers the JAK/STAT-dependent, monocyte-accelerated nonspecific immunity ex vivo similar to those found in COVID-19 patients with mild disease in vivo. However, SARS-CoV did not exhibit this accentuation of innate immunity in the study.
The present findings indicating undetectable SARS-CoV-2 and SARS-CoV infection in PBMCs were partially in contrast to the previous studies demonstrating a detectable infection and susceptibility of human PBMCs to Middle East respiratory syndrome CoV (MERS-CoV) and SARS-CoV.
Further, the authors hypothesized that the virus particles of SARS-CoV-2 link to the PBMCs in an ACE2-independent manner since the virus-infected PBMCs failed to demonstrate detectable ACE2 expression ex vivo.
Together, the study demonstrates that despite the absence of detectable productive SARS-CoV-2 infection, the physical interaction of PBMCs with SARS-CoV-2 led to the significant stimulation of the monocytic fraction, followed by a robust ISG induction ex vivo, which can potentially control the pathogenesis of the infection.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.