Unravelling immunoregulatory circuits of tissue inflammation

National Institute Of Diabetes And Digestive And Kidney Diseases

Investigators

Linked publications & trials

Abstract

In this year, we extended the concept of cell-autonomous Complement activity as driver of cell behavior into non-immune cells and infection. We found that SARS-CoV-2 infection of respiratory epithelial cells (RECs) also induces Complement gene transcription as one of the top pathways in response to the virus and such REC Complement production likely contributes to COVID-19 severity. Thus, the intracellular COMPLEMENT pathway is not immune cell-restricted but also present in multiple cell types, reflecting evolutionary conservation. This work was published in Science Immunology. Briefly, we found that viral sensing in respiratory epithelial cells was followed by recruitment of TFs downstream of the type I interferon (IFN) receptor (driven by JAK1/2-STAT1), and NF-kB. These initiated transcription of both C3 and cell-intrinsic complement factor B (CFB), representing an alternative Complement pathway for C3 protein activation within cells. C3 is potently inflammatory and its processed fragments an independent risk factor for death in COVID-19. These findings are important because they indicate that infected cells at the site of inflammation are a key source of Complement during COVID-19. They also shed light on why COVID-19 is hard to treat using COMPLEMENT inhibitors that only block plasma-derived Complement. Accordingly, we showed that cell-permeable CFB inhibitors, and inhibitors of IFN signaling, alone or with antivirals, abrogated intracellular Complement production. We proposed that combinatorial treatments to normalize this excessive response may be effective for management of hyperinflammation in COVID-19. Since SARS-CoV2-infected lungs are a Complement-rich environment (see above) and we showed that CD4+ T cells are themselves a tissue source of Complement (annual report from 2020), we hypothesized that studying transcriptomes of Complement-activated T cells would reveal the effects of Complement on T cells. We showed that Complement induces a self-contained autocrine/paracrine vitamin D (VitD) system in T cells that is required for appropriate inflammatory T cell shut-down, permitting tissue healing to take place. This paper is currently under revision but available as pre-print. Briefly, Complement induces a cell-intrinsic VitD system permitting T cells to both activate and respond to VitD. Sensing of active VitD causes genome-wide epigenetic changes that generate new and augment existing super-enhancers, and recruit key TFs (principally VDR, c-JUN, STAT3 and BACH2) that shape the VitD response in T cells. We found, again, that BACH2 was a central TF in these events. T cells of patients with COVID-19 showed Th1 hyper-activation and evidence of dysregulation of this VitD shut-down program, indicating either a lack of substrate (VitD deficiency) and/or abnormal regulation of this system. These data may explain the epidemiologic link between vitamin D insufficiency/deficiency and risk of both developing COVID-19 and suffering mortality after infection. They are also noteworthy because they establish cross-regulation between the two main interests of our lab, Complement and the TF BACH2. Whilst working on REC and T cell responses in COVID-19, we noted that some RNAseq reads in SARS-CoV2-infected cells aligned to both human and viral genomes, representing host-virus chimeric (HVC) transcripts. Others interpreted these as evidence of viral integration into the genome, generating great anxiety about the safety of SARS-CoV2 mRNA vaccines. DNA viruses (e.g. HBV and HPV), and retroviruses (e.g. HIV), have elaborate mechanisms for genome integration as part of their lifecycle. Not only is SARS-CoV2 a positive strand RNA virus without this machinery, but this virus has an exclusively cytoplasmic lifecycle, making genomic integration highly unlikely. We thus hypothesized that HVC reads represent in vitro artifacts of RNAseq. By analyzing RNAseq data from virally infected cells and patients with COVID-19, as well as developing a novel method for enriching RNAs containing viral sequences, we concluded that there is no compelling evidence to support integration of SARS-CoV2 into the human genome and that HVCs were most likely artifacts of RNAseq library preparation. This paper, the first to refute SARS-CoV2 integration, was published in the Journal of Virology, and accepted as cover art. These are important findings because they provide reassurance about viral integration and safety of mRNA vaccines. We also collaborated with a number of other labs, leveraging our expertise in gene regulation and transcriptional output. Outputs from these collaborations include co-authot publications in Nature Immunology, Science, Immunity and BMC Genomics.