Lodi, A. | bioRxiv Science

bioRxiv2024

An in vivo 'turning model' reveals new RanBP9 interactions in lung macrophages.

The biological functions of the scaffold protein Ran Binding Protein 9 (RanBP9) remain elusive in macrophages or any other cell type where this protein is expressed together with its CTLH (C-terminal to LisH) complex partners. We have engineered a new mouse model, named RanBP9-TurnX, where RanBP9 fused to three copies of the HA tag (RanBP9-3xHA) can be turned into RanBP9-V5 tagged upon Cre-mediated recombination. We created this model to enable stringent biochemical studies at cell type specific level throughout the entire organism. Here, we have used this tool crossed with LysM-Cre transgenic mice to identify RanBP9 interactions in lung macrophages. We show that RanBP9-V5 and RanBP9-3xHA can be both co-immunoprecipitated with the known members of the CTLH complex from the same whole lung lysates. However, more than ninety percent of the proteins pulled down by RanBP9-V5 differ from those pulled-down by RanBP9-HA. The lung RanBP9-V5 associated proteome includes previously unknown interactions with macrophage-specific proteins as well as with players of the innate immune response, DNA damage response, metabolism, and mitochondrial function. This work provides the first lung specific RanBP9-associated interactome in physiological conditions and reveals that RanBP9 and the CTLH complex could be key regulators of macrophage bioenergetics and immune functions.

cell biology↗

bioRxiv2021

Differential plasmacytoid dendritic cell phenotype and type I Interferon response in asymptomatic and severe COVID-19 infection

SARS-CoV-2 fine-tunes the interferon (IFN)-induced antiviral responses, which play a key role in preventing coronavirus disease 2019 (COVID-19) progression. Indeed, critically ill patients show an impaired type I IFN response accompanied by elevated inflammatory cytokine and chemokine levels, responsible for cell and tissue damage and associated multi-organ failure. Here, the early interaction between SARS-CoV-2 and immune cells was investigated by interrogating an in vitro human peripheral blood mononuclear cell (PBMC)-based experimental model. We found that, even in absence of a productive viral replication, the virus mediates a vigorous TLR7/8-dependent production of both type I and III IFNs and inflammatory cytokines and chemokines, known to contribute to the cytokine storm observed in COVID-19. Interestingly, we observed how virus-induced type I IFN secreted by PBMC enhances anti-viral response in infected lung epithelial cells, thus, inhibiting viral replication. This type I IFN was released by plasmacytoid dendritic cells (pDC) via an ACE-2-indipendent mechanism. Viral sensing regulates pDC phenotype by inducing cell surface expression of PD-L1 marker, a feature of type I IFN producing cells. Coherently to what observed in vitro, asymptomatic SARS-CoV-2 infected subjects displayed a similar pDC phenotype associated to a very high serum type I IFN level and induction of anti-viral IFN-stimulated genes in PBMC. Conversely, hospitalized patients with severe COVID-19 display very low frequency of circulating pDC with an inflammatory phenotype and high levels of chemokines and pro-inflammatory cytokines in serum. This study further shed light on the early events resulting from the interaction between SARS-CoV-2 and immune cells occurring in vitro and confirmed ex vivo. These observations can improve our understanding on the contribution of pDC/type I IFN axis in the regulation of the anti-viral state in asymptomatic and severe COVID-19 patients. Author summarySARS-CoV-2 pandemic has resulted in millions of infections and deaths worldwide, yet the role of host innate immune responses in COVID-19 pathogenesis remains only partially characterized. Innate immunity represents the first line of host defense against viruses. Upon viral recognition, the secretion of type I and III interferons (IFN) establishes the cellular state of viral resistance, and contributes to induce the specific adaptive immune responses. Moving from in vitro evidences on the protective role played by plasmacytoid dendritic cells (pDC)-released type I IFN in the early phase of SARS-CoV-2 infection, here we characterized ex vivo the pDC phenotype and the balance between anti-viral and pro-inflammatory cytokines of COVID-19 patients stratified according to disease severity. Our study confirms in COVID-19 the crucial and protective role of pDC/type I IFN axis, whose deeper understanding may contribute to the development of novel pharmacological strategies and/or host-directed therapies aimed at boosting pDC response since the early phases of SARS-CoV-2 infection.

immunology↗