Project description:Inflammation following SARS-CoV-2 infection is a hallmark of COVID-19 and predictive of morbidity and death. However, the inflammatory pathways contributing to host-defense vs immune-mediated pathology have not been fully elucidated. This duality is clearly seen with type-I interferons (IFN-I) which are critical mediators of innate control of viral infections, but also drive recruitment of inflammatory cells to site of infection, a key feature of severe COVID-19. Here, we modulated IFN-I signaling in rhesus macaques (Macaca mulatta) prior to and during acute SARS-CoV-2 infection (day -1 through day 2 post infection) using a mutated IFNα2 (IFN-modulator; IFNmod) which blocks binding to IFNAR2 and signaling of endogenous IFN-I. IFNmod treatment resulted in a highly significant and consistent reduction in SARS-CoV-2 viral load in BAL, lung, and hilar LN (>3-log difference) and upper airways (nasal swabs). IFNmod also potently reduced inflammatory cytokines and chemokines in BAL, expansion of inflammatory monocytes (CD14+CD16+), and lung pathogenesis. Furthermore, Siglec-1 expression, which has been shown to enhance SARS-CoV-2 infection, was rapidly downregulated in the lung and on monocytes of IFNmod-treated SARS-CoV-2 infected RMs. Notably, while RNAseq analysis showed that IFNmod induced a modest upregulation of antiviral IFN-stimulated genes (ISGs) in uninfected RMs, it resulted in a robust reduction in pathways associated with both antiviral and inflammatory ISGs in SARS-CoV-2-infected RMs. In conclusion, IFNmod treatment provides sufficient levels of type I IFN signaling that inhibit viral replication while also limiting hyperinflammation. IFN-I plays a vital role in regulating SARS-CoV-2 replication, but uncontrolled IFN signaling has a detrimental impact on inflammation and pathogenesis. A better understanding of the role of IFN-I pathways is essential for designing therapies targeting these pathways and aimed at limiting COVID-19 pathogenesis.

Project description:Background: Type I interferons (IFNs) are essential to the clearance of viral diseases, in part by initiating upregulation of IFN regulated genes (IRGs). A clear distinction between genes upregulated directly by virus and genes upregulated by secondary IFN production has not been made. Here we investigated the genes regulated by IFN-a2b compared to the genes regulated by SARS-CoV infection in ferrets. Methods: We characterized early host immune responses in peripheral blood and lung necropsies of ferrets injected with IFN-a2b or infected with SARS-CoV/Tor 2 strain, using microarray analysis on the Affymetrix platform. Results: We identified a common IRG signature that was upregulated in both SARS-CoV infected ferrets as well as in ferrets injected with IFN-a2b. We also identified unique patterns of gene expression for leukocyte activation, cell adhesion and complement pathways between IFN-a2b injection and SARS-CoV infection. Conclusions: Our results define the effects of IFN-a2b on the immune system of ferrets highlighting genes regulated by IFN during SARS-CoV infection. We have shown the similarities and differences of top funcional gene groups as well as pathways that play key roles in early immune responses in ferrets in response to IFN-a2b or SARS-CoV. Key words: ferret, gene expression, SARS, interferon. Keywords: time course In experiments with IFN-a2b, for peripheral blood, 15 ferrets were randomly allocated to 3 groups: Day 0, 5 ferrets (no IFN injection), day 1, 6 ferrets (injected), and day 2, 4 ferrets (injected). For lung necropsies of injected ferrets with IFN-a2b, we used 12 ferrets in 3 groups: 4 ferrets, day 0 (no IFN injection), 4 ferrets, day 1 (injected) and 4 ferrets, day 2 (injected). Experimental groups for SARS-CoV infection was as follows: For peripheral blood, 3 and 4 ferrets for day 0 (no infection) and day 2 (infection) respectively. For lung neceropsies, a total of 9 ferrets in 3 groups, each with 3 replicates for day 0 (no infection), day 1 (infection) and day 2 (infection).

Project description:Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infection causes an immune-mediated disease. We have recently shown that SARS-CoV-induced epithelial Calu-3 cytokines could exacerbate and dampen host inflammatory and T cell responses, respectively, through modulating the functions of macrophages and dendritic cells, thereby suggesting that not only are lung epithelial cells the primary cells of SARS-CoV infection, but they also involve in initiating and orchestrating the host innate and adaptive immunity. Comprehensive evaluation of the complex epithelial signaling to SARS-CoV is, thus, crucial for paving the way to better understand SARS pathogenesis and develop the innovative therapeutics against SARS. Here, based on the microarray-based functional genomics, we reported that 2B4 cells, a clonal derivative of Calu-3 cells, elicited a temporal and spatial activation of nuclear factor (NF)kappaB, activator protein (AP)-1 (ATF2/c-Jun), and interferon regulatory factor (IRF)-3/-7 at 12-, 24-, and 48-hrs post infection (p.i.), respectively, resulting in the activation of many antiviral genes, including interferon (IFN)-Î², -Î»s, SARS-related inflammatory mediators, and various IFN-stimulated genes (ISGs). While elevated responses of IFN-Î² and IFN-Î»s were not detected until 48-hrs p.i., as a consequence of a delayed IRF-3/-7 activation, we showed, for the first time, that both types of IFNs exerted previously under-described non-redundant, complementary, and/or synergistic effects on the epithelial defense against SARS-CoV. Collectively, our results highlight the molecular mechanisms of the sequential activation of virus- and IFN-dependent signaling of lung epithelial cells against SARS-CoV and identify novel cellular targets for future studies, aiming at advancing strategies against SARS. DNA microarrays were performed in the Molecular Genomics (MG) Core Facilities at UTMB. The detailed information about the procedures of microarray analysis has been posted on the MG Core Facilities website (genomics@scms.utmb.edu). To characterize the dynamic, spatial, and temporal changes of the gene expression induced by SARS-CoV, confluent 2B4 cells grown in T-75 flasks were infected with SARS-CoV (MOI=0.1) or remained uninfected (as control) for 12, 24, and 48hrs. Because 2B4 cells were also permissive to the productive infection of Dhori virus (DHOV), a member of the Orthomyxoviridae family within the Thogotovirus genus, resulting in robust responses of IFNs and other pro-inflammatory mediators, we also established parallel cultures of DHOV-infected 2B4 cells (MOI=0.1) for the comparative analysis of global gene expression elicited by SARS-CoV- versus DHOV-infected 2B4 cells. To meet the minimal number required for application of statistical algorithms, we performed the study in triplicate at each time point for mock-, SARS-CoV-, and DHOV-infected cultures, yielding a total of 27 arrays. Briefly, supernatants were harvested from differentially treated cultures at 12-, 24-, and 48-hrs p.i. for subsequent analyses of viral yields and cytokine profiling, whereas the cells were subjected to total RNA extraction by using an RNAqueous-4PCR kit and following the protocol recommended by the manufacturer (Ambion, Austin, TX). Purified RNA samples were sent to our core facility for conversion to cDNA, biotin-labeled, and hybridized to 27 Affymetrix Human Genome U133 Plus 2.0 âGene Chipsâ each of which contained 54,675 probe set identifiers representing more than ~47,400 transcripts that identify ~38,500 well-characterized genes, and various internal controls (Affymetrix, Santa Clara, CA). Mock-infected cells were compared to cells infected with SARS-CoV or DHOV at each time point.

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Modulation of type-I interferon responses in Rhesus Macaque whole blood

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