BioProject: The University of Texas Medical Branch, Galveston

  Source:		PRJNA118845
  Download:	https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE17400

     		Dynamic Innate Immune Responses of Human
			Bronchial Epithelial Cells against SARS-CoV
			and DOHV infection

			The University of Texas Medical Branch, Galveston (Owner) [website] [video]

			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)-β, -λs, SARS-related
			inflammatory mediators, and various IFN-stimulated
			genes (ISGs). While elevated responses of
			IFN-β and IFN-λs 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.
			Overall design: 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 “Gene Chips” 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


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