SARS-CoV Regulates Immune Function-Related Gene Expressions in Human Monocytic Cells

Severe Acute Respiratory Syndrome (SARS) is characterized by acute respiratory distress (ARDS) and pulmonary fibrosis, and the monocyte/macrophage is the key player in the pathogenesis of SARS. In this study, we compared the transcriptional profiles of SARS coronavirus (SARS-CoV) infected monocytic cells against that infected by coronavirus 229E (CoV-229E). Total RNA was extracted from infected DC-SIGN transfected monocytes (THP-1-DC-SIGN) at 6 and 24 h after infection and the gene expression was profiled by oligonucleotide-based microarray. Analysis of immune-related gene expression profiles showed that 24 h after SARS-CoV infection, (i) IFN-alpha/beta-inducible and cathepsin/proteosome genes were down-regulated; (ii) the hypoxia/hyperoxia-related genes were up-regulated; and (iii) the TLR/TLR-signaling, cytokine/cytokine receptor-related, chemokine/chemokine receptor-related, the lysosome-related, MHC/chaperon-related, and fibrosis-related genes were differentially regulated. These results elucidate that monocyte/macrophage dysfunction and dysregulation of fibrosis-related genes are two important pathogenic events of SARS.


Background
During the months of November 2002 to July 2003, the outbreak of Severe Acute Respiratory Syndrome (SARS) greatly impacted the public health around the world. A total of 8096 cases and 774 deaths [1] were reported. About 20% of SARS patients developed acute respiratory distress syndrome (ARDS) [2]. Chest X-rays revealed bilateral diffuse consolidation in these patients [3]. Massive macrophage infiltration is a prominent feature in the lung sections of patients who died of SARS [4]. SARS-CoV infects human monocytes [5] and monocytic cells infected by SARS-CoV produce chemokines that attract the migration of neutrophils, macrophages and activated T lymphocytes [6]. Patients who recovered from SARS often suffered a sequel of pulmonary fibrosis [7] and macrophages play a role in fibroblast accumulation [8]. Thus, it is strongly indicated that immune response plays an important role in the pathogenesis of SARS and that monocyte/macrophage is the key player in the immunopathogenesis of SARS.
Microarray is a recently developed tool that is useful in revealing the host response to an infectious agent at the genomic level. The microarray methodology has been used to show immune cell gene expression profile after infections by Mycobacterium leprae, CMV, HIV, E. coli, Chlamydia pneumoniae, and influenza virus H5N1 [9][10][11][12][13][14]. The gene expression profile in the peripheral blood mononuclear cells of convalescent SARS patients has been reported in a microarray study [15]. The molecular signature and disease severity index thus identified are useful for the diagnosis and prognosis of SARS-CoV infection if another SARS outbreak should occur [15]. However, the expression of immune function-related genes in SARS-CoV-infected monocyte/macrophage has never been revealed In the present study, oligo-microarray was used to profile the expression of immune function-related genes. We used DC-SIGN stably transfected monocytic THP-1 cells as targets to model the alveolar environment, as it has been shown that interstitial alveolar macrophages in the histologically normal adult lung tissue constitutively express DC-SIGN [16]. The gene expression profile induced by SARS-CoV was compared to the human coronavirus 229E (CoV-229E), a group I coronavirus that causes mild common cold, of which the infectivity of DC-SIGN-transfected THP-1 cells is comparable to that of SARS-CoV [6]. Since SARS-CoV induced chemokine gene expression in monocytic cells peaks at as early as 24 h [6], we chose to study the gene expression profiles at both 6 and 24 h after infection. The results of the present study showed that after SARS-CoV infection of monocytes, the expressions of (i) IFN--inducible and cathepsin/proteosome genes were down-regulated; (ii) the hypoxia/hyperoxia-related gene were up-regulated; and (iii) the TLR/TLR-signaling, cytokine/cytokine receptorrelated, chemokine/chemokine receptor-related, the lysosome-related, MHC/chaperonrelated, and fibrosis-related genes were differentially regulated.

RNA extraction
Total RNA was extracted from un-infected, CoV-229E infected, and SARS-CoV infected THP-1-DC-SIGN cells. After chloroform was added to each tube, samples were centrifuged and RNA contents were isolated from the upper aqueous phase. Glycogen and iso-propanol were added to precipitate the total RNA. After wash by 75% ethanol, total RNA was spun down and stored in DEPC-water. Twenty μg of RNAs from SARS-CoV-and CoV-229E-infected DC-SIGN-THP-1 cells and 40 μg of RNAs from uninfected controls were labeled with cyanine 3-dUTP(Cyt3) and cyanine 5-dUTP(Cyt5), respectively. The Cyt3 and Cyt5 labeled samples were then mixed with 1 μ1 of human Cot1-DNA(10μg/μl), polyA (8-10μg/μl) and yeast tRNA (4μg/μl) each to block non-specific binding. The sample mixture was then denatured at 100C for 1 min. Twenty μl of 2X hybridization buffer (50% formamide, 10X SSC, and 0.2% SDS) was added onto each prehybridized slide. Hybridization was performed in a humid chamber in a water bath at 42C overnight. Two arrays for both SARS-CoV and CoV-229E for each time-point were obtained.

Image and Data analysis
After hybridization, the slides were scanned on a GenePix 4000A scanner (Axon Instrument, Foster City, CA). The TIFF images were then analyzed by GenePix Pro software and GPR files were generated. Signal dots were aligned with the grid first. The signal dots that were too small were deleted. In the appearance of bubbles and severe scratches in the slide image, the signal dots were considered invalid. A PMT gain of red light (635nm) intensity and green light (532nm) intensity was adjusted according to the following principles: First, a general normalization method was applied to ensure that the ratio of total correct light intensity to green light intensity was equal to 1:1. Second, all the housekeeping gene-glyceraldehyde-3-phosphate dehydrogenease (GAPDH) dots were adjusted so they appear to be yellow, as GAPDH should not have been up-or downregulated. Microarray chip hybridization results for SARS-CoV and CoV-229E at 24h time point are shown as Figure 1A and 1B, respectively. All the data were uploaded to the US National Cancer Institute Microarray Website for data processing, transformation, and annotation. The transformed numeral data, with gene annotations, were then downloaded from the above website. Genes were highlighted when the average expression ratios (Fold change) were >1.5 (>1.5-fold up-regulation (boldface) or <0.67 (>1.5-fold down-regulation ) (underlined boldface). When the intensity ratio was positive from one replicate and negative from the other replicate, the images from the GPR files were re-checked. If the dada were still inconsistent, they were excluded from further analysis. Heatmap of microarray results is shown. (Figure 1)

Pathway network analysis
Ingenuity pathway analysis software was used to identify specific up-regulated or downregulated gene-to-gene network at 24h after SARS-CoV infection. Data including gene name and fold change levels were input into the software. Genes which are immune response related genes were selected in the pathway analysis. In this analysis, two major immunological pathway networks were identified including IL-8 centered network and proteasome-related gene network. (Figure 2)   (Table 5). It was most apparent at 24 h after infection, that cathepsins A, S, C, H, and D as well as proteosomes 4, 2, 26S ATPase 4 (26s/A4), 9, activator 2, 3, activator 1, 26S non-ATPase8 (26s nA8), and 5

Results
were all down-regulated >1.5-fold in which the changes of cathepsins H and D and proteosomes activator 2, activator 1, 26s nA8 and 5 gene expressions were >2-fold.
These results show that the MHC class II and I antigen processing pathways are inhibited in SARS-CoV infected monocytes.

The expressions of chaperon and MHC-related genes are differentially regulated
To further understand the regulation of genes that are directly involved in antigen processing/presentation, chaperon and MHC-related genes were analyzed. Table 6 shows that CoV-229E induced down-regulation of SEC61B and upregulation of X-box binding  (Table 9). These were hypoxia-inducible 2 and hypoxia up-regulated 1 genes. The oxidative responsive 1 gene up-regulation was observed only at 24 but not at 6 h after infection. These results demonstrate that SARS-CoV infection of monocytes creates a hypoxic environment, which induces the expression of hypoxia-related genes.

Discussion
Monocytes play a key role in mediating inflammatory response in the lungs of SARS patients [4]. Thus, the interaction between SARS-CoV and monocytes is important to the Thus, it appears that by down-regulating the genes that are important to antigen processing and presentation, SARS-CoV suppresses the primary functions of monocyte/macrophage. Since interferon up-regulates MHC related genes, the downregulation of interferon  genes could be partially responsible for the down-regulation of MHC-related genes [19]. It is our speculation that by suppressing the antigenprocessing and presentation functions of monocyte/macrophage, SARS-CoV delays specific T cell activation and thus delays its own clearance.
The clinical picture of SARS is characterized by pulmonary cellular infiltration and lung consolidation [1]. We have demonstrated infiltration of neutrophils, macrophages and T lymphocytes in the lungs of SARS-CoV-infected patient during early phase of infection (Yen 2006 JVI) and CXCL8/IL-8 was produced by SARS-CoV-infected monocytes. The results of microarray analysis in this study showed that in addition to CXCL8/IL-8 and major basic protein homolog (MBPH), is up-regulated by >2-fold (Table 7). Pathway network analysis showed many genes that up-regulate CXCL8/IL-8 were up-regulated after SARS-CoV infection (Figure 2). These include C/EBP delta, CD14, complement C3, and major basic protein homologue PRG3 [36][37][38][39]. A previous study demonstrated that C/EBP delta can substitute for IL-17 to induce neutrophil activation and accumulation [40]. Interestingly, alanyl aminopeptidase (ANPEP, Figure 2 Table 7).
We speculate that through the activity of hyaluonidase 3 and increased RHMM expression, more hyaluronan fragments are produced which contribute to the increase of neutrophil and monocyte infiltration and functions.
The relationship between leukotriene and lung fibrosis has been demonstrated in mice deficient in 5-lipoxygenase knockout mice. The knockout mice which are deficient in leukotriene are protected from lung fibrosis induced by bleomycin ( [46]. No increase of inflammatory cells in the lungs was noted in the knockout mice in contrast to wild-type mice which have abundant leukocytes. Leukotriene A4 (LTXA4) hydroxylase catalyses the production of leukotiene B4 from leukotriene A4 while leukotriene C4 (LTXC4) synthases mediates the production of leukotriene C4 from leukotriene A4 [47,48]. Our results showing down-regulation of LTXC4 synthases (LTXC4 synthase, microsomal glutathione-S-transferase2, microsomal glutathione-S-transferase3) and up-regulation of LTXA4 hydroxylase (Table 7) indicate that there is increased leukotriene B4 accumulation after SARS-CoV infection. Therefore, leukontriene B4, a potent chemoattractant for neutrophils [49], could also be accounted for the increase of neutrophil migration in the lungs.
Prostaglandin E (PGE) is commonly used as a treatment for ARDS [50]. PGE2 inhibits fibroblast proliferation, collagen synthesis and fibroblast chemotaxis [51]. In cycloxygenase-2-deficient mice, the severity of intratracheal bleomycin-induced lung fibrosis is increased due to PGE2 reduction [52]. It is worth noting that both prostaglandin E and prostaglandin D synthetases (PGES and PGDS) are down-regulated in SARS-CoV infected cells (Table 7) implying the PGE2 level is reduced in SARS patients which may contribute to lung fibrosis. Platelet-derived growth factor (PDGF) is a major fibroblast mitogen [53] . It also serves as a chemoattractant for neutrophils, monocytes, and fibroblasts [54]. Therefore, up-regulation of PDGF receptor  by SARS-CoV (Table 7) through PDGF signaling would result in fibroblast proliferation, neutrophil and monocyte infiltration and worsened lung fibrosis [55].
Lung fibrosis is a squeal of severe acute pulmonary disease [7]. It is thus of interest to understand how SARS-CoV affects the expression of fibrosis-related genes. A previous study revealed that collagen III is up-regulated in ARDS and its levels are related to poor prognosis [56]. Proline 2-oxoglutarate 4-dioxygenase (P4HA1), a key enzyme in collagen synthesis is up-regulated but collagen I2 and collagen XVIII1 are downregulated by SARS-CoV infection (Table 8)