AIM: To investigate the role of inflammatory and anti-viral genes in the pathogenesis of SARS.

METHODS: cDNA microarrays were used to screen the gene expression profiles of peripheral blood mononuclear cells (PBMCs) in two SARS patients (one in the acute severe phase and the other in the convalescent phase) and a healthy donor. In addition, real-time qualitative PCR was also performed to verify the reproducibility of the microarray results. The data were further analyzed.

RESULTS: Many inflammatory and anti-viral genes were differentially expressed in SARS patients. Compared to the healthy control or the convalescent case, plenty of pro-inflammatory cytokines such as IL-1, TNF-α, IL-8, and MAPK signaling pathway were significantly upregulated in the acute severe case. However, anti-inflammatory agents such as IL-4 receptor, IL-13 receptor, IL-1Ra, and TNF-α-induced proteins 3 and 6 also increased dramatically in the acute severe case. On the contrary, a lot of IFN-stimulated genes like PKR, GBP-1 and 2, CXCL-10 and 11, and JAK/STAT signal pathway were downregulated in the acute severe case compared to the convalescent case.

CONCLUSION: Gene expression in SARS patients mirrors a host state of inflammation and anti-viral immunity at the transcription level, and understanding of gene expression profiles may make contribution to further studies of the SARS pathogenesis.

A new contagious disease occurred in 2003[1-3], which lasted for at least 6 mo and swept over 29 countries in the world, causing numerous deaths and triggering public panic[4]. However, it took less than 2 mo to successfully identify the causative agent-a novel coronavirus[3,5]. Meanwhile, investigation of the unique pathogenic mechanism of this disease is still challenging and intriguing. Clinical data suggest that it is an abnormal pathological reaction to pulmonary viral infection characterized by acute lung injury[1,2,4,6] that determines the process of the symptoms. Acute lung injury is a multi-factorial, pathophysiological process involving cytokines and adhesion molecules, as well as inflammatory and immune cells[6-8]. Many pro- and anti-inflammatory cytokines such as IL-1, TNF-α, IL-8, IL-4, and IL-10 have been demonstrated to play a pivotal role in the pathogenesis of acute lung injury and severe systemic inflammation[6,8-11]. To determine the role of cytokines in the pathogenesis of SARS, immunological techniques such as RIA, ELISPOT, and ELISA have been employed to measure cytokine alterations in blood samples from SARS patients[12-14]. Jones et al[12] reported that the number of IFN-γ, IL-2, IL-4, IL-10, and IL-12 secreting cells induced by T-cell activators is below normal in many or most patients, while the number of cells which are induced to produce IL-6 and TNF-α by T-cell or monocyte activators is higher than normal in many early SARS patients, and increases in some SARS patients during and after treatment. Wong et al[13] found that Th1 cytokine, inflammatory cytokines such as IL-1, IL-6, and IL-12 and chemokines such as IL-8, MCP-1, and IP-10 are increased. Furthermore, Zhang et al[14] revealed that there is a difference in relationship between IL6, IL-8, TGF-β concentration, and SARS severity (positive for IL-6, but negative for IL-8 and TGF-β). Although these studies have shown the evidence of activated Th1 cell-mediated immunity and the hyper-innate inflammatory response, the role of these cytokines in the pathogenesis of the severe systemic inflammation and the mechanisms underlying the pathogenesis of SARS need to be further studied.

Development of microarray technology has provided a powerful tool for study of the complicated biological process in cells and tissues. cDNA microarray is used to analyze the virus-host cell interactions, and improvements have been achieved in the diagnosis, treatment, and prevention of infectious diseases[15]. Therefore, in this study, we used cDNA microarray to analyze the global gene expression profiles of peripheral blood mononuclear cells (PBMCs) from two SARS patients, one in the acute severe phase and the other in the convalescent phase. The results may make contribution to studies of the SARS pathogenesis.

As an emerging disease, attention has been paid to the high infectivity and virulence of SARS. In the course of the disease, the important observation is lymphopenia and the depletion of T-lymphocyte subsets in most SARS cases, indicating the immunity dysfunction in this readily-transmissible disease, particularly during its early phase[7]. Another characteristic of the disease is acute lung injury accompanied with signs of the systemic inflammation, the duration and intensity of which are closely associated with the severity and prognosis of the disease[1,2,4].

A typical feature of all inflammatory disorders is the excessive recruitment of leukocytes to the inflammation site, which is a well-orchestrated process involving several protein families, including pro-inflammatory cytokines, chemotactic cytokines, and adhesion molecules[9,10]. This process is resolved by anti-inflammatory cytokines such as IL-4, IL-10, IL-13, and TGF-β[10]. The well-known pro-inflammatory cytokines include IL-1 and TNF-α, which are induced as the signals by pattern recognition receptors (like TLRs) and initiate activation of a series of signal transduction networks to release mediators, prompting inflammation and immunity[10,16,17]. In our study, pro-inflammatory cytokines (like IL-1, IL-8, IL-17, IL-18, TNF-α and etc.) were highly expressed in the acute severe case and lowly expressed in the convalescent case. Real-time quantitative PCR of empirically selected genes also showed that pro-inflammatory cytokines were highly expressed. These findings, as expected, are consistent with the clinical stage[2,6,8], though individual variation and sensitivity of examination exist. In addition, anti-inflammatory agents (IL-4, 10, and 13 receptors) and agonists of IL-1 and TNF (type II IL-1 receptor, soluble IL-1 receptor, IL-1Ra, and TNF-α decoy receptor) increased dramatically in the acute severe case, which could constitute a negative feedback to robust inflammation or manifestations of systemic inflammation[16,17]. But whether the alteration is associated with virus replication or interaction between the viral and cellular proteins is to be further elucidated.

Chemokines are also important cytokines involved in inflammation, dendritic cell maturation, neutrophil degranulation, antibody class switching and T-cell activation[18-20]. Furthermore, recent in vitro and in vivo findings support some members of chemokine system like CXCL9, CXCL10, and CXCL11 contribute to the resolution of viral infections[19,20]. Unfortunately, transcripts of IFN-induced chemokines[18,19] (RANTES, CXCL10, and CXCL11) were downregulated in the acute severe case, while inflammatory chemokines such as GRO-1, G-CSF3R, IL-8 and its receptors increased significantly. The expression profiles of chemokines as well as neutrophil predominance in white cells reflect rampant inflammation in the acute severe case[8,9].

Although the acute severe case is treated with IFN-α, poor expression of IFN-stimulated genes[21], TLR3, 7 and immune cell activation markers (CD antigens and MHC molecules, data not shown) may mirror the defect of host anti-viral immunity. Furthermore, some studies indicate that the activation of some pro-inflammatory cytokines such as IL-1, IL-6 or IL-8 signaling pathways interferes with the IFN signaling[22-25], let alone the action of IL-4, IL-10 or IL-13[9]. Outbreak of pro- and anti-inflammatory agents might interfere with the IFN signaling pathway, but direct interaction between coronavirus and IFN system cannot be excluded.

It is intriguing to find that, although the important genes involved in NF-κB signaling pathway were not detected in our microarrays, JAK/STAT signaling pathway was suppressed, another important signaling pathway associated with production of inflammatory cytokines, the MAPK signaling pathway[20,26,27] was upregulated in the acute severe case compared to the healthy control or convalescent case. It was reported that there are expression alterations in the MAPK pathway in different leukocytes from patients with SARS and virus infection and viral proteins exert effects on the MAPK signaling pathway in cell culture models[27-29]. But the relationship between changes of three signaling pathways and the SARS process needs to be further studied.

In conclusion, our results may partially reveal the different expression patterns of inflammatory and immune genes and related signal pathways at different phases of SARS pathological process. The overexpressed pro- and anti-inflammatory cytokines may contribute to acute lung injury and imbalance of homeostasis especially in acute severe phase. Thus our results may hopefully make a contribution to further studies of the SARS pathogenesis.