Cellular signaling events in cytokines and sulphamethoxasole interactions

Abstract

Introduction: The use of sulphonamides as antimicrobial agents has been practiced for decades. It is frequently used with another antimicrobial agent trimethoprim to provide a more effective antimicrobial spectrum in treating bacterial and protozoan infections in immunocompromised patients. During bacterial infection, proinflammatory cytokines including tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-12 as well as anti-inflammatory cytokines including IL-4 and IL-10 are produced by macrophages. The complexity of cytokine interactions provides a favorable environment for the host immune system to fight against pathogens. In previous studies, the metabolites of sulphamethoxazole have been demonstrated to have a role in the antimicrobial effects but without the details on molecular mechanisms. In the present study, we delineated the mechanisms and effects of sulphamethoxazole metabolites on cytokine production. Methods: With the use of primary human differentiated blood macrophages as our model, the cells were treated for 30 minutes with the drug metabolites and followed by the addition of bacterial endotoxin (lipopolysaccharide LPS) for 15 minutes to 3 hours. Cytokine mRNA and protein productions were measured by Quantitative Real-time PCR and ELISA, respectively. For the investigation of signaling events including the role of kinases and transcription factors, cellular and nuclear protein fractions were collected and analysed by specific Western blot assays. Results: The results showed that the LPS-induced cytokines including TNF-α, IL-6 and IL-10 were downregulated by the sulphamethoxazole metabolites both at the transcription and translation levels. Since the expression of cytokines is mediated by the action of signaling kinases such as mitogen-activated protein kinases (MAPK) and transcription factors, we measured the activity status of MAPK in the sulfphamethoxazole metabolites-treated macrophages. The results showed that sulphamethoxazole metabolites abrogated the LPS-induced MAPK phosphorylation, concomitant with their effects on cytokine downregulation. Furthermore, the activation of nuclear factor-κB (NF-κB) induced by LPS was also suppressed by the metabolites. Conclusion: In conclusion, our data elucidated that in addition to their antimicrobial effects, sulphamethoxazole and its metabolites may play a role in limiting the propagation of uncontrolled inflammation, via the suppression of MAPK and NF-κB activities, in microbial infections.