Neutralization of TNF-α and IL-1β Regulates CXCL8 Production through CXCL8/CXCR1 Axis in Macrophages during Staphylococcus aureus Infection

ABSTRACT

Anti-cytokine therapy is widely acknowledged as an anti-inflammatory technique to treat varied infectious diseases. TNF-α and IL-1β are major cytokines that regulate every aspect of the inflammatory process. However, the effects of single or dual cytokine neutralization on S. aureus mediated CXCL8 secretion and CXCR1 expression in murine peritoneal macrophages remained noninvestigated. Thus we aimed to explore the effects of kinetic-dose dependent neutralization of TNF-α and IL-1β using specific anti-cytokine antibodies and its influential impact on the CXCL8/CXCR1 axis at different stages of S. aureus (30, 60, and 90 min) infection. The murine peritoneal macrophages were isolated and infected with viable S. aureus followed by subsequent addition of anti-TNF-α and anti-IL-1β into the medium. The treated cells were centrifuged and lysate and supernatant collected for various experiments. The ROS generation was measured and cytokine production was estimated by ELISA. The expression of TNFR1, IL-1R, CXCR1, signaling molecules (NF-κB and JNK) were evaluated by Western blot. The role of single or dual cytokine neutralization on intracellular bacterial phagocytosis had also been analyzed by confocal microscopy. Dual cytokine neutralization significantly suppressed ROS, cytokines, CXCL8 secretion, and intracellular bacterial count compared to single cytokine neutralization and it was more apparent at 90 min post S. aureus infection. There was a drastic reduction in TNFR1, IL-1R, and CXCR1 expression on macrophage surface due to reduced expression of downstream signaling molecules, NF-κB and JNK. Hence dual cytokine neutralization was more effectual compared to single cytokine neutralization in the downregulation of S. aureus induced CXCR1 expression.

KEYWORDS:

• CXCR1
• cytokine neutralization
• interleukin-1 beta
• Staphylococcus aureus
• tumor necrosis factor-alpha

Acknowledgments

The author thanks the University Grants Commission (UGC), Government of India, New Delhi, India for funding this project under the University for potential of excellence (UPE) scheme in the UGC UPE II grant: Focus area: Modern Biology Group C2: Mechanistic and therapeutic aspects of infectious diseases [Sanctioned No: UGC/856/UPE-2/MOD BIO/2nd Inst/dated 18.09.2017]. The author is indebted to, Department of Science and Technology, Government of India for providing us with the instruments procured under the DST-PURSE program to the Department of Physiology, University of Calcutta.

Declaration of interest

The authors declare no conflicts of interest for the manuscript entitled “Neutralization of TNF-α and IL-1β regulates CXCL8 production through CXCL8/CXCR1 axis in macrophages during Staphylococcus aureus infection” for submission in Immunological Investigations. The authors also state that they do not have a direct financial relationship with the commercial identities mentioned in this manuscript.

Supplementary material

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Additional information

Funding

This work was supported by the University Grants Commission (UGC), Government of India, New Delhi, India [Sanctioned No: UGC/856/UPE-2/MOD BIO/2nd Inst date].