Th-17 regulatory cytokines IL-21, IL-23, and IL-6 enhance neutrophil production of IL-17 cytokines during asthma

ABSTRACT

Background: In a subset of severe asthma patients, chronic airway inflammation is associated with infiltration of neutrophils, Th-17 cells and elevated expression of Th-17-derived cytokines (e.g., interleukin [IL]-17, IL-21, IL-22). Peripheral neutrophils from allergic asthmatics are known to express higher IL-17 cytokine levels than those from healthy subjects, but the regulatory mechanisms involved are not well understood. We hypothesize that Th-17 regulatory cytokines could modulate IL-17 expression in neutrophils. Methods: Peripheral blood neutrophils isolated from asthmatics were stimulated with IL-21, IL-23, and IL-6 cytokines and their ability to produce IL-17A and IL-17F was determined relative to healthy controls. Signal transducer and activator of transcription 3 (STAT3) phosphorylation levels were measured in stimulated neutrophil using flow cytometry. The requirement for STAT3 phosphorylation was determined by blocking its activation using a specific chemical inhibitor. Results: Stimulating asthmatic neutrophils with IL-21, 23, and 6 enhanced the production of IL-17A and IL-17F at significantly higher levels comparatively to healthy controls. Stimulating neutrophils with IL-21, IL-23, and IL-6 cytokines enhanced STAT3 phosphorylation, in all cases. Interestingly, inhibiting STAT3 phosphorylation using a specific chemical inhibitor dramatically blocked the ability of neutrophils to produce IL-17, demonstrating that STAT3 activation is the major factor mediating IL-17 gene expression. Conclusions: These findings suggest that neutrophil infiltration in lungs of severe asthmatics may represent an important source of pro-inflammatory IL-17A and -F cytokines, a production enhanced by Th-17 regulatory cytokines, and thus providing a feedback mechanism that sustains inflammation. Our results suggest that STAT3 pathway could be a potential target for regulating neutrophilic inflammation during severe asthma.

KEYWORDS:

• Asthma
• cytokines
• inflammation
• IL-21
• IL-23
• Neutrophils
• Th-17 cells

Acknowledgements

The authors would like to thank Dr Amer Mahmood from Stem Cell Unit, Department of Anatomy, King Saud University, and Mr Sibtain Afzal for technical support.

Declaration of interest

The author(s) declare that they have no competing interests.

Funding

The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at king Saud University for funding this Research group NO (RGP-1437-026). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Authors' contributions

RH carried out the real-time PCR experiments and FACS analysis experiments, conceived of the study, lead efforts on its design, and coordinated and finalized the manuscript draft. AS carried out phosflow experiments. AJ performed the western analysis and densitometry measures. AVT and AA participated in the design of the study and performed the statistical analysis. SM contributed in recruiting patients to the study. All authors read and approved the final manuscript.