Zfh2 is an in vivo mediator of hypercapnic immune suppression
James Kwon1, Ryan Haake1, Peter H. S. Sporn2 and Greg J. Beitel1
1Dept. of Molecular Biosciences, Northwestern University, Evanston, IL
2Division of Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL
Patients with advanced chronic obstructive pulmonary disease (COPD), the third leading cause of death in the U.S., and other severe lung diseases often develop hypercapnia (an elevated level of CO2 in blood and tissues), which is associated with increased risk of pulmonary infection and mortality. Previously, we reported the results of an S2 cell-based RNAi screen to identify candidate genes that modulate immune suppression by elevated CO2 levels. Knock down of select genes reversed the suppression of Diptericin (Dipt) by CO2, restoring Dipt expression in elevated CO2 towards levels observed in air. We also reported preliminary results suggesting that the transcription factor Zfh2 is an in vivo mediator of hypercapnic immune suppression. We have now confirmed those results by showing that in S. aureus infection assays, flies homozygous for the zfh2[209] mutation show decreased mortality and decreased bacterial loads compared to w[1118] control flies in 13% CO2, but not in air. Importantly, Western blotting shows that in zfh2[209] mutants the level of Zfh2 protein is reduced in the fat body, the major organ of immune response, but not the head or carcass (the body minus the fat body and head). Further evidence that the fat body is a critical site of action for zfh2 was obtained by knocking down zfh2 using the shRNA of zfh2[13305] and either of the fat body drivers CG-Gal4 or C754, which increased survival and decreased bacterial loads in elevated CO2 conditions. Using qPCR and a newly developed ex vivo fat body culture system, we determined that reducing zfh2 levels increased expression of the antimicrobial peptide genes Diptericin, Attacin and Metchnikowin in elevated CO2 conditions, but not in air. Together these results define Zfh2 as the first identified in vivo mediator of hypercapnic immune suppression and the fat body as a critical site of action of hypercapnic immune suppression.