FIGURE 4. zfh2 mediates hypercapnic immune suppression in vivo.

(A) Control (w¹¹¹⁸) flies exposed to hypercapnia (13% CO₂) for 48 h, then inoculated with S. aureus, show reduced survival compared to S. aureus-infected flies exposed only to air (n= 636 for air, 622 for 13% CO₂). For mortality experiments, p-values were calculated using the Gehan-Breslow-Wilcoxon test.

(B) In air, mortality of S. aureus-infection in zfh2^MS209 mutant flies is not different than in w¹¹¹⁸ control flies (n= 636 for w¹¹¹⁸, 358 for w¹¹¹⁸; zfh2^MS209).

(C) When pre-exposed to 13% CO₂, zfh2^MS209 flies exhibit decreased mortality from S. aureus infection compared to w¹¹¹⁸ control flies (n= 622 for w¹¹¹⁸, 336 for w¹¹¹⁸; zfh2^MS209).

(D) zfh2^MS209 flies are partially protected against the increase in mortality of S. aureus infection caused by exposure to elevated CO₂, Genotype: w¹¹¹⁸; zfh2^MS209 (n=358 for air, 336 for 13% CO₂). (Compare to w¹¹¹⁸ control flies in Figure 4A.)

(E) Pre-exposure to 13% CO₂ increases bacterial load in w¹¹¹⁸ but not w¹¹¹⁸; zfh2^MS209 flies. Error bars show mean and SEM of the log₁₀ load values (n= 20 for w¹¹¹⁸ in air and 13% CO₂, 16 for zfh2^MS209 in air and CO₂).

(F) Zfh2 protein levels are reduced in the fat body, but not the carcass or head, of flies with CG-GAL4 driven expression of the UAS-zfh2¹³³⁰⁵ short hairpin RNAi construct (RNAi; genotype w¹¹¹⁸;CG-GAL4/UAS-zfh2¹³³⁰⁵). CG, control genotype w¹¹¹⁸;CG-GAL4/+.

(G) Exposure to 13% CO₂ for 48 h prior to infection increases bacterial load in control (w¹¹¹⁸;CG-GAL4 /+) but not CG-GAL4/UAS-zfh2¹³³⁰⁵ flies. Error bars show mean and SEM of the log₁₀ load values (n= 24 for each condition).

(H) Exposure to 13% CO₂ for 48 h prior to infection increases the mortality of S. aureus infection in control CG-GAL4 flies (w¹¹¹⁸;CG-GAL4/+) (n= 428 for air, 465 for 13% CO₂).

(I) Exposure to 13% CO₂ for 48 h prior to infection does not increase mortality of S. aureus infection in flies in which zfh2 was knocked-down in the fat body (w¹¹¹⁸;CG-GAL4/UAS - zfh2¹³³⁰⁵) (n= 610 for air, 685 for 13% CO₂).

(J,K) Results similar to those in H and I were obtained using another fat-body specific driver, C754-GAL4 (n= 314 for control-air; 357 for control-13% CO₂, 459 for C754-GAL4/UAS-zfh2¹³³⁰⁵-air; n = 488 for C754-GAL4/UAS-zfh2¹³³⁰⁵-13% CO₂).