Infection-experienced HSPCs protect against infections by generating neutrophils with enhanced mitochondrial bactericidal activity

Hematopoietic stem and progenitor cells (HSPCs) respond to infection by proliferating and generating in-demand neutrophils through a process called emergency granulopoiesis (EG). Recently, infection-induced changes in HSPCs have also been shown to underpin the longevity of trained immunity, where they generate innate immune cells with enhanced responses to subsequent microbial threats. Using larval zebrafish to live image neutrophils and HSPCs, we show that infection-experienced HSPCs generate neutrophils with enhanced bactericidal functions. Transcriptomic analysis of EG neutrophils uncovered a previously unknown function for mitochondrial reactive oxygen species in elevating neutrophil bactericidal activity. We also reveal that driving expression of zebrafish C/EBPβ within infection-naïve HSPCs is sufficient to generate neutrophils with similarly enhanced bactericidal capacity. Our work suggests that this demand-adapted source of neutrophils contributes to trained immunity by providing enhanced protection toward subsequent infections. Manipulating demand-driven granulopoiesis may provide a therapeutic strategy to boost neutrophil function and treat infectious disease.


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Figs. S1 to S7

Fig. S1 .
Fig. S1.Neutrophil numbers in EG larvae return to SS levels by 7 dpi, similar numbers of neutrophils are recruited to infections within SS and EG larvae and live confocal imaging of neutrophils following Sal-GFP and S. iniae infection.(A) Flow quantification of neutrophils from Tg(lyz:DsRED2) EG and SS larvae (as selected for in Fig. 1A-D) 1, 2, 3, 4, 5, 6 and 7 days post injection (dpi) with Sal-GFP and PBS at 2 dpi (n=10-15 larvae/sample, 5 experimental replicates).(B) Immunofluorescence detection of neutrophils in the hindbrain ventricles of SS and

Fig. S2 .
Fig. S2.Enhancing for the selection of EG neutrophils through photoconversion of newly generated neutrophils in the CHT.(A) Schematic illustrating strategy to enhance selection of EG neutrophils by photoconverting neutrophils within the CHT region.(B) Live imaging of SS and EG neutrophils within the CHT regions of Tg(mpx:Dendra2) larvae immediately prior to, and following, photoconversion.Red box marks photoconverted region.(C) Frame shots from live time-lapse confocal imaging of photoconverted SS and EG neutrophils within Tg(mpx:Dendra2) larvae showing volumes of intracellular Sal-GFP at the beginning (t=0) and end of the time-lapse experiment.(D) Bacterial killing rates of photoconverted SS and EG neutrophils following Sal-GFP infection.Green data points highlight killing rates of neutrophils as shown in C. Error bars, mean ± SD; * P <0.05; unpaired Student's t-test (D).CFU, colony-forming units.Scale bars, 50 µm in B and 10 µm in C.

Fig. S4 .
Fig. S4.Bacterial burdens in SS and EG larvae in the presence and absence of macrophages, confocal imaging of neutrophils in the absence of macrophages and transplanted neutrophils following Sal-GFP infection.(A) Bacterial burdens within individual L-PBS-injected SS and EG larvae at 3, 6 and 9 hours post secondary injection (hpsi) with Sal-GFP at 4 dpf.(B) Bacterial burdens within individual L-Clod.-injectedSS and EG larvae at 3, 6 and 9 hpsi with Sal-GFP at 4 dpf.(C) Frame shots from live time-lapse confocal imaging of SS and EG neutrophils within L-PBS-and L-Clod.-injectedTg(lyz:DsRED2;mpeg1:EGFP) larvae, showing volumes of intracellular Sal-GFP at the beginning (t=0) and end of the time-lapse experiment.(D) Frame shots from live time-lapse confocal imaging of SS and EG neutrophils transplanted from Tg(lyz:DsRED2) larvae into infection-naïve recipient larvae, showing volumes of intracellular Sal-GFP at the beginning (t=0) and end of the time-lapse experiment.(E) Live confocal imaging of ROS production within individual Sal-GFP-laden SS and EG neutrophils transplanted from Tg(lyz:DsRED2) larvae into infection-naïve recipient larvae, as detected by CellROX fluorescence.Error bars, mean ± SD; ns, not significant, ** P <0.01, *** P <0.001; unpaired Student's t-test (A and B).CFU, colony-forming units.Scale bar 10 µm.

Fig. S5 .
Fig. S5.Confocal imaging of mtROS production within neutrophils, Sal-GFP-laden neutrophils following MitoTEMPO treatment and expression of DEGs of interest timm23a and tfam.(A) Live confocal imaging of mtROS production within individual Sal-GFP-laden SS and EG neutrophils within Tg(lyz:EGFP) larvae, as detected by MitoSOX fluorescence, in the presence of DMSO (control) and MitoTEMPO.White dashed lines outline MitoSOX fluorescence within neutrophils.(B) Frame shots from live time-lapse confocal imaging of SS and EG neutrophils within Tg(lyz:DsRED2) larvae, in the presence of DMSO (control) and MitoTEMPO, showing volumes of intracellular Sal-GFP at the beginning (t=0) and end of the time-lapse