FIGURE SUMMARY
Title

Klebsiella pneumoniae alters zebrafish circadian rhythm via inflammatory pathways and is dependent on light cues

Authors
Ding, H., Chen, X.C., Wan, L., Zhang, Y.Y., Rui, X.H., He, T., Liu, J., Shang, Z.B.
Source
Full text @ Heliyon
Fig. 1

Locomotor ability of zebrafish exposed to Klebsiella pneumoniae decreased under 14 h/10 h light-dark (L/D) conditions. (A) Locomotor ability of juvenile fish in the control and NTUH-2044 groups under L/D conditions. (B) Total travel distance of juvenile zebrafish in light and dark time periods in the control and NTUH-2044 groups. (C) Average swimming speed of juvenile zebrafish in light and dark time periods in the control and NTUH-2044 groups. Each experiment was repeated in triplicate. Data are expressed as the mean ± standard deviation (SD). Data were analyzed using Student's t-test. (***P < 0.001, ns: not significant).

Fig. 2

Changes in behavior of Klebsiella pneumoniae-infected zebrafish under continuous darkness (D/D) conditions. (A) Behavioral rhythm analysis of juvenile fish in the control and NTUH-2044 groups under D/D conditions. (B) Amplitude comparison of juvenile zebrafish in the control and NTUH-2044 groups under D/D conditions. (C) Period comparison of juvenile zebrafish in the control and NTUH-2044 groups under D/D conditions. (D) Phase comparison of juvenile zebrafish in the control and NTUH-2044 groups under D/D conditions. Each experiment was repeated three times. Data are expressed as the mean ± standard deviation (SD). Data were analyzed using Student's t-test. (*P < 0.05, **P < 0.01, ***P < 0.001).

Fig. 3

Expression of core circadian rhythm genes in Klebsiella pneumoniae-infected zebrafish. Expression levels of (A) bmal1b, clock1a, cry1aa, per1b, per2, and aanat2 in the control and NTUH-2044 groups under light/dark (L/D) conditions. Expression levels of (B) bmal1b, clock1a, cry1aa, per2, per1b, and aanat2 in the control and NTUH-2044 treatment groups under D/D conditions. Each experiment was repeated three times. Data are expressed as the mean ± standard deviation (SD). Data were analyzed using Student's t-test. (*P < 0.05, **P < 0.01, ***P < 0.001).

Fig. 4

Inflammatory cell and gene expression levels in zebrafish infected with Klebsiella pneumoniae and treated under light/dark (L/D) conditions. (A) Neutrophil recruitment of Tg(mpx:EGFP) transgenic zebrafish in the control and NTUH-2044 treatment groups under L/D conditions. (B) Number of neutrophils in Tg(mpx:EGFP) transgenic zebrafish compared with those in the control and NTUH-2044 groups under L/D conditions. (C) Macrophage recruitment of Tg(mpeg:EGFP) transgenic zebrafish in the control and NTUH-2044 groups under L/D conditions. (D) Number of macrophages in Tg(mpeg:EGFP) transgenic zebrafish compared with those in the control and NTUH-2044 groups under L/D conditions. qRT-PCR analysis of inflammation-related genes il1b (E), nfkb (F), tnfa (G), and tgfb1a (H) in the control and NTUH-2044 groups under L/D conditions. Each experiment was repeated three times. Data are expressed as the mean ± standard deviation (SD). Data were analyzed using Student's t-test. (ANOVA) (*P < 0.05, **P < 0.01, ***P < 0.001).

Fig. 5

Anti-inflammatory drugs reduce Klebsiella pneumoniae-induced inflammation. Zebrafish were infected with NTUH-K2044 for 48 hpf, followed by treatment with the anti-inflammatory drug G6PDi-1, and inflammation was evaluated at 5 days post-fertilization (dpf). (A) Neutrophil recruitment in Tg(mpx:EGFP) transgenic zebrafish in the control, NTUH-2044, and NTUH-2044+G6PDi-1 treatment groups. (B) Number of neutrophils in Tg(mpx:EGFP) transgenic zebrafish in the control, NTUH-2044, and NTUH-2044+G6PDi-1 treatment groups. (C) Macrophage recruitment of Tg(mpx:EGFP) transgenic zebrafish in the control, NTUH-2044, and NTUH-2044+G6PDi-1 treatment groups. (D) Number of macrophages in Tg(mpx:EGFP) transgenic zebrafish in the control, NTUH-2044, and NTUH-2044+G6PDi-1 treatment groups. qRT-PCR analysis of inflammation-related genes il1b (E), nfkb (F), tnfα (G), and tgfb1a (H) in the control, NTUH-2044, and NTUH-2044+G6PDi-1 treatment groups. Each experiment was performed in triplicate. Data are expressed as the mean ± standard deviation (SD). Data were analyzed by Student's t-test.(ANOVA) (**P < 0.01,***P < 0.001).

Fig. 6

Anti-inflammatory drug rescue of Klebsiella pneumoniae-disrupted circadian rhythm genes. Zebrafish were infected with NTUH-K2044 for 48 hpf, followed by treatment with the anti-inflammatory drug G6PDi-1, and the expression of genes associated with circadian rhythm were evaluated in different groups of zebrafish at 5 dpf. qRT-PCR analysis of zebrafish clock genes per2 (A), cry1ab (B), cry1aa (C), and clock1a (D) in the control, NTUH-2044, and NTUH-2044+G6PDi-1 treatment groups. Each experiment was performed in triplicate. Data are expressed as the mean ± standard deviation (SD). Data were analyzed using Student's t-test. (**P < 0.01, ***P < 0.001).

Fig. 7

Proposed scheme of how Klebsiella pneumoniae alters zebrafish circadian rhythm via inflammatory pathways dependent on light cues.

Acknowledgments
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