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- Fig. 1.
The effect of priming agents on M. marinum loads in adult zebrafish. (A) Outline of the study. Adult wild-type zebrafish were primed with 0.5×107-1×107 colony-forming units (cfu) per fish of heat-killed bacteria or with other priming agents (13.5 µg per fish, except MDP 4.5 µg) 1 or 7 days prior to M. marinum infection (−1/7 day) with an i.p. injection. Sterile 1× PBS was used as an injection control. The following day (0 day), a low dose of M. marinum was injected i.p. into the zebrafish. Internal organs were collected either 2, 4 or 7 weeks post infection (wpi), DNA was extracted and the bacterial counts were measured with M. marinum-specific qPCR. (B) Priming with heat-killed L. monocytogenes (HKLm) reduces mycobacterial loads at 7 wpi. Zebrafish were primed with either PBS (n=8), heat-killed M. marinum (HKMm; n=9), HKLm (n=10), heat-killed S. iniae (HKSi; n=8), heat-killed E. coli (KHEc; n=9), lipopolysaccharide (LPS; n=8), paclitaxel (n=5), muramyl dipeptide (MDP; n=9) or zymosan (n=10) 1 day prior to M. marinum infection (16±4 cfu). Organs were collected at 7 wpi. Priming with HKLm led to a bigger decrease in the mycobacterial loads in adult zebrafish compared to other tested priming agents. Paclitaxel increased the mycobacterial loads in adult zebrafish. Medians are shown in the figure. (C) Priming with HKLm 1 day prior to M. marinum infection increases the frequency of clearance. The fold change in the percentage of fish that were able to clear the M. marinum infection was higher in the group that was primed with HKLm 1 day prior (3.7-fold; PBS: n=12, HKLm: n=13) to infection compared to group that was primed 7 days (2.3-fold; PBS: n=12, HKLm: n=12) before the infection.
- Fig. 2.
Priming with HKLm significantly reduces mycobacterial loads in adult zebrafish via innate responses. (A) Priming of adult zebrafish with 0.5×107 cfu of HKLm 1 day prior to M. marinum infection (27±2 cfu) led to a significant decrease in mycobacterial loads compared to control injection of sterile 1× PBS. The graph shows one representative experiment. Samples were collected at 4 wpi (PBS: n=19, HKLm: n=19). (B) Priming with HKLm 1 day prior to M. marinum infection leads to sterilization of M. marinum in 25% of the wild-type (WT) zebrafish. Clearance percentage in the WT PBS control group was 3.7%. The data were collected from four independent experiments. M. marinum infection doses in the independent experiments were 27±2 cfu, 26±13 cfu, 75±13 cfu and 26±8 cfu. PBS: n=54, HKLm: n=56. (C) Priming of adult zebrafish with HKLm leads to a significant decrease in mycobacterial loads compared to PBS controls already at 2 wpi. Infection dose: 48±8 cfu. PBS: n=11, HKLm: n=12. (D) HKLm priming 1 day prior to M. marinum infection significantly reduced mycobacterial loads in rag1−/− mutant fish compared to the PBS control group at 4 wpi, indicating a role for innate immune responses. The graph contains a combined result from two separate experiments. M. marinum infection doses were 48±8 cfu and 27±2 cfu. (E) Priming with HKLm 1 day prior to M. marinum infection leads to sterilization of M. marinum in 17% of the rag1−/− mutant fish. Clearance percentage in the PBS control group was 0%. Data are pooled from two independent experiments. PBS: n=26, HKLm: n=23. (F,G) HKLm priming did not affect cumulative mortality in WT adult fish (F; PBS: n=152, HKLm: n=170), but caused a trend of reduced mortality in HKLm-injected rag1−/− mutant fish (PBS: n=53, HKLm: n=55). P-values in A, C and D were calculated with a two-tailed non-parametric Mann–Whitney test with GraphPad Prism. Medians for the individual experiments are shown in the figures. The P-values in B, E, F and G were calculated with Fisher's test using GraphPad (QuickCalcs) online software.
- Fig. 3.
Protective immunity against M. marinum is mediated by a protein and/or nucleic acid component of HKLm. (A-C) Zebrafish were primed with different components of HKLm and L. monocytogenes 1 day prior to M. marinum infection and mycobacterial loads were determined with M. marinum-specific qPCR at 4 wpi. (A) Protective immunity is not mediated by a secreted component in the L. monocytogenes growth medium. Infection dose: 26±6 cfu; PBS: n=10, medium: n=16. (B) Protective immunity is mediated by a component that was found in the insoluble phase. Infection dose: 33±11 cfu; PBS: n=10, insoluble: n=12, soluble: n=12. (C) Protective effect of HKLm priming was lost when HKLm was treated with DNase and RNase, or proteinase K. Infection dose: 33±11 cfu; PBS: n=10, DNase and RNase: n=7, proteinase K: n=8. P-values for all experiments were calculated with a two-tailed non-parametric Mann–Whitney test with GraphPad Prism and corrected with the Bonferroni's method. Medians for each experiment are shown.
- Fig. 4.
HKLm treatment does not protect against high-dose or established M. marinum infection. (A,B) Protective effect of HKLm priming is lost with high-dose priming. Fish were injected with a high dose of HKLm (15.6×107 cfu, 30-fold compared to previous dose) 1 day prior to M. marinum infection (34±11 cfu). Priming with a high dose of HKLm did not reduce mycobacterial numbers (A) and led to an increase in the mortality of the fish at 4 wpi (B). PBS: n=10, HKLm: n=10. (C,D) HKLm priming does not protect from high-dose M. marinum infection. Fish were primed with HKLm 1 day prior to high-dose M. marinum infection (4883±919 cfu). No effect was observed on bacterial loads (C) or cumulative end-point mortality (D). PBS: n=16, HKLm: n=17. (E,F) HKLm does not protect against an established M. marinum infection. Fish were injected with HKLm 2 weeks after an M. marinum infection (22±6 cfu). No effect on mycobacterial loads (E) or cumulative end-point mortality (F) was observed. PBS: n=14, HKLm: n=16. P-values for bacterial loads were calculated with a two-tailed non-parametric Mann–Whitney test with GraphPad Prism (A,C,E). Medians for the experiments are shown.
- Fig. 5.
HKLm priming induces mpeg, tnfα and nos2b expression, downregulates sod2 expression in adult zebrafish and leads to decreased oxygen consumption in vitro. (A-H) The expression levels of mpeg (A), mpx (B), ifnγ1-1 (C), ifnγ1-2 (D), arg1 (E), tnfα (F), nos2b (G) and sod2 (H) were measured with qPCR from wild-type fish primed with HKLm or sterile PBS buffer as a control. At 1 day after the priming, the fish were infected with a low dose (67±16 cfu) of M. marinum. Samples for qPCR analysis were collected at 1 dpi. The results were normalized to uninfected wild-type baseline control. PBS: n=10, HKLm: n=11. (I) HKLm priming leads to metabolic changes in vitro. RAW264.7 cells were primed with LPS or HKLm and oxygen consumption was measured 19-24 h after priming. HKLm priming leads to a 1.8-fold decrease (P=0.008) in oxygen consumption compared to control. LPS was used as a positive control (2.3-fold decrease, P=0.0042). PBS: n=9, LPS: n=7, HKLm: n=9. P-values for all experiments were calculated with a two-tailed non-parametric Mann–Whitney test with GraphPad Prism. Medians for each experiment are shown. Bonferroni correction was used in C.
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