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J Immunol
2019 Nov 15;20310:2679-2688. doi: 10.4049/jimmunol.1900459.
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Distinct Host-Mycobacterial Pathogen Interactions between Resistant Adult and Tolerant Tadpole Life Stages of Xenopus laevis.
Rhoo KH
,
Edholm ES
,
Forzán MJ
,
Khan A
,
Waddle AW
,
Pavelka MS
,
Robert J
.
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Mycobacterium marinum is a promiscuous pathogen infecting many vertebrates, including humans, whose persistent infections are problematic for aquaculture and public health. Among unsettled aspects of host-pathogen interactions, the respective roles of conventional and innate-like T (iT) cells in host defenses against M. marinum remain unclear. In this study, we developed an infection model system in the amphibian Xenopus laevis to study host responses to M. marinum at two distinct life stages, tadpole and adult. Adult frogs possess efficient conventional T cell-mediated immunity, whereas tadpoles predominantly rely on iT cells. We hypothesized that tadpoles are more susceptible and elicit weaker immune responses to M. marinum than adults. However, our results show that, although anti-M. marinum immune responses between tadpoles and adults are different, tadpoles are as resistant to M. marinum inoculation as adult frogs. M. marinum inoculation triggered a robust proinflammatory CD8+ T cell response in adults, whereas tadpoles elicited only a noninflammatory CD8 negative- and iT cell-mediated response. Furthermore, adult anti-M. marinum responses induced active granuloma formation with abundant T cell infiltration and were associated with significantly reduced M. marinum loads. This is reminiscent of local CD8+ T cell response in lung granulomas of human tuberculosis patients. In contrast, tadpoles rarely exhibited granulomas and tolerated persistent M. marinum accumulation. Gene expression profiling confirmed poor tadpole CD8+ T cell response, contrasting with the marked increase in transcript levels of the anti-M. marinum invariant TCR rearrangement (iVα45-Jα1.14) and of CD4. These data provide novel insights into the critical roles of iT cells in vertebrate antimycobacterial immune response and tolerance to pathogens.
Figure 1. Survival curves of M. marinumâinoculated adults and tadpoles of X. laevis. (A) Four-month-old adults (n = 5â10 per group) and 3-wk-old tadpoles (developmental stage of 52, n = 10â19 per group) were inoculated with different amounts of M. marinum i.p. (5 Ã 105 CFU for low dose, 1 Ã 106 CFU for medium dose, and 2 Ã 106 CFU for high dose). The survival rate was dependent on the doses of M. marinum based on the Cox proportional hazard model analysis (p < 0.05). Comparisons of survival rates between adult frogs and tadpoles for each dose, as well as median survival times, are listed in Table II. (B) M. marinum loads in different organs from postmortem adults. (C) M. marinum loads of whole individual postmortem tadpoles. Viral loads were determined by real-time PCR using M. marinumâspecific 16S rRNA gene.
Figure 2. Flow cytometric analysis of T cell in spleen and liver of adults and tadpoles. Four-month-old young adults and 3-wk-old tadpoles were i.p. inoculated with 1 × 106 or 3 × 105 CFU of M. marinum, respectively (n = 5–6 per time point). Then, the total lymphocytes at different dpi were stained with Xenopus-specific CD5 mAb and CD8 mAb to analyze two subsets of T cell populations (A and B). After gating on live cells, CD8+CD5+ cells and CD8negCD5+ cells were defined (black boxes).
Figure 3. Comparison of the frequency and the number of CD8 and CD8neg T/iT cells in the spleen of adults and tadpoles following M. marinum inoculation. Using the flow cytometric strategy shown in Fig. 2, the kinetics of CD8 T cell (A) frequency and (B) number were determined in adults (white) and tadpoles (black) at different days postinoculation (n = 6–7 per group from two independent experiments). Furthermore, the kinetics of CD8neg T/iT cell (C) frequency and (D) number were determined. Asterisks indicated statistical significance by the Kruskal–Wallis test, nonparametric. C, uninfected control.
Figure 4. Comparison of the frequency and the number of CD8 and CD8neg T/iT cells in the liver of adults and tadpoles during M. marinum infection. Using the flow cytometric strategy shown in the Fig. 2, the kinetics of CD8 T cell (A) frequency, and (B) number were determined in adults (white) and tadpoles (black) at different days postinoculation (n = 6–7 per group from two independent experiments). Furthermore, the kinetics of CD8neg T/iT cell (C) frequency and (D) number were determined. Asterisks indicated statistical significance by the Kruskal–Wallis test, nonparametric. C, uninfected control.
Figure 6. Determination of M. marinum loads and dissemination using an absolute quantification method and a recovery of live M. marinum in culture from adults and tadpoles of X. laevis. Adult frogs were i.p. inoculated with 1 × 106 CFU of M. marinum, and then M. marinum loads were determined using a real-time PCR with M. marinum–specific 16S rRNA gene (A–D). (A) PLs and (B) liver and (C) spleen tissues were taken at the indicated dpi. The dashed line indicates the level of detection by real-time PCR. To measure only the live and replicating M. marinum, we cultured kanamycin-resistant M. marinum from the inoculated adults and tadpoles at 21 dpi using a Middlebrook 7H10 media supplemented with 50 μg/ml kanamycin. (D) Quantitative measurement of CFU was then normalized to total milligrams of homogenates. Asterisk indicates p < 0.05 by Kruskal–Wallis nonparametric test.
Figure 7. Relative expression of T cell–related immune genes in the liver of M. marinum–inoculated adults and tadpoles. Four-month-old young adults (white bar) and 3-wk-old tadpoles (gray bar) were i.p. inoculated with 1 × 106 or 3 × 105 CFU of M. marinum, respectively (n = 5–6 per time point). Relative gene expression in liver for (A) CD3ε, (B) CD8β, (C) CD4, and (D) iVα45-Jα1.14 was normalized to the housekeeping gene gapdh. (E) Ct values for gapdh of each time point between adults and tadpoles. Asterisks indicate a significant difference by the Kruskal–Wallis test (p < 0.05). C, uninfected control; u.d, undetected value.
Figure 8. Relative expression of immune receptor genes and pro- and anti-inflammatory cytokine genes in the liver of M. marinum–inoculated adults and tadpoles. Four-month-old young adults (white bar) and 3-wk-old tadpoles (gray bar) were i.p. inoculated with 1 × 106 or 3 × 105 CFU of M. marinum, respectively (n = 5–6 per time point). The relative gene expression in the liver was determined for the immune gene receptors (A) CSF-1R (macrophage recruitment marker), (B) G-CSF-R (neutrophil recruitment marker), and (C) CCR2 (inflammatory monocyte marker); for the proinflammatory cytokine genes (D) TNF-α, (E) IL-1β, and (F) iNOS; and for the anti-inflammatory cytokine genes (G) TGF-β and (H) IL-10. All the data were normalized to the housekeeping gene gapdh. Asterisks indicate significant difference by the Kruskal–Wallis test (p < 0.05). C, uninfected control.
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