McGuire CC and Robert JR (2022), Developmental exposure to thyroid disrupting ch...

XB-ART-59461

Curr Res Toxicol 2022 Jan 01;3:100094. doi: 10.1016/j.crtox.2022.100094.

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Developmental exposure to thyroid disrupting chemical mixtures alters metamorphosis and post-metamorphic thymocyte differentiation.

McGuire CC, Robert JR.

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While there is some evidence to suggest that disruption of the thyroid hormone (TH)-axis during perinatal development may weaken T cell immunity later in life, data are currently lacking on whether environmentally relevant thyroid disrupting chemicals (TDCs) can induce similar outcomes. To fill this gap in knowledge, X. laevis tadpoles were exposed to an environmentally relevant mixture of TDCs, either during early tadpole development, or immediately before and during metamorphosis, to assess T cell differentiation and anti-viral immune response against FV3 infection after metamorphosis. Extending our previous study showing a delay in metamorphosis completion, here we report that TDC exposure prior to metamorphosis reduced the frequency of surface MHC-II + splenic lymphocytes and weakened some aspects of the anti-viral immune response. TDC exposure during metamorphosis slowed post-metamorphic migration of the thymus reduced the renewal of cortical thymocytes and splenic CD8 + T cells. The results indicate that TDC exposure during perinatal development may perturb the formation of T cell immunity later in life.

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Genes referenced: myh6

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	Fig. 1. Quantification of post-metamorphic thymus migration. Stage 60 X. laevis tadpoles were exposed to 1 μg/L of the mixture for 21 days. 21 days after the exposure (roughly 28 days post metamorphosis), froglets were imaged for the relative location of their thymic tissue (A). The distance between the thymus and the posterior end of the arm connecting to the froglet’s shoulder were quantified by ImageJ using number of pixels (B). N = 5 for each treatment and * denotes statistical significance using student’s t-test, error bars denote SEM.
	Fig. 2. Quantification of immature cortical thymocytes. Stage 60 X. laevis metamorphic tadpoles were exposed to 1 μg/L of the mixture for 21 days. At days 7, 14 and 21 post-metamorphosis, froglet thymocytes were evaluated for CD8, CTX (cortical thymocyte-specific antigen of Xenopus) and EdU (5-ethynyl-2′-deoxyuridine) fluorescence via flow cytometry. Representative gating for the frequency of CD8 + and CTX+ (A) and proliferative EdU + CD8+/CTX + thymocytes (B) are shown. The CD8+/CTX + thymocytes were then quantified for total cell number (C) and total number of proliferative EdU + CD8+/CTX + thymocytes (D). N = 4 independent samples of 3 pooled tadpoles, * denotes statistical significance using 2-way ANOVA and Tukey’s post-test.
	Fig. 3. Quantification of mature medullar thymocytes. Stage 60 X. laevis tadpoles were exposed to 1 μg/L of the mixture for 21 days. At days 7, 14 and 21 post-metamorphosis, froglet thymocytes were evaluated for CD8, CD5 and EdU fluorescence via flow cytometry. Representative gating for the frequency of CD8 + and CD5+ (A) and proliferative EdU + CD8+/CTX + thymocytes (B) are shown. The CD8+/CD5 + thymocytes were then quantified for total cell number (D) and total number of proliferative EdU + CD8+/CD5 + thymocytes (D). N = 4 per group (treatment and timepoints are separate groups), * denotes statistical significance using 2-way ANOVA and Tukey’s post-test.
	Fig. 4. Quantification of splenic CD8 + T cells. Stage 60 X. laevis tadpoles were exposed to 1 μg/L of the mixture for 21 days. At days 7, 14 and 21 post-metamorphosis, froglet thymocytes were evaluated for CD8, CD5 and EdU fluorescence via flow cytometry. Representative gating for the frequency of CD8 + and CD5+ (A) and proliferative EdU + CD8+/CTX + splenocytes (B) are shown. The CD8+/CD5 + thymocytes were then quantified for total cell number (C) and total number of proliferative EdU + CD8+/CD5 + thymocytes (D). N = 4 per group (treatment and timepoints are separate groups). * Denotes statistical significance using 2-way ANOVA and Tukey’s post-test.
	Fig. 5. Anti-viral response against FV3 challenge. Stage 52 tadpoles were exposed to 0.1 and 1 μg/L of the mixture for 3 weeks and then maintained in clean water for 6 months to reach full maturity. Frogs were then infected with FV3, and kidneys were harvested 3 days post infection. (A): Quantification of FV3 genome copy number in uninfected and infected animals. (B) Relative mRNA expression of anti-viral cytokines Type I IFN and TNFα. N = 3 per group. * Denotes statistical significance using one-way ANOVA and Tukey’s post-test among infected groups. # Denotes statistical significance using one-way ANOVA and Tukey’s post-test between uninfected DMSO treated control and DMSO -treated FV3 infected animals.
	Fig. 6. CD8 + T cell proliferation during FV3 infection. Stage 52 tadpoles were exposed to 1 μg/L of the mixture for 3 weeks and then maintained in clean water for 6 months to reach full maturity. Frogs were then infected with FV3, and splenocytes were harvested and prepared for flow cytometry 6 days post infection. Data presented includes quantification of frequency of CD8 + T cells at steady state (A), and proliferative CD8 + T cells during FV3 infection (B). N = 3 per group, * denotes statistical significance using one-way ANOVA (with logit transformation of % data) and Tukey’s post test for (A) and 2-way ANOVA and Tukey’s post-test for (B). Statistical significance in B is denoted by different lowercase letters, where means that differ significantly have different letters from one another, while means that do not significantly differ have the same letter.
	Fig. 7. Quantification of MHC-II + splenic lymphocytes. Stage 52 tadpoles were exposed to 0.1 and 1 μg/L of the mixture for 3 weeks and then maintained in clean water for 6 months to reach full maturity. Splenocytes were then prepared for flow cytometry and gated lymphocytes evaluated for expression of MHC-II and IgM. Representative gating is displayed for each group (A), with quantification of the frequency of total MHCII + lymphocytes (B). N = 3 per group, * denotes statistical significance using one-way ANOVA (with logit transformation of % data) and Tukey’s post-test.

References [+] :

Albornoz, Gestational hypothyroidism increases the severity of experimental autoimmune encephalomyelitis in adult offspring. 2013, Pubmed