Developmental thyroid disruption causes long-term impacts on immune cell function and transcriptional responses to pathogen in a small fish model

Abstract

Current evidence suggests thyroid hormones (THs) impact development of the immune system, but few studies have explored the connection between the thyroid and immune systems, especially in fish. This is important as some environmental contaminants disrupt TH homeostasis and may thus have negative impacts on the immune system. To determine the long-term consequences of early life stage (ELS) hypothyroidism on immune function, fathead minnows were exposed to the model thyroid hormone suppressant propylthiouracil (PTU) from < 1 to 30 days post hatch. Fish were transferred to clean water and raised to adulthood (5-7 months post hatch) at which time, several aspects of immune function were evaluated. Ex vivo assessment of immune cell function revealed significant decreases (1.2-fold) in the phagocytic cell activity of PTU-treated fish relative to the controls. Fish were also injected with Yersinia ruckeri to evaluate their in vivo immune responses across a suite of endpoints (i.e., transcriptomic analysis, leukocyte counts, spleen index, hematocrit, bacterial load and pathogen resistance). The transcriptomic response to infection was significantly different between control and PTU-treated fish, though no differences in bacterial load or pathogen resistance were noted. Overall, these results suggest that early life stage TH suppression causes long-term impacts on immune function at the molecular and cellular levels suggesting a key role for TH signaling in normal immune system development. This study lays the foundation for further exploration into thyroid-immune crosstalk in fish. This is noteworthy as disruption of the thyroid system during development, which can occur in response to chemicals present in the environment, may have lasting effects on immune function in adulthood.

Figure 1

General experimental design of the current study. Fathead minnow larvae (< 1 day post hatch, dph) were exposed to either a low (25 mg/L) or high (70 mg/L) concentration of propylthiouracil (PTU), a model thyroid hormone suppressant, through 30 dph. Thyroid hormone (TH) suppression was confirmed via immunofluorescent labeling of thyroxine and TH-sensitive gene expression analysis at 7 and 30 dph, respectively. In addition, growth (i.e., mass, length) was assessed at both time points. Upon reaching adulthood at ~ 11 months post hatch (mph), ex vivo cellular immune function, specifically respiratory burst and phagocytic cell activity, was assessed. The in vivo immune response was determined via the assessment of a suite of immune-related endpoints across multiple levels of biological organization (i.e., transcriptomic analysis, leukocyte counts, spleen index, hematocrit, bacterial load, and pathogen resistance) following injection with Yersinia ruckeri.

Figure 2

Thyroxine (T4) content in the follicular cells of control and propylthiouracil (PTU)-exposed larvae at seven days post hatch as measured by integrated density (n = 10). Values represent mean integrated density in PTU-exposed larvae relative to control larvae. Error bars represent standard error.

Figure 3

Representative images of immunofluorescent labeling of thyroxine (T4) in control (A), low concentration (B) and high concentration propylthiouracil-exposed larvae (C).

Figure 4

Hepatic expression of deiodinase 2 (di2, A) and transthyretin (ttr, B) in control and propylthiouracil (PTU)-exposed larvae at 30 days post hatch (n = 8). Values represent mean fold change in PTU-exposed larvae relative to control larvae. Error bars represent standard error. Different letters indicate statistically significant differences between treatment groups.

Figure 5

Mean phagocytic cell activity in renal cells of control and propylthiouracil (PTU)-exposed fish (n = 5/group). Error bars represent standard error. Asterisk indicates statistically significant difference between control and PTU treatment groups.

Figure 6

Survival curves of control and propylthiouracil (PTU)-exposed fish following pathogen injection (n = 3 trials, each consisting of 12 fish/group).