Abstract
Mucosal surfaces such as fish gills interface between the organism and the external environment and as such are major sites of foreign Ag encounter. In the gills, the balance between inflammatory responses to waterborne pathogens and regulatory responses toward commensal microbes is critical for effective barrier function and overall fish health. In mammals, IL-4 and IL-13 in concert with IL-10 are essential for balancing immune responses to pathogens and suppressing inflammation. Although considerable progress has been made in the field of fish immunology in recent years, whether the fish counterparts of these key mammalian cytokines perform similar roles is still an open question. In this study, we have generated IL-4/13A and IL-4/13B mutant zebrafish (Danio rerio) and, together with an existing IL-10 mutant line, characterized the consequences of loss of function of these cytokines. We demonstrate that IL-4/13A and IL-4/13B are required for the maintenance of a Th2-like phenotype in the gills and the suppression of type 1 immune responses. As in mammals, IL-10 appears to have a more striking anti-inflammatory function than IL-4-like cytokines and is essential for gill homeostasis. Thus, both IL-4/13 and IL-10 paralogs in zebrafish exhibit aspects of conserved function with their mammalian counterparts.
Highlights
We demonstrate that IL-4/13A and IL-4/13B are required for the maintenance of a Th2-like phenotype in the gills and the suppression of type 1 immune responses
The expression of il4/13b mRNA was found increased in il4/13b2/2 larvae (Fig. 1E), suggesting that the effects of the mutation might only be evident at the translational level and that increased mRNA might be an attempt to compensate for loss of function of the IL-4/13B protein. Quantitative PCR (qPCR) analysis revealed that the expression of il4/13a was significantly reduced in il4/13a;b2/2 embryos (Fig. 1F), whereas the expression of il4/13b was significantly increased (Fig. 1G), in agreement with what was observed in the single mutants
No significant changes were observed in the levels of il1b, tnfa, and il6 mRNA encoding inflammatory cytokines but an increase of ifng1–2 mRNA was detected in il10e46/e46 gills compared with wildtype (Fig. 4E)
Summary
Whereas a modest increase of tnfa, il6, and ifng1–2 mRNA encoding proinflammatory cytokines was observed in il4/13a2/2 and il4/13b2/2 single mutants, more pronounced upregulation was observed in il4/13a;b2/2 double mutants (Fig. 2A), indicating that il4/13a and il4/13b genes have a fundamental albeit redundant role in suppressing inflammation. Differentially expressed genes were identified in the gills of mutant animals compared with wildtype (Supplemental Table I).
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