B cell signatures of BCWD-resistant and susceptible lines of rainbow trout: A shift towards more EBF-expressing progenitors and fewer mature B cells in resistant animals

Abstract

Bacterial cold water disease (BCWD) is a chronic disease of rainbow trout, and is caused by the Gram-negative bacterium Flavobacterium psychrophilum (Fp), a common aquaculture pathogen. The National Center for Cool and Cold Water Aquaculture has bred two genetic lines of rainbow trout: a line of Fp-resistant trout (ARS-Fp-R or R-line trout) and a line of susceptible trout (ARS-Fp-S, or S-line). Little is known about how phenotypic selection alters immune response parameters or how such changes relate to genetic disease resistance. Herein, we quantify interindividual variation in the distribution and abundance of B cell populations (B cell signatures) and examine differences between genetic lines of naive animals. There are limited trout-specific cell surface markers currently available to resolve B cell subpopulations and thus we developed an alternative approach based on detection of differentially expressed transcription factors and intracellular cytokines. B cell signatures were compared between R-line and S-line trout by flow cytometry using antibodies against transcription factors early B cell factor-1 (EBF1) and paired domain box protein Pax5, the pro-inflammatory cytokine IL-1β, and the immunoglobulin heavy chain mu. R-line trout had higher percentages of EBF(+) B myeloid/ progenitor and pre-B cells in PBL, anterior and posterior kidney tissues compared to S-line trout. The opposite pattern was detected in more mature B cell populations: R-line trout had lower percentages of both IgM(+) mature B cells and IgM-secreting cells in anterior kidney and PBL compared to S-line trout. In vitro LPS-activation studies of PBL and spleen cell cultures revealed no significant induction differences between R-line and S-line trout. Together, our findings suggest that selective resistance to BCWD may be associated with shifts in naive animal developmental lineage commitment that result in decreased B lymphopoiesis and increased myelopoiesis in BCWD resistant trout relative to susceptible trout.

A BCWD-resistant line of rainbow trout exhibits higher abundance of IgT+ B cells and heavy chain tau transcripts compared to a susceptible line following challenge with Flavobacterium psychrophilum

“…Only one significant difference was observed in Mock-challenged fish, and for IgM ++ ISCs only, in the spleen: MS fish had more than twice the abundance of IgM ++ ISCs than R line fish (0.89% and 0.43% respectively; Table I), in agreement with our earlier study (Zwollo et al, 2015). After Fp-challenge, FpS fish had a significantly higher abundance of IgM ++ ISCs in their AK compared to FpR fish, similar to the IgM + B cell pattern in Mock-challenged fish (Table I), The opposite pattern was observed for PCF cells, with a higher abundance of IgM ++ ISCs in FpR fish compared to FpS fish.…”

“…In a previous study, we had shown that S-line fish had a significantly higher abundance of IgM + B cells in their AK compared to R line fish (Zwollo et al, 2015). Here, we confirm that pattern in PBL, albeit that no such significant differences were detected in AK or SPL (Table I).…”

“…In a previous study, we reported that naïve R-line fish had a lower abundance of IgM + B cells and IgM ++ ISCs compared to naïve S-line fish (Zwollo et al, 2015). The results from the current study confirm these findings, and provide evidence that R-line fish have a higher abundance of IgT + B cells compared to S line fish, both before and after challenge, and that IgT abundance and secHCtau expression correlate with lower pathogen loads in challenged fish.…”

“…Resistance to Fp has also been correlated with naive animal spleen size (Hadidi et al, 2008), with Fp-resistant trout having larger spleens (Wiens et al, 2013b)(Wiens et al, 2013c), although its functional significance remains unclear, as splenectomized animals do not show changes in resistance to Fp (Wiens et al, 2015). We have also reported on differences in humoral immunity between the lines, with naïve R-line trout having lower abundance of IgM + and IgM ++ cells (ISCs) compared to S-line fish (Zwollo et al, 2015). …”

Innate immune cell signatures in a BCWD-Resistant line of rainbow trout before and after in vivo challenge with Flavobacterium psychrophilum

“…Three additional M-CSFR-like sequences have since been identified in the rainbow trout (Berthelot et al, 2014). Lastly, antibodies against trout cytokine IL-1 β can aid in the identification of myeloid-lineage cells in combination with other markers (Zwollo et al, 2015). …”