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

Abstract

Phenotypes of myeloid-lineage cells remain poorly understood in the rainbow trout, and were the focus of this study, including effects of in vivo challenge to Flavobacterium psychrophilum (Fp), the cause of Bacterial Cold Water Disease (BCWD). A genetic line was used that is highly resistant to BCWD (R-line) as well as a susceptible control line (S-line). Using flow cytometry, we describe two Pax5-negative, myeloid-lineage populations: Population 1 consisted of small cells with high SSC and strong staining for Q4E, MPO, Pu1, EBF, and IL- 1β, which we named "neutrophil-like" cell. Population 2 had high Q4E, but weaker MPO, Pu1, EBF, and IL-1β staining. Five days after Fp-challenge, both genetic lines had a reduced abundance of neutrophil-like cells in anterior kidney, PBL, and spleen. Pop. 2 abundance was reduced in anterior kidney, and increased in spleen. S-line fish responded more strongly to Fp-challenge compared to R-line fish. Challenged fish with a higher abundance of neutrophil-like cells had significantly lower Fp-loads after challenge, suggesting that these cells aid in the resistance to BCWD.

Figure 1.

Gene expression of M-CSFR in 4 rainbow trout immune tissues (AK, PBL, SPL, and PK) from YC13 and/or YC15, using RT-qPCR in a Taqman assay. M-CSFR fold-change values relative to the S AKYC13 average are indicated on the Y-axis. R-line fish (R) are shown in grey, S-line fish (S) in black. Average +/− SE. *: p<0.05; ** p<0.01. N values: AK YC13: 8R, 8S; AK YC15:7R, 7S; PBL YC15:12R, 11S; SPL YC15:13R, 15S; PK YC13: 10R, 10S.

Figure 2.

Examples of contour graphs from two-color flow cytometry, using samples from mock R-line fish. Q4E on the Y-axis and Pu1, IL-1β, EBF, Pax5.PD, or MPO on the X-axis. Boxes label the populations of interest using numbers 1–5, as defined in Table I. A. AK. B. PBL.

Figure 3.

Contour graphs comparing cell size (FSC, Y-axis) and cell abundance of MPO⁺ and Pu1⁺ stained cells between naïve trout and human wbcs from blood, using flow cytometry. Left top and bottom contours: Percoll-purified (50%−60%) trout PBLs. Middle top and bottom contours: Percoll-purified (50%−60%) human PBLs. Right top and bottom contours: purified human neutrophils. Percentage of cells in each quadrant are indicated by numbers. Circled populations: MPO⁺, neutrophil-like populations. Boxed populations: Pu1⁺ populations.