Genes responsive to ASAL
We identified a set of 1239 nonredundant informative probes that responded significantly (FDR 1%) to ASAL at least at one time point and one temperature (i.e., the union of the 711 and 666 ASAL-responsive probes at 6HPI or 24HPI, respectively) (see Supplemental Tables S1A–S1D). To further confirm our data set, we compared the probe identifiers and (or) gene names of 20K microarray probes identified as ASAL-responsive to those reported in
Booman et al. (2011) and
Feng et al. (2009) for Atlantic cod stimulated with formalin-killed atypical ASAL at 10 °C. Of the 12 QPCR-validated ASAL-responsive genes presented in
Booman et al. (2011), we identified eight in the present data set as the same 20K microarray probes used for QPCR primer design in the previous study (fold-changes for the comparisons in which these genes were identified can be found in
Table 1, and their expression levels in all samples can be found in
Fig. 4). In addition to these genes, ferritin heavy subunit and
IRF1, which were SSH-identified and QPCR-validated as ASAL-responsive in
Feng et al. (2009), were among the ASAL-responsive probes in the present study (fold-changes for the comparisons in which these genes were identified can be found in
Table 1 and their expression levels in all samples can be found in
Fig. 4). Based on these results, we are confident that our data set is accurate and that the genes reported in this manuscript represent a trustworthy catalog of ASAL-responsive (i.e., antibacterial) genes in Atlantic cod.
Ferritin and
hepcidin are genes known to be involved in iron homeostasis (
Solstad et al. 2008;
Feng et al. 2009) and represent strong candidates for future marker development of robust immune response against ASAL. They were both significantly induced by IP injection with ASAL in the current study, and their response to bacterial antigens has been validated by QPCR in two previous studies (
Feng et al. 2009;
Booman et al. 2011). Furthermore, studies have shown that iron homeostasis plays an important role in ASAL pathogenesis in Atlantic cod and Atlantic salmon (
Reith et al. 2008;
Feng et al. 2009;
Beaz-Hidalgo and Figueras 2013). Many studies have also shown that the genes
SCYA123 and
IL8 can be important in the response of Atlantic cod to both bacteria and viruses (
Seppola et al. 2008;
Borza et al. 2010;
Feng et al. 2009;
Rise et al. 2010;
Booman et al. 2011;
Holen et al. 2012;
Hori et al. 2012). However, to our knowledge, this is the first time that several other known Atlantic cod antiviral genes, including
DHX58,
sacsin, and
IRF10 (
Rise et al. 2008;
Hori et al. 2012), have been shown to also be responsive to stimulation with bacterial antigens.
Temperature effects on the cod’s immune response
In the current study, we show that elevated temperature only had a minimal effect on the Atlantic cod spleen transcriptome response to ASAL at 6HPI and 24HPI. Only 15 genes were differentially expressed between fish held at 10 °C versus 16 °C at 6HPI, and only two genes were identified when this comparison was made at 24HPI (
Fig. 1;
Table 2). This result is in distinct contrast to what our research group previously reported for pIC stimulation. In
Hori et al. (2012), 290 and 339 genes were differentially expressed between pIC-stimulated fish held at 10 °C versus 16 °C at 6HPI and 24HPI, respectively. Further, we found that there were 296 significant ASAL-responsive probes when comparing ASAL 6HPI@10 °C versus PBS 6HPI@10 °C, and 506 probes when comparing ASAL 6HPI@16 °C versus PBS 6HPI@16 °C. This is in contrast to the pIC data published in
Hori et al. (2012) where there were only 41 significant pIC-responsive probes when comparing pIC 6HPI@10 °C versus PBS 6HPI@10 °C, and 656 probes when comparing pIC 6HPI@16 °C versus PBS 6HPI@16 °C.
This difference between the two studies was also observed in the results from the clustering and GO term analyses. Fish injected with pIC at 10 °C and sampled at 6HPI had spleen transcript expression profiles that clustered with those of fish that were not stimulated with pIC (i.e., showed a nonstimulated transcript expression signature) (
Hori et al. 2012), a result that was further confirmed in the current study by clustering these samples based on the nonredundant lists of pIC-responsive probes (
Figs. 2B and
3B). This transcript expression clustering result was not seen in ASAL-stimulated fish held at 10 °C and sampled at 6HPI. Rather, all ASAL-stimulated fish grouped together when clustered based on a nonredundant set of ASAL-responsive genes (i.e., showed a distinct antibacterial transcript expression signature), and clustered separately from nonstimulated fish at both time points (
Figs. 2A and
3A). In
Hori et al. (2012), several GO terms were enriched in the list of pIC-responsive genes at 16 °C compared with those at 10 °C for the 24HPI sampling point and these included unfolded protein binding, regulation of interferon-gamma-mediated signaling, and regulation of cytokine-mediated signaling pathway. In the current study, a comparable analysis showed that there were no GO terms enriched in the list of ASAL-responsive genes at 16 °C compared with those at 10 °C. Overall, these findings clearly demonstrate that, in terms of regulation of gene expression, the Atlantic cod immune response following pIC injection is much more temperature-dependent than is this species’ immune response to ASAL.
Some genes known to be important in the fish innate immune response were responsive to both ASAL (present study) and pIC (
Hori et al. 2012). Of these, interferon regulatory factors (
IRFs) 1 and 10, interleukin-8 variant 5 (
IL8),
SCYA123, and
DHX58 (alias
LGP2)(
Rise et al. 2008;
Feng et al. 2009;
Rise et al. 2010;
Polinski et al. 2013) all showed a clear shift in the timing of up-regulation when pIC-injected fish were held at 16 °C versus at 10 °C, with the fish held at 16 °C showing a significantly higher response than fish held at 10 °C at 6HPI and the opposite being true at 24HPI (
Hori et al. 2012). In contrast, only two of these genes,
SCYA123 and
IL8, showed a difference in the timing of up-regulation at the different temperatures following ASAL injection (
Table 2). Microarray probe 44453 representing
SCYA123 was ∼6-fold more highly expressed in ASAL 6HPI@16 °C as compared with ASAL 6HPI@10 °C (
Table 2). Microarray probe 38638 representing
IL8 was ∼5-fold more highly expressed in ASAL 24HPI@10 °C as compared with ASAL 24HPI@16 °C (
Table 2). Although the overall impact of elevated temperature on the spleen transcriptome response to ASAL was less than the impact of elevated temperature on the pIC response, the temperature-dependence of
SCYA123 and
IL8 transcript expression responses to bacterial antigens (i.e., ASAL) could still impact the ability of the fish to respond to bacterial pathogens.
Interestingly, our finding that the cod’s splenic immune gene expression response to pathogenic bacterial antigens (i.e., ASAL) was only minimally affected by increased temperature is not consistent with much of the literature in this research area. For example, while differences in the up-regulation of known inflammatory genes (e.g.,
TNFα and
IL8) in bluefin tuna (
Thunnus maccoyii) peripheral blood leukocytes (PBLs) stimulated with LPS were not found at 6HPI, 12HPI, or 24HPI when the fish were held at elevated versus optimal temperatures, differences were apparent at 3HPI (
Polinski et al. 2013). IP injection of
Yersinia ruckeri bacterin into rainbow trout (
Oncorhynchus mykiss) at elevated temperature (15 °C and 25 °C vs. 5 °C) resulted in significantly increased and earlier expression of several key immune related genes (e.g.,
IL1β,
IL10,
IFNγ) in the spleen (
Raida and Buchmann 2007). In addition,
Lund et al. (2008) reported an increase in mortality of Atlantic cod challenged with four strains of atypical
A. salmonicida when inoculated at 14 °C versus 10 °C. Furthermore, vaccination of Atlantic salmon against ASAL leads to a greater antibody production at 10 °C compared with 2 °C, but with similar levels of pathogen protection (
Eggset et al. 1997). Finally, rainbow trout challenged with the myxozoan
Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease (PKD), experienced greater mortality at 19 °C compared with either 16 °C or 14 °C (
Bettge et al. 2009). Collectively, these results suggest that the impact(s) of elevated temperature on fish immunity and immune-related gene expression may be related to the fish species, the tissue type, the antigen or pathogen, and (or) the particular temperature range over which responses are compared.
In conclusion, we have shown that the overall impact of moderately elevated temperature on the Atlantic cod immune-relevant gene expression response to ASAL is less than the impact of elevated temperature on this species’ response to pIC. This is a novel finding and could affect the Atlantic cod’s ability to fight bacterial infections when exposed to elevated temperatures. In keeping with the hypothesis that modulation of immune-related gene transcription can affect immune robustness of fish,
Jeffries et al. (2012) suggested that the differential expression of key immune genes may underlie the survival of wild sockeye salmon exposed to elevated temperatures. However, the literature is equivocal with regard to the effects of temperature on fish responses to bacterial pathogens (including
A. salmonicida), and thus, experiments must be performed to determine if differences in the expression profiles observed in
Hori et al. (2012) and the present manuscript correlate significantly with mortality during live pathogen challenges at elevated temperatures.