Sex- and region-specific differences in CNS cytokine expression at baseline
Immune signaling is important for early development, and sex-specific differences have been evidenced in peripheral and CNS immune signaling under normal conditions [20]. Therefore, we wanted to examine whether cytokine levels exhibited sex-specific differences at baseline, under saline control conditions, during early postnatal development in Lewis and BN rat strains. To evaluate this, and all subsequent cytokine comparisons, we used a bead-based Luminex assay to assess the levels of a set of 10 analytes including a subset of Th-related cytokines, specifically IFN-γ, IL-4, IL-17, and IL-10, as well as inflammatory chemokines in peripheral blood and within different brain regions of experimental animals. Animals were exposed to peripheral immune challenge in adjuvant, adjuvant-only, or saline on P10, and samples were collected 2 and 5 days post-challenge in male and female Lewis and BN rats (Fig. 1a). Data presented for baseline sex comparisons were collapsed between both time points of collection, P12 and P15, as no statistically significant differences were observed between the two time points for saline control conditions.
Notable sex-specific differences in baseline cytokine levels were seen in the cortex and to a lesser extent the hippocampus. In the cortex, baseline sex differences were observed in several important Th-type cytokines, such as IL-4 (p < 0.001), IL-10 (p = 0.01), and IL-17 (p < 0.05) as well as IL-β (p < 0.001), with males exhibiting an increased level of these cytokines compared to females (Fig. 1b). Interestingly, these results were only true for Lewis rats and not observed in the BN rat strain, with the exception of higher IL-1β (p < 0.001) in male BN rats compared to females. When considering cytokine levels in the hippocampus, baseline sex-specific effects were more limited. Similar to the cortex, a significant difference was observed for IL-1β (p = 0.01) in Lewis rats. Additionally, a sex-specific increase in IFN-γ (p < 0.05) was also seen in the hippocampus of Lewis rats, with males displaying a higher level of IFN-γ than females (Fig. 1b).
Minimal sex-specific differences were seen in serum cytokine levels at baseline, with the only significant finding being an effect of sex on the level of MCP-1 (monocyte chemoattractant protein 1) in Lewis rats, with males displaying a greater level of MCP-1 than females (p = 0.004; Additional file 1: Figure S1A). No sex-specific differences were observed in the cerebellum under saline control conditions (Additional file 1: Figure S1D). In multiple brain regions, baseline differences in GM-CSF levels were noted but data were not included in the final analysis due to several samples having values below the LOD, thus skewing group differences (Additional file 1).
Brain region-specific differences in the level of several cytokines were also noted. In serum, levels of the majority of analytes were found to be significantly different than concentrations of these same cytokines measured in brain regions of the corresponding animals (Fig. 1c). Specifically, the levels of MCP-1 (p < 0.001), IL-1β (p < 0.001), IL-4 (p < 0.001), and IL-17 (p < 0.001) were observed to be higher in serum than the cortex, hippocampus, and cerebellum, while the level of IL-10 (p < 0.001) appeared lower in the serum compared to selected brain regions (Fig. 1c).
Differences were also observed when comparing cytokine levels between brain regions with main findings including lower levels of many analytes in cortical lysates. The levels of IL-1α (p < 0.001), IL-1β (p < 0.001), IFN-γ (p < 0.001), IL-4 (p < 0.001), and IL-6 (p < 0.001) were seen to be lower in the cortex, compared to both the hippocampus and cerebellum (Fig. 1c), while IL-10 (p < 0.05) was significantly lower in the cortex compared to the hippocampus only, and cortical IL-17 (p < 0.001) was less than that measured in the cerebellum. Additionally, the hippocampal level of TNF-α was significantly lower in the hippocampus compared to other brain regions (p < 0.001; Fig. 1c).
Effect of peripheral immune challenge on serum cytokines and chemokines
Next, to broadly characterize the innate and adaptive immune responses to an early postnatal peripheral immune challenge, we assessed post-challenge cytokine levels between strain, sex, region, and time point of collection as described above and outlined in Fig. 1a. Consistent with the age of the rat pups, the effect of treatment on the serum cytokine response was relatively mild (Additional file 2: Figure S2A and Additional file 3: Figure S3A). Over half of the serum samples had levels of IFN-γ, IL-1α, IL-6, and TNF-α below the LOD, and these cytokines were therefore excluded from further analysis. For serum analytes that were above the LOD and were found to be differentially regulated in response to treatment, effects were broadly similar across strains with some sex-specific skewing. For GM-CSF, significant main effects of treatment were seen in both Lewis and BN rat strains (Additional file 2: Figure S2A). Specifically, post hoc testing revealed sex-dependent increases in the level of GM-CSF in serum 5 days post-challenge in males of both strains (Lewis, p < 0.001; BN, p = 0.005; Fig. 2a), with significant increases also seen under adjuvant conditions in male Lewis rats (p = 0.001). In contrast, female BN rats exhibited significant increases in GM-CSF in response to adjuvant treatment compared to saline controls at the same time point (p < 0.001; Fig. 2a). Of note, a significantly higher level of GM-CSF was seen under adjuvant conditions compared to immune challenge in female BN rats at 5 days post-challenge (Fig. 2a). However, this was the only instance of such a finding in the study and is likely due to several serum GM-CSF values falling below the level of detection in these animals. A main effect of treatment was observed in the level of the chemokine MCP-1 in both Lewis and BN rats (Additional file 2: Figure S2A). Higher levels of MCP-1 were detected under adjuvant-only conditions at 5 days post-challenge in female BN rats (p = 0.02) and at 2 days post-challenge in female Lewis rats (p = 0.001), or 2 days following mixed immune challenge in Lewis males (p = 0.016; Fig. 2b).
Innate immune cell-related cytokine response in the CNS
To understand the CNS immune response to an acute peripheral immune challenge, we evaluated cytokine and chemokine levels in tissue lysates from the cortex, hippocampus, and cerebellum of male and female Lewis and BN rats. The most striking results and greatest inflammatory response to the mixed immune challenge were seen in the cortex. In both male and female Lewis and BN rats, a significant main effect of treatment was observed for several cytokines associated with the innate immune response in the cortex at 2 and 5 days following treatment (Additional file 2: Figure S2B). Specifically, IL-1α, IL-1β, and IL-6 were significantly upregulated following either peripheral immune challenge or adjuvant-only exposure in both strains (Fig. 3a). Similar effects of treatment were noted for GM-CSF and MCP-1, both important innate immune cell recruitment and activation molecules (Fig. 3a). Representative cortical innate immune cytokine data are collapsed across sex and time point of collection as a response to treatment appeared similar between these conditions. The only exception to this pattern was the lack of increased MCP-1 expression in the cortex in male BN rats exposed to either peripheral immune challenge or adjuvant-only (Additional file 3: Figure S3B).
In the hippocampus, a main effect of treatment was also seen for the majority of cytokines (Additional file 2: Figure S2C). A significant increase in GM-CSF compared to saline controls was observed at both 2 and 5 days post-challenge across experimental conditions, strains, and sexes (Fig. 3b, Additional file 3: Figure S3C). In both sexes, significant increases in IL-1α (p < 0.05) and IL-1β (p < 0.001) were noted in BN rats, while a significant increase in IL-1β (p < 0.001), but not IL-1α, was observed in Lewis rats at 2 but not 5 days post-challenge (Fig. 3b). Elevated levels of MCP-1 were seen only in female Lewis rats at 2 days (p = 0.01) and 5 days (p = 0.034) post-challenge (Fig. 3b). Interestingly, immune challenge decreased hippocampal IL-6 relative to saline control in female rats of both strains (p < 0.05; Fig. 3c). Other cytokines, such as IL-1α and IL-1β, were also decreased by peripheral immune challenge or exposure to adjuvant-only at 5 days post-challenge (Additional file 3: Figure S3C). The effect of peripheral immune challenge on the cytokine response in the cerebellum was both weak and varied (Additional file 2: Figure S2D), with the exception of a significant increase in GM-CSF, similar to that seen in the hippocampus and cortex (Additional file 3: Figure S3D).
Th-type cytokine responses in the CNS
Due to evidence suggesting that the immune responses in Lewis and BN rats are skewed toward a Th1- or Th2-specific response, respectively [21, 22], a primary aim of this study was to evaluate the contribution of a different genetic immune background on the response to immune challenge. In cortical lysates from both strains, a main effect of treatment was observed for all canonical Th-type cytokines measured (Additional file 2: Figure S2B). Interestingly, the level of IFN-γ (a major Th1 cytokine) in the cortex following mixed immune challenge was significantly elevated in males of both strains (p < 0.001), with similar effects at 2 and 5 days post-challenge (Fig. 4a). In female rats of either strain, cortical IFN-γ levels were significantly increased at 5 days (p < 0.01) but not at 2 days post-challenge (Fig. 4a). In contrast to the cortical response, peripheral immune challenge had little or no significant effect on IFN-γ levels in the hippocampus and cerebellum of Lewis or BN rats (Additional file 3: Figures S3C and D). Striking strain and sex differences were seen in the response to immune challenge in cortical levels of IL-4, an indicator of Th2-type responses. Specifically, peripheral immune challenge significantly increased cortical IL-4 levels in female Lewis rats at 2 days (p = 0.003) and 5 days (p < 0.001) post-challenge, while exposure to adjuvant-only treatment only resulted in elevated cortical IL-4 levels at 5 days post-exposure (p < 0.001; Fig. 4b). In contrast, compared to saline controls, cortical IL-4 levels were significantly decreased in female BN rats in response to mixed immune challenge (p < 0.01) or adjuvant (p < 0.01), or unchanged in male rats of either strain (Fig. 4b; Additional file 3: Figure S3B). Coincident with these responses, post hoc analysis revealed a lack of effect of either immune challenge or adjuvant-only on IL-17 levels in the BN rat cortex at both 2 and 5 days post-challenge. However, cortical IL-17 levels were significantly elevated compared to controls in response to mixed immune challenge in male Lewis rats (p < 0.001) and in response to either mixed immune (p < 0.05) or adjuvant treatment (p < 0.05) in female Lewis rats (Fig. 4c). Cortical IL-10 levels were similar between strains, although significantly increased levels of IL-10 in response to experimental manipulation were seen only in male rats, with no apparent effects in females (Fig. 4d). Data for cortical IL-17 and IL-10 levels were collapsed between day of collection due to minimal differences seen for those two analytes over time (Additional file 3: Figure S3B).
In the hippocampus, male BN rats exhibited significant increases in IFN-γ (p < 0.001), IL-4 (p = 0.035), IL-10 (p = 0.037), and IL-17 (p < 0.001) in response to treatment at 2 days post-immune challenge (Fig. 4e). Interestingly, there were no significant increases in the levels of these cytokines in the hippocampus of female BN rats or either sex of Lewis rats (Additional file 3: Figure S3C). While a main effect of treatment on Th-type responses in the cerebellum was apparent under certain conditions (Additional file 2: Figure S2D), post hoc analysis revealed effects of treatment on cytokine production in the cerebellum to be largely non-significant across most conditions (Additional file 3: Figure S3D).
Evaluation of CNS cellular immune response
To understand the effects of mixed immune challenge or adjuvant-only exposure on the brain-specific cellular response, the number of GFAP immunopositive cells and the average intensity of GFAP immunofluorescence were evaluated as indicators of astrogliosis, whereas the total number of IBA-1 immunopositive cells and percentage of IBA-1 immunopositive cells also immunoreactive for CD68 were quantified to assess the microglial response.
GFAP is an intermediate filament expressed mainly in astrocytes that is upregulated under conditions of hypertrophy and activation [24]. In both the cortex and hippocampus, GFAP average fluorescence intensity was significantly increased within both strains at 2 and 5 days post-challenge. In Lewis rats, across all conditions, the average intensity of GFAP immunoreactivity was significantly higher in the hippocampus than in the cortex, whereas no significant regional differences were observed in BN rats (Fig. 5a–c). In addition to brain region-specific strain differences, sex-specific effects of both mixed immune challenge and adjuvant-only exposure on average intensity of GFAP immunofluorescence were observed. While exposure to either treatment significantly increased the average intensity of GFAP immunofluorescence in male and female BN rats across brain regions at 2 and 5 days post-challenge (Fig. 5d), exposure to either treatment in Lewis rats significantly increased the average intensity of GFAP immunofluorescence in males only (Fig. 5e). Due to best-fit statistical modeling used for IHC analysis, conditions were collapsed between groups when no differences were observed. With regard to GFAP intensity as noted here, a similar response to immune challenge was seen across the brain region within a rat strain.
A significant increase in the number of GFAP immunopositive cells was also seen in response to mixed immune challenge or adjuvant-only exposure, but these effects varied between brain regions and strains. In the cortex, GFAP immunopositive cell counts were increased following mixed immune challenge or adjuvant-only exposure in both strains at 2 and 5 days post-challenge (Fig. 5f, g) with the exception of female BN rats that exhibited no significant change in GFAP immunopositive cell counts at 2 days post-mixed immune challenge (Fig. 5g). In contrast, more consistent sex and strain differences were seen in the hippocampus. In Lewis rats, males exhibited a significant increase in the number of GFAP immunopositive cells in the hippocampus 2 and 5 days post-challenge following mixed immune challenge or adjuvant-only exposure, whereas female Lewis rats showed no differences in response to either challenge (Fig. 5f). In the BN strain, the numbers of GFAP immunopositive cells in the hippocampus significantly increased in both sexes 2 and 5 days post-challenge, with the exception of male BN rats that exhibited no effect at 5 days post-challenge (Fig. 5g).
The cellular neuroinflammatory response was further evaluated by immunostaining for IBA-1 in the cortex and hippocampus. IBA-1 is a pan-macrophage/monocyte marker used broadly in the brain to identify microglia [25]. Interestingly, in contrast to increased brain GFAP immunofluorescence intensity and number of GFAP immunopositive cells seen across many conditions, the number of IBA-1 immunopositive cells in the cortex and hippocampus were either unchanged or significantly decreased in these regions in response to mixed immune challenge or adjuvant-only exposure, compared to saline controls (Fig. 6a, b). In BN rats, a reduction in the number of IBA-1 immunopositive cells was observed in the cortex and hippocampus of male rats exposed to either mixed immune challenge or adjuvant-only and in the cortex and hippocampus of females treated with adjuvant-only (Fig. 6b). In contrast, there was no difference in the number of IBA-1 immunopositive cells in female BN rats exposed to mixed immune challenge or Lewis rats under either treatment condition compared to saline controls (Fig. 6b, c).
Additionally, colocalization of CD68 immunoreactivity with IBA-1 was used as a measure of microglial activation in the brain, as CD68 is a lysosomal marker used broadly to indicate phagocytic activity in macrophages [26]. Following the analysis of the response to either mixed immune challenge or adjuvant exposure, there were no significant differences between treatment conditions, strain, or sex with respect to IBA-1/CD68 colocalization (Fig. 6d).