DHA supplementation inhibited the early but not later response to MCMV infection
To explore the effects of DHA on the susceptibility of mice to MCMV infection, 5-week-old C57BL/6 mice were first fed a special DHA-enriched or control diet for three weeks. After that, the mice were challenged with 3×104 PFU of MCMV in 200 µL of PBS by intraperitoneal injection and sacrificed on day 3, 7 or 21 (Fig. 1A). As the main markers of illness severity following MCMV infection, weight loss, the virus replication level, the visceral coefficient, and the local tissue mRNA levels of inflammatory cytokines were determined at the indicated time postinfection [17, 18]. We found that body weight loss was significantly lower on day 3 but higher on days 5 and 7 after MCMV infection in DHA-fed mice than in control mice. However, there was no significant difference in body weight loss between control and DHA-fed mice on days 9, 14 and 21 after MCMV infection (Fig. 1B).
The spleen and liver are the two main target organs of MCMV after intraperitoneal infection [19], and our data showed that the levels of MCMV IE-1 DNA were increased in both the spleen and liver tissues on day 7 but not day 3 postinfection. At the dose of MCMV given, no MCMV IE-1 DNA was detected on day 21 postinfection in the spleen or liver in either the control or DHA-fed mice (Fig. 1C).
Viral infection is often accompanied by hepatosplenomegaly [20, 21]. Regarding the spleen/body weight and liver/body weight index, neither the spleen/body weight nor the liver/body weight was significantly different between control and DHA-fed mice on day 3 postinfection, but liver/body weight was significantly lower and spleen/body weight was decreased in DHA-fed mice compared with control mice on day 7 postinfection (Fig. 1D). Interestingly, in control mice, both spleen/body weight and liver/body weight index were significantly increased on day 7 postinfection compared with day 3 postinfection. When these two indexes on days 7 and day 3 postinfection in DHA-fed mice were compared, neither index was dramatically increased in DHA-fed mice compared with control mice (Fig. 1D). H&E staining showed that the pathology of both the spleen and liver was not obviously different between control and DHA-fed mice on day 7 postinfection (Fig. 1E).
All these findings demonstrated that DHA supplementation impaired the early response to MCMV infection without affecting final MCMV clearance at the later stage.
DHA feeding affected NK cell homeostasis, maturation and selective Ly49H expansion in the response to MCMV infection
As the NK cell number peaks on day 7 after MCMV infection [22], we next explored the homeostasis and maturation of NK cells on day 7 after MCMV infection. We analyzed the proportion and numbers of NK cells in the BM, spleen, pLNs, and liver tissue. Compared with those in control mice, both the proportion and total numbers of NK cells (CD3−CD19−NK1.1+NKp46+ among CD45+ cells) were significantly decreased in the spleen but not other organs or tissues, although a similar trend without statistical significance was observed in the BM (Fig. 2A).
In response to MCMV infection, NK cells undergo accelerated phenotypic maturation [22]. When the maturation of NK cells was investigated based on the expression of CD27 and CD11b [23], terminal matured NK cells, indicated as CD27−CD11b+ NK cells, were significantly reduced in the BM, spleen and liver, whereas immature NK cells, indicated as CD27+CD11b− NK cells, were significantly increased in the spleen and liver of DHA-fed mice compared with control mice (Fig. 2B). We also determined the expression levels of KLRG1, another marker of NK cell terminal maturation [24], and found that it was also elevated on NK cells after MCMV infection [22]. Our data showed that compared with control mice, DHA-fed mice exhibited significantly reduced KLRG1+ NK cells in the BM, spleen and liver (Fig. 2C). Regarding the specific phase driven by Ly49H recognition [15], our data showed that DHA-fed mice had a significantly reduced ratio and total number of Ly49H+ NK cells in the spleen compared with those in control mice (Fig. 2D). However, there was no significant difference in the expression of CD11b, 27, KLRG1 or Ly49H on day 21 postinfection between control and DHA-fed mice (Fig. 2E).
All these data indicate that DHA supplementation affected NK cell homeostasis and repressed NK cell maturation and Ly49H+ NK cell expansion in the main organs targeted in MCMV, such as the spleen and liver, at the early stage but not the later stage of MCMV infection.
DHA-fed mice showed an enhanced capacity for IFN-γ production and degranulation by NK cells during MCMV infection
Early during the course of infection, NK cells exert antiviral effects through direct toxicity and secretion of IFN-γ [25]. Therefore, we next tested the ratio of IFN-γ+ NK cells after in vitro stimulation with PMA and ionomycin and determined the levels of the NK cell degranulation-related molecules perforin and granzyme B [26]. Our data revealed that the overall proportion and mean fluorescence intensity (MFI) of IFN-γ-secreting NK cells were increased in DHA-fed mice compared with control mice (Fig. 3A). The overall proportion and MFI of granzyme B, but not perforin, among splenic total NK cells were also increased in DHA-fed mice compared with control mice (Fig. 3B-C). These data suggest that DHA may improve NK cell effector function, although it represses NK cell expansion and maturation during MCMV infection.
DHA feeding improved the cellular metabolic status and mitochondrial activity of NK cells during MCMV infection
To explore the potential mechanism underlying the enhanced NK cell effector function induced by in vivo DHA supplementation, we next tested whether DHA feeding interfered with NK cell metabolic status, a basic process critical for facilitating robust NK cell effector functions [27]. NK cell activation results in an increase in the rates of both glycolysis and mitochondrial oxidative phosphorylation (OXPHOS). The expression levels of dedicated transporters, including the transferrin receptor CD71 and amino acid transporter CD98 [27], which control cellular access to nutrients, were substantially increased on the surface of NK cells from the DHA-fed mice compared with control mice on day 7 postinfection (Fig. 4A, B). Glucose uptake, indicated by the fluorescent glucose analog 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxyglucose (2-NBDG) [28], was also increased in NK cells from the DHA-fed mice compared with control mice on day 7 postinfection (Fig. 4C).
Mitochondria, the essential hub of metabolic activity, are critical for OXPHOS activity and powerhouses of immunity [29]. In general, healthy mitochondria generate a proper membrane potential for the movement of substrates from the cytosol into the mitochondrial matrix for OXPHOS [30]. We found that on day 7 postinfection, splenic NK cells from DHA-fed mice had an increased overall mitochondrial content and increased mitochondrial membrane potential, as indicated by flow cytometric labeling with MitoTracker and TMRE, respectively (Fig. 4D, E).
Taken together, these findings suggest an increased overall rate of cellular metabolism and increased mitochondrial activity in NK cells from DHA-fed mice.
DHA feeding impaired the cellularity and activation of T cells in the spleen
As the above data showed that DHA feeding inhibited NK cell expansion, we decided to determine whether DHA feeding would affect cells of the adaptive immune response, such as T cells and B cells. The data showed that DHA feeding resulted in reduced proportions and numbers of total T cells and subsets of CD4+ and CD8+ T cells but had not obvious effect on B cell number (Fig. 5A).
To further determine whether DHA feeding affects T cell activation, we also measured the ratio of CD62LhiCD44lo cells and CD62LloCD44hi cells, which represent naïve and activated T cells, respectively [31], among CD4+ and CD8+ T cells in both control and DHA-fed mice. We found an increased proportion of CD44−CD62L+ cells but a decreased proportion of CD62L+CD44− cells among both CD4+ T cells and CD8+ T cells in DHA-fed mice compared with controls (Fig. 5B).
These data demonstrate that DHA feeding also impaired T cell expansion and activation.
DHA feeding influenced the mRNA expression of inflammatory mediators in the spleens of MCMV-infected mice
Coordinated secretion of cytokines and chemokines occurs in the target organ during MCMV infection [32]. To explore whether DHA feeding could influence the expression of various inflammatory mediators, the mRNA levels of these inflammatory mediators in splenic tissue were determined. The data showed significantly increased mRNA levels of IL-4 but significantly decreased mRNA levels of TNF-α in the spleens of DHA-fed mice compared with controls. The mRNA levels of MIP-1α (also called CCL3) and MCP-1 (also called CCL2) tended to be lower in the spleens of DHA-fed mice compared with controls, although the difference was not statistically significant (Fig. 6). Overall, these data suggest that DHA feeding led to a relatively immunosuppressive microenvironment on day 7 after MCMV infection.