Fig 2: DHA feeding improved IFN-? production and degranulation by NK cells in the spleen at day 7 postinfection. A Flow cytometric analysis of the ratio and the geometric mean fluorescence intensity (gMFI) of IFN-?+ splenic NK cells (CD3-CD19-NK1.1+NKp46+) from control versus DHA-fed mice following stimulation with PMA and ionomycin in the presence of GolgiPlug and GolgiStop for 6 h. B, C Flow cytometric analysis depicting the frequencies and gMFI of perforin (B) and granzyme B (GZMB) (C) by splenic NK cells (CD3-CD19-NK1.1+NKp46+) from control versus DHA-fed mice at day 7 postinfection. D Purified splenic NK cells from both DHA-fed and control mice at day 7 after MCMV infection were cocultured with CTV-labeled YAC-1 (left) or MCMV-infected 3T3 cells (right) for 6 h at the E:T ratio of 16:1. Representative flow cytometry plots of CTV+ singlets are shown on the left. The percentages of NK cell-specific lysis are shown on the right, which were calculated by the following formula: [(% CTV + 7-AAD+ cell specific lysis - %CTV + 7-AAD+ cell spontaneous lysis)/ (100 - % CTV + 7-AAD+cell spontaneous lysis)] × 100. For each experiment, n = 9 pooled from 3 independent experiments (A–C); n = 5 pooled from 2 independent experiments (D). Each symbol represents an individual mouse, and the blue dots and red square represent control and DHA-enriched diet-fed mice, respectively. All experiments were replicated 3 times. Error bars represent interquartile ranges; *p < 0.05; **p < 0.01

Fig 3: The effects of DHA feeding on the cellularity and activation of T and B cells in the spleen at day 7 postinfection. A Flow cytometric analysis and enumeration of total T cells (CD45+CD3-CD19-), CD4+ T cells (CD45+CD3+CD19-CD4+CD8-), CD8+ T cells (CD45+CD3+CD19-CD4-CD8+) and B cells (CD45+CD3-CD19+) in the spleens of control versus DHA-fed mice. B Flow cytometric analysis and cumulative data indicating the expression of CD62L and CD44 in CD4+ T and CD8+ T cells between control and DHA-fed mice. For each experiment, n = 5 to 9 pooled from 2 independent experiments (A, B). Each symbol represents an individual mouse, and the blue dots and red square represent control and DHA-enriched diet-fed mice, respectively. Error bars represent interquartile ranges; *p < 0.05; **p < 0.01; ***p < 0.001

Fig 4: Minocycline alters the immune system response in the hippocampus and cortex. a Gating strategy. b–g Quantification of absolute numbers in the ipsilateral hippocampus and cortex. b CD45+ cells, c CD3+ cells, d monocytes (Ly6Chigh), e microglia (CD45lowCD11b+), f MHCII+ microglia, and g TLR4+ microglia. Mean values are plotted ± SD, One-way ANOVA followed by Tukey multiple comparison post hoc test were used to determine statistical differences; *p < 0.05. n = 8–10 per group. Major histocompatibility complex II (MHCII). Toll-like receptor 4 (TLR4)

Fig 5: CD3 is expressed on RAW cells. (A) RNA of human T cells and monocyte-derived macrophages (MDM) and RAW cells were obtained to assess relative expression (R.E) of CD3-S by qPCR. Graphic shows murine and human data; however, each one was normalized to its corresponding endogenous control. (B) Immunoblot analysis of CD3-S identification in RAW cells, human monocytes (CD14+), MDM, human CD4+ T cells, murine splenocytes (S), and murine bone marrow cells (BM). ß-actin was used as a loading control. (C) Representative dot plot showing analysis strategy to identify intra- and extracellular CD3 expression in RAW cells by flow cytometry. (D) Frequency of RAW cells positive for F4/80 and CD3-S obtained by cell surface staining and evaluated by flow cytometry. (E) Frequency of RAW cells positive for intracellular or extracellular CD3 or both. (F) Representative immunoblot analysis of samples immunoprecipitated with a system of protein G coupled to magnetic beads and monoclonal anti-CD3. n = 5 independent experiments (for flow cytometry), n = 3 independent experiments by duplicate (for qPCR), (B,F), a representative image of 3 independent experiments. Bars represent the mean of means ± SEM (to (A)) or the mean value ± SD (to (D,E)). One-way ANOVA followed by the Bonferroni test were used for qPCR analyses. ** p < 0.01.