Proc Natl Acad Sci USA 85:6232C6236


Proc Natl Acad Sci USA 85:6232C6236. of brefeldin A for 6 h and assayed for IFN- expression by circulation cytometry. (d to f) Brain cells were incubated with S510 peptide in the presence of brefeldin A for 6 h and assayed for IFN- expression by circulation cytometry. Download FIG?S2, TIF file, 2.4 MB. Copyright ? 2021 Verma et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license. FIG?S3. Anti-CCR2 mAb treatment inhibits macrophage infiltration into the CNS after rJHMV contamination. Mice were treated with either rat IgG or anti-CCR2 (MC21) mAb. Data show the absence of CD45hi CD11b+ Ly6G? cells, demonstrating successful depletion of macrophages. Data represent results for 5 mice per group. A Mann-Whitney U test was used to analyze the data. **, 0.0001. (e) Representative confocal images showing computer virus distribution in the NSC 228155 brain, as assessed by staining for N protein. (f) Summary data, analyzed using Mann-Whitney U assessments. Five sections from 3 individual mice were included in the analysis. Data are representative of results from 3 mice per group (means SEM). Bar, 50?m. In agreement with the lack of clinical indicators, Lyz2-DP1?/? mice showed at least 10-fold-lower computer virus titers than CX3CR1-DP1?/?, DP1?/?, or WT mice, with CX3CR1-DP1?/? and DP1?/? mice harboring the highest virus loads (Fig.?1d) at 5?days postinfection (dpi), the KIFC1 peak of computer virus replication. Computer virus titers were also lower at 3?dpi in Lyz2-DP1?/? than in WT mice, demonstrating the early control of contamination in mice lacking macrophage-specific DP1 expression. To further examine the distribution of computer virus in the brain, we performed confocal microscopy. While we observed no differences in computer virus localizations within the brain, Lyz2-DP1?/? mice exhibited significantly fewer infected cells at each site of contamination, as assessed by nucleocapsid (N) protein staining (Fig.?1e and ?andf).f). In contrast, in agreement with the observed increase in viral loads, brains from CX3CR1-DP1?/? and NSC 228155 DP1?/? mice showed increased staining for computer virus antigen (Fig.?1e and ?andf).f). These data show that while PGD2/DP1 signaling on microglia is usually protective, its signaling on macrophages/neutrophils appears to have a deleterious effect. DP1 signaling modulates cytokine and chemokine expression by myeloid cells. Given the well-described anti-inflammatory role of DP1 signaling and the increases in virus loads in the global or microglia-specific absence of DP1, we reasoned that this PGD2/DP1 signaling axis diminished the antivirus immune response (Fig.?2). In JHMV-infected DP1?/? mice, contrary to this expectation, we observed diminished expression of molecules such as RIG-I, CCL2, CXCL10, IL-6, MDA5, and IFN-, with effects being most prominent in microglia. Similarly, contamination of CX3CR1-DP1?/? mice resulted in diminished expression of RIG-I, IL-6, MDA5, and CXCL10 in microglia, compared to WT mice, although CXCL10 did not reach statistical significance (= 0.09). DP1 signaling in CX3CR1-DP1?/? macrophages was normal, and consistent with this, there were no differences in the macrophage-specific expression of these proinflammatory molecules. On the other hand, the absence of DP1 signaling in macrophages experienced little effect on the expression of these molecules in either macrophages or microglia, with the exception of a reduction in IFN- expression in macrophages. Together, these results suggest that DP1 signaling in microglia is critical for normal cytokine and chemokine expression by these cells, likely contributing to poor outcomes. However, the effects of these molecules in Lyz2-DP1?/? mice on expression were limited, suggesting that they did not make major contributions to the improved outcomes observed after rJHMV contamination. Open in a separate window FIG?2 Cytokine and chemokine expression in infected mice. Cytokine and chemokine mRNA expression was examined in sorted microglia NSC 228155 (black) and macrophages (blue) from rJHMV-infected brains of WT, Lyz-DP1?/?, CX3CR1-DP1?/?, and DP1?/? mice at 3?dpi. Gene expression was normalized to HPRT. Pooled data from three impartial experiments with 10 to 12 mice per group were analyzed by a Mann-Whitney U test (means SEM). *, 0.0001. NSC 228155 Macrophages lacking DP1.