Fig 2: qPCR analysis in RNA isolated from the cerebral cortex. (A) PLX treatment caused a significant reduction of microglial receptors (Csf1r, P2ry12, Trem2, and Itgam) in both Sham and SCI groups. (B) Inflammatory cytokine IL-1α and Lrrc25 expression. (C-D) Astrocytes marker Gfap and the genes that are associated with astrocyte function including Vim, Gbp2, Osmr, C4a, and Entpd2 remained unchanged in both SCI and PLX groups. All data are presented as independent data points. N = 4 mice/group. *p < 0.05, ++++p < 0.0001, vs. Sham/Veh group; ####p < 0.0001 vs. SCI/WT. Two-way ANOVA following Tukey's multiple comparisons test.

Fig 3: Pre-depletion of microglia with PLX5622 reduces infiltrating cells and reactive oxygen species (ROS) production after SCI. (A) PLX5622 diet or vehicle chow were fed to animals three weeks before injury and remained for 2 days after injury. (B) Body weight was recorded both prior and after injury. All injured mice appeared significant loss of body weight. PLX had no effects on animal weight both prior and after injury. n = 30 mice/group before SCI and 12 (Sham/Veh), 12 (Sham/PLX), 18 (SCI/Veh), and 18 (SCI/PLX) mice after SCI. (C) Food intake was recorded both prior and after injury. In all injured mice, food consumption decreased significantly. PLX had no effects on food consumption both prior and after injury. n = 15 mice/group (Before injury) and 10 (Sham/Veh), 10 (Sham/PLX), 15 (SCI/Veh), and 15 (SCI/PLX) mice (After SCI). (D) Representative dot plots show the relative immune cell composition in the impact site of the spinal cord at two days post-injury. (E) The number of living CD45intCD11b+Ly6C- microglia, CD45hiCD11b+ infiltrating myeloid cells, CD45hiCD11b+Ly6C+Ly6G- monocytes, and CD45hiCD11b+Ly6C+Ly6G+ neutrophils are quantified. n = 6 (Sham/Veh), 6 (Sham/PLX), 7 (SCI/Veh), and 7 (SCI/PLX). (F) Representative histograms show the relative production of ROS in microglia, bulk infiltrating myeloid cells, monocytes, and neutrophils as measured by DHR123. (G) The mean fluorescence intensity (MFI) of dihydrorhodamine 123 (DHR123) was quantified for all cells. For all flow cytometry experiments. n = 6 (Sham/Veh), 6 (Sham/PLX), 7 (SCI/Veh), and 7 (SCI/PLX). (H) Quantification of the myeloid marker CD11b and the pro-inflammatory cytokine TNFα by qPCR analysis at 2 days post-injury. Total RNA was extracted from sham or injured spinal cord. n = 4 (Sham/Veh), 5 (Sham/PLX), 5 (SCI/Veh), and 6 (SCI/PLX). **p < 0.01, ***p < 0.001, ****p < 0.0001, ++++p < 0.0001, vs. Sham/Veh group. #p < 0.05, ##p < 0.01, ##p < 0.001, ####p < 0.0001, vs. SCI/Veh group. 2-way ANOVA following Tukey's multiple comparisons test.

Fig 4: Hypoxia-induced expression of IL-33 is mostly confined to retinal endothelial cells.C57BL/6 mice pups were exposed to OIR and at P15 eyes from pups in normoxia or OIR were enucleated, retinas isolated, minced, digested and single cell retinal suspension were prepared in FACS buffer. The retinal cells were first incubated with indicated dyes and antibodies. After washings, the cells were resuspended into sorting buffer, added SYTOX blue and subjected to FACS analysis. a Live cells from both normoxia and P15 were gated as calcein blue positive cells. CD11b positive and CD45 low cells were gated as microglia. CD31 positive and ACSA1 negative cells were gated endothelial cells. ACSA1 positive and CD31 negative cells are gated as astrocytes. CD146 positive and CD140b positive cells are gated as pericytes. b Calcein blue positive cells from both normoxia and OIR were gated as live cells and the NeuO+ cells were gated as neurons. c Total cellular RNA was isolated from all the sorted cells and analyzed for IL-33 and GAPDH mRNA levels by QRT-PCR. The bar graphs show the quantitative analysis of three independent experiments (n = 6 animals/group/experiment), expressed as Mean ± SD. *P < 0.05 vs normoxia.

Fig 5: Intraperitoneal Ly6G+CD11b+ population predominantly consists of CD244+ cells. The samples were collected from the MC38-based peritoneal dissemination (PD) model (n=6) as described in Fig. 2. The ratios of CD244+ cells among CD11b+F4/80−Ly6CmidLy6G+ cells were evaluated by flow cytometry. (A) Representative flow cytometric data of CD244+ cells among the CD11b+F4/80−Ly6CmidLy6G+ cells in the peritoneal cavity. (B) Time-course enumeration of the ratio of CD244+ cells among Ly6G+ cells. (C) Ratios of CD244+ cells among Ly6G+ cells in the peritoneal cavity, spleen, and peripheral blood on Day 20 after tumor inoculation. Values are expressed as mean values with standard errors. One-way ANOVA with Holm's post-hoc test was performed. *P<0.05, **P<0.01.