Fig 2: Suppressive neutrophils in LCMV clone 13 infection have an oxidative metabolic phenotype(A–D) Representative flow cytometry plots and histograms depicting (A) MitoTracker Green, (B) tetramethylrhodamine ethyl ester (TMRE), (C) 2-NDBG, and (D) BODIPY C-16 staining in Ly6C+ Ly6G- monocytes and Ly6C+ Ly6G+ neutrophils from 14 d.p.i. with chronic LCMV clone 13. Cells were gated on CD11b+ F4/80- single cells. Data are shown as mean ± SEM. *p < 0.05 by paired t test. n = 5 for each condition. Data were collected from 2 independent experiments.

Fig 3: Splenectomy reduces cardiac infiltration of monocytes and heart failure in 5/6Nx mice.a Induction of GPR68 expression in circulating monocytes in 5/6Nx mice. Flow cytometry analysis was performed to detect high-GPR68-expressing CD11b+/Ly6C+ cells in the spleen, blood, and bone marrow of Sham and 5/6Nx mice. b Decrease in the number of cardiac-infiltrated CD11b+/Ly6C+ cells in splenectomized (Splx)-5/6Nx mice. Left panel shows the flow cytometry histogram of CD11b+/Ly6C+ cells isolated from the cardiac ventricle of Sham, 5/6Nx, Splx, and Splx-5/6Nx mice. The number of CD11b+/Ly6C+ cells, indicated by red circles, was quantified and the data are shown in the right panel. c Serum concentrations of BNP in Sham, 5/6Nx, Splx, and Splx-5/6Nx mice. d The mRNA levels of inflammatory cytokines (Tnf-a and IL-6) and fibrosis-related factors (aSma and Col1a2) in the cardiac ventricle of Sham, 5/6Nx, Splx, and Splx-5/6Nx mice. The mean value of the sham-operated wild-type group was set as 1.0. e Double immunofluorescence labeling of GPR68 with F4/80 in the ventricle slices prepared from Sham, 5/6Nx, Splx, and Splx-5/6Nx mice. High-GPR68-expressing cells are double-labeled (yellow) with F4/80. The scale bar indicates 50 µm. f Decrease in the number of cardiac-infiltrated high-GPR68-expressing monocytes in Splx-5/6Nx mice. For all panels, graphs show the mean ± SD of individual mice in independent experiments. Statistical significance was determined using two-way ANOVA with Tukey–Kramer post hoc tests (b–d, f). Numbers and P-values are shown in each graph. Source data are provided as a Source Data file.

Fig 4: Combining chemotherapy with blocking of complement signaling attenuates dox-induced immune modulation.a qRT-PCR analysis of the anaphylatoxin receptors c3ar and c5ar1 expression in sorted lung cell populations (endothelial cells, epithelial cells, immune cells and fibroblasts). n = 35; 11; 20 and 17 for CD45+, PDGFRa+, CD31+ and EpCAM+ respectively. Bars represent range of data points, line represents mean in relative expression to housekeeping gene (ubc); P-values were calculated using one-way ANOVA followed by Tukey multiple comparisons test were performed. b c3ar and c5ar1 expression in granulocytic-MDSCs and mono-MDSCs isolated from lungs. n = 25 and 23 for mMDSCs and gMDSCs. Bars represent range of data points, line represents meanin relative expression to housekeeping gene (rsp23); P-values were calculated using two-tailed Mann–Whitney test. c Immunofluorescence of C3aR-FITC and C5aR1-PE in M-MDSCs (left) and G-MDSCs (right) sorted from lungs, and placed on microscope slides by cytospin. DAPI-blue. Scale bar, 7.5 µm. Data is representative of one independent experiment with n = 5. d Two tailed Pearson correlation between lung mRNA levels of c3 with c3ar (green, r = 0,7156, p = 0,0003) or c5ar1 (red, r = 0,6026, p = 0.0038). e Experimental scheme: following orthotopic injection of 4T1 cells and primary tumor resection, mice were treated with doxorubicin (5 mg/kg) in combination with C3aR antagonist (SB290157- 10 mg/kg) or with C5aR1 antagonist (PMX53- 1 mg/kg), or vehicle (administered i.p). Lung metastasis was monitored by CT imaging. f Complement score calculated from total lung mRNA at early metastatic stage, 24 h following treatment with last dose. n = 16 in each group. Bars represent range of data points, line represents mean. P-values were calculated using Brown–Forsythe ANOVA multiple comparison test. g–k Flow cytometry analysis of lung infiltrating immune cells at early-metastatic stage. g Total immune cells (CD45+ cells), data presented as mean ± s.e.m (h) G-MDSCs (CD45+CD11b+SiglecF-Ly6G+Ly6Cint), i Eosinophils (CD45+CD11b+Ly6G-CD11c-SiglecF+), j Mono-MDSCs (CD45+CD11b+SiglecF-Ly6G-Ly6C+), k PD1+ T cells. n = 14, 14, 15 and 15 mice in PBS, DOX, DOX + SB290157, and DOX + PMX53, respectively. h–k Bars represent range of data points, line represents mean. g–k P-values were calculated using Kruskal–Wallis test for multiple comparisons. Data are representative of three independent experiments and are presented as mean normalized to control. Graphical summary was designed using BioRender. Source data are provided as a Source Data file.

Fig 5: Role of high-GPR68-expressing monocytes in CKD-induced cardiac inflammation and fibrosis.a Schematic experimental procedure for allogeneic transplantation of monocytes into Splx-5/6Nx mice. Monocytes infected with lentivirus-expressing control or anti-Gpr68 miRNA were intravenously injected into Splx-5/6Nx mice every 3 days for 2 weeks after the 5/6 nephrectomy operation. b Upper panels show immunofluorescence labeling of GPR68 (red) in the cardiac ventricle slices prepared from Splx-5/6Nx mice at 2 weeks after initiating the injection of monocytes infected with lentivirus-expressing control or anti-Gpr68 miRNA. Green fluorescence is derived from the lentiviral GFP reporter. The scale bar indicates 20 µm. Lower panels show the flow cytometry histogram of GFP fluorescence of CD11b+/Ly6C+ cells in the heart. GFP-positive cells were detected only in monocyte transplanted mice. c Serum BNP concentrations in Splx-5/6Nx mice at 2 weeks after initiating the injection of control or GPR68-downregulated monocytes. d Downregulation of GPR68 in monocytes ameliorated CKD-induced cardiac fibrosis. The left panel shows Masson’s trichrome staining of tissue fibrosis in blue. Scale bars indicate 1 mm (upper panel) and 50 µm (lower panel). The right panel shows the quantification of the fibrosis area under light microscopy. e, f The mRNA levels of inflammatory cytokines (Tnf-a and IL-6) and fibrosis-related factors (aSma and Col1a2) in the cardiac ventricle of Splx-5/6Nx mice at 2 weeks after initiating the injection of control or GPR68-downregulated monocytes. g Serum urea nitrogen levels in Splx-5/6Nx mice at 2 weeks after initiating the injection of control or GPR68-downregulated monocytes. For all panels, graphs show the mean ± SD of individual mice in independent experiments. Statistical significance was determined using one-way ANOVA with Tukey–Kramer post hoc tests (c–g). Numbers and P-values are shown in each graph. Source data are provided as a Source Data file.