The C3–C3aR axis modulates trained immunity in alveolar macrophages
- John T. Milliken Department of Medicine, Washington University School of Medicine, United States
- Department of Medicine, University of California Los Angeles David Geffen School of Medicine, United States
- Lung Imaging Laboratory, Division of Pulmonary, Critical Care, & Sleep Medicine, Department of Medicine, Icahn School of Medicine at Mount Sinai, United States
- Department of Anesthesia, University of California, Los Angeles David Geffen School of Medicine, United States
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, United States
- Department of Stem Cell Biology & Regenerative Medicine, Icahn School of Medicine at Mount Sinai, United States
- Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, United States
Figures
Figure 1 with 1 supplement
C3 deficiency predisposes to impaired pulmonary trained immunity.
(A) UMAP (Uniform Manifold Approximation and Projection) plots showing the identity of each cell cluster in human bronchoalveolar lavage (BAL) from BCG- and saline (Sal)-treated donors at Day 2 (BCG n = 17,173; Sal n = 23,718) and Day 7 (BCG n = 21,527; Sal n = 29,540) post-vaccination. N = 3 volunteers in each group. Cell clusters include: macrophage (Mo), activated macrophage (AcMo), non-resident macrophage (nrMo), NK cells, γδ T cells, plasmacytoid DC (pDC), conventional DC (cDC1, cDC2), migratory DC (McDC), B cells, MAIT cells, CD4, CD8, CD4/8, Cytotoxic-like (Ctx), terminally differentiated effector memory CD45RA-re-expressing CD8 T cells (EMRA CD8), naïve T cells (NvCD4, NvCD8), T regulatory cells (Treg), neutrophils, mast cells, doublets, ciliated bronchial epithelial cells (CIBE), secretory bronchial epithelial cells (ScBE), and unidentified cells (Unknown). Data from Marshall et al., 2025. (B) Dot plot and bar chart showing C3 expression across all human alveolar macrophages (Mφ; Mo, AcMo, nrMo pooled) across conditions. Dot size reflects the percentage of cells expressing C3; dot color reflects mean normalized expression. Bar chart shows percentage of C3-expressing Mφ per condition. (C) As in (B), for C3AR1. BCG vaccination increases the proportion of C3AR1-expressing alveolar macrophages at both timepoints relative to saline controls. (D) Schematic representing the training of mice via the intranasal route with heat-killed Pseudomonas aeruginosa (HKPA) and subsequent restimulation with lipopolysaccharide (LPS), followed by BAL and cytokine analysis. Created with BioRender. (E) WT untrained compared against WT-trained BAL levels of CXCL1, CXCL2, IL-6, and TNFα. (F) Comparison of BAL C3a levels, similar to (E). (G) WT-trained versus C3-deficient (C3KO)-trained BAL concentrations of IL-6 and TNFα. WT-trained levels derived from (E) for comparison with C3KO-trained mice. Data were compared with two-sided unpaired t-tests with (E, G) or without (F) Holm–Šidák correction for multiple hypothesis testing. Each point represents a measurement from one mouse with at least n = 4 in each group, mean ± SD shown. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 1—figure supplement 1
C3 deficiency predisposes to impaired pulmonary trained immunity.
(A) Stacked bar charts showing macrophage subtype composition per donor across conditions. Proportions of resident alveolar macrophages (Mo), activated macrophages (AcMo), and non-resident monocyte-derived macrophages (nrMo) are shown for saline (Sal) and BCG-treated donors at Day 2 (G1 donors) and Day 7 (G2 donors). Data from Marshall et al. that included 91,958 cells from 12 samples [Day 2: Saline (3); BCG (3), Day 7: Saline (3); BCG (3)] consisting of BCG and saline-treated individuals at Days 2 and 7 after inhalation. (B) Dot plots showing C3 expression across human alveolar macrophage subtypes (Mo, AcMo, nrMo) in saline and BCG-treated donors at Days 2 and 7. Dot size reflects the percentage of cells expressing C3; dot color reflects mean normalized expression. Data from Marshall et al. (C) As in (B), for C3AR1. Comparison of protein (D), neutrophils (E), TNFα (F), and CXCL1 (G) in the BAL of wild-type (WT), C3-deficient (C3−/−), and C3aR-deficient (C3aR−/−) mice at 14 days after training via the intranasal route with heat-killed Pseudomonas aeruginosa (HKPA). Data were compared with two-sided unpaired t-tests. Each point represents a measurement from one mouse with at least n = 4 in each group, mean ± SD shown. *p < 0.05, ns, non-significant.
Figure 2 with 1 supplement
C3 deficiency results in impaired trained immune responses in ex vivo alveolar macrophages (AMs).
(A) Schematic representing in vitro training of AMs with HKPA, with later stimulation by lipopolysaccharide (LPS) and subsequent cytokine analysis of the supernatants. Created with BioRender. Effects of HKPA-induced training in vitro on IL-6 and TNFα in supernatant from (B) WT AM, and (C) their comparison with C3KO-trained AMs. (D) Schematic representing in vitro training of AMs with heat-killed Candida albicans (HKCA), with subsequent restimulation by LPS and cytokine analysis of the supernatants. Created with BioRender. (E) Effects of HKCA-induced training in vitro on CXCL1, CXCL2, IL-6, and TNFα in supernatant from WT AM. (F) Comparison of C3a levels post-HKCA training, similar to (B). (G) Comparison of IL-6 and TNFα post-HKCA training in WT versus C3KO AMs. WT-trained levels derived from (D) for comparison with C3KO-trained AMs. Data were compared with two-sided unpaired t-tests with (B, C, E, G) or without (F) Holm–Šidák correction for multiple hypothesis testing. Each point is a technical replicate made by pooling AMs from at least n = 4 mice in each group, with mean ± SD shown, and each experiment was repeated twice. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 2—figure supplement 1
C3 deficiency results in impaired trained immune responses in lipopolysaccharide (LPS)-stimulated alveolar macrophages (AMs).
(A) Schematic representing in vitro training of AMs with HKPA, followed by ex vivo stimulation using LPS and analyses of the cells. (B) Phagocytic capacity of in vitro HKPA-trained or untrained mouse AMs from WT and C3KO mice, measured by mean fluorescence intensity (MFI) of pHrodo-labeled E. coli bioparticles in live cells. (C) Reactive oxygen species (ROS) production in vitro in HKPA-trained or untrained mouse AMs from WT and C3KO mice, measured by CellRox MFI in live cells. (D) Schematic representing in vivo training of mouse lungs with HKPA, followed by ex vivo stimulation of harvested AM using LPS and subsequent cytokine analysis of the supernatants. Supernatants were collected at 24 hr post-LPS stimulation for cytokine analysis. TNFα and IL-6 concentrations in supernatants from untrained and HKPA-trained WT and C3KO AMs are shown. Data shown as mean ± SD; each point represents one biological replicate. Statistical comparisons by one-way ANOVA with multiple comparisons correction. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Illustrations in this figure from NIAID NIH BioArt Source Mouse (bioart.niaid.nih.gov/bioart/279), Flask (bioart.niaid.nih.gov/bioart/303), and 96 Well Plate (bioart.niaid.nih.gov/bioart/7).
Figure 3 with 1 supplement
Alveolar macrophages take up C3 from airspaces of live alveoli in situ.
(A–G) Cartoons in A, B show the experimental design of the confocal imaging studies shown in C−G. As indicated in A, B, we microinstilled alveolar airspaces of live, intact, perfused mouse lungs sequentially with: cell-permeant calcein red-orange dye (CR); Alexa Fluor 647 (AF), AF-tagged C3 (C3–AF), or AF-tagged C3a (C3a-AF); and phycoerythrin (PE)-tagged anti-CD11c Ab. The confocal image in C shows C3-AF fluorescence (yellow) in alveolar airspaces and CR fluorescence (magenta) in airspace-facing cells, including the alveolar epithelium and alveolar macrophages. alv, example airspace; mv, microvessel. Confocal images in D show the same alveoli, but CD11c fluorescence (cyan) now marks CD11c+ cells. Arrowheads point out example CD11c+ cells with intracellular C3-AF fluorescence. High power confocal images of CD11c+ cells (E–F) and group data (G) show C3-AF accumulated in cytosols of CD11c+ cells over time. In G, circles indicate mean ± SEM fluorescence in all of the CD11c+ cells present in imaging fields of at least 30 alveoli; n = 4 microinstillations in 2 lungs per group; *p < 0.05 versus C3-AF by ANOVA with post hoc Tukey testing. C3-AF, C3a-AF, and AF fluorescence in airspaces was normalized to C3-AF, C3a-AF, and AF fluorescence in glass micropipettes. Scale bars: 100 (C, D) and 10 (E, F) µm.
Figure 3—figure supplement 1
C3 uptake in human precision-cut lung slices (hPCLS).
(A) Schematic for obtaining human PCLS. Schematic obtained from NIAID Visual & Medical Arts. (10/7/2024). Human Lungs. NIAID NIH BIOART Source. bioart.niaid.nih.gov/bioart/231. (B) Representative image showing C3-AF647 (green) incubated with CD68+ macrophages (red). Inset shows individual colors. DAPI: blue. (C) Representative image showing C3a-AF647 (green) incubated with CD68+ macrophages (red). Inset shows individual colors. DAPI: blue. (D) Comparison of individual data points from n = 3 hPCLS, 3 technical replicates per donor. Y-axis represents the mean fluorescence within the region of interest (ROI) defined by CD68. Data shown as mean ± SD. Statistical comparisons by one-way ANOVA with multiple comparisons correction. ***p < 0.001.
Figure 4
C3 uptake enhances trained immune responses in ex vivo alveolar macrophages (AMs) via the C3a receptor (C3aR).
(A) Schematic representing in vitro training of AMs with heat-killed Candida albicans (HKCA), with pre-treatment of C3 or C3a prior to induction of training, and later stimulation by lipopolysaccharide (LPS) and subsequent cytokine analysis of the supernatants. Created with BioRender. (B) Effects of adding C3 prior to training on IL-6 and TNFα levels from C3KO AMs and their comparison with WT-trained AMs. (C) Effects of adding C3a prior to training, similar to (B). (D) Schematic representing addition of the C3aR antagonist prior to C3 treatment and in vitro training of AMs with HKCA, with later stimulation by LPS and subsequent cytokine analysis of the supernatants. Created with BioRender. (E) Effects of C3aR antagonism on IL-6 and TNFα levels from trained WT and C3KO AMs treated with exogenous C3. (F) Comparison of IL-6 levels post-HKCA-training in C3aR-deficient (C3aRKO), C3KO, and WT AMs treated with exogenous C3. Data were compared using one-way ANOVA with Dunnett’s post hoc tests (B, C, F) or two-sided unpaired t-testing with Holm–Šidák correction for multiple testing (E). Each point is a technical replicate made by pooling AMs from at least n = 4 mice in each group, with mean ± SD shown, and each experiment was repeated twice. *p < 0.05, **p < 0.01.
Figure 5
C3–C3aR axis is required for glycolysis as a part of trained immune responses in alveolar macrophages (AMs).
(A) Principal component analysis (PCA, left) and EnrichR analysis of 391 genes (right, Supplementary file 1) downregulated in heat-killed Candida albicans (HKCA)-trained C3KO versus WT AMs by filtering genes (FDR step up ≤0.05). Arrow shows metabolism gene set in EnrichR; bars ranked by p-value. (B) Schematic representing in vitro training of AMs with HKCA. Created with BioRender. (C) Extracellular acidification rate (ECAR) from Seahorse analysis representing full glycolytic activity, and basal and maximum glycolysis in untrained and HKCA-trained WT and C3KO AMs. (D) Schematic representing addition of the C3aR antagonist (SB290157) prior to C3 treatment and in vitro training of AMs with HKCA. Created with BioRender. (E) Seahorse analysis in the presence and absence of exogenous C3 supplementation and C3aR antagonism. Each point is a technical replicate of pooled AMs from at least n = 4 mice in each group, with mean ± SD shown. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 when analyzed using one-way ANOVA with Dunnett’s post hoc tests.
Author response image 1
C3a-C3aR colocalization in mouse ex vivo cultured alveolar macrophages (mexAM).
mexAMs were harvested and cultured as per the protocol from Gorki et al. (2022). Cells were incubated in a Millicell EZ Slide 8-well glass chamber slide overnight to allow for adherence, then fixed, permeabilized, and incubated with anti-C3a conjugated to AF555 (blue, Hycult HM1072), anti-C3aR conjugated to AF647 (red, Hycult HM1123), and anti-LAMP1 (green, Cell Signaling 99437) overnight at 4°C. Slides were washed 3X in PBS (5 min each) and mounted overnight at 4°C in ProLong Diamond Antifade Mountant with DAPI (white). Images were acquired on a Zeiss LSM 880 confocal microscope at 63X. At least 6 cells per condition imaged. Experiments were conducted in duplicate (technical replicates) and repeated (for biological replicates). Scale bar, 2 μm.
Additional files
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Supplementary file 1
Table of pathways based on Reactome analysis with a list of overlapping genes when comparing downregulated transcripts between trained C3-deficient and wildtype primary alveolar macrophages.
- https://cdn.elifesciences.org/articles/104977/elife-104977-supp1-v1.zip
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MDAR checklist
- https://cdn.elifesciences.org/articles/104977/elife-104977-mdarchecklist1-v1.docx
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Source data 1
The source data includes the raw data used to generate the figures included in the manuscript.
- https://cdn.elifesciences.org/articles/104977/elife-104977-data1-v1.xlsx
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The C3–C3aR axis modulates trained immunity in alveolar macrophages
eLife 14:RP104977.
https://doi.org/10.7554/eLife.104977.3
