C3 deficiency predisposes to impaired pulmonary trained immunity and diminished protection from lung injury.

(A) Schematic representing the training of mice via the intranasal route with heat-killed Pseudomonas aeruginosa (HKPA) and subsequent restimulation with lipopolysaccharide (LPS), followed by bronchoalveolar lavage (BAL) and cytokine analysis. Created with BioRender.

(B) WT untrained compared against WT-trained BAL levels of CXCL1, CXCL2, IL-6, and TNFα.

(C) Comparison of BAL C3a levels, similar to (B).

(D) WT-trained versus C3-deficient (C3KO)-trained BAL concentrations of IL-6 and TNFα. WT-trained levels derived from (B) for comparison with C3KO-trained mice.

(E) Concentrations of protein and RAGE from WT-trained versus C3KO-trained mice.

(F) Representative histopathological slides of lungs showing increased tissue damage in C3KO-trained versus WT-trained mice (N=5 in each group), high power view from selected area.

Data were compared with two-sided unpaired t-tests with (B, D) or without (C, E) Holm-Šidák correction for multiple hypothesis testing. Each point represents a measurement from one mouse, n = 5-10 for each group with mean ± SD shown. *p < 0.05, **p < 0.01, ***p < 0.001.

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 LPS and subsequent cytokine analysis of the supernatants. Created with BioRender.

(B-C) 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 HKCA, with subsequent restimulation by LPS and cytokine analysis of the supernatants. Created with BioRender.

(E) Effects of heat-killed Candida albicans (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.

C3 uptake enhances trained immune responses in ex vivo alveolar macrophages via the C3a receptor (C3aRs).

(A) Schematic representing in vitro training of AMs with HKCA, with pre-treatment of C3 or C3a prior to induction of training, and later stimulation by 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 (D). 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.

C3-C3aR axis is required for glycolysis as a part of trained immune responses in alveolar macrophages.

(A) Principal component analysis (PCA, left) and EnrichR analysis of 391 genes (right, Table S1) downregulated in HKCA-trained C3KO vs WT AM 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 using an unpaired t-test.