In vitro protocol demonstrating five functional steps of trained immunity in mice: Implications on biomarker discovery and translational research

Abstract

We developed an in vitro methodology to study trained immunity using murine bone-marrow-derived macrophages stimulated with β-glucan and lipopolysaccharide (LPS). Longitudinal analysis of interleukin (IL)-6 and tumor necrosis factor (TNF) production demonstrates that trained macrophages secrete higher cytokine levels following primary stimulation with β-glucan compared to unstimulated macrophages (step 1). After a resting period, trained macrophages return to basal levels of cytokine production (step 2) but rapidly produce enhanced levels of IL-6 and TNF after secondary stimulation with LPS, compared to macrophages individually stimulated with either β-glucan (step 3) or LPS (step 4) alone. The combined cytokine production of macrophages after single stimulation with β-glucan (stimulus 1) and LPS (stimulus 2) is significantly lower than the cytokine levels produced by trained macrophages sequentially stimulated with both β-glucan and LPS (stimulus 1 + 2) (step 5). These results experimentally reproduce the distinctive functional stages that macrophages undergo during the training process.

Keywords: CP: Immunology; SAA3; T cell proliferation; mTOR; mouse strains; sample cryopreservation; trained immunity.

Figure 1.. Kinetics of cytokine production demonstrate the functional steps of trained immunity

(A) Schematic illustration of the in vitro trained-immunity protocol using bone-marrow monocytes from C57BL/6 mice. (B) Representation of four steps that define trained immunity. During the first stimulus, trained macrophages increase their functional immune status (step 1), which returns to the basal level following removal of the stimulus (step 2). In response to a secondary challenge, the function of trained macrophages is enhanced compared to macrophages after the primary (step 3) or the secondary challenge (step 4) alone. Time-course quantification of IL-6 and TNF production from naive, activated, and trained macrophages by ELISA on day 0 (unstimulated), prior to medium change on day 3 (stimulus 1), prior to LPS stimulation on day 6 (resting), and 6 h after LPS stimulation on day 6 (stimulus 2). Data are presented as mean ± SEM (n = 3 mice per group of three independent experiments; one-way ANOVA; **p ≤ 0.01, ***p ≤ 0.005, ****p ≤ 0.001). (C) Representation of the fifth step that defines trained immunity. The combined production of IL-6 and TNF following stimulus 1 (trained macrophages on day 3) and stimulus 2 (activated macrophages on day 6) was compared to the cytokine levels produced by trained macrophages after stimulus 1 + 2 (trained macrophages on day 6). Paired t test; *p ≤ 0.05, ****p ≤ 0.001.

Figure 2.. Trained macrophages undergo metabolic reprogramming

(A and B) Seahorse assay analysis of (A) glycolytic metabolism and (B) mitochondrial respiration in naive, activated, and trained macrophages. Data are presented as mean ± SEM (n = 4 mice per group of three independent experiments; one-way ANOVA with Tukey’s multiple comparisons test; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.005, ****p ≤ 0.001). (C) Quantification of intracellular itaconic and uric acid by mass spectrometry analysis. Data are presented as mean ± SEM (n = 4 mice per group of two independent experiments; one-way ANOVA; *p ≤ 0.05, ****p ≤ 0.001). (D) Quantification of lactate production by colorimetric assay. Data are presented as mean ± SEM (n = 4 mice per group; one-way ANOVA; ***p ≤ 0.005).

Figure 3.. Transcriptomic and epigenetic analyses identify SAA3 as biomarker of training

(A) Volcano plots of expressed genes in naive, activated, and trained macrophages. (B) ATAC-seq signal within differentially accessible regions (DARs) showing differences in the open-chromatin landscape among the three conditions. (C) ATAC-seq genome tracks of Il6, NF-κB1, Cxcl10, and Saa3. (D) Quantification of CXCL10 and SAA3 production from naive, activated, and trained macrophages after 6 h of LPS stimulation by ELISA. Data are presented as mean ± SEM (n = 4 mice per group of three independent experiments; one-way ANOVA; ***p ≤ 0.005, ****p ≤ 0.001). (E) Representative immunofluorescence images of CXCL10 and SAA3 in macrophages from naive, activated, and trained macrophages (n = 4 mice per group of two independent experiments. 5× magnification; scale bar, 50 μm). (F) Schematic illustration of the in vivo trained-immunity protocol. IL-6, TNF, CXCL10, and SAA3 protein quantification in the serum of naive, activated, and trained mice was measured after 6 h of LPS injection. Data are presented as mean ± SEM (n = 8 mice per group; one-way ANOVA with Tukey’s multiple comparisons test; *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.001).

Figure 4.. Trained macrophages upregulate signals 1, 2, and 3 and induce T cell proliferation

(A) t-Distributed Stochastic Neighbor Embedding analysis of co-stimulatory molecules CD80, CD86, OX40L, and PDL1 in naive, activated, and trained macrophages measured by flow cytometry (n = 6 mice per group). (B) Representative histograms and quantification of proliferating CFSE-labeled CD4⁺ and CD8⁺ T cells co-cultured with naive, activated, and trained macrophages. Data are presented as mean ± SEM (n = 4 mice per group of three independent experiments; one-way ANOVA; **p ≤ 0.01, ***p ≤ 0.005, ****p ≤ 0.001). (C) Representative histograms and quantification of proliferating CFSE-labeled CD4⁺ and CD8⁺ T cells co-cultured with trained macrophages using a Boyden chamber (Transwell) with 0.4-μm membrane pore size. Data are presented as mean ± SEM (n = 4 mice of two independent experiments; paired t test; ****p ≤ 0.001).

Figure 5.. Effects of sample cryopreservation and mouse genetics on the induction of training

Time-course quantification of IL-6 and TNF production by macrophages (A) differentiated from freshly isolated or cryopreserved monocytes, (B) from C57BL/6 or BALB/C mice, and (C) from wild-type or mTOR-deficient mice. Data are presented as mean ± SEM (n = 3 mice per group of three independent experiments; one-way ANOVA or paired t test; **p ≤ 0.01, ***p ≤ 0.005, ****p ≤ 0.001).