The generation of macrophages with anti-inflammatory activity in the absence of STAT6 signaling

Abstract

Macrophages readily change their phenotype in response to exogenous stimuli. In this work, macrophages were stimulated under a variety of experimental conditions, and phenotypic alterations were correlated with changes in gene expression. We identified 3 transcriptionally related populations of macrophages with immunoregulatory activity. They were generated by stimulating cells with TLR ligands in the presence of 3 different "reprogramming" signals: high-density ICs, PGE2, or Ado. All 3 of these cell populations produced high levels of transcripts for IL-10 and growth and angiogenic factors. They also secreted reduced levels of inflammatory cytokines IL-1β, IL-6, and IL-12. All 3 macrophage phenotypes could partially rescue mice from lethal endotoxemia, and therefore, we consider each to have anti-inflammatory activity. This ability to regulate innate-immune responses occurred equally well in macrophages from STAT6-deficient mice. The lack of STAT6 did not affect the ability of macrophages to change cytokine production reciprocally or to rescue mice from lethal endotoxemia. Furthermore, treatment of macrophages with IL-4 failed to induce similar phenotypic or transcriptional alterations. This work demonstrates that there are multiple ways to generate macrophages with immunoregulatory activity. These anti-inflammatory macrophages are transcriptionally and functionally related to each other and are quite distinct from macrophages treated with IL-4.

Keywords: RNA-seq; alternatively activated; endotoxemia; regulatory; transcriptomics.

Figure 1.. Regulatory activation provides protection from lethal endotoxemia.

(A) BALB/c mice received 1 × 10⁶ resting, nonstimulated macrophages (Mϕ, filled circles) intraperitoneally or macrophages stimulated in vitro with LPS in combination with Ova-IC (blue squares), PGE₂ (red squares), or Ado (green squares) or macrophages treated with IL-4 (yellow triangles), 3 h before challenge with a lethal dose of endotoxin (10 mg/kg). The survival of the mice was recorded every 8 h over the next week. (B) A similar survival experiment was carried out in WT mice that received 1 × 10⁶ L+I macrophages from WT (blue squares) or stat6^−/− (olive squares) mice before endotoxin challenge. Each graph represents data of 2 independent experiments with 10 mice/experiment for each condition. The data from the control group and WT RMϕ-IC were shared between the 2 graphs. ***P < 0.0001, and **P = 0.0006, Kaplan-Meier estimates obtained for mice treated with resting macrophages versus other macrophage treatments.

Figure 2.. R-Mϕ induction is independent of STAT6 signaling pathway.

BALB/c WT and stat6^−/− BMDMs were treated with 10 ng/ml LPS alone or a combination of LPS and 25 µl Ova/anti-Ova ICs (L+I), 200 nM PGE₂ (L+P), 200 μM Ado (L+A), or 20 ng/ml IL-4 for 16 h. The levels of IL-10 (A and E), IL-12/23p40 (B and F), IL-1β (C and G), and IL-6 (D and H) were measured in their supernatants by ELISA. Error bars indicate means ± sem of 3 independent experiments. ***P < 0.001, **P < 0.01, and *P < 0.05. Representative RT-PCR analysis of alternate activation markers relmα and ym1 from WT or stat6^−/− BMDMs stimulated for 4 h with their respective stimuli before RNA isolation. The gapdh is used as the internal control (I).

Figure 3.. Regulatory activation results in alterations in cytokine/chemokine profiles.

(A) Chemokine and cytokine secretion by BMDMs was measured by a proteome profiler membrane antibody array. Supernatants from nonstimulated macrophages were compared with macrophages treated with 10 ng/ml LPS or a combination of LPS and 100 µl Ova-IC (L+I), 200 nM PGE₂ (L+P), 200 μM Ado (L+A), or 20 ng/ml IL-4 for 12 h. The proteins that are of interest to this study are indicated in circles, and the letters and numbers are provided to identify the position of the analyte in the membrane. The profiling was done on pooled supernatants collected from 3 separate experiments from 3 different mice. (B). Mean fold differences in intensity of the duplicate samples for relevant analytes are compared with supernatants from nonstimulated. The alphanumeric values within parentheses indicate their position in the membrane array. BCA-1, B Cell-attracting chemokine-1; TCA-3, T cell activation-3; KC, keratinocyte-derived chemokine.

Figure 4.. Global RNA expression profiles of macrophage samples.

RNA-seq was carried out on an Illumina platform, comparing nonstimulated (NS) murine peritoneal macrophages and macrophages exposed to LPS, L+I, L+P, L+A, or IL-4. A Principal Component Analysis (PCA) plot (A) and heat map of a hierarchical clustering analysis that uses the Euclidean distance metric (B) are shown. (A) In the PCA plot, each symbol represents an experimental sample with symbol color indicating macrophage treatment condition (NS, gray; LPS, orange; L+I, light blue; L+A, medium blue; L+P, navy blue; and IL-4, green), and symbol shape indicates batch. (B) Colors along the top of the heat map indicate the treatment condition (same color codes as in A), and colors along the left side of the heat map indicate the batch/experimental date. (C and D) Overlap of differentially expressed genes up-regulated (C) or down-regulated (D) by >2-fold relative to nonstimulated macrophages are displayed in Venn diagrams. Each large-colored square represents the treatment condition (same color codes as in A). The changes associated with Diseases and Functions in R-Mϕ were predicted by the IPA program. Genes that showed a changed in L+I or L+A of at least 2-fold when compared with LPS were selected to identify pathways associated with regulatory functions (E and F). Fold changes were uploaded to IPA, and the Diseases and Functions predicted to be altered based on a significant Z-score were selected for these graphs. A Z-score above 1.65 (activated) or below −1.65 (inhibited) is considered statistically significant.

Figure 5.. Glucose and lactate production in R-Mϕ.

BMDMs were left unstimulated or stimulated with a combination of 10 ng/ml LPS alone or LPS in combination with 25 µl Ova-IC (L+I), 200 nM PGE₂ (L+P), 200 μM Ado (L+A), or 20 ng/ml IL-4 for 24 h. (A) Glucose consumption was determined 24 h poststimulation by an enzymatic assay, as described in Materials and Methods. (B) l-Lactate concentrations in the supernatants were obtained 8 h poststimulation by use of the NADH-coupled enzyme reaction that reduces tetrazolium salt to formazan, which is measured at an absorbance of 490 nm. The error bars represent means ± sem values calculated from values of 4 separate experiments. ***P < 0.001, **P < 0.01, and *P < 0.05.

Figure 6.. Regulatory gene induction is independent of STAT6 signaling.

RT-PCR analyses were carried out at indicated time-points on cDNA obtained from BALB/c WT and stat6^−/− BMDMs, which were left unstimulated or stimulated with 10 ng/ml LPS alone or a combination of LPS and 100 µl Ova-IC (L+I), 200 nM PGE₂ (L+P), 200 μM Ado (L+A), or 20 ng/ml IL-4 (A–F). Each data point represents mean values ± sem from duplicate values of 4 separate experiments. (G) The relative expression of genes up-regulated after treatment of macrophages with L+I is shown. (H) The relative expression of genes up-regulated after treatment of macrophages with IL-4 is shown. Each error bar represents mean values ± sem from duplicate values of 4 separate experiments. Surface expression of CCR1 on cells stimulated under the same conditions was assessed by flow cytometry by use of a PE-conjugated antibody to CCR1, 24 h after stimulation. A representative histogram (I) and the fold induction in CCR1 mean fluorescence intensity (MFI) over nonstimulated cells are represented from 5 separate experiments (J). The mean values ± sem are depicted, and *P < 0.05, and **P < 0.01. Regulatory gene induction was also assessed in the peritoneal macrophages (PMϕ) of Balb/c (I–L) or C57BL/6 mice (M and N) mice injected intraperitoneally with 500 μg LPS alone or in combination with 400 µl Ova-IC (L+I), 50 μg PGE₂ (L+P), 50 μg Ado (L+A), or 50 μg IL-4. Macrophages were isolated 6 h later, and surface expression of CCR1 on macrophages was assessed by flow cytometry. Cells were gated on their expression of F4/80 (allophycocyanin) but not for other cell markers (CD3, CD4, CD19, and FITC). (I and M) The expression of regulatory (ccr1 and IL-10)- or alternate (ym1)-activated gene induction was evaluated by real-time PCR (J–L and N). Data were obtained from injections of 3 different mice. The numbers on the left side of the histogram denote the percentage of positive cells. *P < 0.05; **P < 0.01.

Figure 7.. Cytokine profile of human macrophages under regulatory stimulation conditions.

Human monocyte-derived macrophages were cultured at the concentration of 5 × 10⁵ cells/500 μL medium and were left unstimulated or stimulated with 30 ng/ml (A) or 10 ng/ml LPS alone or a combination of LPS and 25 µl Ova/anti-Ova ICs (L+I), 200 nM PGE₂ (L+P), 200 μM Ado (L+A), or 20 ng/ml IL-4 for 8 (A) or 24 h (B–G). Cytokines in A were measured by use of a sandwich ELISA kit. The levels of all other indicated cytokines were measured by use of the bioplex assay. The horizontal bar represents the mean value, and the asterisks represent the significance of the observed values compared with LPS-treated cells. ***P < 0.001, **P < 0.01, and *P < 0.05.