Hemolysis transforms liver macrophages into antiinflammatory erythrophagocytes
Marc Pfefferlé, Giada Ingoglia, Christian A. Schaer, Ayla Yalamanoglu, Raphael Buzzi, Irina L. Dubach, Ge Tan, Emilio Y. López-Cano, Nadja Schulthess, Kerstin Hansen, Rok Humar, Dominik J. Schaer, Florence Vallelian
Marc Pfefferlé, Giada Ingoglia, Christian A. Schaer, Ayla Yalamanoglu, Raphael Buzzi, Irina L. Dubach, Ge Tan, Emilio Y. López-Cano, Nadja Schulthess, Kerstin Hansen, Rok Humar, Dominik J. Schaer, Florence Vallelian
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Research Article Hematology

Hemolysis transforms liver macrophages into antiinflammatory erythrophagocytes

Abstract

During hemolysis, macrophages in the liver phagocytose damaged erythrocytes to prevent the toxic effects of cell-free hemoglobin and heme. It remains unclear how this homeostatic process modulates phagocyte functions in inflammatory diseases. Using a genetic mouse model of spherocytosis and single-cell RNA sequencing, we found that erythrophagocytosis skewed liver macrophages into an antiinflammatory phenotype that we defined as MarcohiHmoxhiMHC class IIlo erythrophagocytes. This phenotype transformation profoundly mitigated disease expression in a model of an anti-CD40–induced hyperinflammatory syndrome with necrotic hepatitis and in a nonalcoholic steatohepatitis model, representing 2 macrophage-driven sterile inflammatory diseases. We reproduced the antiinflammatory erythrophagocyte transformation in vitro by heme exposure of mouse and human macrophages, yielding a distinctive transcriptional signature that segregated heme-polarized from M1- and M2-polarized cells. Mapping transposase-accessible chromatin in single cells by sequencing defined the transcription factor NFE2L2/NRF2 as a critical driver of erythrophagocytes, and Nfe2l2/Nrf2 deficiency restored heme-suppressed inflammation. Our findings point to a pathway that regulates macrophage functions to link erythrocyte homeostasis with innate immunity.

Authors

Marc Pfefferlé, Giada Ingoglia, Christian A. Schaer, Ayla Yalamanoglu, Raphael Buzzi, Irina L. Dubach, Ge Tan, Emilio Y. López-Cano, Nadja Schulthess, Kerstin Hansen, Rok Humar, Dominik J. Schaer, Florence Vallelian

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Figure 6

Heme/NRF2 signaling drives erythrophagocyte-like polarization of BMDMs.

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Heme/NRF2 signaling drives erythrophagocyte-like polarization of BMDMs. ...
(A) RT-qPCR expression data for heme-response genes in Nrf2+/+ and Nrf2–/– BMDMs after heme-albumin (200 μM) or albumin (control) treatment for 6 hours (mean of n = 3 replicates). (B) Scatter plot of differentially expressed genes (logratio > 0.5) of heme-albumin– versus albumin-treated BMDMs followed by LPS (4 hours). Data represent the log10 of the expression ratio as mean from n = 4 gene array experiments. A transcription factor enrichment analysis for the red-labeled gene cluster is displayed in Supplemental Figure 6A. (C) Factorial layout and predicted phenotype outcomes of gene array experiments with BMDMs from Nrf2+/+ and Nrf2–/– mice that were treated with or without heme-albumin and LPS. Effects on differential gene expression for heme in WT cells (effectheme) and for the Nrf2 gene in heme-exposed cells (effectNrf2) were calculated by the displayed formulae. (D) Correlation of the GSEA hallmark gene set enrichment scores for effectheme and effectNrf2. The top 3 hallmark gene sets are highlighted (P < 0.001). (E) Overlaid GSEA enrichment score plots for the hallmark gene sets highlighted in D. (F) Heatmap of the ECAR of Nrf2+/+ and Nrf2–/– BMDMs. BMDMs were treated for 4 hours with albumin (control) or with 200 μM heme-albumin before the Seahorse experiment. Cells were analyzed at baseline and after treatment with oligomycin. Mean values from 3 measurements per condition (yellow = low ECAR, red = high ECAR). (G) LPS-stimulated Il6 and Il12b mRNA expression in Nrf2+/+ and Nrf2–/– BMDMs after pretreatment with albumin or with heme-albumin (+h) for 4 hours (n = 6–9), measured by RT-qPCR. (H) Il12b, Nos2, and Nqo1 mRNA in WT BMDMs treated with LPS, LPS+heme, or with the Nrf2 activators RA-839 and ML-334 measured by RT-qPCR. Mean of 3 independent experiments. **P < 0.01 by ANOVA with Tukey’s post hoc test for G. NS, not significant.