Chronic TLR7 and TLR9 signaling drives anemia via differentiation of specialized hemophagocytes

Abstract

Cytopenias are an important clinical problem associated with inflammatory disease and infection. We show that specialized phagocytes that internalize red blood cells develop in Toll-like receptor 7 (TLR7)-driven inflammation. TLR7 signaling caused the development of inflammatory hemophagocytes (iHPCs), which resemble splenic red pulp macrophages but are a distinct population derived from Ly6C^hi monocytes. iHPCs were responsible for anemia and thrombocytopenia in TLR7-overexpressing mice, which have a macrophage activation syndrome (MAS)-like disease. Interferon regulatory factor 5 (IRF5), associated with MAS, participated in TLR7-driven iHPC differentiation. We also found iHPCs during experimental malarial anemia, in which they required endosomal TLR and MyD88 signaling for differentiation. Our findings uncover a mechanism by which TLR7 and TLR9 specify monocyte fate and identify a specialized population of phagocytes responsible for anemia and thrombocytopenia associated with inflammation and infection.

Figure 1.. TLR7 promotes RPM-like hemophagocyte development in vitro.

(A) Representative flow cytometric staining of CMPs cultured with SCF or SCF+R848. (B-D) RNA-Seq analysis of CD11b⁺F4/80⁺ macrophages sorted from CMPs differentiated in R848 or MCSF. (B) Of the 22,707 total protein coding genes, 813 were upregulated and 1,020 were downregulated in R848-differentiated macrophages compared to MCSF-differentiated macrophages (≥2-fold, FDR≥0.05). Three independent biological replicates were sequenced for each condition. (C) Red bars indicate the percent of genes in a tissue macrophage core transcriptional signature (14) that were significantly increased in R848-differentiated compared to MCSF-differentiated macrophages. Black line indicates –log p-value calculated using exact hypergeometric probability with a normal approximation. (D) Heat map of increased genes (≥2-fold, FDR≥0.05) in R848-differentiated compared to MCSF-differentiated macrophages in RPM core signature. (E) R848-differentiated macrophages were treated with or without cytochalasin D (CytoD) then allowed to phagocytose CFSE-labeled RBC for 15 min at indicated ratios. Percent of CD11b⁺F4/80⁺ macrophages that had phagocytosed RBC is shown. Data are representative of four experiments, n=3 technical replicates per condition/experiment. Mean values+SD (E) are shown.

Figure 2.. TLR7 signaling drives formation of hemophagocytes distinct from RPMs in vivo.

(A-D) Splenocytes from WT and TLR7.1 mice were surface-stained for CD11b, CD45.2, F4/80, Ly6G and Siglec-F, and then intracellularly stained with mAb to Ter-119 to detect leukocytes that had phagocytosed RBCs. (A) Representative flow cytometry pre-gated as live singlets, CD45.2⁺ cells. Prior to intracellular staining with fluorescently labeled anti-Ter-119, some samples were blocked with unconjugated Ter-119 (control). Other samples were not blocked prior to staining (Ter-119). (B) Frequency and number of CD45.2⁺Ly6G⁻Siglec-F⁻Ter-119⁺ cells, and mean fluorescence intensity (MFI) of Ter-119 staining in CD45.2⁺Ter-119⁺ cells were then quantified. Data are representative of four experiments. (C) CD11b and F4/80 expression on CD45.2⁺Ly6G⁻Siglec-F⁻Ter-119⁺ splenocytes. iHPCs are in blue and RPMs are in red. Data are representative of four experiments. (D) RPMs were gated as CD45.2⁺Ly6G⁻Siglec-F⁻Ter-119⁺ cells that were CD11b^lo/intF4/80^hi and iHPCs were gated as CD45.2⁺Ter-119⁺ cells that were CD11b^int/hiF4/80^lo. Histograms of indicated surface staining are shown. Data are representative of four experiments. (E) Percentage of RPMs or iHPCs that were Ter-119⁺ from WT and TLR7.1 mice quantified by flow cytometry. Data are representative of four experiments, n=5 per group. (F and G) RPMs and iHPCs were FAC-sorted and stained by H&E. Intracellular RBCs were quantitated by microscopy. (G) Phagocytic index (number of intracellular RBCs per 100 cells) (left) and percentage of cells with at least one RBC (right) were calculated. Data are representative of two experiments, n=3–4 per group. (B) mean±SEM, (F and G) mean+SEM, (B) each symbol represents an individual mouse. *p<0.05, ** p<0.01, ***p<0.001, ****p<0.0001, two-tailed, unpaired Student’s t-test (B). one-way ANOVA with Tukey’s post test (E).

Figure 3.. iHPCs differentiate in response to cell-intrinsic TLR7 signals.

A) The ratio of TLR7.1 to WT BM-derived cells in mixed bone marrow chimeras of indicated populations after reconstitution. Data are representative of two experiments with n=8–10 per experiment. B) The ratio of WT to Tlr7^−/− BM-derived cells in mixed bone marrow chimeras injected with the TLR7 agonist R848 (right) or PBS (left) for 13 days. Data are representative of two experiments with n=3–5 per group per experiment. (A, B) mean±SEM, (A, B) each symbol represents an individual mouse. *p<0.05, ** p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA with Dunnett’s post test (A and B).

Figure 4.. iHPCs are derived from Ly6C^hi monocytes.

(A) Splenic monocytes (live singlets, CD11b⁺F4/80⁻Ly6G⁻Ly6C^hi or CCR2⁺ cells) (black) and iHPCs (live singlets, F4/80^loLy6G⁻Ter-119⁺VCAM1^lo or CD31^hi cells) (blue) were assessed for the expression of the cell surface proteins indicated (solid lines) compared to fluorescence minus one (FMO) control stains (dashed lines). Data are representative of three experiments. (B) Bone marrow Ly6C^hi monocytes were sorted from WT B6 mice and cultured for 21 hours with media alone (--) or with R848. Spic, Pecam1, Ccr2, and Ly6c1 transcripts were quantified by qPCR. Data are representative from five experiments with n=3 per experiment. (C, D) Ccr2-DTR⁻, WT/Ccr2-DTR⁺, TLR7.1/Ccr2-DTR⁻, and TLR7.1/Ccr2-DTR⁺ mice (n=5–7 mice per group) were injected with DT every other day for 17 days. (C) Representative flow cytometry of Ter-119⁺ hemophagocytes pre-gated on live singlets, CD45.2⁺Ly6G⁻Siglec-F⁻ cells from the spleens of TLR7.1/Ccr2-DTR⁻ and TLR7.1/Ccr2-DTR⁺ mice determined by flow cytometry. (D) Frequency and number of the indicated cell populations were quantitated from the spleens of Ccr2-DTR⁻, Ccr2-DTR⁺, TLR7.1/Ccr2-DTR⁻, and TLR7.1/Ccr2-DTR⁺ mice by flow cytometry. Data are combined from three experiments. (E) Bone marrow Ly6C^hi monocytes were sorted from WT B6 mice and cultured for 21 hours with media alone (--), R848, LPS, or CpG. Spic, Pecam1, Ccr2, and Ly6c1 transcripts were quantified by qPCR. Data are representative from two experiments. (F and G) RNA-Seq analysis of RPMs and Ly6C^hi monocytes sorted from spleens of WT B6 mice and TLR7.1 mice and iHPCs from spleens of TLR7.1 mice. n=5 (TLR7.1 iHPC), n=4 (WT and TLR7.1 RPM), n=6 (WT and TLR7.1 Mono). (F) PCA of indicated populations. (G) Heat map of DEG between the three populations (RPMs, Ly6C^hi monocytes, and iHPCs) sorted from TLR7.1 mice. Mean values+SEM (D), ± SEM (B, E). *p<0.05, ** p<0.01, ***p<0.001, ****p<0.0001, two-tailed, unpaired Student’s t-test (B) and Mann–Whitney U test (D).

Figure 5.. Monocyte-derived iHPCs drive anemia.

(A) RBC count, hemoglobin levels, and hematocrit from 3-month-old WT and TLR7.1 mice. Each symbol represents an individual mouse, n=8–12 mice per group. (B) Correlation between RBC count and number (left) and frequency (right) of splenic Ter-119⁺ iHPCs in TLR7.1 mice. (C) RBC count of TLR7.1 WT, TLR7.1 Spic^+/−, TLR7.1 Spic^−/−, and control mice that were bled prior to 8 weeks and between 9 and 13 weeks of age. n=7–14 mice per group. (D) TLR7.1/Ccr2-DTR⁻ and TLR7.1/Ccr2-DTR⁺ mice were treated with DT every other day for 17 days beginning when RBC count was below 8. RBC count was measured at indicated times. n=5–7 mice per group. (E) Ccr2-DTR⁻ and Ccr2-DTR⁺ mice were treated with DT every day for 6 days and CpG daily starting one day after beginning DT treatment. RBC count was measured prior to and at the end of treatment. Data are representative of two experiments, n=3 (No Tx) and n=7 (CpG) mice per group. (F) Platelet counts in TLR7.1/Ccr2-DTR⁻ and TLR7.1/Ccr2-DTR⁺ mice treated as in (D). Mean values ±SEM (A, C-F) are shown. (A and B) each symbol represents an individual mouse.*p<0.05, ** p<0.01, ***p<0.001, ****p<0.0001, ns not significant Mann–Whitney (A), Wilcoxon paired t-test of Days −1 and +17 for TLR7.1 experiment and −1 and +5 for CpG injection (D-F).

Figure 6.. iHPC differentiation depends on IRF5.

(A) Bone marrow Ly6C^hi monocytes were sorted from WT and Irf5^−/− mice and cultured for 21 hours with media alone (--) or with R848. Spic, Pecam1, Ccr2, and Ly6c1 transcripts were quantified by qPCR. Data are representative from two experiments with n=2 to 4 per group per experiment. (B and C) WT and Irf5^−/− mice were injected with R848 i.p. daily for 2 days. Splenocytes were analyzed by flow cytometry. (B) Representative flow plots of WT and Irf5^−/− CD11b⁺CD31⁺ iHPCs (gated on live singlets, CD45.2⁺F4/80⁻Ly6G⁻Siglec-F⁻ cells). (C) Frequency (left) and number (right) of iHPCs in WT and Irf5^−/− spleens. Data are representative from two experiments with n=4 per group per experiment. Mean values±SEM (A and C) are shown. (A and C) each symbol represents an individual mouse. *p<0.05, ** p<0.01, ***p<0.001, ****p<0.0001, two-tailed, unpaired Student’s t-test (A and C).

Figure 7.. iHPC development during malaria infection is dependent on Myd88 and endosomal TLRs.

(A-E) B6 mice were injected with 1×10⁶ P. yoelii 17XNL-infected RBCs (closed squares) or PBS (open circles) and analyzed at indicated days (A, B, D) or day 12 of infection (C). (A) Parasitemia as measured by flow cytometry of RFP-expressing P. yoelii 17XNL, and RBC and platelet count during the course of infection. (B) Percent (left) and number (right) of intracellular Ter-119⁺ cells of total CD45⁺ splenocytes. (C) Gated CD31⁺CD45.2⁺Ly6G⁻Siglec-F⁻Ter-119⁺ iHPCs (blue gate) and RPMs (red gate) on day 12 of infection. (D) iHPC frequency (left) and number (right) during the course of infection. (E) Correlation of RBC count and iHPC number on all days. Data are representative of two experiments, n=3 (PBS), 5–6 (P. yoelii 17XNL) mice per group. (F and G) WT B6 and Myd88^−/− mice were infected with 1×10⁶ P. yoelii 17XNL-infected RBCs and analyzed at day 12 of infection (F) or the indicated days (G). (F) Gated CD11b⁺CD31⁺ iHPCs (Gated on live singlets, CD45.2⁺ F4/80⁻Ly6G⁻ Siglec-F⁻) on day 12 of infection. (G) iHPC frequency and number per spleen at day 12 of infection (left); RBC count and parasitemia at the indicated days (right). Data are representative of two experiments, n= 5 (WT), and n= 4–5 (Myd88^−/−) mice per group. (H, I) The ratio of WT to Unc93b1^−/− (H) or WT to Myd88^−/−Trif^−/− (I) BM-derived cells in mixed bone marrow chimeras of indicated populations before and on day 8 after infection with 1×10⁶ P. yoelii 17XNL-infected RBCs. Ly6C^hi monocytes pre-infection are from blood (open circles). Ly6C^hi monocytes post-infection (black circles) and iHPCs post-infection (blue circles) are from spleen. Data are representative of two experiments. Mean values±SEM (A, B, D, G, H, and I) are shown. (E, G left, H, and I) Each symbol represents an individual mouse. *p<0.05, ** p<0.01, ***p<0.001, ****p<0.0001, two-tailed, unpaired Student’s t-test (A, B, and G), Linear regression (E), one-way ANOVA with Tukey’s post test (H, I).