Dysfunction of CD169+ macrophages and blockage of erythrocyte maturation as a mechanism of anemia in Plasmodium yoelii infection

Abstract

Plasmodium parasites cause malaria with disease outcomes ranging from mild illness to deadly complications such as severe malarial anemia (SMA), pulmonary edema, acute renal failure, and cerebral malaria. In young children, SMA often requires blood transfusion and is a major cause of hospitalization. Malaria parasite infection leads to the destruction of infected and noninfected erythrocytes as well as dyserythropoiesis; however, the mechanism of dyserythropoiesis accompanied by splenomegaly is not completely understood. Using Plasmodium yoelii yoelii 17XNL as a model, we show that both a defect in erythroblastic island (EBI) macrophages in supporting red blood cell (RBC) maturation and the destruction of reticulocytes/RBCs by the parasites contribute to SMA and splenomegaly. After malaria parasite infection, the destruction of both infected and noninfected RBCs stimulates extramedullary erythropoiesis in mice. The continuous decline of RBCs stimulates active erythropoiesis and drives the expansion of EBIs in the spleen, contributing to splenomegaly. Phagocytosis of malaria parasites by macrophages in the bone marrow and spleen may alter their functional properties and abilities to support erythropoiesis, including reduced expression of the adherence molecule CD169 and inability to support erythroblast differentiation, particularly RBC maturation in vitro and in vivo. Therefore, macrophage dysfunction is a key mechanism contributing to SMA. Mitigating and/or alleviating the inhibition of RBC maturation may provide a treatment strategy for SMA.

Keywords: erythropoiesis; malaria; mouse; phagocytosis; reticulocyte.

Fig. 1.

H&E staining of the BM and spleen from noninfected (NI) and P. yoelii yoelii 17XNL–infected mice. The procedures for tissue fixation and processing are as described in the Materials and Methods. Magnification and scale bars are as indicated. (A) BM tissue from an NI mouse. (B–E) BM tissues from 17XNL-infected mice on day 1 (B, D1), day 4 (C, D4), day 10 (D, D10), and day 18 (E, D18) pi, all at 20× magnification. (F) Partial image from E under 100× magnification. Note: increasing amounts of white spaces (sinusoids without mature RBCs) from days 1 to 18 pi. (G) Images of whole spleens from individual NI mice and 17XNL-infected mice on day 18 (D18) pi. (H) Plot of spleen weights in grams from NI and 17XNL-infected mice on day 4 and 18 pi. Mann–Whitney U test (n = 4), **P < 0.01; ***P < 0.001. (I–K) Images of H&E-stained whole spleens from NI (I), 17XNL-infected mice on day 1 pi (J, D1) and day 18 pi (K, D18) under 2× magnification. (L–P) Images of mouse spleens from NI mice (L) and 17XNL-infected mice on day 1 pi (M, D1), day 4 pi (N, D4), day 10 pi (O, D10), and day18 pi (P, D18) under 40× magnification. Black arrows indicate white pulp regions. (Q) Images of H&E-stained spleen from a 17XNL-infected mouse on D18 under 100× magnification. Light blue circles indicate cell clusters suggesting EBIs with none or a few mature erythrocytes. Yellow arrows point to malaria parasite pigments seen as brown color.

Fig. 2.

Flow cytometry analysis of erythroblast subpopulations of noninfected (NI) and P. yoelii yoelii 17XNL-infected mice. (A and B) Counts (A) and frequencies (B) of different developmental erythropoietic cell populations (EryA, EryB, and EryC) from the blood of NI and 17XNL-infected mice on day 1 (D1), day 4 (D4), day 10 (D10), day 18 (D18), and day 22 (D22) pi. (C and D) The same cell counts and frequencies as in A and B but from BM. (E and F) The same cell counts and frequencies as in A and B but from the spleen. Kruskal–Wallis test (n = 3): *P < 0.05; **P < 0.01 (matching the colors of the bars).

Fig. 3.

Dynamics of cytokines and chemokines during 17XNL infection. Plasma levels of cytokines and chemokines from noninfected (NI) and 17XNL-infected mice were measured using a mouse cytokine kit (Invitrogen, Waltham, MA, USA) and Luminex 200 instrument according to the manufacturer’s instructions (Invitrogen). Names of cytokines and chemokines are labeled in each subfigure. Kruskal–Wallis test (n = 5 for 17XNL infected mice and n = 3 for NI mice); *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 4.

Dynamics of erythroblast island (EBI) macrophages from the spleen and BM during 17XNL infection. Cells from the spleen and BM of noninfected (NI) and P. yoelii yoelii 17XNL-infected mice (17XNL) were isolated on days 4, 10, 18, and 40 pi and were stained using antibodies specific for surface proteins for flow cytometry analysis as indicated. (A–E) Percentages of live cells expressing F4/80⁺EPOR⁺CD106⁺CD169⁺ (indicative of EBI macrophages) or individual markers in the BM of NI and 17XNL-infected mice. (F–J) Percentages of live cells expressing F4/80⁺/EPOR⁺CD106⁺CD169⁺ (indicative of EBI macrophages) or individual markers in the spleen of NI and infected mice. Mann–Whitney U test (n = 5): *P < 0.05; **P < 0.01.

Fig. 5.

Erythroblast island (EBI) differentiation in vitro and in vivo supported by macrophages from noninfected mice (NI) and 17XNL-infected mice. (A–H) Macrophages from NI and 17XNL-infected mice day 15 pi were seeded on coverslips in culture plates for 1 to 2 d to allow adherence to the coverslips. MEL cells (1 × 10⁶) were added to the cultures, and images of cell clusters were taken on day 4 and day 6 after the addition of MEL cells. Blue arrows point to nuclei of macrophages, red arrows point to malaria Hz pigments, and black arrows indicate differentiating erythroblasts. (A–D) Images from day 4 post addition of MEL cells; (E–H) images from day 6 post addition of MEL cells. (A, B, E, and F) Cell clusters using splenic cells from NI mice; (C, D, G, and H) cell clusters from 17XNL-infected splenic macrophages. (I and J) Percentages of apoptotic and necroptotic cells, respectively, from the NI and 17XNL-infected spleens (SP) and BM. (K and L) Representative images of EBIs isolated from NI mice. (M and N) Representative images of EBIs from infected mice on day 14 pi. (O) Plots of erythroblast counts per macrophage from NI and 17XNL-infected mice on days 4, 10, 18, and 42 pi. Mann–Whitney U test (n = 5); *P < 0.05; ***P < 0.001; ns, not significant.

Fig. 6.

Macrophage depletion and reconstitution with BM and spleen cells show defective macrophages in erythropoiesis. (A) Diagram of the experimental procedure for macrophage depletion and reconstitution. NI, noninfected; pi, postinfection; Macro reconst, macrophage reconstitution; PYR, pyrimethamine; PHZ, phenylhydrazine. 1, Infect mice (n = 5) with 17XNL parasites and set up NI control (n = 5); 2, Day 16 pi, collect cells from the BM and spleen; 3, One day before cell harvest, treat mice (n = 10) with clodronate to deplete macrophages; 4, Inject macrophages from step 2 into mice with depleted macrophages; 5, Treat mice with PYR in drinking water; 6, Treat mice with PHZ to stimulate erythropoiesis and measure hemoglobin level and blood cells on the day of PHZ treatment and every other day after. (B) PYR in drinking water (7 mg/L) completely killed parasites in the reconstituted mice. (C and D) Cell counts and percentages of macrophages recovered from the spleen and BM after DiR (1,1′-Dioctadecyl-3,3,3′,3′-Tetramethylindotricarbocyanine Iodide) staining in vitro and injection (iv, 1 × 10⁶) into mice. Cells from the spleen and BM were counted using flow cytometry. (E) Hemoglobin levels (grams/deciliter) day 0 to day 12 post-PHZ treatment. Clo_17XNL BM, clodronate treated and reconstituted with BM cells (4 × 10⁷) from 17XNL-infected mice; Clo_NI BM, clodronate treated and reconstituted with BM cells (4 × 10⁷) from NI mice; Lipo control, liposome in PBS (no macrophage depletion); Clo_no rec, clodronate treated without reconstitution. (F) The same treatments as in (E) but reconstituted with splenic cells (Sp). (G and H) the same experiments as in (E) and (F), but blood cells were counted. (I and J) The same treatments as in (E) and (F), but reticulocytes were counted on days 8 and 10 post-PHZ treatment. Mann–Whitney test U (n = 4 to 5); *P < 0.05.

Fig. 7.

Anti-CD169 antibody treatment reduces erythroblast binding to macrophages and erythrocyte maturation. (A and B) CD169 expression on F4/80⁺EPOR⁺ macrophages from the BM and spleen of noninfected (NI) and 17XNL-infected mice, as detected using flow cytometry. (C) The numbers of MEL cells bound to BM macrophages isolated from NI and infected mice. The MEL cells were added to macrophage cultures with or without the treatment of anti-CD169 or control antibodies (anti-fluorescein). Macrophages with three or more MEL cells were counted randomly. (D) The numbers of erythroblasts bound to macrophages isolated from the BM of NI and infected mice. EBIs were isolated from NI and infected mice, treated with EDTA, and cultured in the presence of anti-CD169, control antibodies, or no antibody (None) for 24 h after washing off EDTA. Erythroblasts were counted as in C. (E and F) Anti-CD169 antibody treatment in vivo reduces reticulocyte to erythrocyte maturation in the BM and spleen. Mice were injected with anti-CD169 or control antibodies on days 3 and 5 pi. The percentages of EryA, EryB, and EryC populations were counted and calculated using flow cytometry as described in the Materials and Methods. (G) The numbers of erythroblasts bound to macrophages isolated from the BM of mice treated with anti-CD169 or control antibodies. The assays were done as in D, except for no in vitro antibody treatment. Mann–Whitney test U (n ≥ 5); *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 8.

A proposed mechanism of malarial anemia by inhibition of erythroblast maturation and macrophage dysfunction. (A) In noninfected mice, there is normal erythropoiesis in the BM without splenomegaly. (B) In malaria, parasite-infected mice, phagocytosis of iRBCs and nRBCs triggers immune responses, cytokine production, dysfunction of macrophages, and a blockage of erythroblast maturation in the BM, leading to anemia. (C) The reduction in RBCs induces extramedullary erythropoiesis in the spleen with replication of erythroblasts and expansion of erythroblast islands (EBIs). Similar to BM, phagocytosis of iRBCs triggers cytokine production and modifies macrophage functions, manifested as increased expression of F4/80 and decreased expression of molecules such as CD169, which also impair EBI macrophage’s ability to bind erythroblasts and support erythrocyte maturation (EryB to EryC transition). As a feedback mechanism, the continuous decline in RBCs signals for more erythroblasts and EBI expansion, leading to splenomegaly. Clearance of parasites restores macrophage’s ability to support RBC maturation and resolution of anemia.