Imbalanced M1 and M2 Macrophage Polarization in Bone Marrow Provokes Impairment of the Hematopoietic Microenvironment in a Mouse Model of Hemophagocytic Lymphohistiocytosis

Abstract

Lipopolysaccharide (LPS) treatment induced hemophagocytic lymphohistiocytosis in senescence-accelerated mice (SAMP1/TA-1), but not in senescence-resistant control mice (SAMR1). SAMP1/TA-1 treated with LPS exhibited functional impairment of the hematopoietic microenvironment, which disrupted the dynamics of hematopoiesis. Macrophages are a major component of the bone marrow (BM) hematopoietic microenvironment, which regulates hematopoiesis. Qualitative and quantitative changes in activated macrophages in LPS-treated SAMP1/TA-1 are thought to contribute to the functional deterioration of the hematopoietic microenvironment. Thus, we examined the polarization of pro-inflammatory (M1) and anti-inflammatory (M2) macrophages, and the dynamics of macrophage production in the BM of SAMP1/TA-1 and SAMR1 after LPS treatment. After LPS treatment, the proportions of M1 and M2 macrophages and the numbers of macrophage progenitor (CFU-M) cells increased in both SAMP1/TA-1 and SAMR1. However, compared to the SAMR1, the increase in the M1 macrophage proportion was prolonged, and the increase in the M2 macrophage proportion was delayed. The increase in the number of CFU-M cells was prolonged in SAMP1/TA-1 after LPS treatment. In addition, the levels of transcripts encoding an M1 macrophage-inducing cytokine (interferon-γ) and macrophage colony-stimulating factor were markedly increased, and the increases in the levels of transcripts encoding M2 macrophage-inducing cytokines (interleukin (IL)-4, IL-10, and IL-13) were delayed in SAMP1/TA-1 when compared to SAMR1. Our results suggest that LPS treatment led to the severely imbalanced polarization of activated M1/M2 macrophages accompanied by a prolonged increase in macrophage production in the BM of SAMP1/TA-1, which led to the impairment of the hematopoietic microenvironment, and disrupted the dynamics of hematopoiesis.

INTRODUCTION

Hemophagocytic lymphohistiocytosis (HLH) is a hyper-inflammatory syndrome caused by incessant activation of lymphocytes and macrophages that results in organ damage, including that to the hematopoietic organs.^1,2) HLH is classified largely into primary HLH caused by inherited defects in the functions of natural killer cells and cytotoxic T cells, and secondary HLH caused by severe infection, inflammation, and malignancies.^1,2) We recently demonstrated that repeated lipopolysaccharide (LPS) treatment induces HLH-like features in senescence-accelerated mice (SAMP1/TA-1), but not in senescence-resistant control mice (SAMR1).³⁾

Hematopoiesis in the bone marrow (BM) is strictly regulated by the hematopoietic microenvironment, which includes stromal cells, via various diffusible factors and direct cellular interactions with adhesion molecules. Stromal cells are a heterogeneous population of cells comprising fibroblasts, macrophages, fat cells, osteoblasts, and endothelial cells.^4–6) We previously reported that SAMP1/TA-1 mice exhibited accelerated senescence-related stromal cell impairments that contribute to hematopoietic stem cell aging.^7–11) Furthermore, we found that inflammation induced by LPS exacerbated the senescence-related stromal cell impairments in the SAMP1/TA-1 mice, resulting in disruption of the dynamics of hematopoiesis, such as the suppression of B-lymphopoiesis, erythropoiesis, and thrombopoiesis, which leads to conditions such as severe and refractory pancytopenia.^3,12) Thus, SAMP1/TA-1 mice are useful for investigating the mechanism of the unexpected inflammation-induced “impairment” of stromal cell functions in HLH. In fact, line-selective macrophage activation with anti-CD40 antibody induced HLH in mice, and macrophage depletion with clodronate liposomes completely prevented the anti-CD40-induced HLH, suggesting that macrophages play a key role in the pathophysiology of HLH.¹³⁾

Monocyte-derived macrophages and resident macrophages are well known to be major components of stromal cells.^14,15) Activated functional macrophages commonly exist in two distinct subsets, M1 and M2 macrophages, which have opposing functions: M1 macrophages are pro-inflammatory, while M2 macrophages are anti-inflammatory. In addition, the balance of M1 and M2 macrophages governs the inflammatory process.^16,17) M2 macrophages promote, while M1 macrophages inhibit the self-renewal and expansion of hematopoietic stem cells.¹⁸⁾ It was recently reported that poor graft function in patients after allogenic bone marrow transplantation may be due to imbalanced M1/M2 macrophage polarization, i.e., an increase in M1 macrophages and a decrease in M2 macrophages, and dysfunction of macrophages in the hematopoietic microenvironment.¹⁹⁾ Thus, qualitative and quantitative changes in activated macrophages in the BM of LPS-treated SAMP1/TA-1 mice may contribute to the functional deterioration of stromal cells. In this study, to determine whether the impairment of stromal cell function may be due to imbalanced polarization of M1 and M2 macrophages, we compared the changes in the polarization of the M1 and M2 macrophages and the dynamics of macrophage production in BM between SAMR1 and SAMP1/TA-1 mice after LPS treatment. In addition, the levels of various cytokines that induce M1 or M2 macrophage activation and monocyte-macrophage production in BM were examined.

MATERIALS AND METHODS

Mice

SAMP1/TA-1 mice were bred and maintained in an experimental facility at Nihon University School of Medicine.^7,8) Senescence-resistant control mice (SAMR1/TaSlc) were purchased from Japan SLC, Inc. (Hamamatsu, Japan). Eight- to 12-week-old SAMR1 and SAMP1/TA-1 male mice were used in the experiments. All protocols involving laboratory mice were reviewed and approved by the Nihon University Animal Care and Use Committee (Experimental Code: AP19MED019-2). The approved experimental protocols were performed humanely in strict accordance with the Nihon University Rules Concerning Animal Care and Use.

LPS Treatment

Mice were injected intravenously with 25-µg doses of LPS (LPS055:B5, Sigma Chemical Co., St. Louis, MO, U.S.A.).^3,12) Control groups of SAMR1 or SAMP1/TA-1 mice were treated with the same volume of pyrogen-free saline.

Preparation of Femoral BM Cells

BM cells were obtained from the femora of three mice per experimental group at each time point. BM cell suspensions were prepared by repeatedly flushing cells from the femora using Iscove’s Modified Dulbecco’s Medium (Invitrogen Corp., Carlsbad, CA, U.S.A.), and the cells were dispersed by repeated passaging through a 23-gauge hypodermic needle. The cells in the resulting individual cell suspensions then were counted using a Sysmex PocH-100 iV Diff hematology analyzer (Sysmex Co., Kobe, Japan).

Flow Cytometry Analysis for M1 and M2 Macrophage Polarization in the BM of SAMR1 and SAMP1/TA-1 Mice

Harvested BM cells were washed with phosphate-buffered saline (PBS), and passed through a 35-µm filter (Cell Strainer, Falcon 352235; Becton Dickinson Labware, Franklin Lake, NJ, U.S.A.) to remove the bone debris and aggregated cells. Cells (2 × 10⁶) were suspended in 0.5 mL of PBS, and incubated with fluorescein isothiocyanate (FITC)-anti-mouse F4/80 monoclonal antibody (Clone EPR20548; Abcom, Cambridge, U.K.) or PE-anti-mouse F4/80 monoclonal antibody (Clone EPR20548; Abcom) for 30 min at 4 °C. F4/80 monoclonal antibody was used as a maker of macrophage/monocyte cells.²⁰⁾ In some experiments, PE-anti-mouse CD11b monoclonal antibody (Clone M1/70; BD Pharmingen™, San Diego, CA, U.S.A.) was used to confirm the macrophage population among the BM cells by a double-staining method with FITC-anti-mouse F4/80 monoclonal antibody (Supplementary Fig. 1). Cells were washed with PBS twice, then resuspended in 100 µL of PBS. Next, to detect inducible nitric oxide synthase (iNOS) and CD206, an Intracellular Fixation and Permeabilization Buffer Set (Catalog number: 88-8824; Thermo Fisher Scientific, Rockford, IL, U.S.A.) was used according to the manufacturer’s instructions.²¹⁾ The washed cells were fixed by adding 100 µL of Fixation Buffer, and incubated for 30 min at room temperature in the dark. Then, 2 mL of Permeabilization Buffer was added, and the cells were centrifuged at 500 × g for 5 min at room temperature. After the cells were resuspended in 100 µL of Permeabilization Buffer, PE-conjugated rat anti-mouse iNOS monoclonal antibody (Clone CXNFT, Catalog No. 25-5920-82; Thermo Fisher Scientific) or FITC-conjugated rat anti-mouse CD206 monoclonal antibody (Clone MR5D3, Catalog number MA5-16870; Thermo Fisher Scientific) was added, and the cells were incubated for 30 min at room temperature in the dark. Subsequently, 2 mL of Permeabilization Buffer was added, and the cells were centrifuged at 500 × g for 5 min at room temperature. The supernatant was then discarded, and the cells were resuspended in 2 mL of Permeabilization Buffer, and centrifuged at 500 × g for 5 min at room temperature. After the cells were resuspended in PBS, they were analyzed by flow cytometry (Cytomics FC500; Beckman Coulter, Brea, CA, U.S.A.) for the direct detection of F4/80 and iNOS double positive M1 macrophages and F4/80 and CD206 double positive M2 macrophages.²¹⁾ The percentage of M1 macrophages was determined as follows (Fig. 1A): the number of F4/80 and iNOS double positive cells/the number of F4/80-positive cells × 100 (%)

Fig. 1. The Proportions of M1 and M2 Macrophages in the BM of SAMR1 and SAMP1/TA-1 Mice after LPS Treatment

By flow cytometry, M1 macrophages were detected as F4/80 and iNOS double-positive cells, and M2 macrophages were detected as CD206 and F4/80 double-positive cells (Supplementary Fig. 1). The percentage of M1 macrophages was determined as follows (A): the number of F4/80 and iNOS double positive cells/the number of F4/80-positive cells ×100 (%). The percentage of M2 macrophages was determined as follows (B): the number of F4/80 and CD206 double positive cells/the number of F4/80 positive cells ×100 (%). The changes in the proportions of M1 macrophages (A) and M2 macrophages (B) were evaluated in the BM of SAMR1 mice (black bars) and SAMP1/TA-1 mice (red bars) at 1, 2, 3, 5, 7, and 14 d after treatment with 25 µg of LPS. The absolute numbers of M1 and M2 cells per femur were calculated, and are shown in Figs. 1C and D. Figure 1E shows the ratio of M2/M1 macrophages after LPS treatment. Samples of macrophages from the femoral BM were obtained from three mice per group for each time point. Each bar represents the mean ± standard deviation. ^§ p < 0.05 vs. control (day 0); * p < 0.05 vs. SAMR1 mice.

The percentage of M2 macrophages was determined as follows (Fig. 1B): the number of F4/80 and CD206 double positive cells/the number of F4/80 positive cells × 100 (%)

The total number of nucleated cells in the femur of each mouse was counted, and the absolute numbers of M1 and M2 cells were calculated (number of M1 or M2 cells per femur). The changes in the absolute numbers of M1 and M2 cells in BM after LPS treatment are shown in Figs. 1C and D. The ratios of M2/M1 macrophages (percentage of M2 macrophages/percentage of M1 macrophages) after LPS treatment were calculated, and are shown in Fig. 1E.

Gene Expression Assay

The levels of transcripts encoding cytokines were determined by real-time PCR using the Applied Biosystems 7500 Fast Sequence Detection System (Applied Biosystems, Foster City, CA, U.S.A.). Briefly, total RNA from BM cells was isolated using ISOGEN reagent (Nippon Gene Co., Ltd., Toyama, Japan). The mRNA was reverse-transcribed using Superscript III (Life Technologies, Carlsbad, CA, U.S.A.) and oligo-dT (Promega Corp., Madison, WI, U.S.A.). The transcript levels were determined by real-time PCR using the TaqMan™ Universal Fast PCR master mix (Applied Biosystems) and gene-specific primers. Specific primers and probes for murine genes encoding interferon (IFN)-γ, interleukin (IL)-4, IL-10, IL-12a, IL-13, IL-18, macrophage colony-stimulating factor (M-CSF), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Applied Biosystems, and used as previously described.^3,12) These factors were selected, because IFN-γ, IL-4, IL-10, and IL-13 are specific regulatory factors that induce M1 or M2 macrophage activation,¹⁵⁾ IL-12 and IL-18 are regulatory factors that induce IFN-γ production,²²⁾ and M-CSF is a specific regulatory factor that mediates the proliferation and differentiation of macrophage progenitor (CFU-M) cells.^23,24)

Progenitor Cell Colony Assay

CFU-M cells were assayed using MethoCult M3231 (StemCell Technologies, Inc., Vancouver, Canada) supplemented with 20 ng/mL recombinant mouse M-CSF (ProSpec-Tany TechnoGene Ltd., Ness Ziona, Israel). Cells were cultured in a humidified incubator at 37 °C with 5% CO₂. Aggregates of 50 or more cells were counted as colonies using phase contrast microscopy (Olympus CKX 41, Olympus Corp., Tokyo, Japan) on day 7 for the CFU-M cells.

Statistical Analysis

Data are expressed as the mean ± standard deviation. Analyses between the non-treated group and LPS-treated group of SAMR1 and SAMP1/TA-1 mice were performed using two-tailed unpaired Student’s t tests. Data analyses between SAMR1 and SAMP1/TA-1 mice were performed using ANOVA with the Bonferroni test. Differences were considered statistically significant at p < 0.05.

RESULTS

Changes in the Polarization of M1 and M2 Macrophages in the BM of SAMR1 and SAMP1/TA-1 Mice after LPS Treatment

Figure 1 shows the changes in the proportions of M1 and M2 macrophages in SAMR1 and SAMP1/TA-1 mice after LPS treatment. With flow cytometry, M1 macrophages were detected as F4/80 and iNOS double-positive cells, and M2 macrophages were detected as CD206 and F4/80 double-positive cells (Supplementary Fig. 1). Figures 1A and B show the changes in the proportions of M1 and M2 macrophages in SAMR1 and SAMP1/TA-1 mice after LPS treatment. We calculated the absolute numbers of M1 cells and M2 cells in BM (Figs. 1C, D). The patterns of fluctuations in the absolute numbers of M1 and M2 monocytes/macrophages after LPS treatment were similar to the patterns of fluctuations in the percentages of M1 and M2 monocytes/macrophages (Figs. 1A, B). The proportion of M2 macrophages was higher than that of M1 macrophages in both non-treated SAMP1/TA-1 and SAMR1 mice (Figs. 1A–E). In addition, the proportion of M2 macrophages was higher in non-treated SAMP1/TA-1 mice than in non-treated SAMR1 mice (Figs. 1B, D, E). When treated with LPS, the proportion of M1 macrophages in SAMR1 mice rapidly increased from day 0 to day 1, and returned to the pretreatment level by day 2 (Figs. 1A, C). In contrast, the proportion of M1 macrophages in SAMP1/TA-1 mice increased continuously from day 0 to day 3, followed by a gradual decrease, but it continued to remain higher than the pretreatment level by day 14 (Figs. 1A, C). After treatment with LPS, the proportion of M2 macrophages in SAMR1 mice increased continuously from day 0 to day 3, then decreased, returning to the pretreatment level by day 14 (Figs. 1B, D). The proportion of M2 macrophages in SAMP1/TA-1 mice also began to increase continuously from day 1 until day 14 after LPS treatment (Figs. 1B, D). Changes in the ratio of M2/M1 macrophages was represented in Fig. 1E. The ratio of M2/M1 macrophages in SAMR1 mice showed that M2 macrophages were notably dominant during the 14 d after LPS treatment (Fig. 1E). In contrast, the dominance of M2 macrophages was attenuated in SAMP1/TA-1 mice after LPS treatment (Fig. 1E).

Changes in the Levels of Transcripts Encoding Regulatory Cytokines in the BM of SAMR1 and SAMP1/TA-1 Mice after LPS Treatment

LPS and IFN-γ induce M1 macrophages, while IL-4, IL-10, and IL-13 induce M2 macrophages.¹⁵⁾ IL-12 and IL-18 induce IFN-γ production.²²⁾ We evaluated the levels of transcripts encoding these cytokines, as mentioned above. Figure 2 shows the values of non-treated SAMR1 mice, which were arbitrarily set to a value of 1 (100%). In the non-treated SAMP1/TA-1 mice, the levels of transcripts encoding IFN-γ, IL-4, IL-10, IL-12a, IL-13, and IL-18 were 463, 406, 283, 66, 26, and 72%, respectively, of the levels of the non-treated SAMR1 mice (Fig. 2). In SAMP1/TA-1 mice, the levels of transcripts encoding IFN-γ dramatically increased starting from 3 to 6 h after LPS treatment, then they returned to the pretreatment levels by day 3. In SAMR1 mice, a slight, but significant, increase in the levels of transcripts encoding IFN-γ was observed 6 h after LPS treatment. The increase in the levels of transcripts encoding IFN-γ was limited in SAMR1 mice when compared to SAMP1/TA-1 mice. In both SAMR1 and SAMP1/TA-1 mice, a slight, but significant, increase in the levels of transcripts encoding IL-12a was observed during the first 3 h after LPS treatment. The increase in the levels of transcripts encoding IL-12a was limited in SAMP1/TA-1 mice when compared to SAMR1 mice. In contrast, no significant increase in the levels of transcripts encoding IL-18 was observed in both SAMR1 and SAMP1/TA-1 mice during the first 3 h after LPS treatment.

Fig. 2. Changes in the Relative Levels of Transcripts Encoding IFN-γ, IL-4, IL-10, IL-12a, IL-13, and IL-18 in the BM of SAMR1 and SAMP1/TA-1 Mice after LPS Treatment

The levels of transcripts encoding an M1 macrophage-inducing cytokine (IFN-γ) or M2 macrophage-inducing cytokines (IL-4, IL-10, and IL-13) were evaluated in SAMR1 mice (black bars) and SAMP1/TA-1 mice (red bars) at 1, 3 and 6 h, and 1, 3 and 7 d after treatment with 25 µg of LPS. In addition, the levels of transcripts encoding IFN-γ-inducing cytokines (IL-12a and IL-18) were evaluated in SAMR1 mice (black bars) and SAMP1/TA-1 mice (red bars) at 1 and 3 h after treatment with 25 µg of LPS. Samples of femoral BM were obtained from three mice per group for each time point. Each bar represents the mean ± standard deviation. The values of non-treated SAMR1 mice were arbitrarily set to a value of 1. ^§ p < 0.05 vs. control (day 0); * p < 0.05 vs. SAMR1 mice.

In SAMR1 mice, the levels of transcripts encoding IL-4, IL-10, and IL-13 rapidly increased during the first hour after LPS treatment, and they subsequently decreased; this increase in the transcript levels after LPS treatment was delayed in SAMP1/TA-1 mice when compared to SAMR1 mice.

Changes in the Numbers of Femoral CFU-M Cells and the Gene Expression Levels of M-CSF in the BM of SAMR1 and SAMP1/TA-1 Mice after LPS Treatment

The numbers of CFU-M cells in the BM were evaluated in SAMR1 and SAMP1/TA-1 mice after LPS treatment (Fig. 3A). The numbers of CFU-M cells in the femoral BM of non-treated SAMR1 and SAMP1/TA-1 mice were 49497 ± 3250 and 33108 ± 3847, respectively. The number of femoral CFU-M cells in SAMR1 mice after LPS treatment increased to 167% of that of the non-treated control mice by day 3 (p < 0.05), followed by a decrease to 89% of that of the non-treated control mice by day 7. The number of femoral CFU-M cells in SAMP1/TA-1 mice after LPS treatment increased to 141% of that of the non-treated control mice by day 3 (p < 0.05), and remained unchanged thereafter.

Fig. 3. Changes in the Numbers of CFU-M Cells and in the Relative Levels of Transcripts Encoding M-CSF in the BM of SAMR1 and SAMP1/TA-1 Mice after LPS Treatment

Changes in the numbers of CFU-M cells were determined in SAMR1 mice (black bars) and SAMP1/TA-1 mice (red bars) at 3 and 7 d after treatment with 25 µg of LPS (A). The results are expressed as the percentage of the control. The levels of transcripts encoding M-CSF were evaluated in SAMR1 mice (black bars) and SAMP1/TA-1 mice (red bars) at 1, 3 and 6 h, and 1, 3 and 7 d after treatment with 25 µg of LPS (B). The values of non-treated SAMR1 mice were arbitrarily set to a value of 1. Samples of femoral BM were obtained from three mice per group for each time point. Each bar represents the mean ± standard deviation. ^§ p < 0.05 vs. control (day 0); * p < 0.05 vs. SAMR1 mice.

Figure 3B shows the levels of transcripts encoding M-CSF in the BM of SAMR1 and SAMP1/TA-1 mice after LPS treatment, and the values of non-treated SAMR1 mice were arbitrarily set to a value of 1 (100%). The levels of transcripts encoding M-CSF in the non-treated SAMP1/TA-1 mice were 125% of the levels of the non-treated SAMR1 mice. The levels of transcripts encoding M-CSF in SAMP1/TA-1 mice dramatically increased from 1 to 3 h after LPS treatment. However, the increase in the levels of transcripts encoding M-CSF after LPS treatment was significantly smaller in SAMR1 mice than in SAMP1/TA-1 mice.

DISCUSSION

In the mice without LPS treatment, the proportion of M2 macrophages was higher in SAMP1/TA-1 mice than in SAMR1 mice (Fig. 1). The levels of M2 macrophage-inducing cytokines, such as IL-4 and IL-10, were also higher in SAMP1/TA-1 mice than in SAMR1 mice (Fig. 2). It has been reported that SAMP1 mice are in a hyperoxidative stress state.²⁵⁾ Thus, low-grade chronic inflammation induced by hyperoxidative stress may contribute to the activation of anti-inflammatory macrophages (M2 macrophages) to suppress the inflammation even in SAMP1/TA-1 mice without LPS treatment. It was reported that in mice with latent chronic inflammation, such as IL-6 transgenic mice and mice primed by a viral Toll-like receptor agonist, LPS treatment induced HLH-like features, as was seen in the SAMP1/TA-1 mice.^26,27) Thus, a chronic inflammatory condition may be an essential pathophysiological factor for inducing HLH-like features with hyper-inflammation in LPS-treated SAMP1/TA-1 mice.

When treated with LPS, the levels of transcripts encoding IFN-γ were markedly increased in SAMP1/TA-1 mice when compared to SAMR1 mice (Fig. 2). IL-12 and IL-18 are cytokines that induce the production of IFN-γ.²²⁾ In SAMP1/TA-1 mice after LPS treatment, the increase in the levels of transcripts encoding IL-12a was limited, and no increase in the levels of transcripts encoding IL-18 was observed. Thus, it is considered that IL-12 and IL-18 are not involved in the production of IFN-γ in SAMP1/TA-1 mice after LPS treatment. It has been shown that M1 macrophages are induced by LPS and/or IFN-γ, and the activated M1 macrophages then secrete more IFN-γ, which further induces even more M1 macrophages.¹⁵⁾ It is possible that this positive feedback may have prolonged the increase in the proportion of M1 macrophages that was seen in SAMP1/TA-1 mice when compared to SAMR1 mice. The increases in the levels of transcripts encoding IL-4, IL-10, and IL-13 in SAMP1/TA-1 mice were delayed when compared to those in SAMR1 mice (Fig. 2). It has been suggested that M2 macrophages are induced by IL-4, IL-10, or IL-13, and the activated M2 macrophages then secrete more IL-10, which further induces even more M2 macrophages.¹⁵⁾ Thus, the delayed LPS-induced production of IL-4, IL-10, and IL-13 in SAMP1/TA-1 mice may have resulted in a delayed and prolonged increase in the proportion of M2 macrophages.

M1 and M2 macrophage polarization is a tightly controlled process that involves a set of signaling pathways, and transcriptional and posttranscriptional regulatory networks.¹⁷⁾ An imbalance in M1 and M2 macrophage polarization is associated with inflammation and various disorders.¹⁷⁾ After LPS treatment, M2 macrophages were notably dominant for 14 d in SAMR1 mice, and the M2 macrophage-dominated polarization was attenuated in SAMP1/TA-1 mice (Figs. 1B, D). These results suggest that the M2 macrophages, which are anti-inflammatory, did not sufficiently reduce the LPS-induced prolongation of inflammation in SAMP1/TA-1 mice.

BM-derived monocyte-macrophages have the capacity to replenish resident macrophages, and maintain homeostasis in various tissues following inflammation.¹⁵⁾ When treated with LPS, the levels of transcripts encoding M-CSF were markedly increased in SAMP1/TA-1 mice as compared to SAMR1 mice (Fig. 3B). Furthermore, the increase in the number of CFU-M cells was concomitantly prolonged in SAMP1/TA-1 mice (Fig. 3A). It is considered that the number of CFU-M cells may reflect the production of monocyte-macrophages. These data suggest that the production of newly generated monocyte-macrophages in BM after LPS treatment is augmented more persistently in SAMP1/TA-1 mice than in SAMR1 mice.

In hematopoietic tissues, monocyte-macrophages are an essential component of the hematopoietic microenvironment for regulating hematopoietic cell proliferation and differentiation via the production of various soluble factors. Luo et al. reported that M2 macrophages promote, while M1 macrophages inhibit, the self-renewal and expansion of hematopoietic stem cells from mouse BM in vitro, and that these opposing effects of M1 and M2 macrophages on mouse BM are attributable to the differential expression of nitric oxide synthase 2 and arginase 1.¹⁸⁾ These data suggest that M1 and M2 macrophages regulate hematopoietic stem cells in a direct manner. Zhao et al. recently reported that patients with poor graft function after bone marrow transplantation showed a significant increase in M1 macrophages, and a marked reduction in M2 macrophages in BM.¹⁹⁾ Taken together, it is possible that severely imbalanced M1 and M2 macrophage polarization in SAMP1/TA-1 mice treated with LPS provokes the functional impairment of the hematopoietic microenvironment.

Clodronate liposomes are useful as a tool to deplete macrophages in in vivo studies.²⁸⁾ Line-selective macrophage activation with anti-CD40 antibody induced HLH in mice, and macrophage depletion with clodronate liposomes completely prevented the anti-CD40-induced HLH, which suggests that macrophages play a key role in the pathophysiology of HLH.¹⁸⁾ Therefore, further studies using clodronate liposomes may enable the elucidation of the role of macrophages in the hematopoietic microenvironment in HLH.

Acknowledgments

This work was supported by JSPS KAKENHI Grant No. JP18K06846.

Author Contributions

M.Y., S.A., I.T., T.H., and H.H. conducted the experiments. M.Y., S.A., I.T., Y.H., T.H., H.H., and S.H. wrote the manuscript. All authors participated in the study design, analysis and interpretation of the data, and review of the manuscript.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

REFERENCES

• 1) Henter JI, Horne A, Aricó M, Egeler RM, Filipovich AH, Imashuku S, Ladisch S, McClain K, Webb D, Winiarski J, Janka G. HLH-2004: diagnostic and therapeutic guideline for hemophagocytic lymphohistiocytosis. Pediatr. Blood Cancer, 48, 124–131 (2007).
• 2) Janka GE. Familial and acquired hemophagocytic lymphohistiocytosis. Annu. Rev. Med., 63, 233–246 (2012).
• 3) Tsuboi I, Harada T, Hirabayashi Y, Aizawa S. Senescence-accelerated mice (SAMP1/TA-1) treated repeatedly with lipopolysaccharide develop a condition that resembles hemophagocytic lymphohistiocytosis. Haematologica, 104, 1995–2005 (2019).
• 4) Dexter TM, Allen TD, Lajtha LG. Condition controlling the proliferation of hematopoietic stem cells in vitro. J. Cell. Physiol., 91, 335–344 (1977).
• 5) Mori KJ, Fujitake H, Okubo H, Dexter TM, Ito Y. Quantitative development of adherent cell colonies in bone marrow cell culture in vitro. Exp. Hematol., 7, 171–176 (1979).
• 6) Aizawa S, Yaguchi M, Nakano M, Toyama K, Inokuchi S, Imai T, Yasuda M, Nabeshima R, Handa H. Hematopoietic supportive function of human bone marrow stromal cell lines established by a recombinant SV40-adenovirus vector. Exp. Hematol., 22, 482–487 (1994).
• 7) Takeda T, Hosokawa M, Takeshita S, Irino M, Higuchi K, Matsushita T, Tomita Y, Yasuhira K, Hamamoto H, Shimizu K, Ishii M, Yamamuro T. A new murine model of accelerated senescence. Mech. Ageing Dev., 17, 183–194 (1981).
• 8) Tsuboi I, Morimoto K, Horie T, Mori KJ. Age-related changes in various hematopoietic progenitor cells in senescence-accelerated (SAM-P) mice. Exp. Hematol., 19, 874–877 (1991).
• 9) Tsuboi I, Morimoto K, Hirabayashi Y, Li G-X, Aizawa S, Mori KJ, Kanno J, Inoue T. Senescent B lymphopoiesis is balanced in suppressive homeostasis: a decrease in interleukin-7 and transforming growth factor-beta levels in stromal cells of senescence-accelerated mice. Exp. Biol. Med. (Maywood), 229, 494–502 (2004).
• 10) Tsuboi I, Hirabayashi Y, Harada T, Koshinaga M, Kawamata T, Kanno J, Inoue T, Aizawa S. Role of hematopoietic microenvironment in prolonged impairment of B cell regeneration in age-related stromal-cell impaired SAMP1 mouse: effect of a single dose of 5-fluorouracil. J. Appl. Toxicol., 28, 797–805 (2008).
• 11) Ho YH, Méndez-Ferrer S. Microenvironmental contributions to hematopoietic stem cell aging. Haematologica, 105, 38–46 (2020).
• 12) Tsuboi I, Harada T, Hirabayashi Y, Aizawa S. Dynamics of hematopoiesis is disrupted by impaired hematopoietic microenvironment in a mouse model of hemophagocytic lymphohistiocytosis. Ann. Hematol., 99, 1515–1523 (2020).
• 13) Ingoglia G, Yalamanoglu A, Pfefferlé M, Dubach IL, Schaer CA, Valkova K, Hansen K, Schulthess N, Humar R, Schaer DJ, Vallelian F. Line-selective macrophage activation with an anti-CD40 antibody drives a hemophagocytic syndrome in mice. Blood Adv., 4, 2751–2761 (2020).
• 14) McCabe A, MacNamara KC. Macrophage: Key regulator of steady-state and demand-adapted hematopoiesis. Exp. Hematol., 44, 213–222 (2016).
• 15) Seyfried AN, Maloney JM, MacNamara KC. Macrophage orchestrate hematopoietic programs and regulate HSC function during inflammatory stress. Front. Immunol., 11, 1499 (2020).
• 16) Sica A, Mantovani A. Macrophage plasticity and polarization in vivo veritas. J. Clin. Invest., 122, 787–795 (2012).
• 17) Wang N, Liang H, Zen K. Molecular mechanisms that influence the macrophage M1-M2 polarization balance. Front. Immunol., 5, 614 (2014).
• 18) Luo Y, Shao L, Chang J, Feng W, Liu YL, Cottler-Fox MH, Emanuel PD, Hauer-Jensen M, Bernstein ID, Liu L, Chen X, Zhou J, Murray PJ, Zhou D. M1 and M2macrophages differentially regulate hematopoietic stem cell self-renewal and ex vivo expansion. Blood Adv., 2, 859–870 (2018).
• 19) Zhao HY, Lyu ZS, Duan CW, Song Y, Han TT, Mo XD, Wang Y, Xu LP, Zhang XH, Huang XJ, Kong Y. An unbalanced monocyte macrophage polarization in the bone marrow microenvironment of patients with poor graft function after allogeneic haematopoietic stem cell transplantation. Br. J. Haematol., 182, 679–692 (2018).
• 20) Kaur S, Raggatt LJ, Millard SM, Wu AC, Batoon L, Jacobsen RN, Winkler IG, MacDonald KP, Perkins AC, Hume DA, Levesque JP, Pettit AR. Self-repopulating recipient bone marrow resident macrophages promote long-term hematopoietic stem cell engraftment. Blood, 132, 735–749 (2018).
• 21) Harada T, Tsuboi I, Hino H, Yuda M, Hirabayashi Y, Hirai S, Aizawa S. Age-related exacerbation of hematopoietic organ damage induced by systemic hyper-inflammation in senescence-accelerated mice. Sci. Rep., 11, 23250 (2021).
• 22) Tominaga K, Yoshimoto T, Torigoe K, Kurimoto M, Matsui K, Hada T, Okamura H, Nakanishi K. IL-12 synergizes with IL-18 or IL-1beta for IFN-gamma production from human T cells. Int. Immunol., 12, 151–160 (2000).
• 23) Murray PJ, Allen JE, Biswas SK, et al. Macrophage activation and polarization: nomenclature and experimental guidelines. Immunity, 41, 14–20 (2014).
• 24) Petvises S, Periasamy P, O’Neill HC. MCSF drives regulatory DC development in stromal co-cultures supporting hematopoiesis. BMC Immunol., 19, 21 (2018).
• 25) Chiba Y, Shimada A, Kumagai N, Yoshikawa K, Ishii S, Furukawa A, Takei S, Sakura M, Kawamura N, Hosokawa M. The senescence-accelerated mouse (SAM): A higher oxidative stress and age-dependent degenerative diseases model. Neurochem. Res., 34, 679–687 (2009).
• 26) Strippoli R, Carvello F, Scianaro R, De Pasquale L, Vivarelli M, Petrini S, Bracci-Laudiero L, De Benedetti F. Amplification of response to toll-like receptor ligands by prolonged exposure to interleukin-6 in mice: implication for the pathogenesis of macrophage activation syndrome. Arthritis Rheum., 64, 1680–1688 (2012).
• 27) Wang A, Pope SD, Weinstein JS, Yu S, Zhang C, Booth CJ, Medzhitov R. Specific sequences of infectious challenge lead to secondary hemophagocytic lymphohistiocytosis-like disease in mice. Proc. Natl. Acad. Sci. U.S.A., 116, 2200–2209 (2019).
• 28) Kozicky LK, Sly LM. Depletion and reconstitution of macrophages in mice. Methods Mol. Biol., 1960, 101–112 (2019).