MΦ Depletion Research Articles

CD169+ Macrophages Regulate Erythropoiesis Under Homeostasis, Recovery From Erythron Injury and in JAK2V617F-Induced Polycythemia Vera

AbstractAbstract 80The role of macrophages (MΦ) in erythropoiesis was suggested several decades ago with the description of “erythroblastic islands” in the bone marrow (BM) composed of a central MΦ surrounded by developing erythroblasts. This hypothesis was strengthened by in vitro observations using cell culture systems showing that MΦ promote erythroblast proliferation and survival. However, the in vivo role of MΦ in erythropoiesis under homeostasis or disease remains unclear. Central MΦ reportedly express CD169 (or Sialoadhesin), an antigen that specifically marks tissue resident MΦ among mononuclear phagocytes of the bone marrow and spleen. Specific depletion of CD169+ MΦ markedly reduced erythroblasts in the BM (40.4+1.8%) but did not result in overt anemia under homeostasis, likely due to concomitant compensatory splenic erythropoiesis and alterations in RBC clearance. However, MΦ depletion significantly impaired erythroid recovery from PHZ-induced hemolytic anemia (reticulocytes: 8.2-fold lower, p<0.01 and hematocrit: 2-fold lower, p<0.01 on day 6 post-PHZ challenge) and acute blood loss (reticulocytes: 3.2-fold lower, p<0.001 and hematocrit: 1.6-fold lower, p<0.001 on day 4 post-phlebotomy). Furthermore, depletion of CD169+ MΦ in the BM and spleen impaired erythroblast recovery seven days after bone marrow transplantation (BM: 8.2-fold lower, p<0.01 and spleen: 120-fold lower, p<0.05 on day 7 post-BMT) and delayed recovery of reticulocyte numbers (4-fold lower, p<0.001 on day 10 post-BMT) and hematocrit (1.1-fold lower, p<0.05 on day 14 post-BMT). Mechanistically, we observed a rapid drop in reticulocyte hemoglobin content (CHr) in CD169+ MΦ-depleted animals starting four days post-BMT, but iron supplementation was unable to correct the impaired expansion of erythroblasts, suggesting other mechanisms. We determined that VCAM-1 expressed by BM CD169+ MΦ and BMP4 derived from splenic red pulp macrophages were critical for the efficient recovery of the erythron after BMT. Moreover, depletion of host-derived, radioresistant macrophages shortly after transplantation was sufficient to delay erythroblast recovery, implicating a critical role for this population until donor-derived macrophages can repopulate post-BMT. In addition, we characterized a CD169+ VCAM1+ MΦ population in human BM aspirates that represents the first step in clinically targeting the analogous BM resident macrophage population in humans. Since CD169+ MΦ support recovery after erythropoietic injury, we hypothesized that MΦ depletion could potentially normalize the erythron in a JAK2V617F-driven murine model of polycythemia vera (PV). Indeed, we observed that MΦ depletion in PV mice reduced erythroblasts in the BM (1.6-fold lower, p<0.05 after 4 weeks of depletion) and spleen (14-fold lower, p<0.01 after 4 weeks of depletion). This reduction of the expanded PV erythron was associated with an efficient (within 20 days of MΦ depletion) and durable (up to 40 days after last depletion) normalization of the hematocrit. A rapid and durable reduction in CHr was observed after MΦ depletion in PV mice, but systemic iron chelation did not produce the same effect as MΦ depletion, further confirming the contribution of additional mechanisms. MΦ depletion abrogated the induction of BMP4 (3.4-fold lower, p<0.001) and stress erythropoiesis (stress BFU-E: 790-fold reduction, p<0.05) in the spleen. Importantly, MΦ depletion reduced the number of erythropoietin-independent colonies in the spleen of PV mice (endogenous BFU-E: 29-fold lower, p<0.05 and endogenous CFU-E: 1400-fold lower, p<0.05), indicating that erythropoiesis in PV, unexpectedly, remains under the control of MΦ in the BM and splenic microenvironments. Altogether, these studies strongly support the notion that CD169+ MΦ promote erythrocyte development and that modulation of the MΦ compartment represents a novel strategy to treat erythropoietic disorders. Disclosures:No relevant conflicts of interest to declare.

Bone marrow CD169+ macrophages promote the retention of hematopoietic stem and progenitor cells in the mesenchymal stem cell niche

Hematopoietic stem cells (HSCs) reside in specialized bone marrow (BM) niches regulated by the sympathetic nervous system (SNS). Here, we have examined whether mononuclear phagocytes modulate the HSC niche. We defined three populations of BM mononuclear phagocytes that include Gr-1hi monocytes (MOs), Gr-1lo MOs, and macrophages (MΦ) based on differential expression of Gr-1, CD115, F4/80, and CD169. Using MO and MΦ conditional depletion models, we found that reductions in BM mononuclear phagocytes led to reduced BM CXCL12 levels, the selective down-regulation of HSC retention genes in Nestin+ niche cells, and egress of HSCs/progenitors to the bloodstream. Furthermore, specific depletion of CD169+ MΦ, which spares BM MOs, was sufficient to induce HSC/progenitor egress. MΦ depletion also enhanced mobilization induced by a CXCR4 antagonist or granulocyte colony-stimulating factor. These results highlight two antagonistic, tightly balanced pathways that regulate maintenance of HSCs/progenitors in the niche during homeostasis, in which MΦ cross talk with the Nestin+ niche cell promotes retention, and in contrast, SNS signals enhance egress. Thus, strategies that target BM MΦ hold the potential to augment stem cell yields in patients that mobilize HSCs/progenitors poorly.

Role Of Macrophages On The Benefits Of O??G On Susceptibility To Infection Following Exercise Stress

Exhaustive exercise has been associated with increased risk for respiratory infection. Oat β-glucan (OβG), a soluble fiber with mild immunostimulant activity, can offset the decrease in macrophage (MΦ) antiviral resistance and increased risk of infection associated with exercise stress. PURPOSE To determine the role of lung MΦs on the benefits of OβG on susceptibility to infection following exercise stress. METHODS Exercise mice were run to fatigue on a treadmill for 3 consecutive days prior to infection. OβG was fed in the drinking water for 10 consecutive days prior to infection. Mice were administered 100μl of clodronate (Ex-H2O-CL2MDP, Ex-OβG-CL2MDP, Con-H2O-CL2MDP and Con-OβG-CL2MDP) or phosphate buffered saline (Ex-H2OPBS, Ex-OβG-PBS, Con-H2O-PBS and Con-OβG-PBS) encapsulated in liposomes, via an intra-nasal route on the 1st day of exercise and the 4th day following infection. MΦs ingest the liposomes and are killed by the clodronate through apoptosis. This procedure has previously been found to deplete approximately 75% of lung MΦs. Fifteen minutes following exercise or rest on the last day of training, mice were intranasally inoculated with HSV-1 (1.28 × 105 PFUs). They were monitored twice daily for morbidity, mortality and symptom severity for 21 days. RESULTS As we have previously shown, exercise increased morbidity, mortality and symptom severity versus control, and this increase was attenuated in OβG fed animals. MΦ depletion negated the beneficial effects of OβG. CONCLUSION These data suggest that MΦs play an important role in the mechanism whereby OβG exerts its beneficial effects on reducing susceptibility to infection following exhaustive exercise. Funded by the American College of Sports Medicine.

Role Of Macrophages On The Benefits Of OβG On Susceptibility To Infection Following Exercise Stress

Exhaustive exercise has been associated with increased risk for respiratory infection. Oat β-glucan (OβG), a soluble fiber with mild immunostimulant activity, can offset the decrease in macrophage (MΦ) antiviral resistance and increased risk of infection associated with exercise stress. PURPOSE To determine the role of lung MΦs on the benefits of OβG on susceptibility to infection following exercise stress. METHODS Exercise mice were run to fatigue on a treadmill for 3 consecutive days prior to infection. OβG was fed in the drinking water for 10 consecutive days prior to infection. Mice were administered 100μl of clodronate (Ex-H2O-CL2MDP, Ex-OβG-CL2MDP, Con-H2O-CL2MDP and Con-OβG-CL2MDP) or phosphate buffered saline (Ex-H2OPBS, Ex-OβG-PBS, Con-H2O-PBS and Con-OβG-PBS) encapsulated in liposomes, via an intra-nasal route on the 1st day of exercise and the 4th day following infection. MΦs ingest the liposomes and are killed by the clodronate through apoptosis. This procedure has previously been found to deplete approximately 75% of lung MΦs. Fifteen minutes following exercise or rest on the last day of training, mice were intranasally inoculated with HSV-1 (1.28 × 105 PFUs). They were monitored twice daily for morbidity, mortality and symptom severity for 21 days. RESULTS As we have previously shown, exercise increased morbidity, mortality and symptom severity versus control, and this increase was attenuated in OβG fed animals. MΦ depletion negated the beneficial effects of OβG. CONCLUSION These data suggest that MΦs play an important role in the mechanism whereby OβG exerts its beneficial effects on reducing susceptibility to infection following exhaustive exercise. Funded by the American College of Sports Medicine.

Role of Macrophages in Elevated IgA and IL-6 Production by Peyer's Patch Cultures Following Acute Oral Vomitoxin Exposure

Oral vomitoxin (VT) exposure in mice results in elevated cytokine gene expression, increased production of IgA, and IgA nephropathy. To determine the potential role of macrophages (Mφ) in these effects, anex vivomodel was devised whereby Peyer's patch (PP) and spleen cells were prepared from mice 2 h after oral exposure to 0 or 25 mg/kg body wt VT, cultured, and then evaluated for IgA and cytokine IL-6 production. Both PP and, to a lesser extent, spleen cells from treatment mice produced more IgA over a 7-day period than did corresponding control cells when cultured without a costimulus or in the presence of either phorbol myristate acetate plus ionomycin (PMA + ION) or lipopolysaccharide (LPS); IgA elevation was most marked in LPS-treated cultures. The VT effect was completely ablated in PP cultures that were depleted of Mφ but not in Mφ-depleted spleen cultures. VT exposure similarly increased production of IL-6, an important helper factor for IgA secretion, in LPS-stimulated PP and spleen cell cultures. IL-6 production was also ablated by Mφ depletion. A potential costimulatory role for Mφ was further suggested because both IgA and IL-6 production increased when Mφ-depleted PP cells from VT-treated animals were cocultured with peritoneal Mφ from VT-treated animals. Similar effects were observed when an analogousex vivoapproach was used with purified PP B cells and peritoneal Mφ. PP B cells from control animals also secreted elevated levels of IgA when cocultured with splenic CD4+cells from VT-treated animals, thus confirming previous studies showing that T cell help also contributes to increased IgA production. Potential roles for soluble mediators and cell contact in this process were suggested when IgA production was measured in cultures of PP cells separated from VT-treated Mφ by a semipermeable membrane. Taken together, these and previous results suggest that Mφ may play a key mechanistic role in elevated IgA production and IgA nephropathy in VT-exposed mice.