polarized macrophages in choroidal neovascularization
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Role of polarized macrophages in the pathogenesis of choroidal neovascularization
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polarized macrophages in choroidal neovascularization
- 1. Polarized macrophages in the pathogenesis of choroidal neovascularization Yasuo Yanagi Associate Professor, Duke NUS medical School Singapore National Eye Centre, Singapore Eye Research Institute
- 2. 2 Choroidal neovascularization and inflammation CNV maturation, fibrosis CNV: Immature vessel + early AMD complement in drusen Chronic subclinical inflammation Inflammation Vessel maturation, inflammation↓ = remission “wound repair response”* *: Grossniklaus HE Am J Ophthalmol 1992 Inflammatory cells, RPE cells Glial cells, endothelial cells Recurrent inflammation RPE/choriod interface prone to inflammation Background Treatment
- 3. 3 Lymphocyte ・ has no effect 1) ・ IL-17,IFN-g-producing Tcells : pro-angiogenic 2) Granulocyte (neutrophil) ・ promotes CNV formation 3) Monocyte/macrophage ・ Pro-angiogenic 4,5) ・ Anti-angiogenic 6,7) 1. Tsutsumi-Miyahara C, et al. Br J Ophthalmol 2004 2. Cruz-Guilloty F, et al. Plos One 2014 3. Zhou J, et al. Mol Vis 2005 4. Sakurai E, et al. Invest Ophthalmol Vis Sci 2003 5. Espinosa-Heidmann DG, et al. Invest Ophthalmol Vis Sci 2003 6. Ambati J, et al. Nat Med 2003 7. Apte RS, et al. PLoS Med 2006 Inflammatory cell types involved in CNV Background
- 4. 4 WBC Lymphocyte Granulocyte Monocyte macrophage T cell B cell CD4+ CD8+ B220+ eoshinophil basophil neutrophil CD11b+Ly6G+ Ly6Chi (classical =M1) Ly6Cint (intermediate) Ly6Clo (alternative =M2) CD11b+F4/80+ Cell surface markers for immune cells (in mice) Background
- 5. 5 C57BL/6 mouse Inflammatory cells in mouse CNV model T cells: CD3+CD4+/CD3+CD8+ B cells: B220+ Granulocytes：CD11b+F4/80-Ly6G+ Macrophages/monocytes： Ly6Chi, Ly6Clo and Ly6Cint/loCD64+ Retina/RPE/choroid Ly6chi = Classically activated mφ Ly6clow = Alternatively activated mφ
- 6. 6 Tan X et al., PLoS ONE 2016 ＊ ＊ 0 0.2 0.4 0.6 0.8 1 1.2 1.4 Control 1 3 7 Proportionsofinflammatorycellsubtypes(%) ＊ Inflammatory cells in mouse CNV model CD4+ T cells B cells CD8+ T cells
- 7. 7 Tan X et al., PLoS ONE 2016 ＊ 0 0.2 0.4 0.6 0.8 1 1.2 1.4 Control 1 3 7 Proportionsofinflammatorycellsubtypes(%) Inflammatory cells in mouse CNV model Granulocytes
- 8. 8 Tan X et al., PLoS ONE 2016 ＊ ＊ 0 0.2 0.4 Control 1 3 7 Proportionsofinflammatorycellsubtypes(%) ＊ ＊ Inflammatory cells in mouse CNV model Ly6c low macrophages Ly6c hi macrophages Ly6c int/low/CD64 macrophages = alternatively activated Mφ = classically activated Mφ
- 9. 9 Laser-induced CNV in Rag2, CD4 and CCR2 KO mice Contribution of macrophages, but not B cells and T cells, is crucial for CNV. 0 20000 40000 60000 control Rag2 0 15000 30000 control CD4KO RAG (recombination-activating gene) 2 KO Control Rag2KO 0 20000 40000 60000 control CCR2KO Control CD4KO CD4 KO CNVarea(μm2)CNVarea(μm2) CCR2KO CNVarea(μm2)
- 10. 10 HE control CD3: T cells CD68: macrophages Courtesy of Dr. Hiroyuki Nakashizuka Immunostaining of CNV samples
- 11. 11 CD163: altenatively activated Mφ Control CD68: classically activated Mφ HE Both M1- and M2- macrophage markers in CNV CNV specimen obtained during surgery
- 12. 12 High expression of VEGF164 and VEGFR1 in Ly6chi macrophages Ly6c CD11b 0 1 2 3 4 5 6 7 8 9 0 5 10 15 20 25 30 35 VEGFR1VEGF-A Laser Induced CNV C57BL/6 mouse Cell sorting and RT-PCR M1 mφM2 Mφ M1 Mφ M2 Mφ M1 mφM2 Mφ CNV Choroid RPE cells M1 macrophage M2 macrophage
- 13. 13 Ly6chi monocyte in Peripheral blood Tan X et al., in preparation Inflammatory monocyte is increased in peripheral blood after CNV induction Laser Induced CNV C57BL/6 mouse CNV induction promotes Ly6chi monocytes release CNV Choroid RPE cells Inflammatory monocyte → M1 macrophage ? M2 macrophage Inflammatory monocyte 0 0.5 1 1.5 2 Control 3 7ProportionofLy6Chimonocytes(%)
- 14. 14 Ocular macrophages Peripheral blood monocytes Decreased mobilization of Classically activated monocytes in CCR2 KO mice Classically activated Classically activated Alternatively activated Alternatively activated
- 15. Splenic denervation inhibits CNV growth 0 5000 10000 15000 20000 25000 30000 control β3 blocker Sympathetic Denervation Ly6chigh inflammatory monocyte 0 20000 40000 60000 80000 100000 120000 140000 160000 Sham treatment Sympathetic Denevevation Sympathetic Devervation ＋adoptive transfer CNVarea(μm2) CNVarea(μm2) b3-adrenoreceptor antagonist treatment * * Inhibition of bone marrow derived HSPCs release Sympathetic nerve b3 adrenoreceptor Inflammatory monocyte Bone marrow and spleen = reservoir for monocytes
- 16. 16 Ocular and peripheral blood monocytes in splenic denervated mice * * Days after laser 0 0.1 0.2 0.3 Control 3 ProportionofLy6Chimacrophages(%) 100% SplenectomyDenerva on Ly5.1 Counts 64.2%70.3% Sham Ocular Ly6chi macrophages Decreased mobilization of inflammatory Ly6chi monocytes
- 17. Conclusion Bone marrow Systemic circulation Inflammatory monocyte CNV Choroid Ly6chi macrophage M2 macrophage b3 adrenoreceptor Mobilization of bone marrow derived monocytes is controlled by factor(s) present in spleen VEGF: angiogenesis Inflammatory cytokine: inflammation SpleenInflammatory monocyte mobilization Sympathetic response
Editor's Notes
- Inflammation has been found to play an important role in the development of choroidal neovascularization (CNV). Firstly, in early age-related macular degeneration (AMD), complement component components can be observed in drusen. and the complement pathways can upregulate the expression of cytokines and their receptors and the recruitment of inflammatory leukocytes, both of which play an important role in the formation of choroidal neovascularization (CNV). Secondly, histological studies of CNV revealed the involvement of macrophages and other immune cells besides RPE, glial cells and endothelial cells. Such inflammation is considered to subside with treatment, but persistent inflammation at the RPE/choroid interface is considered to trigger recurrence.
- However, regarding the roles of each type of immune cells, previous investigations are not consistent. A few studies have also suggested that T cells contribute to CNV formation. There is a report showing that granulocyte may promote CNV formation. Regarding monocyte and macrophages, the results are controversial; a depletion of macrophage was found to correlate with reduced CNV formation. Conversely, there are reports that intraocular injection of macrophages actually reduces CNV size
- The discordant results of the previous results may be due to the difference in the markers used in those studies. In our studies, we first used flow cytometry to evaluate temporal changes in the proportions of lymphocytes, granulocytes, and macrophages in the posterior segment of the eye. This list the markers used in the current study. Importantly, macrophages are known to polarize into specific subtypes and play divergent roles depending on the context. However, we notice that in most of previous studies, the phenotype of the cells were not sufficiently characterized, thus calling them M1 or M2 (or classically and alternatively actoavted) is not precise. We used Ly6G/Ly6C system in combination with other markers to characterize macrophages.
- We induced CNV lesions by laser photocoagulation, and we first investigated changes in the proportions of intraocular inflammatory cells during CNV formation following laser injury by flow cytometry. These are the markers we used for T cells, B cells, Granulocytes and monocytes. By using Ly6C and CD64 for intraocular macrophages, we could identify three relatively clear ocular subpopulations (Ly6Chi, Ly6Clo and Ly6Cint/loCD64+). Basically, Ly6Chi macrophages are considered as classically activated macrophages, and Ly6clow macrophages are alternatively activated macrophages T cells CD3+CD4+/CD3+CD8+ B cells B220+ Granulocytes：CD11b+F4/80-Ly6G+ Macrophages/monocytes：CD11b+Ly6G-F4/80+Ly6Chi/int/lo
- As shown here, the proportion of CD4+ T cells recruited into the posterior segment of the eye relative to the total, increased progressively from day 3 to day 7 after laser injury. In contrast, there were no changes in the proportion of CD8+ T cells after injury.
- The proportions of granulocytes also increased significantly at day 3 after laser injury and dropped thereafter.
- The proportions of two intraocular macrophage subtypes (that is, classically and alternatively activated macrophages) increased significantly at day 3 after laser injury and dropped thereafter. Ly6Cint/loCD64+ subtype showed a higher percentage at day 7 after laser injury.
- Then, using a laser-induced CNV model we examined lesion size in mice lacking inflammatory cells or macrophage-depleted mice. Firstly, to determine the involvement of lymphocytes in CNV, we compared CNV areas in Rag2–/– and CD4–/–mice to their respective WTs. Rag2 deficient mice lack T and B lymphocytes due to an inability to initiate rearrangement of the T cell receptor and Ig loci, CD4-deficient mice lack mature T lymphocyttes. There was no significant difference in lesion size between WT and Rag2–/– mice or CD4–/– mice 7 days after laser injury Next, to examine the effect of monocytes/macrophages, we compared lesion size between CCR2−/− mice and WT mice and found that lesion area was significantly reduced in CCR2−/− mice (62.3% ± 6.1%) compared with WT mice 7 days after laser injury (Fig. 3A and 3C). Moreover, when macrophages were depleted by clodronate liposomes, lesion area was also significantly reduced (by 45.9 % ± 5.8%) compared with controls. Taken together, contribution of macrophages, but not B cells and T cells, is crucial for CNV.
- Furthermore, in surgically excised membranes from AMD patients, we did not detect any T cells, whereas CD68 macrophages were abundant as previously reported.
- As shown here, both classically and alternatively activated macrophages are present in human CNV lesion. However, the role of polarized macrophages has previously not been thoroughly investigated.
- So as a first step to investigate the roles of classically and alternatively activated macrophages, we examined the expression of Vegfa164 and Vegfr1 in intraocular classically activated M1 macrophages and alternatively activated M2 macrophages with or without laser treatment by means of RT-PCR. Vegfa164 expression was barely detected in intraocular M1 or M2 macrophages without laser treatment. In contrast, Vegfa164 and VEGFR1expression levels were upregulated in intraocular M1 cells after laser injury. In contrast, neither Vegfr1 nor VEGFR1 was not detected in intraocular M2 cells with or without laser treatment
- Considering that circulating inflammatory cells migrate into the lesion, we next asked whether there were any changes in the inflammatory cells circulating in peripheral blood. Whereas there was no significant difference in the proportions of alternatively activated macrophages/monocytes in the blood, classically activated macrophages/monocytes circulating in peripheral blood increased until day 7 after laser treatment.
- Lets get the story back to CCR2 knockout mice. To clarify the mechanism of reduced CNV size in CCR2 knockout mice, we assessed the changes in intraocular macrophage and peripheral blood monocyte subtype frequencies after laser injury. As expected, classically activated macrophages both in the eye and peripheral blood were significantly reduced in CCR2-/- mice compared with controls without laser treatment, at 3 days and 7 days after laser injury. Interestingly, there were no differences in intraocular alternatively activated monocytes between two groups with or without laser injury, again supporting the role of classically activated monocytes in the activity of CNV.
- Bone marrow and spleen was demonstrated to function as a reservoir for monocytes. We thought these findings indicate that monocytes/macrophages derived from bone marrow and/or the spleen, may contribute to CNV formation. So using mice treated with β3 receptor antagonist and splenic-denervated mice, we sought to confirm the involvement of bone marrow- and spleen-derived monocytes/macrophages, in exacerbating CNV lesion size. The results demonstrated that lesion size was significantly reduced in both groups of mice (65.3% ± 1.0%, 39.5% ± 5.6%, and 52.6% ± 11.9%, respectively) compared with controls 7 days after laser injury. Finally, to investigate the involvement of Ly6Chi macrophage/monocyte infiltration into the posterior segment of eye in CNV formation, we injected classically activated monocytes harvested from spleen into splenic-denervated mice before laser photocoagulation. The reduction in lesion size in splenic-denervated mice compared with controls was significantly increased in the adoptive transfer model of laser-induced CNV.
- Lastly, we injected Ly6Chi cells harvested from the spleens of Ly5.1 mice into Ly5.2 mice, and compared the proportion of donor-derived intraocular Ly6Chi macrophages among control, splenic-denervated mice and splenectomized mice. Compared with controls, though donor-derived Ly6Chi cells were infiltrated into the eyes of recipient mice, the proportion of intraocular spleen-derived Ly6Chi cells was reduced in splenic-denervated and splenectomized mice after laser injury. However, there was no difference in the proportion of Ly6chi monocytes in the peripheral blood in splenic denervated mice.
- So to conclude my talk, importantly, our results demonstrated that classically activated macrophages/monocytes exacerbates CNV in mice, first. Second, activated monocytes are derived from bone marrow and spleen-derived Ly6Chi cells might play a role in the development of CNV even though they were not directly recruited into the peripheral blood. Lastly, sympathetic activity might contribute to CNV via the recruitment of macrophages to the eye. Although splenectomy is not a realistic treatment, we believe that treatment with systemic b blockers to decrease local inflammation might be feasible.
- This work was conducted by our Ph.D student, Dr. Tan. I would like to express my utmost gratitude to Dr. Tan for her contribution to this work.