Immune modulation by MANF promotes tissue repair and regenerative success in the retina

Abstract

Regenerative therapies are limited by unfavorable environments in aging and diseased tissues. A promising strategy to improve success is to balance inflammatory and anti-inflammatory signals and enhance endogenous tissue repair mechanisms. Here, we identified a conserved immune modulatory mechanism that governs the interaction between damaged retinal cells and immune cells to promote tissue repair. In damaged retina of flies and mice, platelet-derived growth factor (PDGF)-like signaling induced mesencephalic astrocyte-derived neurotrophic factor (MANF) in innate immune cells. MANF promoted alternative activation of innate immune cells, enhanced neuroprotection and tissue repair, and improved the success of photoreceptor replacement therapies. Thus, immune modulation is required during tissue repair and regeneration. This approach may improve the efficacy of stem-cell-based regenerative therapies.

Fig. 1. MANF is a hemocyte-derived damage response factor and promotes retinal repair in Drosophila

A, Experimental design and current model for hemocyte mediated retinal repair in Drosophila. B, Left, representative image of hemocyte smears from 3^rd instar larvae (HmlΔ:;Gal4; UAS::GFP) detecting MANF (red) in Hml>GFP+ cells. GFP, green; DAPI, blue. Scale bar 5μm. Right, western blot analysis of MANF and GFP proteins in cellular and plasma fractions from hemolymph of 3^rd instar larvae (HmlΔ::Gal4; UAS::GFP). C, Relative mRNA levels of MANF detected by RT-qPCR in hemocyte samples collected from 3^rd instar larvae of the designated genotypes and treatments (n≥5 for all conditions). For UV treatments, larvae were exposed to 50mJ of UV at 2^nd instar stage and hemocytes collected 24 hours after. D, Left, relative mRNA levels of MANF detected by RT-qPCR in hemocyte samples collected from 3^rd instar larvae overexpressing Pvf-1 in the retina (n≥5 for all conditions). Right, western blot analysis of MANF (intracellular in hemocytes and secreted into the hemolymph) and Actin (intracellular in hemocytes) proteins in whole hemolymph collected from 3^rd instar larvae overexpressing Pvf-1 in the retina. Bottom graph: average relative levels of MANF in whole hemolymph samples normalized to actin. E–F, Left, representative images of adult eye phenotypes from flies with the designated genotypes, after exposure of the right eye of P24 pupae to 17.5mJ of UV light. Right, average relative size of the UV-treated eye when compared to the untreated eye of the same fly (6<n<17 for each genotype, each dot represents one fly). For all quantifications error bars represent s.e.m. and p-values are from student’s t-test.

Fig. 2. MANF-dependent hemocyte activation is required for neuroprotection in Drosophila

A,C Representative IHC images of hemocyte smears from 3^rd instar larvae of the designated genotypes and treatments, detecting Atilla protein in red. Hml+ cells are identified by GFP expression, green; DAPI, blue. Scale bar 5μm. For UV treatments, larvae were exposed to 50mJ of UV at 2^nd instar stage and hemocytes collected 24 hours later. In (A) All analysis performed after 24h culture in control media (WT, UV 50mJ and UAS:MANF) or media supplemented with hrMANF protein. In (C), hemocytes were assayed directly after collection and were not cultured (images and left graph) or assayed as in (A). Right graphs: percentage of Atilla+ cells in the hemocyte population collected from 3^rd instar larvae of the designated genotypes and treatments is shown (n≥3 for each genotype/treatment). B,D, Relative mRNA levels of Arg detected by RT-qPCR in hemocyte samples collected from 3^rd instar larvae of the designated genotypes (n≥3 for all conditions). E, Representative images of adult eyes from flies with the designated genotypes, after exposure of the right eye of P24 pupae to 17.5mJ of UV light. Right, average relative size of the UV-treated eye when compared to the untreated eye of the same fly (5<n<20 for each genotype, each dot represents one fly). For all quantifications error bars represent s.e.m. and p-values are from student’s t-test. (35) and (36) correspond to two independent dsRNAi expressing lines targeting KdelR transcripts.

Fig. 3. PDGF-A/MANF damage-associated paracrine signaling is conserved in mammals

A, Cellular layers in the mouse eye. Panels B–H are from C57BL/6 mice. B,D, IHC showing expression of PDGF-A, CD11b and MANF after light exposure or in controls. See also Fig. S4A–B. C, Retinal mRNA levels of MANF (RT-qPCR) relative to controls (n=3). E,F, IHC showing expression of CD11b (E and F) and MANF (F), one day after intravitreal injection of mrPDGF-AA or vehicle (PBS). Details in (F) highlight CD11b+ cells detected in the vitreous (right) and choroid blood vessels (left) and MANF co-expression. (E) Average number of CD11b+ cells in the vitreous (mrPDGF-AA, n=5; PBS, n=6; 3 sections per eye for each animal, each dot represents one animal). G, H TUNEL staining, two days after light exposure: G, after intravitreal injection of anti-PDGFRα antibody or vehicle (Goat IgG) or H, in Manf +/− and Manf +/+ littermates. Average number of TUNEL+ nuclei is quantified (G: No light: anti-PDGFRα, n=5; IgG, n=5, n=3; Light exposure: anti-PDGFRα, n=6; IgG, n=5; H: No light: Manf +/+, n=5; Manf +/−, n=3; Light exposure: Manf +/+, n=5; Manf +/−, n=5. 12 sections per eye for each animal, each dot represents one animal). I, Retina of BALB/cJ mice, stained with TUNEL, two days after intravitreal injection of hrMANF or vehicle (PBS) and exposure to 5klux of bright light for 1h. Average number of TUNEL+ nuclei per retinal field is shown (hrMANF, n=8; PBS, n=8; each dot represents one retinal field). J, Retina of P28 Crx^tvrm65 mice, stained with DAPI, fourteen days after intravitreal injection of hfib-MANF or hfib-Cntrl. Red dashed lines indicate the thickness of the ONL after hfib-MANF delivery for comparison. Quantification of photoreceptor preservation as % of nuclei rows in ONL relative to untreated controls (hfib-MANF, n=8; hfib-Cntrl, n=8; 5 sections per eye, untreated controls for relative quantifications, n=4, 5 sections per eye; each dot represents one animal). For all quantifications error bars represent s.e.m. and p-values are from student’s t-test. Scale bars are 20μm.

Fig. 4. MANF-dependent immune modulation mediates retinal neuroprotection

A, IHC showing expression of CD11b, MANF and Ym1 in P28 Crx^tvrm65 mice 14 days after intravitreal injection of hfib-MANF or hfib-Cntrl. Arrowheads indicate co-expression. B, Average number of Ym1+ or Arg1+ cells, per eye cryosection, in P28 or P21 Crx^tvrm65 mice, 14 or 7 days after intravitreal injection of hfib (hfib-MANF, n=6; hfib-Cntrl, n=6; 5 sections per eye; each dot represents one animal) or recombinant protein (hrMANF, n=6; PBS, n=6; each dot represents one section). C, Left, average number of Arg1+ cells per eye cryosection, in BALB/cJ mice, 2 days after intravitreal injection of hrMANF or vehicle (PBS) and light exposure (hrMANF, n=7; PBS, n=7; 3 sections per eye; each dot represents one animal). Right, percentage of CD11b+/MANF+ cells in the retina of BALB/cJ mice after the same treatment (hrMANF, n=13 sections; PBS, n=5 sections, each dot represents one section). See also Fig. S6A. D, Average number of Ym1+ cells, per eye cryosection, in C57BL/6 mice, one day after intravitreal injection of mrPDGF-AA or vehicle (mrPDGF-AA, n=5,; PBS, n=6; 3 sections per eye, each dot represents one animal). See also Fig. S6B. E,F,H Relative mRNA levels (RT-qPCR) in BM-macrophages from wt (E, n=3) or Cx3Cr1-deficient (H, n=3) mice, stimulated with hrMANF or vehicle (PBS) or Raw macrophages transfected with MANF targeting siRNA pool or a non-targeting siRNA pool (F, n=5). See also Fig. S9. G, I, TUNEL staining, 2 days after intravitreal injection of hrMANF or vehicle (PBS) and light exposure of CD11b:DTR (G) or Cx3Cr1^{tg(YFP-CRE-ER)} (Cx3Cr1 −/−) mice (I). Average number of TUNEL+ nuclei is shown (G, no light: PBS, n=3; DT, n=4. light: PBS, n=3; DT, n=4, DT+hrMANF, n=5; 4 sections per eye. I, No light: Cx3Cr1+/−, n=4; Cx3Cr1−/−, n=8. Light: Cx3Cr1+/−, n=6; Cx3Cr1-/: PBS, n=7; hrMANF, n=6; 12 sections per eye, each dot represents one animal). For all quantifications error bars represent s.e.m. and p-values are from student’s t-test. Sale bars are 20μm.

Fig. 5. MANF enhances the efficiency of retinal regenerative therapies

A, Cartoon representing the trans-corneal subretinal injection method. B, IHC showing expression of CD11b, MANF and GFP at an integration site of Nrl-GFP donor photoreceptors one week after transplantation. C, Average number of MANF+CD11b+ cells/field in integration sites vs. sites of no integration (10 fields per condition, all fields contained cells in the subretinal space, each dot represents one field). D, Quantification of integration into wild-type (wt, n=8) or Cx3Cr1−/− (n=6) mice, analyzed by IHC for GFP expression, 7 days after subretinal injection of P7 Nrl-GFP donor photoreceptors (PhR). Each dot represents one animal. E, Quantification of integration in C57BL/6 mice, analyzed by IHC for GFP expression, 7 days after subretinal injection of Nrl-GFP donor photoreceptors (PhR) supplemented with hrMANF protein (n=10, P14; n=7, P21) or vehicle (PBS, n=8, P7 and P14; n=9, P21). Each dot represents one animal. See also Fig. S8A for representative images of P21 transplants. F, Representative images and quantification of integration in wild-type (wt, n=8, same as in Fig. 5D) or Crx^tvrm65 mice, analyzed by IHC for GFP expression, 7 days after subretinal injection of P7 Nrl-GFP donor photoreceptors (PhR) supplemented with hrMANF protein (hrMANF, n=4) or vehicle (PBS, n=4). Each dot represents one animal. G, Examples of ERG-waves obtained in MANF supplemented (blue) and PBS supplemented (black) transplants of P7 Nrl-GFP PhRs in Crx^tvrm65 mice. H, maximal b-wave amplitudes measured 1–4 weeks after sub-retinal injections of P7 Nrl-GFP PhRs supplemented with MANF (n=3–7 at each time point), PBS (n=4–6 at each time point) and of eyes that did not receive a transplant (n=6–10), all in Crx^tvrm65 host. Each dot represents one animal. See also Fig. S8B for b waves after hrMANF or PBS injection without cells and Fig. S8C for b waves of wt eyes. p-values are from a 2-way ANOVA analysis. For all quantifications error bars represent s.e.m. p-values in C–F are from student’s t-test. Scale bars are 20μm.

Fig. 6. Model for the evolutionarily conserved immune modulatory function of MANF and its implication in tissue repair and regeneration

A, In Drosophila (left) or mouse (right) the damaged retina secretes Pvf-1/PDGF-A which acts on innate immune cells – hemocytes in Drosophila or microglia/macrophages in mice. MANF derived from innate immune cells (or other sources) promotes phenotypic changes – atilla and arginase expression in hemocytes or alternative activation of microglia/macrophages – which are part of the mechanism involved in tissue protection. B, MANF supplementation is an enhancer of retinal regenerative therapies by increasing the integration efficiency of exogenously supplied photoreceptors for retinal repair.