Alpha-1 Antitrypsin Attenuates M1 Microglia-Mediated Neuroinflammation in Retinal Degeneration

Abstract

Neurodegenerative diseases are a set of disorders characterized by progressive neuronal death and are associated with microglia-mediated neuroinflammation. Recently, neuroinflammation is proposed as a promising therapeutic target for many neurodegenerative diseases. Alpha-1 antitrypsin (AAT) is recognized as a novel immunomodulatory agent in autoimmune diseases and transplantation, however, its impact on neuroinflammation and neurodegeneration remains unknown. This study aims to explore the effects of AAT on microglia-mediated neuroinflammation and retinal degeneration in rd1 mouse model. We found reduced expression of AAT in rd1 retina, and AAT supplement exhibited certain protective effect on retinal degeneration, presenting with increased amount of photoreceptor nuclei, and amplified wave amplitudes in electroretinogram analysis. Of note, AAT shifted microglia phenotype from pro-inflammatory M1 (CD16/CD32⁺, iNOS⁺) to anti-inflammatory M2 (CD206⁺, Arg1⁺) both in vivo and in vitro, underscoring the concept of immunomodulation on microglia polarization by AAT during neurodegeneration. Furthermore, AAT suppressed the activation of STAT1, promoted the expression of IRF4 while inhibited IRF8 expression, indicating the involvement of these signaling pathways in AAT immunomodulation. Collectively, our data provided evidence for a novel protective role of AAT through immunomodulation on microglia polarization. Attenuating neuroinflammation by AAT may be beneficial to retard neurodegeneration in rd1 mice.

Keywords: alpha-1 antitrypsin; immunomodulation; microglia polarization; neuroinflammation; rd1 mice.

Figure 1

Alpha-1 antitrypsin (AAT) expression decreased in rd1 mice. (A,B) Real time PCR (A) and western blot (B) results revealed that the expression of AAT decreased in retina from rd1 mice compared with C57 controls at P14. ***p < 0.001 (two-tailed unpaired t-test). (C). There were some cells co-staining with AAT (red) and IBA1 (green) in C57-retina, whereas lack of AAT was observed in the retina from rd1 mice at P14. (D) Immunofluorescence on retinal section showed that AAT was mainly distributed in the inner nuclear layer in C57 mice, while the rd1 mice lacked AAT expression. (E,F) In retinal whole mounts, expression of AAT was decreased as the disease progressed from P4 to P20 in rd1 mice, whereas C57 mice presented stable expression of AAT during the same time. Of note, AAT was co-labeled mostly with CD68 (E) and CD11b (F), also markers for microglia in the retina, slightly with Brn3a⁺ retinal ganglion cell (F), but not with GFAP⁺ astrocytes/Müller cells (E). Scare bar, 50 µm.

Figure 2

Alpha-1 antitrypsin (AAT) reduced retinal degeneration in rd1 mice. (A) The scanning model used in the study. Twenty-five linear scans within 6 × 3 mm² area adjacent to optic nerve disk were obtained at nasal and temporal retinae. (B) The average retinal thickness was measured in circle area with a radius of 1.5 mm, centering at 3 mm away from optic nerve disk. (C) The retinal structure was well-organized with multiple layers in the C57 mice (n = 6), while the layers of ONL, ELM, and IS/OS junction were poorly visible in the PBS-treated rd1 mice at P16. However, the AAT-treated rd1 displayed a visible low-reflecting ONL layer. Abbreviations: NFL, nerve fiber layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; ELM, external limiting membrane; IS/OS, inner segment/outer segment; RPE, retinal pigment epithelium. The retinal volume (D) and thickness (E) were increased in the AAT-treated rd1 mice compared to PBS-treated ones. n = 9 mice for each group. ***p < 0.001 (two-tailed unpaired t-test).

Figure 3

Alpha-1 antitrypsin (AAT) alleviated the decrement of retinal thickness in rd1 mice. (A) Panoramic view of the central [Location 1], mid-peripheral [Location 2], and peripheral [Location 3] areas in the retinal paraffin sections by H&E staining. The representative images showed denser nuclei in the outer nuclear layer (ONL) and thicker retinal thickness of the rd1 mice treated with AAT compared to those with PBS treatment at P16. Scare bar, 50 µm. (B) The amounts of cellular nuclei in ONL at indicated locations were elevated significantly after AAT treatment in comparison with PBS controls. (C) The total thicknesses of retina were increased in the center, mid-periphery, and periphery areas in AAT-treated rd1 mice, compared with those in PBS-treated controls. Six sections of each retina and three retina of each group were used for analysis. *p < 0.05, **p < 0.01, ***p < 0.001 (two-tailed unpaired t-test).

Figure 4

Alpha-1 antitrypsin (AAT) supplement protected retinal function in rd1 mice. (A,B) Retinal function was measured at P16 by electroretinogram using single-flash recordings at light intensities of 3.0 and 10.0 log cd•s/m², respectively. C57 mice presented with typical a- and b-wave responses (n = 6), while the PBS-treated rd1 mice showed nearly undetectable amplitude of a- or b-waves under a variety of scotopic testing conditions. (C,D) AAT treatment induced mild increase in a-wave but significant elevation of b-wave amplitudes in the rd1 mice. n = 9 mice for each group. *p < 0.05, **p < 0.01, ***p < 0.001 (two-tailed unpaired t-test).

Figure 5

Protective effect of alpha-1 antitrypsin (AAT) on degenerative rods. (A) The representative images of immunostaining on retinal sections revealed that rhodopsin-positive rod photoreceptors were scattered in the PBS-treated rd1 mice, but widely distributed and well-organized in the outer nuclear layer after AAT treatment. The TUNEL⁺ apoptotic cells decreased in AAT supplement group, whereas the amount of IBA1⁺ microglia also decreased comparing with the PBS-treated ones. The location “Center” refers to the panoramic view area 1 in Figure 3A, and “periphery” refers to the area 3. Scare bar, 50 µm. Statistical analysis of the TUNEL⁺ (B) and IBA1⁺ cells (C) revealed that all their amounts significantly decreased either in the center or periphery. Six sections of each retina and three retina of each group were used for analysis. **p < 0.01, ***p < 0.001 (two-tailed unpaired t-test).

Figure 6

Alpha-1 antitrypsin (AAT) suppressed pro-inflammatory M1 microglial polarization during retinal degeneration. (A). In retinal whole mounts, the amount of CD16/32⁺IBA1⁺ M1 microglia in the central, mid-peripheral, and peripheral retina decreased significantly after AAT supplement. Scare bar, 50 µm. Depicted is mean ± SEM of three fields/eyes from six eyes. *p < 0.05 (two-tailed unpaired t-test). (B) In the retinal section, CD16/32⁺IBA1⁺ microglia prominently accumulated in the outer nuclear layer in PBS treatment group whereas the amount of CD16/32⁺IBA1⁺ pro-inflammatory microglia significantly decreased in the AAT-treated group. Scare bar, 20 µm. (C) In the cultured microglia under oxidative stress by hydrogen peroxide stimulation, AAT supplement significantly suppressed the pro-inflammatory M1 phenotype of microglia, presenting with less CD16/32⁺ cells co-stained with IBA1⁺ cells. Scare bar, 20 µm.

Figure 7

Microglia skewed toward anti-inflammatory M2 phenotype in the presence of alpha-1 antitrypsin (AAT) supplement. (A) In the retinal whole mounts, the amount of CD206⁺IBA1⁺ microglia significantly increased in AAT-treated group compared with the PBS-treated mice, particularly in the central and mid-peripheral retina. (B) Arg1⁺ cells, another M2 microglia marker, were absent in the PBS-treated rd1 retina, while after AAT supplement, these M2 microglia appeared and most concentrated in the mid-peripheral areas. Scare bar, 50 µm. Depicted is mean ± SEM of three fields/eyes from six eyes. *p < 0.05, **p < 0.01 (two-tailed unpaired t-test). (C) Immunostaining results showed that iNOS was elevated after hydrogen peroxide stimulation on cultured BV2 microglia, in particular expressed in the cytoplasm. In the AAT-treated microglia, not only the expression of iNOS significantly decreased, but also apparent upregulation of Arg1 expression was observed. Scare bar, 20 µm. (D) Western blot revealed that AAT downregulated the expression of iNOS while upregulated the Arg1 expression, the counteracting factor of iNOS.

Figure 8

Alpha-1 antitrypsin (AAT) modulated IRF4/8 activation and phosphorylation of STAT1 in vivo and in vitro. (A,B) Western blot revealed that in AAT-treated retina of rd1 mice, the IRF8 expression significantly decreased while the expression of IRF4 increased slightly. Moreover, the STAT1 signaling was suppressed after AAT supplement. (C,D) In vitro, hydrogen peroxide stimulation upregulated the expression level of IRF8 and promoted phosphorylation of STAT1 in the primary cultured microglia, while AAT treatment reversed the expression trend with decreased IRF8 and STAT1 and increased IRF4. n = 3. *p < 0.05, **p < 0.01, ***p < 0.001 (two-tailed unpaired t-test).