Microbiol Immunol. 2016 Sep;60(9):617-25. doi: 10.1111/1348-0421.12405.

Immunosuppression, peripheral inflammation and invasive infection from endogenous gut microbiota activate retinal microglia in mouse models.

Maneu V1, Noailles A2, Gómez-Vicente1, Carpena N3, Cuenca N2, Gil ML3, Gozalbo D3.

1 Department of Optics, Pharmacology and Anatomy, University of Alicante, Pabellón 13, Carretera San Vicente del Raspeig s/n, 03690-San Vicente del Raspeig, Alicante, Spain.
2 Department of Physiology, Genetics and Microbiology, University of Alicante, Pabellón 13, Carretera San Vicente del Raspeig s/n, 03690-San Vicente del Raspeig, Alicante, Spain.
3 Department of Microbiology and Ecology, University of Valencia, Facultad de Farmacia, Avda Vicent Andrés Estellés s/n, 46100-Burjassot, Spain.



Although its actual role in the progression of degenerative processes is not fully known, the persistent activated state of retinal microglia and the concurrent secretion of inflammatory mediators may contribute to neuronal death and permanent vision loss. Our objective was to determine whether non-ocular conditions (immunosuppression and peripheral inflammation) could lead to activation of retinal microglia. Mouse models of immunosuppression induced by cyclophosphamide and/or peripheral inflammation by chemically induced sublethal colitis in C57BL/6J mice were used. Retinal microglia morphology, spatial distribution and complexity, as well as MHCII and CD11b expression levels were determined by flow cytometry and confocal immunofluorescence analysis with anti-CD11b, anti-IBA1 and anti-MHCIIRT1B antibodies. Retinas of mice with double treatment showed changes in microglial morphology, spatial distribution and expression levels of CD11b and MHCII. These effects were higher than those observed with any treatment separately. In addition, we also observed in these mice: (i) translocation of endogenous bacteria from gut to liver, and (ii) upregulation of TLR2 expression in retinal microglia. Using a mouse model of immunosuppression and gut colonization by Candida albicans, translocation of fungal cells was confirmed to occur in wild type and, to a higher extent, in TLR2 KO mice, which are more susceptible to fungal invasion; interestingly microglial changes were also higher in TLR2 KO mice. Hence, non-ocular injuries (immunosuppression, peripheral inflammation and invasive infection from endogenous gut microbiota) can activate retinal microglia and therefore could affect the progression of neurodegenerative disorders and should be taken into account to improve therapeutic options. © 2016 The Societies and John Wiley & Sons Australia, Ltd.

KEYWORDS: colitis; fungal colonization; immunosuppression; retinal microglia

PMID: 27466067



In ocular neurodegenerative disorders, such as diabetic retinopathy or glaucoma, a highly intricate situation takes place involving a plethora of cytokines and other factors, some of them harmful and other protective, ending in a biased effect towards neuronal death and permanent vision loss. In order to improve therapeutic approaches we need to better understand all the factors involved in the progression of the disease (1). It is now accepted that an excessive or prolonged activation of microglial cells, both in the brain and retina, can be one of the primary factors that may lead to chronic inflammation and irreversible neuronal death in degenerative disorders (2, 3). Microglial cells act as a phagocytic cell population, with a relevant role in both physiological and pathological conditions. When an infection or another harmful stimulus that challenges the homeostatic state occurs, microglial cells turn into an activated state, displaying a variety of distinct functional phenotypes, and showing an increase in the expression of several surface markers, such as major histocompatibility complex class II molecules, which are expressed only in activated microglial cells (Figure 1). In this activated state microglial cells can proliferate, migrate to the site of the stimulus, show greater phagocytic capacity, and secrete inflammatory mediators, although their protective or harmful role still remains controversial (2).



Figure 1. Confocal immunofluorescence image of a transversal retinal section showing the distribution and morphology of microglial cells in the retina from a rat model of retinitis pigmentosa (P23H), a retinal neurodegenerative disease associated with chronic microglia activation. Microglial cells (which are spread through all the retinal layers) were stained with anti-Iba1 antibody (green), and the activation marker anti-MHC class-II RT1B antibody (red); activated microglia correspond to Iba1 and MHCII double positive cells (yellow). Nuclei were counterstained with TO-PRO-3 iodide (blue). GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bar 40 μm.

Systemic infections, immunosuppression, and peripheral inflammation, can increase microglia activation in the brain, and there is recent evidence suggesting a relationship between persistent immune activation and neurodegenerative disorders. Previous reports have demonstrated that systemic infection can also activate the retinal microglia, which can recognize microbial ligands through pattern-recognition receptors (PRRs) (4,5). Therefore, immunosuppression and peripheral inflammation might also be considered as risk factors for patients with ocular neurodegenerative disorders. Besides, most disseminated infections come from an endogenous origin, involving the translocation of the pathogen across the gut mucosa to the bloodstream, a process that is favoured by immunosuppression, gut colonization and inflammatory processes affecting gut mucosa (6). Therefore, we have determined whether immunosuppression and gut inflammation, which are often associated to a systemic infection from endogenous origin, could induce retinal microglia activation and consequently should be taken into account as risk factors in the progression of degenerative diseases.

Our results suggest that immunosuppression, either by immunosuppressive diseases or medical treatments, as well as peripheral inflammation, may contribute to retinal microglia activation. In the case of immunosuppression and peripheral inflammation, systemic infection could provide a good explanation for microglial changes in the retina, since microbial translocation from the gut microbiota was detected. Microbial ligands also could induce retinal microglia changes in immunosuppressed mice, as gut microbiota-mediated signalling promotes the hematopoietic differentiation of myeloid cells in the bone marrow of healthy individuals and microbiota-driven myelopoiesis requires recognition of microbiota-derived products by Toll-like receptors (7). Recent results obtained in our laboratory indicate that systemic treatment with PRR-ligands is able to alter mouse retinal cell populations (unpublished). Hence, non-ocular injuries, as a general immunosuppressive state, a peripheral inflammation, and/or infectious processes, might be underestimated risk factors that could affect the evolution of neurodegenerative disorders. This could be most relevant in elderly patients with a high prevalence of chronic side pathologies. The knowledge of the factors involved in the highly intricate situation that exists in a degenerative pathology should improve the therapeutic options for the affected patients.


Figure 2. The authors, from the University of Valencia (Figure 2A, upper) and from the University of Alicante (Figure 2B), with other lab members.  Figure 1 A, from left: J. Megías, V. Maneu, M.L. Gil, D. Gozalbo, C. Bono and A. Martínez. Figure 1B, from left (standing): V. Maneu, G. Esquiva, N. Cuenca, P. Lax, L. Campello, E. de Juan,  and  A. Noailles; from left (seated): V. Gómez, A. Angulo, L. and L. Fernández.



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