Free
Retina  |   June 2014
Retinal Microglia Are Activated by Systemic Fungal Infection
Author Affiliations & Notes
  • Victoria Maneu
    Departamento de Óptica, Farmacología y Anatomía, Universidad de Alicante, Alicante, Spain
    Instituto Teófilo Hernando de I+D del Medicamento, Universidad Autónoma de Madrid, Madrid, Spain
  • Agustina Noailles
    Departamento de Fisiología, Genética y Microbiología, Universidad de Alicante, Alicante, Spain
  • Javier Megías
    Departamento de Microbiología y Ecología, Universitat de València, Burjassot, Spain
  • Violeta Gómez-Vicente
    Departamento de Fisiología, Genética y Microbiología, Universidad de Alicante, Alicante, Spain
  • Núria Carpena
    Departamento de Microbiología y Ecología, Universitat de València, Burjassot, Spain
  • M. Luisa Gil
    Departamento de Microbiología y Ecología, Universitat de València, Burjassot, Spain
  • Daniel Gozalbo
    Departamento de Microbiología y Ecología, Universitat de València, Burjassot, Spain
  • Nicolás Cuenca
    Departamento de Fisiología, Genética y Microbiología, Universidad de Alicante, Alicante, Spain
    Instituto multidisciplinar para el estudio del medio Ramón Margalef, Universidad de Alicante, Alicante, Spain
  • Correspondence: Victoria Maneu, Departamento de Óptica, Farmacología y Anatomía, Pabellón 13, Universidad de Alicante, Carretera San Vicente del Raspeig s/n, 03690, San Vicente del Raspeig (Alicante); vmaneu@ua.es
  • VM and AN contributed equally to the work presented here and should therefore be regarded as equivalent authors. 
Investigative Ophthalmology & Visual Science June 2014, Vol.55, 3578-3585. doi:10.1167/iovs.14-14051
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Victoria Maneu, Agustina Noailles, Javier Megías, Violeta Gómez-Vicente, Núria Carpena, M. Luisa Gil, Daniel Gozalbo, Nicolás Cuenca; Retinal Microglia Are Activated by Systemic Fungal Infection. Invest. Ophthalmol. Vis. Sci. 2014;55(6):3578-3585. doi: 10.1167/iovs.14-14051.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose.: We determined whether systemic fungal infection could cause activation of retinal microglia and, therefore, could be potentially harmful for patients with retinal degenerative diseases.

Methods.: Activation of retinal microglia was measured in a model of sublethal invasive candidiasis in C57BL/6J mice by confocal immunofluorescence and flow cytometry analysis, using anti-CD11b, anti-Iba1, anti-MHCII, and anti-CD45 antibodies.

Results.: Systemic fungal infection causes activation of retinal microglia, with phenotypic changes in morphology, surface markers expression, and microglial relocation in retinal layers.

Conclusions.: As an excessive or prolonged microglial activation may lead to chronic inflammation with severe pathological side effects, causing or worsening the course of retinal dystrophies, a systemic infection may represent a risk factor to be considered in patients with ocular neurodegenerative diseases, such as diabetic retinopathy, glaucoma, age-related macular degeneration, or retinitis pigmentosa.

Introduction
Microglial cells are part of the mononuclear phagocyte population of the central nervous system (CNS) and the retina, and have a relevant role in both physiologic and pathologic conditions. In a healthy retina, microglia are in a “resting state,” in which, while scanning the environment, they secrete neurotrophic factors that support surrounding neurons' survival. After injurious stimuli, such as ocular infections, trauma, or neurodegeneration processes, microglial cells acquire an activated state, in which they proliferate, migrate to damaged sites, undergo morphologic transformation into amoeboidic phagocytes, and secrete molecules that initiate tissue repair mechanisms. 14 Hence, at first stages of a neurodegenerative process, microglia initiate repair mechanisms and are considered to have a neuroprotective role. On the other hand, excessive or prolonged microglial activation leads to chronic inflammation, due to the continuous secretion of neurotoxic agents that cause severe pathologic side effects, retinal damage, and neuronal apoptosis. 2,3,5  
To date, several CNS injuries, such as infections, neurodegenerative diseases, chemical injury, or aging, have been correlated with microglial activation and high levels of proinflammatory cytokines. 3,4,6,7 In Parkinson's disease, there is an increased number of activated microglia in the brain, accompanied by increased expression of proinflammatory cytokines. 8,9 Also in Alzheimer's disease, activated microglia have been associated with the amyloid plaques in the brain. 10,11 Moreover, microglial activation has been demonstrated in several other neurodegenerative diseases, such as amyotrophic lateral sclerosis or Huntington's disease. 3 Retinal neurodegenerative diseases are associated with chronic microglial activation and neuroinflammation as well, as is the case for retinitis pigmentosa, 12 age-related macular degeneration (AMD), 13,14 or glaucoma. 15,16 In the degenerating retina, endogenous signals activate microglial cells leading to local proliferation, migration, enhanced phagocytosis, and secretion of cytokines, chemokines, and neurotoxins. These immunologic responses and the loss of limiting control mechanisms may contribute significantly to retinal tissue damage and proapoptotic events in retinal degenerations. 1,2,12  
Therefore, it can be assumed that different stimuli that induce prolonged microglial activation can be potentially harmful for neuronal tissue. In this sense, systemic infections can induce microglial activation in the brain and may be considered as a risk factor in neurodegenerative diseases. It has been described that microglia are the most abundant defense population in the brain, showing a 3-fold increase after 7 days of infection in a model of systemic invasive candidiasis. 17 The study of microglial homeostasis in the retina meets some technical limitations, such as the heterogeneous distribution of microglial cells in the retinal tissue and the lack of experimental tools, which prevent an accurate quantification and molecular analysis of this specialized neuronal macrophage population. 1 The aim of this work was to determine whether systemic fungal infection might cause activation of retinal microglia to discern whether it should be considered as a risk factor for patients affected with ocular neurodegenerative diseases. 
Materials and Methods
Mice
Female C57BL/6J mice (8–10 weeks old) purchased from Harlan Ibérica (Barcelona, Spain) were bred and maintained under specific pathogen-free conditions at the University of Valencia animal facilities. The protocol was approved by the Committee on the Ethics of Animal Experiments of the University of Valencia (Permit Number, A1264596506468). All animals were handled in accordance with current regulations for the use of laboratory animals (National Institutes of Health [NIH], ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and European Directive 2010/63/UE) to minimize animal suffering and limit the number used for the experiments. 
Yeast Strain and Infection Model
Viable and inactivated Candida albicans ATCC 26555 yeasts were used. Yeasts were obtained as reported previously. 18,19 Briefly, starved yeast cells were inoculated (200 μg, dry weight, of cells per mL) in a minimal synthetic medium and incubated for 3 hours at 28°C to obtain yeasts. Only yeast cells without germ tubes were observed at 28°C. Viable yeasts were resuspended in PBS at the desired concentration and used in infection experiments (see below). For inactivation, yeast cells were resuspended (20 × 106 cells/mL) in 4% (wt/vol) paraformaldehyde (fixation buffer; eBioscience, San Diego, CA, USA) and incubated for 1 hour at room temperature. After treatment, fungal cells were washed extensively in PBS and brought to the desired cell density in PBS. For infection assays, mice were injected (day 1) with a sublethal dose (6 × 105 viable yeasts in 100 μL of PBS) of C. albicans , by intravenous administration, according to the method described previously by our group. 20,21 Alternatively, mice were injected with three doses of 10 × 106 inactivated yeasts on days 1, 2, and 3. In both cases the retinas were analyzed on day 4. Untreated mice were used as controls. 
To assess C. albicans invasion of retinal tissue in infected animals, retinal cryosections were stained with Calcofluor White M2R 0.1% (Sigma-Aldrich, Munich, Germany) for 15 minutes, coverslipped, and observed under a fluorescence microscope. Alternatively, whole retinas were dissociated, resuspended in 100 μL of PBS, plated in Sabouraud-Dextrose agar, and cultured for 48 hours at 37°C to allow colony formation. 
Immunohistochemistry
For immunohistochemistry assays, enucleated eyes were fixed using 4% paraformaldehyde in 0.1 M phosphate buffer pH 7.4 (PB), for 1 hour at room temperature. To maintain retinal orientation, a suture was placed on the superior pole of each eye before enucleation. Following several washes with PB, the eyes sequentially were cryoprotected in 15% and 20% (wt/vol) sucrose for 1 hour, and 30% sucrose overnight. Next day, the cornea, lens, and vitreous were dissected out, and the eyecups were embedded in Tissue-Tek (Sakura Finetek, Torrance, CA, USA), and frozen in liquid N2. Then, 16-μm vertical retinal cryosections along the nasotemporal axis were mounted on slides (Superfrost Plus; Menzel GmbH & Co. KG, Braunschweig, Germany), washed, and incubated with blocking solution (5% normal donkey serum in PB containing 0.5% Triton X-100) for 1 hour at room temperature. Immunostaining with a cocktail of mouse anti-MHC class-II RT1B monoclonal antibody, 1:50 (clone OX-6; AbD Serotec, Kidlington, United Kingdom), and rabbit anti-Iba1 polyclonal antibody, 1:500 (Wako Chemicals, Neuss, Germany) was performed overnight at room temperature in a wet chamber. After incubation with the primary antibody, retinal sections were washed and incubated in a mix of secondary antibodies: Alexa Fluor 555-conjugated donkey anti-mouse IgG (Molecular Probes, Eugene, OR, USA) and Alexa Fluor 488-conjugated donkey anti-rabbit IgG, both at a 1:100 dilution, for 1 hour. Finally, the sections were washed and mounted with Citifluor (Citifluor Ltd., London, UK) for visualization under a laser scanning confocal microscope (TCS SP2; Leica Microsystems, Wetzlar, Germany). Immunohistochemical controls were performed by omission of either the primary or the secondary antibodies. Only retinal sections that contained the optic nerve were used for cell scoring. 
Flow Cytometry Analysis
First, the eyes were enucleated and the retinas were dissected carefully. Retinas were placed in 1 mL of PBS buffer and were disaggregated by gently pipetting up and down. The cell suspension was filtered through a 30-μm filter from BD Biosciences (San Diego, CA, USA) to avoid cell clumps. Disaggregated cells were labelled with rat FITC- or APC-conjugated anti-CD11b antibody at 1:200 dilution (Clone M1/70; eBioscience). For the detection of microglial activation, disaggregated retinal cells were triple-stained with a cocktail of rat APC-conjugated anti-CD11b antibody, PE-conjugated anti-MHC class II antibody (clone M5/114.15.2; Milteny Biotec, Bergish Gladbach, Germany) and FITC-conjugated anti-mouse CD45 (clone 104.2, Milteny Biotec). Labelled cells from each mouse retina were analyzed separately. Analyses were performed in a FACSCanto cytometer (BD Biosciences) and the data were analyzed with FACSDiva software (BD Biosciences). To quantify the area corresponding to retinal microglial cells in density plots, CD11b-positive cells were represented in forward scatter (FSC)-FITC and the total area was measured for each retinal sample. 
Statistical Analysis
All experiments were performed in at least three animals. Student's t-test was applied using GraphPad software (GraphPad Software, Inc., San Diego, CA, USA). Results are expressed as mean ± SD, and P < 0.05, P < 0.01, P < 0.001 values were considered significant. 
Results
In control mice, microglial cells labelled with anti-Iba1 antibody appeared with the usual morphology of resting microglia in a healthy retina (Figs. 1A, 1C, 1G, 1I). These quiescent cells had a small, round soma with various branching processes and showed no reactivity against MHC class II antibody (Figs. 1B, 1C, 1H, 1I). Regarding cell distribution, microglial cells were located mainly in the inner plexiform layer (IPL) and the outer plexiform layer (OPL) in control mice (Figs. 1A–C, 1G–I). 
Figure 1
 
Confocal immunoflouorescence images of retinal microglial cells from C57BL/6J mice from a control mouse showing typical ramified morphology (AC, GI) and a mouse infected with viable yeasts of C. albicans ATCC26555 by intravenous administration showing activated microglia with enlarged soma and short, thick processes (DF, JL). Retinal sections were stained with anti-Iba1 antibody (A, D, G, J), anti-MHC class-II RT1B antibody (B, E, H, K), or both (C, F, I, L) showing the coexpression of MHC class-II RT1B and Iba1 in microglial cells of infected retinas. Arrows indicate microglial cell soma. Arrowheads point to nonspecific immunostaining of vessels with the secondary antibody. IS, inner segments, GCL, ganglion cell layer. Scale bars: 20 μm (AF), 10 μm (GL).
Figure 1
 
Confocal immunoflouorescence images of retinal microglial cells from C57BL/6J mice from a control mouse showing typical ramified morphology (AC, GI) and a mouse infected with viable yeasts of C. albicans ATCC26555 by intravenous administration showing activated microglia with enlarged soma and short, thick processes (DF, JL). Retinal sections were stained with anti-Iba1 antibody (A, D, G, J), anti-MHC class-II RT1B antibody (B, E, H, K), or both (C, F, I, L) showing the coexpression of MHC class-II RT1B and Iba1 in microglial cells of infected retinas. Arrows indicate microglial cell soma. Arrowheads point to nonspecific immunostaining of vessels with the secondary antibody. IS, inner segments, GCL, ganglion cell layer. Scale bars: 20 μm (AF), 10 μm (GL).
In the retinas of infected mice, after three days of infection, ramified cells experienced a transformation through gradations of “activated” states, exhibiting changes in shape and distribution: the cellular soma was enlarged, showing coarse and swollen appearance, processes were shortened and thickened with little branches (Figs. 1D–F, 1J–L). Some of the microglial cells showed an amoeboid shape (Figs. 2A–C). In these retinas, microglial cells were immunoreactive to anti-MHC class II antibody, which colocalized with Iba1 in most cells (Figs. 1E, 1F, 1K, 1L). Specifically, we found colocalization of Iba1 and MHCIIRT1B antibodies in all amoeboid cells, including amoeboid microglial cells surrounding blood vessels (Figs. 2A–C). 
Figure 2
 
Distribution and morphology of microglial cells in the retinas of infected mice. Retinal sections were immunostained with anti-Iba1 antibody (A, D, G, J), anti-MHC class-II RT1B antibody (B, E, H, K), or both (C, F, I, L). Arrowheads (AC) and arrows (AC) detail the characteristic morphology of Iba1 and MHCII double positive microglial cells in infected animals, with amoeboid or ovoid forms that were widely distributed in all retinal layers. Small arrows (AC) point to Iba1+ cells that surrounded blood vessels. Small arrows (DF) point to Iba1 + cells leaving blood vessels and getting into the retinal tissue. Arrows (DF) mark the nonspecific immunostaining of blood vessels with the secondary antibody. The arrowhead (DF) points to an Iba1+/MHCII+ microglial cell with a typical activated morphology. The arrowhead (GI) points to an Iba1+ cell within a blood vessel observed in a transversal section. Small arrows (GI) point to immunoreactive cells that seem to be perivascular macrophages as described by Guillemin and Brew. 25 Arrows (GI) mark the nonspecific immunostaining of blood vessels with the secondary antibody. The small arrow (JL) details the part of an immunopositive Iba1 microglial cell that was leaving a blood vessel. The arrowhead (JL) points to the part of the same microglial Iba1+ cell that still was into the blood vessel. Arrows (JL) mark the nonspecific immunostaining of blood vessels with the secondary antibody. Scale bars: 20 μm (AC), 10 μm (DL).
Figure 2
 
Distribution and morphology of microglial cells in the retinas of infected mice. Retinal sections were immunostained with anti-Iba1 antibody (A, D, G, J), anti-MHC class-II RT1B antibody (B, E, H, K), or both (C, F, I, L). Arrowheads (AC) and arrows (AC) detail the characteristic morphology of Iba1 and MHCII double positive microglial cells in infected animals, with amoeboid or ovoid forms that were widely distributed in all retinal layers. Small arrows (AC) point to Iba1+ cells that surrounded blood vessels. Small arrows (DF) point to Iba1 + cells leaving blood vessels and getting into the retinal tissue. Arrows (DF) mark the nonspecific immunostaining of blood vessels with the secondary antibody. The arrowhead (DF) points to an Iba1+/MHCII+ microglial cell with a typical activated morphology. The arrowhead (GI) points to an Iba1+ cell within a blood vessel observed in a transversal section. Small arrows (GI) point to immunoreactive cells that seem to be perivascular macrophages as described by Guillemin and Brew. 25 Arrows (GI) mark the nonspecific immunostaining of blood vessels with the secondary antibody. The small arrow (JL) details the part of an immunopositive Iba1 microglial cell that was leaving a blood vessel. The arrowhead (JL) points to the part of the same microglial Iba1+ cell that still was into the blood vessel. Arrows (JL) mark the nonspecific immunostaining of blood vessels with the secondary antibody. Scale bars: 20 μm (AC), 10 μm (DL).
In infected mice, microglial cells were detected not only in the plexiform layers of the retina, but also in the nuclear layers, the outer nuclear layer (ONL), and the inner nuclear layer (INL; Figs. 1D–F, 1J–1L). Amoeboid microglial cells were observed in the ONL, between the outer segments (OS) and the RPE, and surrounding blood vessels (Figs. 2A–C). We also found Iba1+/MHCIIRT1B+ amoeboid microglial cells in the IPL (Figs. 2D–F), and Iba1+ cells getting out of blood vessels (Figs. 2G–I). Cells within the blood vessels appeared as Iba1+/MHCIIRT1B−, while the ones within the retinal tissue were Iba1+/MHCIIRT1B+ (Figs. 2D–F). Microglial Iba1+ and MHCIIRT1B+ processes can be observed around the blood vessels in Figures 2G through 2I and in Figures 2J through 2L. 
In flow cytometry analyses, the whole microglial population of the retina was identified by its immunoreactivity against CD11b antibody in control and infected mice (Fig. 3A). Triple immunostaining showed a significant increase in the expression of MHCII and CD45 antigens in microglial cells of infected mice (1.91- and 3.22-fold increase in fluorescence mean intensity respectively; Figs. 3A, 3B), as well as an increase in the CD11b fluorescence mean intensity value (3.13-fold) with respect to the mean value in control mice (Fig. 3B). In infected mice, a CD11b+ CD45++ population was observed. Microglia activated state also was detected by quantifying the area of the retinal microglial cell population identified in FSC-CD11b-FITC density plots. In control mice, CD11b-positive cells appeared as a homogeneous population in FSC-CD11b-FITC dot and density plots, while retinal microglia of infected mice appeared as a much more heterogeneous population. The area value of activated microglia in infected mice was 3.5-fold increased with respect to control (Fig. 3C). 
Figure 3
 
Effect of fungal systemic infection on mouse retinal microglia, assessed by flow cytometry. (A) Retinal cells from C57BL/6J control and infected mice were triple labelled with a cocktail of APC-conjugated anti-CD11b, PE-conjugated anti-MHC class-II, and FITC-conjugated anti-mouse CD45 antibodies, and analyzed by flow cytometry (106 cells were analyzed in each assay). The CD11b-positive cells were gated (the percentage of gated cells is indicated). Double plots of CD11b+ cells presenting CD11b/MHCII and CD11b/CD45 double staining are shown in each case. Results show data from a single experiment representative of eight independent assays. (B) Histograms showing mean fluorescence intensity values of microglial cells (CD11b-positive population) labeled with anti-CD11b, anti-MHCII, and anti-CD45 antibodies (106 cells were analyzed in each assay) from control and infected mice. (C) Contour plots representing FSC against CD11b expression of gated cells. Results show data from a single representative experiment. Histograms show mean values ± SD of the CD11b-positive population area delimited in FSC-CD11b contour plots. **P < 0.01, ***P < 0.001, Student's t-test.
Figure 3
 
Effect of fungal systemic infection on mouse retinal microglia, assessed by flow cytometry. (A) Retinal cells from C57BL/6J control and infected mice were triple labelled with a cocktail of APC-conjugated anti-CD11b, PE-conjugated anti-MHC class-II, and FITC-conjugated anti-mouse CD45 antibodies, and analyzed by flow cytometry (106 cells were analyzed in each assay). The CD11b-positive cells were gated (the percentage of gated cells is indicated). Double plots of CD11b+ cells presenting CD11b/MHCII and CD11b/CD45 double staining are shown in each case. Results show data from a single experiment representative of eight independent assays. (B) Histograms showing mean fluorescence intensity values of microglial cells (CD11b-positive population) labeled with anti-CD11b, anti-MHCII, and anti-CD45 antibodies (106 cells were analyzed in each assay) from control and infected mice. (C) Contour plots representing FSC against CD11b expression of gated cells. Results show data from a single representative experiment. Histograms show mean values ± SD of the CD11b-positive population area delimited in FSC-CD11b contour plots. **P < 0.01, ***P < 0.001, Student's t-test.
The percentage of CD11b cells was increased in the retinas of infected mice. As deduced by the flow cytometry analysis, the microglial population in control mice constituted 0.10% ± 0.07% of total cells analyzed, whereas in infected mice the CD11b-positive population raised to 0.61% ± 0.01% (Fig. 3A). In agreement with the flow cytometry data, the immunohistochemistry also revealed an increase in microglial cell number, as the number of Iba1+ cells per retinal section was 10.1 ± 1.4 in control mice versus 13.9 ± 0.9 in infected mice (Fig. 4A). A quantitative analysis of immunohistochemical sections showed that the number of Iba1+ cells associated with blood vessels was 26.7% in infected mice, while in control mice no Iba1+ cells were associated with vessels (Fig. 4B). 
Figure 4
 
Histograms showing (A) Iba1+ cells associated with vessels in infected and control animals, (B) Iba1+ cells per mm2 of retina from retinal sections around the optic nerve head, and (C) total Iba1+ cell number in the ciliary body of control and infected mice. **P < 0.01, Student's t-test.
Figure 4
 
Histograms showing (A) Iba1+ cells associated with vessels in infected and control animals, (B) Iba1+ cells per mm2 of retina from retinal sections around the optic nerve head, and (C) total Iba1+ cell number in the ciliary body of control and infected mice. **P < 0.01, Student's t-test.
In the ciliary body of control mice, we found Iba1+/MHCII− cells with ramified morphology (Figs. 5A, 5B), while in infected mice some Iba1+/MHCII+ cells with a typical activated amoeboid morphology were found (Figs. 5C, 5D). Also, in the ciliary body of infected mice, blood-borne monocytes were found within blood vessels (Figs. 5E, 5F). A significant increase in the number of Iba1+ cells was observed in the ciliary body of infected animals (12.9 ± 0.1 cells/ciliary body section) compared to untreated controls (8.8 ± 0.8 cells/ciliary body section, Fig. 4C). 
Figure 5
 
Confocal immunoflouorescence images of Iba1+ cells in the ciliary body from a C57BL/6J control mouse (A, B) and a mouse infected with viable yeasts of C. albicans ATCC26555 (CF). Sections were stained with anti-Iba1 and anti-MHCII RT1B antibodies (A, C, E), or anti-MHCII RT1B antibody (B, D, F). Arrows (CF) point to MHCII+ microglial cells. (E, F) Detail of a ciliary body of an infected mouse. Arrows point to Iba1+/MHCII+ cells within blood vessels. Scale bars: 20 μm.
Figure 5
 
Confocal immunoflouorescence images of Iba1+ cells in the ciliary body from a C57BL/6J control mouse (A, B) and a mouse infected with viable yeasts of C. albicans ATCC26555 (CF). Sections were stained with anti-Iba1 and anti-MHCII RT1B antibodies (A, C, E), or anti-MHCII RT1B antibody (B, D, F). Arrows (CF) point to MHCII+ microglial cells. (E, F) Detail of a ciliary body of an infected mouse. Arrows point to Iba1+/MHCII+ cells within blood vessels. Scale bars: 20 μm.
The injection of three doses of 10 × 106 inactivated yeasts on days 1 to 3 did not elicit a significant increase in the retinal microglia population. The FITC-CD11b fluorescence mean value appeared similar to the one in control, uninjected mice (not shown). 
In our conditions, we could not detect C. albicans invasion of retinal tissue, neither by Calcofluor White M2R staining of retinal sections nor by incubating retinal tissue of infected mice in Sabouraud-Dextrose plates for 48 hours. 
Discussion
We studied the effect of a systemic fungal infection on retinal microglia in a mouse model of systemic candidiasis used previously in our laboratory. 20,21 The microglia population was identified by its immunoreactivity against Iba1 protein, as it is localized specifically in microglia and not found in neurons, astrocytes, or oligodendroglia. 22,23 The MHC class II antigens and the leukocyte common antigen CD45 were used to detect microglial activated states, as their upregulation is well known to occur in an activated state. 2427 In control mice, microglial cells appeared with the characteristic morphology, reactivity, and distribution of microglial cells in a healthy state, 2 while microglia in the retinas of infected mice appeared in a greater number and with different activated states, from cells with enlarged soma, shortened and thickened processes to cells with a real amoeboid shape; thus, in accordance with an advanced activation stage, in which microglia turn into phagocytic microglia, morphologically defined by the complete absence of cellular processes. 1,2  
Hence, our observations indicated that a systemic fungal infection causes retinal microglia activation and an increase of microglial cells in the retina. Our result is in agreement with a recent publication by Zinkernagel et al., 28 who found that systemic cytomegalovirus infection caused migration movements of the microglial cells in the retina, also showing signs of morphologic activation. 28  
The increased microglial cell number in infected mice can be due to the proliferation of retinal microglia and/or the arrival of infiltrating macrophages, as proliferation and the arrival of new blood-derived precursors that transform into phagocytes have been described in pathological situations. 1,2938 Zinkernagel et al. 28 conclude that the increased number of retinal microglia cells after a systemic cytomegalovirus infection, can be attributable just to proliferation in situ. Although the increase in the CD11b+, MHCII+, and CD45+ populations we observed in infected mice also could be consistent with microglial proliferation, the appearance of a high-expressing CD45 population in infected mice favors the arrival of macrophages or infiltrating perivascular microglial cells. 25,39 This is supported further by immunohistochemical analyses, as some of Iba1-expressing cells appeared associated with blood vessels and a number of these seemed to be getting out of the vessels to reach the retinal tissue. In some cases, Iba1 and MHCII double positive cells were observed around blood vessels, probably corresponding to perivascular macrophage cells. 24,25,39 Moreover, we observed a statistically significant increased Iba1+ population in the ciliary body of infected mice, some of them within blood vessels, which supports the idea of the presence of blood-borne macrophages. 31  
The fact that inactivated yeasts did not elicit a significant increase in the retinal microglia population showed that only viable microorganisms and not inactivated yeasts cause microglial activation, probably due to a rapid clearance of inactivated yeasts by phagocytotic cells. 
We could not detect C. albicans invasion of retinal tissue, which supports the idea that the activation of retinal microglial cells is due to a massive cytokine production that occurs during systemic infections. This finding is in agreement with that of Gallego et al., 16 who showed in a mouse model of glaucoma that, after inducing ocular hypertension with laser in one eye, the glial cells in the contralateral eye also appeared activated, maybe by immunologically mediated processes. However, a possible direct interaction of microglia with circulating soluble fungal ligands, such as β-glucan and mannan, 40 also may contribute to their activation. 
As is well known, toll-like receptors (TLRs), mainly TLR2 and TLR4, are critical for cytokine production in response to C. albicans , and Dectin-1, the β-glucan receptor, is required for phagocytosis of fungal cells, and also collaborates with TLR2 in cytokine production. 41,42 Retinal microglia, like microglia of the CNS and other immune cells, probably recognize pathogen-associated molecular patterns (PAMPs) through TLRs and Dectin-1. Several studies have shown that TLRs and Dectin-1 have an important role in microglial-mediated neuroinflammation and CNS injury following infection; β-glucan activates CNS microglia in a Dectin-1-dependent manner, suggesting that this receptor may have an important role in antifungal immunity in the CNS. 43,44 Moreover, we have demonstrated previously that TLR2 and Dectin-1 are expressed in mouse retinal microglia, and that Dectin-1 is involved in the phagocytosis of C. albicans . 45 Therefore, the possibility that fungal-derived ligands cause microglia activation following direct recognition by Pattern Recognition Receptors should not be ruled out. 
Although microglial activation is associated commonly with neuroprotection, excessive or prolonged activation may lead to chronic inflammation, with severe pathologic side effects. 5,46 In the retina, chronic microglia activation has been related to various neurodegenerative diseases, likely contributing to retinal tissue damage and proapoptotic events. 1,2 Despite its relevance, the study of microglial homeostasis in the retina has the challenge of the lack of experimental tools to analyze its activation in a simple and rapid way. In this work, we described the retinal microglial activation induced by systemic infection using immunohistochemistry and flow cytometry analysis. Previous reports have shown that microglia in the brain can be activated by systemic fungal infections, 17 as well as by systemically administered LPS 47,48 and systemic viral infections. 28 Our results showed that a systemic infection by C. albicans greatly activates microglia in the retina, as indicated by the changes in microglial cell morphology and distribution; upregulation of MHC class II and CD45, as well as CD11b antigens; and increase of the microglial population. Furthermore, our results demonstrated that quantifying the area corresponding to retinal microglial cells in FSC-CD11b-FITC density plots constitutes a useful tool to detect microglial activation. 
In conclusion, we have demonstrated that a systemic fungal infection activates the microglia in the retina, indicating that it may be considered as a risk factor for patients affected with ocular neurodegenerative diseases, such as diabetic retinopathy, glaucoma, AMD, or retinitis pigmentosa. 
Acknowledgments
The authors thank the Servicio Central de Soporte a la Investigación Experimental (SCSIE) of the University of Valencia, for technical assistance. 
Supported by project grants from the Spanish Ministry of Economy and Competitiveness-FEDER BFU2012-36845, Instituto de Salud Carlos III RETICS RD12/0034/0010, and ONCE (NCu); Instituto de Salud Carlos III PI080556 (DG); and MICINN-Juan de la Cierva Postdoctoral Fellowship JCI-2009-05224 (VG-V). 
Disclosure: V. Maneu, None; A. Noailles, None; J. Megías, None; V. Gómez-Vicente, None; N. Carpena, None; M.L. Gil, None; D. Gozalbo, None; N. Cuenca, None 
References
Karlstetter M Ebert S Langmann T. Microglia in the healthy and degenerating retina: insights from novel mouse models. Immunobiology . 2010; 215: 685–691. [CrossRef] [PubMed]
Langmann T. Microglia activation in retinal degeneration. J Leukoc Biol . 2007; 81: 1345–1351. [CrossRef] [PubMed]
Polazzi E Microglia Monti B. and neuroprotection: from in vitro studies to therapeutic applications. Prog Neurobiol . 2010; 92: 293–315. [CrossRef] [PubMed]
Harry GJ. Microglia during development and aging. Pharmacol Ther . 2013; 139: 313–326. [CrossRef] [PubMed]
Czeh M Gressens P Kaindl AM. The yin and yang of microglia. Dev Neurosci . 2011; 33: 199–209. [CrossRef] [PubMed]
Benveniste EN. Inflammatory cytokines within the central nervous system: sources, function, and mechanism of action. Am J Physiol . 1992; 263: C1–C16. [PubMed]
Lucin KM Wyss-Coray T. Immune activation in brain aging and neurodegeneration: too much or too little? Neuron . 2009; 64: 110–122. [CrossRef] [PubMed]
Hirsch EC Hunot S. Neuroinflammation in Parkinson's disease: a target for neuroprotection? Lancet Neurol . 2009; 8: 382–397. [CrossRef] [PubMed]
Tansey MG McCoy MK Frank-Cannon TC. Neuroinflammatory mechanisms in Parkinson's disease: potential environmental triggers, pathways, and targets for early therapeutic intervention. Exp Neurol . 2007; 208: 1–25. [CrossRef] [PubMed]
Bornemann KD Wiederhold KH Pauli C Abeta-induced inflammatory processes in microglia cells of APP23 transgenic mice. Am J Pathol . 2001; 158: 63–73. [CrossRef] [PubMed]
Griffin WS Liu L Li Y Mrak RE Barger SW. Interleukin-1 mediates Alzheimer and Lewy body pathologies. J Neuroinflammation . 2006; 3: 5. [CrossRef] [PubMed]
Gupta N Brown KE Milam AH. Activated microglia in human retinitis pigmentosa, late-onset retinal degeneration, and age-related macular degeneration. Exp Eye Res . 2003; 76: 463–471. [CrossRef] [PubMed]
Ardeljan D Chan CC. Aging is not a disease: distinguishing age-related macular degeneration from aging. Prog Retin Eye Res . 2013; 37: 68–89. [CrossRef] [PubMed]
Penfold PL Madigan MC Gillies MC Provis JM. Immunological and aetiological aspects of macular degeneration. Prog Retin Eye Res . 2001; 20: 385–414. [CrossRef] [PubMed]
Bosco A Crish SD Steele MR Early reduction of microglia activation by irradiation in a model of chronic glaucoma. PLoS One . 2012; 7: e43602. [CrossRef] [PubMed]
Gallego BI Salazar JJ de Hoz R IOP induces upregulation of GFAP and MHC-II and microglia reactivity in mice retina contralateral to experimental glaucoma. J Neuroinflammation . 2012; 9: 92. [CrossRef] [PubMed]
Lionakis MS Lim JK Lee CC Murphy PM. Organ-specific innate immune responses in a mouse model of invasive candidiasis. J Innate Immun . 2011; 3: 180–199. [CrossRef] [PubMed]
Yanez A Murciano C O'Connor JE Gozalbo D Gil ML. Candida albicans triggers proliferation and differentiation of hematopoietic stem and progenitor cells by a MyD88-dependent signaling. Microbes Infect . 2009; 11: 531–535. [CrossRef] [PubMed]
Murciano C Yanez A Gil ML Gozalbo D. Both viable and killed Candida albicans cells induce in vitro production of TNF-alpha and IFN-gamma in murine cells through a TLR2-dependent signalling. Eur Cytokine Netw . 2007; 18: 38–43. [PubMed]
Villamon E Gozalbo D Roig P O'Connor JE Fradelizi D Gil ML. Toll-like receptor-2 is essential in murine defenses against Candida albicans infections. Microbes Infect . 2004; 6: 1–7. [CrossRef] [PubMed]
Murciano C Villamon E Gozalbo D Roig P O'Connor JE Gil ML. Toll-like receptor 4 defective mice carrying point or null mutations do not show increased susceptibility to Candida albicans in a model of hematogenously disseminated infection. Med Mycol . 2006; 44: 149–157. [CrossRef] [PubMed]
Imai Y Ibata I Ito D Ohsawa K Kohsaka S. A novel gene iba1 in the major histocompatibility complex class III region encoding an EF hand protein expressed in a monocytic lineage. Biochem Biophys Res Commun . 1996; 224: 855–862. [CrossRef] [PubMed]
Ito D Imai Y Ohsawa K Nakajima K Fukuuchi Y Kohsaka S. Microglia-specific localisation of a novel calcium binding protein, Iba1. Brain Res Mol Brain Res . 1998; 57: 1–9. [CrossRef] [PubMed]
Dick AD Ford AL Forrester JV Sedgwick JD. Flow cytometric identification of a minority population of MHC class II positive cells in the normal rat retina distinct from CD45lowCD11b/c+CD4low parenchymal microglia. Br J Ophthalmol . 1995; 79: 834–840. [CrossRef] [PubMed]
Guillemin GJ Brew BJ. Microglia, macrophages, perivascular macrophages, and pericytes: a review of function and identification. J Leukoc Biol . 2004; 75: 388–397. [CrossRef] [PubMed]
Kreutzberg GW. Microglia: a sensor for pathological events in the CNS. Trends Neurosci . 1996; 19: 312–318. [CrossRef] [PubMed]
Sedgwick JD Schwender S Imrich H Dorries R Butcher GW ter Meulen V. Isolation and direct characterization of resident microglial cells from the normal and inflamed central nervous system. Proc Natl Acad Sci U S A . 1991; 88: 7438–7442. [CrossRef] [PubMed]
Zinkernagel MS Chinnery HR Ong ML Interferon gamma-dependent migration of microglial cells in the retina after systemic cytomegalovirus infection. Am J Pathol . 2013; 182: 875–885. [CrossRef] [PubMed]
Ajami B Bennett JL Krieger C McNagny KM Rossi FM. Infiltrating monocytes trigger EAE progression, but do not contribute to the resident microglia pool. Nat Neurosci . 2011; 14: 1142–1149. [CrossRef] [PubMed]
Sasahara M Otani A Oishi A Activation of bone marrow-derived microglia promotes photoreceptor survival in inherited retinal degeneration. Am J Pathol . 2008; 172: 1693–1703. [CrossRef] [PubMed]
Joly S Francke M Ulbricht E Cooperative phagocytes: resident microglia and bone marrow immigrants remove dead photoreceptors in retinal lesions. Am J Pathol . 2009; 174: 2310–2323. [CrossRef] [PubMed]
Ritter MR Banin E Moreno SK Aguilar E Dorrell MI Friedlander M. Myeloid progenitors differentiate into microglia and promote vascular repair in a model of ischemic retinopathy. J Clin Invest . 2006; 116: 3266–3276. [CrossRef] [PubMed]
Simard AR Soulet D Gowing G Julien JP Rivest S. Bone marrow-derived microglia play a critical role in restricting senile plaque formation in Alzheimer's disease. Neuron . 2006; 49: 489–502. [CrossRef] [PubMed]
Kezic J Xu H Chinnery HR Murphy CC McMenamin PG. Retinal microglia and uveal tract dendritic cells and macrophages are not CX3CR1 dependent in their recruitment and distribution in the young mouse eye. Invest Ophthalmol Vis Sci . 2008; 49: 1599–1608. [CrossRef] [PubMed]
Xu H Chen M Mayer EJ Forrester JV Dick AD. Turnover of resident retinal microglia in the normal adult mouse. Glia . 2007; 55: 1189–1198. [CrossRef] [PubMed]
Kaneko H Nishiguchi KM Nakamura M Kachi S Terasaki H. Characteristics of bone marrow-derived microglia in the normal and injured retina. Invest Ophthalmol Vis Sci . 2008; 49: 4162–4168. [CrossRef] [PubMed]
Graeber MB Streit WJ. Microglia: biology and pathology. Acta Neuropathol . 2010; 119: 89–105. [CrossRef] [PubMed]
Ransohoff RM Perry VH. Microglial physiology: unique stimuli, specialized responses. Annu Rev Immunol . 2009; 27: 119–145. [CrossRef] [PubMed]
Gregerson DS Yang J. CD45-positive cells of the retina and their responsiveness to in vivo and in vitro treatment with IFN-gamma or anti-CD40. Invest Ophthalmol Vis Sci . 2003; 44: 3083–3093. [CrossRef] [PubMed]
Held J Kohlberger I Rappold E Busse Grawitz A, Hacker G. Comparison of (1->3)-beta-D-glucan, mannan/anti-mannan antibodies, and Cand-Tec Candida antigen as serum biomarkers for candidemia. J Clin Microbiol . 2013; 51: 1158–1164. [CrossRef] [PubMed]
Gil ML Gozalbo D. Role of toll-like receptors in systemic Candida albicans infections. Front Biosci (Landmark Ed) . 2009; 14: 570–582. [CrossRef] [PubMed]
Goodridge HS Underhill DM. Fungal recognition by TLR2 and Dectin-1. Handb Exp Pharmacol . 2008; 87–109.
Shah VB Huang Y Keshwara R Ozment-Skelton T Williams DL Keshvara L. Beta-glucan activates microglia without inducing cytokine production in Dectin-1-dependent manner. J Immunol . 2008; 180: 2777–2785. [CrossRef] [PubMed]
Shah VB Williams DL Keshvara L. Beta-glucan attenuates TLR2- and TLR4-mediated cytokine production by microglia. Neurosci Lett . 2009; 458: 111–115. [CrossRef] [PubMed]
Maneu V Yanez A Murciano C Molina A Gil ML Gozalbo D. Dectin-1 mediates in vitro phagocytosis of Candida albicans yeast cells by retinal microglia. FEMS Immunol Med Microbiol . 2011; 63: 148–150. [CrossRef] [PubMed]
Magrone T Marzulli G Jirillo E. Immunopathogenesis of neurodegenerative diseases: current therapeutic models of neuroprotection with special reference to natural products. Curr Pharm Des . 2012; 18: 34–42. [CrossRef] [PubMed]
Halder SK Matsunaga H Ishii KJ Akira S Miyake K Ueda H. Retinal cell type-specific prevention of ischemia-induced damages by LPS-TLR4 signaling through microglia. J Neurochem . 2013; 126: 243–260. [CrossRef] [PubMed]
Tremblay S Miloudi K Chaychi S Systemic inflammation perturbs developmental retinal angiogenesis and neuroretinal function. Invest Ophthalmol Vis Sci . 2013; 54: 8125–8139. [CrossRef] [PubMed]
Figure 1
 
Confocal immunoflouorescence images of retinal microglial cells from C57BL/6J mice from a control mouse showing typical ramified morphology (AC, GI) and a mouse infected with viable yeasts of C. albicans ATCC26555 by intravenous administration showing activated microglia with enlarged soma and short, thick processes (DF, JL). Retinal sections were stained with anti-Iba1 antibody (A, D, G, J), anti-MHC class-II RT1B antibody (B, E, H, K), or both (C, F, I, L) showing the coexpression of MHC class-II RT1B and Iba1 in microglial cells of infected retinas. Arrows indicate microglial cell soma. Arrowheads point to nonspecific immunostaining of vessels with the secondary antibody. IS, inner segments, GCL, ganglion cell layer. Scale bars: 20 μm (AF), 10 μm (GL).
Figure 1
 
Confocal immunoflouorescence images of retinal microglial cells from C57BL/6J mice from a control mouse showing typical ramified morphology (AC, GI) and a mouse infected with viable yeasts of C. albicans ATCC26555 by intravenous administration showing activated microglia with enlarged soma and short, thick processes (DF, JL). Retinal sections were stained with anti-Iba1 antibody (A, D, G, J), anti-MHC class-II RT1B antibody (B, E, H, K), or both (C, F, I, L) showing the coexpression of MHC class-II RT1B and Iba1 in microglial cells of infected retinas. Arrows indicate microglial cell soma. Arrowheads point to nonspecific immunostaining of vessels with the secondary antibody. IS, inner segments, GCL, ganglion cell layer. Scale bars: 20 μm (AF), 10 μm (GL).
Figure 2
 
Distribution and morphology of microglial cells in the retinas of infected mice. Retinal sections were immunostained with anti-Iba1 antibody (A, D, G, J), anti-MHC class-II RT1B antibody (B, E, H, K), or both (C, F, I, L). Arrowheads (AC) and arrows (AC) detail the characteristic morphology of Iba1 and MHCII double positive microglial cells in infected animals, with amoeboid or ovoid forms that were widely distributed in all retinal layers. Small arrows (AC) point to Iba1+ cells that surrounded blood vessels. Small arrows (DF) point to Iba1 + cells leaving blood vessels and getting into the retinal tissue. Arrows (DF) mark the nonspecific immunostaining of blood vessels with the secondary antibody. The arrowhead (DF) points to an Iba1+/MHCII+ microglial cell with a typical activated morphology. The arrowhead (GI) points to an Iba1+ cell within a blood vessel observed in a transversal section. Small arrows (GI) point to immunoreactive cells that seem to be perivascular macrophages as described by Guillemin and Brew. 25 Arrows (GI) mark the nonspecific immunostaining of blood vessels with the secondary antibody. The small arrow (JL) details the part of an immunopositive Iba1 microglial cell that was leaving a blood vessel. The arrowhead (JL) points to the part of the same microglial Iba1+ cell that still was into the blood vessel. Arrows (JL) mark the nonspecific immunostaining of blood vessels with the secondary antibody. Scale bars: 20 μm (AC), 10 μm (DL).
Figure 2
 
Distribution and morphology of microglial cells in the retinas of infected mice. Retinal sections were immunostained with anti-Iba1 antibody (A, D, G, J), anti-MHC class-II RT1B antibody (B, E, H, K), or both (C, F, I, L). Arrowheads (AC) and arrows (AC) detail the characteristic morphology of Iba1 and MHCII double positive microglial cells in infected animals, with amoeboid or ovoid forms that were widely distributed in all retinal layers. Small arrows (AC) point to Iba1+ cells that surrounded blood vessels. Small arrows (DF) point to Iba1 + cells leaving blood vessels and getting into the retinal tissue. Arrows (DF) mark the nonspecific immunostaining of blood vessels with the secondary antibody. The arrowhead (DF) points to an Iba1+/MHCII+ microglial cell with a typical activated morphology. The arrowhead (GI) points to an Iba1+ cell within a blood vessel observed in a transversal section. Small arrows (GI) point to immunoreactive cells that seem to be perivascular macrophages as described by Guillemin and Brew. 25 Arrows (GI) mark the nonspecific immunostaining of blood vessels with the secondary antibody. The small arrow (JL) details the part of an immunopositive Iba1 microglial cell that was leaving a blood vessel. The arrowhead (JL) points to the part of the same microglial Iba1+ cell that still was into the blood vessel. Arrows (JL) mark the nonspecific immunostaining of blood vessels with the secondary antibody. Scale bars: 20 μm (AC), 10 μm (DL).
Figure 3
 
Effect of fungal systemic infection on mouse retinal microglia, assessed by flow cytometry. (A) Retinal cells from C57BL/6J control and infected mice were triple labelled with a cocktail of APC-conjugated anti-CD11b, PE-conjugated anti-MHC class-II, and FITC-conjugated anti-mouse CD45 antibodies, and analyzed by flow cytometry (106 cells were analyzed in each assay). The CD11b-positive cells were gated (the percentage of gated cells is indicated). Double plots of CD11b+ cells presenting CD11b/MHCII and CD11b/CD45 double staining are shown in each case. Results show data from a single experiment representative of eight independent assays. (B) Histograms showing mean fluorescence intensity values of microglial cells (CD11b-positive population) labeled with anti-CD11b, anti-MHCII, and anti-CD45 antibodies (106 cells were analyzed in each assay) from control and infected mice. (C) Contour plots representing FSC against CD11b expression of gated cells. Results show data from a single representative experiment. Histograms show mean values ± SD of the CD11b-positive population area delimited in FSC-CD11b contour plots. **P < 0.01, ***P < 0.001, Student's t-test.
Figure 3
 
Effect of fungal systemic infection on mouse retinal microglia, assessed by flow cytometry. (A) Retinal cells from C57BL/6J control and infected mice were triple labelled with a cocktail of APC-conjugated anti-CD11b, PE-conjugated anti-MHC class-II, and FITC-conjugated anti-mouse CD45 antibodies, and analyzed by flow cytometry (106 cells were analyzed in each assay). The CD11b-positive cells were gated (the percentage of gated cells is indicated). Double plots of CD11b+ cells presenting CD11b/MHCII and CD11b/CD45 double staining are shown in each case. Results show data from a single experiment representative of eight independent assays. (B) Histograms showing mean fluorescence intensity values of microglial cells (CD11b-positive population) labeled with anti-CD11b, anti-MHCII, and anti-CD45 antibodies (106 cells were analyzed in each assay) from control and infected mice. (C) Contour plots representing FSC against CD11b expression of gated cells. Results show data from a single representative experiment. Histograms show mean values ± SD of the CD11b-positive population area delimited in FSC-CD11b contour plots. **P < 0.01, ***P < 0.001, Student's t-test.
Figure 4
 
Histograms showing (A) Iba1+ cells associated with vessels in infected and control animals, (B) Iba1+ cells per mm2 of retina from retinal sections around the optic nerve head, and (C) total Iba1+ cell number in the ciliary body of control and infected mice. **P < 0.01, Student's t-test.
Figure 4
 
Histograms showing (A) Iba1+ cells associated with vessels in infected and control animals, (B) Iba1+ cells per mm2 of retina from retinal sections around the optic nerve head, and (C) total Iba1+ cell number in the ciliary body of control and infected mice. **P < 0.01, Student's t-test.
Figure 5
 
Confocal immunoflouorescence images of Iba1+ cells in the ciliary body from a C57BL/6J control mouse (A, B) and a mouse infected with viable yeasts of C. albicans ATCC26555 (CF). Sections were stained with anti-Iba1 and anti-MHCII RT1B antibodies (A, C, E), or anti-MHCII RT1B antibody (B, D, F). Arrows (CF) point to MHCII+ microglial cells. (E, F) Detail of a ciliary body of an infected mouse. Arrows point to Iba1+/MHCII+ cells within blood vessels. Scale bars: 20 μm.
Figure 5
 
Confocal immunoflouorescence images of Iba1+ cells in the ciliary body from a C57BL/6J control mouse (A, B) and a mouse infected with viable yeasts of C. albicans ATCC26555 (CF). Sections were stained with anti-Iba1 and anti-MHCII RT1B antibodies (A, C, E), or anti-MHCII RT1B antibody (B, D, F). Arrows (CF) point to MHCII+ microglial cells. (E, F) Detail of a ciliary body of an infected mouse. Arrows point to Iba1+/MHCII+ cells within blood vessels. Scale bars: 20 μm.
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×