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

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 determin…

Characterization of a new murine retinal cell line (MU-PH1) with glial, progenitor and photoreceptor characteristics

Unlike fish and amphibians, mammals do not regenerate retinal neurons throughout life. However, neurogenic potential may be conserved in adult mammal retina and it is necessary to identify the factors that regulate retinal progenitor cells (RPC) proliferative capacity to scope their therapeutic potential. Müller cells can be progenitors for retinal neuronal cells and can play an essential role in the restoration of visual function after retinal injury. Some members of the Toll-like receptor (TLR) family, TLR2, TLR3 and TLR4, are related to progenitor cells proliferation. Müller cells are important in retinal regeneration and stable cell lines are useful for the study of retinal stem cell bi…

TLR2 modulates gut colonization and dissemination of Candida albicans in a murine model

Invasive candidiasis often arises from translocation of endogenous yeasts from the gastrointestinal tract to the bloodstream. Here we describe that both wild type and TLR2−/− mice strains, orally administered with Candida albicans yeasts, display similar sustained high level of gut colonization when oral antibacterial treatment is present, while removal of antibiotic treatment causes a progressive clearance of yeasts in control but not in TLR2−/− mice. Fungal invasion of internal organs, following immunosuppression of colonized mice, was increased in TLR2−/− mice. These results point out to a role of TLR2 in gut protection against colonization and endogenous invasion by C. albicans. This wo…

Retinal microglia are activated by systemic fungal infection

Purpose: To determine 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 (i) confocal immunofluorescence and (ii) 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 re-location in retinal layers. Conclusions: As an excessive or prolonged microglial activation may …

Role of IFN-gamma in immune responses to Candida albicans infections

Candida albicans is the most frequent etiologic agent that causes opportunistic fungal infections called candidiasis, a disease whose systemic manifestation could prove fatal and whose incidence is increasing as a result of an expanding immunocompromised population. Here we review the role of interferon-gamma (IFN-γ) in host protection against invasive candidiasis. This cytokine plays an essential role in both the innate and adaptive arms of the immune response to candidiasis. We focus on recent progress on host-pathogen interactions leading to the production of IFN-γ by host cells. IFN-γ is produced by CD4 Th1, CD8, γδ T, and natural killer (NK) cells, essentially in response to both IL-12…

The Yeast External Invertase as a Reporter to Study Regulation ofCandida Albicans Promoter Sequences inSaccharomyces Cerevisiae

Molecular cloning and characterization of aCandida albicansgene (EFB1) coding for the elongation factor EF-1β

A Candida albicans gene homologous to Saccharomyces cerevisiae elongation factor 1 beta was isolated by screening a genomic DNA library using a C. albicans cDNA as a probe. This cDNA was previously obtained by immunoscreening of an expression library with polyclonal antibodies raised against candidal cell wall components. Sequence analysis of the cDNA and the whole C. albicans gene (EMBL accession number X96517) revealed an intron-interrupted open reading frame of 639 base pairs that encodes a 213 amino acid protein. Exon sequences are highly homologous (74%) to S. cerevisiae EFB1, whereas intron sequence is less conserved (34% identity), and the predicted amino acid sequence shares about 7…

Molecular Cloning of aCandida albicans Gene (SSB1) Coding for a Protein Related to the Hsp70 Family

We have cloned and sequenced a Candida albicans gene (SSB1) encoding a potential member of the heat-shock protein seventy (hsp70) family. The protein encoded by this gene contains 613 amino acids and shows a high degree (85%) of sequence identity to the ssb subfamily (ssb1 and ssb2) of the Saccharomyces cerevisiae hsp70 family. The transcribed mRNA (2·1 kb) is present in similar amounts both in yeast and germ tube cells of C. albicans. The sequence data has been deposited in the GenBank data library under the Accession Number X97723. © John Wiley & Sons, Ltd.

In vitro differentiation of murine hematopoietic progenitor cells toward the myeloid lineage occurs in response to Staphylococcus aureus and yeast species

We have studied the effect of inactivated microbial stimuli (Candida albicans, Candida glabrata, Saccharomyces boulardii, and Staphylococcus aureus) on the in vitro differentiation of lineage negative (Lin−) hematopoietic progenitor mouse cells. Purified Lin− progenitors were co-cultured for 7 days with the stimuli, and cell differentiation was determined by flow cytometry analysis. All the stimuli assayed caused differentiation toward the myeloid lineage. S. boulardii and particularly C. glabrata were the stimuli that induced in a minor extent differentiation of Lin− cells, as the major population of differentiated cells corresponded to monocytes, whereas C. albicans and S. aureus induced …

Candida albicansstimulatesin vivodifferentiation of haematopoietic stem and progenitor cells towards macrophages by a TLR2-dependent signalling

Toll-like receptors (TLRs) are expressed by haematopoietic stem and progenitor cells (HSPCs), and may play a role in haematopoiesis in response to pathogens during infection. We have previously demonstrated that (i) inactivated yeasts of Candida albicans induce in vitro differentiation of HSPCs towards the myeloid lineage, and (ii) soluble TLR agonists induce in vivo their differentiation towards macrophages. In this work, using an in vivo model of HSPCs transplantation, we report for the first time that HSPCs sense C. albicans in vivo and subsequently are directed to produce macrophages by a TLR2-dependent signalling. Purified lineage-negative cells (Lin(-)) from bone marrow of C57BL/6 mic…

Identification ofCandida albicansclinical isolates by PCR amplification of anEFB1gene fragment containing an intron-interrupted open reading frame

The use of a single pair of primers, deduced from the intron and exon nucleotide sequences of the Candida albicans EFB1 gene, in polymerase chain reaction (PCR) assays performed with whole cells of both laboratory strains and clinical isolates of Candida species, resulted in the species-specific amplification of a 785 bp DNA fragment in C. albicans strains. Clinical C. albicans isolates were tested, and 85 out of 86 generated the expected PCR-amplified product; other Candida species, both laboratory strains and clinical isolates, as well as laboratory strains belonging to other fungal genera, including medically relevant taxa, failed to amplify any DNA fragment. In addition, unusual C. albi…

Complementation of Saccharomyces cerevisiae mutationsin genes involved in translation and protein folding (EFB1 and SSB1)with Candida albicans cloned genes

We have demonstrated that the expression of Candida albicans genes involved in translation and protein folding (EFB1 and SSB1) complements the phenotype of Saccharomyces cerevisiae mutants. The elongation factor 1beta (EF-1beta) is essential for growth and efb1 S. cerevisiae null mutant cells are not viable; however, viable haploid cells, carrying the disrupted chromosomal allele of the S. cerevisiae EFB1 gene and pEFB1, were isolated upon sporulation of a diploid strain which was heterozygous at the EFB1 locus and transformed with pEFB1 (a pEMBLYe23 derivative plasmid containing an 8-kb DNA fragment from the C. albicans genome which contains the EFB1 gene). This indicates that the C. albic…

Dectin-1 mediatesin vitrophagocytosis ofCandida albicansyeast cells by retinal microglia: Figure 1

We have investigated the expression of TLR2 and Dectin-1 in retinal microglia and their involvement in Candida albicans phagocytosis using a cytometric approach. The expression of both receptors has been demonstrated in CD11b+ retinal cells. Phagocytosis of pHrodo-labelled C. albicans yeasts by microglial CD11b+ cells of C57BL/6 mice was inhibited both by the Dectin-1 antagonist laminarin and anti-Dectin-1 antibodies, whereas phagocytosis of yeasts by retinal microglia of TLR2 KO mice was unaffected. These data indicate that phagocytosis of C. albicans yeasts by retinal microglia is mediated by Dectin-1, whereas TLR2 does not play a significant role in this process.