Abstract
BackgroundWe have previously shown that, in response to microbial infection, activated Müller glia secrete inflammatory cytokines/chemokines and exhibit antimicrobial properties. The aim of this study is to understand the mechanisms and the key components involved in this response.MethodsImmortalized human retinal Müller glia (MIO-M1 cells) were challenged with Staphylococcus (S) aureus, the leading cause of severe intraocular infection followed by RT2 profile PCR array analysis. The expression of human β-defensin 1 (HBD1), 2 (HBD2), 3 (HBD3), hepcidine and cathelicidin LL37 was checked by RT-PCR and quantified by Taqman® qPCR. The expression of AMPs was confirmed at protein level by dot-blot analysis. The production of ROS was measured by dicholoro-dihydro-fluorescein diacetate (DCFH-DA) staining by flow cytometry as well as fluorescence microscopy. The level of nitric oxide (NO) was measured by measuring a stable metabolite, nitrite using the Griess reagent. In vitro killing assay was performed by Live/Dead® BacLight™ staining as well as by dilution plating in suspension and adherent conditions following S. aureus infection. Phagocytosis was measured by CFU enumeration following infection.ResultsPCR array data showed that, in comparison to uninfected control cells, bacterial challenge significantly (> two-fold) induced the expression of 26 genes involved in cytokine/chemokine, antimicrobials, Toll-like receptor, apoptotic, and NF-κB signaling. RT-PCR analysis showed time-dependent increased expression of HBD1, HBD2, HBD3, LL-37, and hepcidin mRNA in bacteria-challenged Müller glia. The expression of these antimicrobial molecules was also increased at the protein level in the culture supernatant, as detected by dot-blot analysis. Additionally, the bacteria-stimulated Müller glia were found to produce reactive oxygen (ROS) and reactive nitrogen (RNS) species. In vitro, killing assays revealed that Müller glia exhibited bactericidal activity against S. aureus in both adherent and suspension cultures. Furthermore, our data demonstrated that Müller glia can phagocytize and kill the bacteria in a time-dependent manner.ConclusionsThese data suggest that retinal Müller glia behave like classical innate immune cells by producing a variety of antimicrobial molecules in response to bacterial challenge, suggesting their pivotal role in retinal innate defense.
Highlights
We have previously shown that, in response to microbial infection, activated Müller glia secrete inflammatory cytokines/chemokines and exhibit antimicrobial properties
S. aureus evokes antibacterial gene expression in retinal Müller glia In order to determine the innate response of Müller glia towards bacterial pathogens, we performed Human Antibacterial Response RT2 ProfilerTM PCR arrays
Using a gentamicin protection assay (Figure 8), our data showed that at eight hours, a significantly increased number of bacteria were internalized by Discussion In this study, we demonstrated that, in addition to the inflammatory response, the activated Müller glia exhibit antibacterial responses, as evidenced by the increased expression and production of antimicrobial peptide (AMP), reactive oxygen species (ROS), and nitric oxide (NO), a phenotype commonly displayed by mucosal epithelial cells
Summary
We have previously shown that, in response to microbial infection, activated Müller glia secrete inflammatory cytokines/chemokines and exhibit antimicrobial properties. Müller cells have many local functions; they stabilize the retinal architecture, provide an orientation scaffold, give structural and metabolic support to retinal neurons and blood vessels and prevent aberrant photoreceptor migration into the sub-retinal space [3,4] Apart from these important support functions, our recent studies have implicated their role in retinal innate defense against microbial infection, such as bacterial endophthalmitis [5]. We previously showed that Müller glia express all known human Toll-like receptors (TLRs), the best-characterized receptors present on innate immune cells and involved in antimicrobial innate defense [15] They are located strategically, that is their end feet are in inner limiting membrane (ILM), next to the vitreous cavity, the site where bacteria proliferate in endophthalmitis [5]. The Müller glia possesses the ability to respond to a variety of infectious and non-infectious stimuli
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
