A putative role for inflammation in Retinitis Pigmentosa 38




Deborah S.Lew

Introduction

Retinitis Pigmentosa (RP) are a group of inherited retinal degenerative diseases for which more than forty-five have been identified. Patients began to experience night blindness, followed by a sight in tunnel vision due to the lost of their peripheral photoreceptors, eventually leading to total blindness about the age of thirty-five or forty years. In this study, we focus of one of the most severe form of RP (RP38) due to a mutation in the Mertk gene. Mertk is a receptor found in the retinal-pigmented epithelium (RPE) involved in the very important process of daily phagocytosis of the photoreceptors. When this function is impaired the retina degenerate leading to lost of vision. It is still unclear why this particular form of the disease is such severe. Recent studies have shown that inflammation could exacerbate the degeneration of the retina. Usually inflammatory cells can be detected in degenerating retinas of mice, but if they could be detected in young mice, when the retina is still intact, that could explain part of the severity of the disease. A recent study has been able to detect inflammatory cells in aged Mertk knock-out (KO) mice by immunostaining of cryosection of the retina, when the retina is already degenerated. Here, we wanted to develop a molecular technique to detect inflammatory in the retina as a complementary tool to immunostaining, that would help detect the presence or not of inflammatory cells in younger mice, in order to answer the question of the severity.

Material & methods

Animals:
WT and Mertk KO mice from the same background (129T2/SvEmsJ) were killed by CO2 asphyxiation at same hour of the day, at the age of three-month-old and five-week-old. Eyeballs were carefully enucleated and rinsed in HBSS without Ca2+ and Mg2+. The lens and vitreous humor were dissected out. Retinas were delicately separated from the eyecups containing the RPE/choroid layers. For one sample four retinas were pooled together in RLT lysis buffer plus β-mercaptoethanol (Quiagen kit) and frozen at -80° C until further RNA extraction.

RNA extraction:
All lysate samples were purified from genomic DNA by a Qiashredder column from Qiagen. RNA was then extracted following the Qiagen RNeasy plus mini kit, following the manufacturer protocol. Purity and concentration of each sample were analyzed by spectrophotometry, and a 5ng/ul solution stock were made and stored at -20° C.

RT-PCR:
RT-PCR was performed using the Qiagen One-Step RT-PCR kit. Ten nanograms of RNA were amplified in a 20ul reaction with 0.5uM primers for the following genes: Nox2, IBA1, Brn3a and Rplp0. Products were then analyzed in a 1% or 2% gel agarose (Supplementary Fig.1S), and sequenced by Sanger sequencing by Genewiz.

Results

Khono et al (2016) were able to detect inflammatory positive cells (IBA1+) by immunostaining in cryosection of eyes from eight and five-week-old Mertk KO mice. To add another tool than immunostaining to the detection of inflammatory cells we wanted to be able to detect their presence by molecular techniques. For that, we designed primers for IBA1 protein, as they used this protein as an immunostaining marker in their study; and Nox2 protein, which is a well-known marker for a specific pro-inflammatory subtype of macrophages. We used primers for the retina ganglion cell gene Brn3a as a retinal specificic control and primers for the ribosomic protein Rplp0 as a housekeeping gene control. As we did not know the limit of detection for this method, and as Khono et al. were able to detect positive IBA1 cells by immunostaining at age of five and eight weeks but with a stronger detection at eight-week-old, we decided to first try this new molecular method in mice of three-month-old where the inflammation should be even stronger increasing our chance of detection. Both IBA1 and Nox2 markers were detected at increased level in Mertk KO mice but no in the WT mice (Fig.1).
Then, to test further the level of detection of this new tool, we tested our primers in five-week-old mice. Once again, both IBA1 and Nox2 were detected in higher level in the Mertk KO mice compared to the WT mice (Fig.2). These results suggest that inflammatory positive cells can be detected in the retina of mice by RT-PCR, and IBA1 and Nox2 are good markers for that purpose.

Discussion

In this study, we developed a new molecular tool to detect inflammatory cells in the retina of mice. As expected from the study of Khono et al., we were able to detect inflammatory cells in the retinas of three-month and five-week-old Mertk KO mice. We showed that IBA1 and Nox2 could be used as specific markers for the presence of inflammatory cells (Fig.1 and 2), and that Brn3a was a good marker for retinal specificity (Fig.1S). Because of the immune privilege of the eye, we were not expecting any cells from the immune system in the retinas of the WT mice, indeed Nox2 that characterized a specific pro-inflammatory subtype of macrophages was not detected in the WT mice of both age.
This study provided a new tool for detecting inflammatory cells in the degenerating retina of a mouse model for retinitis pigmentosa 38. Further study to detect their presence in younger mice, when the retina is not yet altered could explain in part the severity of this disease. If retinal cells are secreting inflammatory cytokines when the retina is still intact attracting inflammatory cells; the presence of those inflammatory cells could exacerbate the degeneration and therefore increase the severity of the disease. Comparing the age of appearance of inflammatory cells between this model and another mouse model for a more classical type of retinitis pigmentosa would also be very interesting. Another interesting point to decipher would be to determine whether the macrophages detected are inflammatory cells coming from the circulating pool or whether they are activated microglia. The development of this molecular tool for the detection of inflammatory cells, in addition with immunostaining, will help answers those questions.

References

Chen et al., Ocul.Immunol.Inflamm., 2002, 10(1):27-39. Distribution, markers, and functions of retinal microglia.

Cunha-Vaz et al., Eur. J. Ophthalmol., 2011, Suppl 6:S3-9. doi:10.5301 EJO.2010.6049. Blood-retinal barrier.

D’Cruz et al., Hum.Mol.Genet., 2000, 9(4):645-51. Mutation of the receptor tyrosine kinase gene Mertk in the retinal dystrophic RCS rat.

Hartong et al., Lancet, 2006, 368(9549):1795-809. Retinitis Pigmentosa.

Khono et al., J Immunol, 2014, 192(8):3816-3827. CCL3 production by microglial cells modulates disease severity in murine models of retinal degeneration.

Mackay DS et al., Mol Vis, 2010, 16:369-77. Novel mutations in MERTK associated with childhood onset rod-cone dystrophy.

Strauss, Physiol.Rev, 2005, 85(3):845-81. The retinal pigment epithelium in visual function.

Whitcup et al., Int. J. Inflam., 2013, 2013:724648. Inflammation in retinal disease.

Young & Bok, J. Cell. Biol., 1969, 42(2):392-403. Participation of the retinal pigment epithelium in the rod outer segment renewal process.

Zhou & Caspi, F1000 Biol. Rep., 2010, 2:3. Ocular immune privilege.

Figures


Figure 1-Detection of inflammatory cells by RT-PCR in three-month-old mice. A. Nox2 macrophage specific primer. A high level of the macrophage specific transcript Nox2 is only found in the KO mice. B. IBA1 macrophages specific primer. Increase of IBA1 transcript level is only found in the KO mice. C. Retinal specific marker Brn3a. Same level of Brn3a transcript is found in WT and KO mice. D. Housekeeping gene. Rplp0 used as a loading control show same amount of transcripts both in WT and KO mice; * shows expected size. WT: Wild-type mouse; KO: Mertk Knock-out mice.


Figure 2-Detection of inflammatory cells by RT-PCR in five-week-old mice. A. Nox2 macrophage specific primer. A high level of the macrophage specific transcript Nox2 is only found in the KO mice. B. IBA1 macrophages specific primer. Increase of IBA1 transcript level is only found in the KO mice. C. Retinal specific marker Brn3a. Same level of Brn3a transcript is found in WT and KO mice. D. Housekeeping gene. Rplp0 used as a loading control show same amount of transcripts both in WT and KO mice. WT: Wild-type mouse; KO: Mertk Knock-out mice


Figure 3-Fig.1S: Validation of the primers. A. Macrophage specific primers. Two pairs of primers for Nox2 and IBA1 were tested in duplicata on WT macrophages B. Housekeeping gene. Rplp0 primers tested in duplicata in WT macrophages. C. Retinal specific marker Brn3a. Three pairs of primers were tested in duplicata in WT macrophages and WT retinas. WT: Wild-type mouse; M: Macrophages.


Abstract
Retinitis Pigmentosa (RP) are a group of inherited retinal degenerative diseases, that lead to a progressive vision loss starting from the periphery of the retina. Researchers have identified more than 45 genes involved in RP. Here we focus on one of the most severe forms of RP: RP 38, which is caused by a mutation in the Mertk receptor gene. The Mertk receptor is required for the daily phagocytosis of the photoreceptor outer segment extremities. Without daily phagocytosis, photoreceptors accumulate oxydative reactives and degenerate. Recent studies have used immunostaining to show positive inflammatory cells in the retinas of five-week-old Mertk KO mice. Finding inflammatory cells in younger mice could help explain the severity of the disease. Here we sought to develop molecular tools to detect inflammatory cells in the retina of mice. We selected three markers: two markers that are known to be macrophage specific (Nox2 and IBA1), and one marker that is a retinal ganglion cell specific gene (Brn3a). Rplp0, a ribosomic protein, has been used as housekeeping gene. Because of the immune privilege in the eye we did not expect detection of macrophage specific markers (Nox2 or IBA1) in the WT retinas but did expect detection of the macrophage specific markers in Mertk KO mice. Confirming our prediction we observed no or very few Nox2 or IBA1 transcripts in WT mice respectfully, but a high level of these transcripts in the one-month-old and three-month-old Mertk KO mice. Additionally, we determined that Brn3a is a good retina specific marker. Taken together these results suggest that Nox2 and IBA1 can be used to detect inflammatory cells in retinas by RT-PCR, and Brn3a can be used as a retinal specific molecular. These methods will be useful for investigating the particular severity of Retinitis Pigmentosa 38.

Full Paper

Acknowledgments

I would like to thank Dr. Finnemann for providing the mice. I would also like to greatly thank Catharina Grubaugh and Tony Evans for their amazing dedication and support. Finally I would like to thank Dr. Rubin for his guidance and for letting me do at the end of each meeting a “one more last RT”.


This document was last modified 05/18/2016.
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