RD causes a marked inflammatory response with increased cytokine and chemokine expression in both human and experimental models
To characterize the inflammatory response to RD, we first analyzed the expression profile of cytokines in vitreous samples from 41 patients with RD and from 34 control patients with macular hole. The mean extent of detachment in the RD group was 2.1 ± 0.8 quadrants with a macular involvement in 34.1% of cases and some degree of retinal wrinkling and folding (grade B or C proliferative vitreoretinopathy) in 36.6% of cases. The mean duration of symptoms before surgery was 7.7 ± 7.3 days with a median of 4.5 days [1–30]. Using a Human Cytokine 27-plex Assay we showed that the cytokines IL-1ra, IL-6, IL-7, IL-8, IFN-γ (Fig. 1A), the chemokines CCL2, CCL3, CCL4, CXCL10 and CCL11 (Fig. 1B) and the growth factor G-CSF (Fig. 1C) were significantly increased in the vitreous from RD patients. In contrast, the levels of IL-10, IL-13 and VEGF were not statistically different between the two groups (Fig. 1A-C). The remaining cytokines, such as IL-4, and IL-17 of the assay were not detectable.
We next evaluated the expression of the cytokines by RT-qPCR of mouse retinal control tissues and retinas harvested after four days of experimental RD. This time point was chosen for analysis as it was similar to the median duration of symptoms in our clinical study and because photoreceptor cell death peaks at around three days after RD in both experimental models and human samples [4, 27–29]. The transcription levels of nine out of eleven mediators found to be elevated in human vitreous from RD patients (except for Ccl11 and Il-8 that does not exist in mice) were significantly upregulated in detached mouse retinas compared to controls (Fig. 1D-F).
In summary, our findings confirm that RD induces a marked inflammatory response in human patients, which is closely mimicked by experimental murine retinal detachment. The cytokine profil is suggestive of an infiltration of mainly myeloid cells, but the increased levels of IFN-γ might be suggestive of T helper type 1 (Th1) cells recruitment.
RD-associated leukocyte infiltration is associated with cone loss
In the healthy retina, microglial cells (MCs) populate the inner retina, but the photoreceptor cell layer and subretinal space are devoid of any immune cells . In RD, MPs have been shown to accumulate in the detached area and are highly associated with TUNEL-positive nuclei in the inner aspects of the outer nuclear layer (ONL), where rod nuclei are located [10, 12, 13]. Using flow cytometry and a gating strategy (Fig. 2A) adapted from O’Koren et al. , we here analyzed the leukocyte population in healthy and detached (day 1, day 3, day 7) mouse retinas. In healthy retinas, we only detected MCs (CD11b+CD45low) that steadily increased to quadruple their numbers at the end of the observation period (Fig. 2B). The myeloid cell population (CD11b+CD45high) sharply increased after RD, peaking at 24 h and remained strongly elevated throughout (Fig. 2B). Interestingly, we also found a sizeable population of T-cells (CD45+CD3+) that infiltrated the retina at day 3 and remained elevated, although to a lesser extent, at day 7 (Fig. 2B). A more detailed analysis of the myeloid population reveals a rapid, but short-lived recruitment of neutrophils (CD11b+CD45highLy6G+) and monocytes (CD11b+CD45highLy6GnegLy6Chigh) at day 1 (Fig. 2C). The number of macrophages (CD11b+CD45highLy6GnegLy6Clow) mainly increased at day 3 and stayed elevated, likely reflecting the differentiation of monocytes (Mos) into macrophages (Fig. 2C). Together, these results demonstrate that RD induces the infiltration of T-cells, monocyte derived-macrophages (Mφs) and accumulation of MCs, and that MPs, MCs and Mφs, represent the main accumulating immune cells.
Next, we quantified the presence of MPs (MCs and Mφs) and the cone population on IBA-1 (MP marker)-, peanut agglutinin (PNA cone outer segment marker)-, cone arrestin (CAR, cone marker) triple-stained retinal flat-mounts (Fig. 2D). Confocal microscopy confirmed that subretinal MPs are not observed in normal mice and highlight the elongated shape of the cone outer segments (Fig. 2D). Interestingly, despite the peak of infiltrating myeloid cells measured in the whole retina at day 1, IBA-1+MPs only accumulated in the subretinal space by day 3 and continued to rise to reach a plateau at day 7 (Fig. 2E). Although cone outer segments seemed shortened at day 1 (Fig. 2D), their number only decreased significantly in the following days and was reduced by approximately 50% at day 7 (Fig. 2D, F and G) mirroring the subretinal MPs accumulation.
Taken together, our results demonstrate that RD leads to a rapid infiltration of myeloid cells, followed by T-cells and a protracted increase of the numbers of MCs and Mφs that started accumulating in the subretinal space by day 3. We also showed that this accumulation was strongly associated with cone death.
TSP-1 inhibits RD-induced subretinal MPs infiltration and associated cone loss
Thrombospondin-1 (TSP-1) is an extracellular matrix molecule that is produced by a wide variety of cell types, notably the RPE, inflammatory and resident macrophages . We and others have shown that it physiologically prevents age-related subretinal MP accumulation and inhibits excessive subretinal MP infiltration and choroidal neovascularization in the context of age-related macular degeneration [32–34] and controls T-cell response . To further explore the role of infiltrating MPs in RD-associated cone loss, we induced RD with diluted sodium hyaluronate that contained or not recombinant TSP-1 (100 µg/ml). Using flow cytometry and the same gating strategy as in Fig. 2 (Fig. 3A), we found that recombinant TSP-1 significantly reduced the number of T-cells, Mos, and Mφs but had no effect on the MC population (Fig. 3B). Accordingly, RT-qPCR analysis showed that recombinant TSP-1 significantly reduced the transcription levels of Il-6, Ifn-γ, Ccl2, Ccl3 and Ccl4 (Fig. 3C). Quantification of IBA-1+MPs and PNA+CAR+cones on triple-stained retinal flat-mounts at day 7 (Fig. 3D) showed that recombinant TSP-1 also significantly decreased subretinal MP accumulation (Fig. 3E) and increased cone survival in detached retinas compared with PBS controls (Fig. 3F and 3G).
Together, these findings show that the pharmacologically induced reduction of the population of infiltrating MPs and T-cells and cytokines expression significantly protects against RD-induced cone loss, suggesting that infiltrating T-cells, Mos, and Mφs directly contribute to cone loss in RD.
Insulin is essential for cone survival in vitro and delays RD-induced cone loss in vivo
Cones are highly dependent on glucose for metabolic activity and long term survival [15, 16]. It has been shown that insulin signaling pathways play a key role in cone glucose uptake [20, 21] and that activation of these pathways by systemic injection of insulin promotes cone survival in a mouse model of RP [18, 19].
In RD, cone glucose availability could become critical as highly metabolic active immune cells compete for fuel in the photoreceptor cell layer . Additionally, inflammatory cytokines such as IL-6, and IFN-γ might impede with insulin signaling, similar to the mechanism of type 2 diabetes .
To investigate whether diminished insulin signaling could contribute to RD-associated cone loss in adult retinas, we first examined the effect of insulin on 5 day retinal explants with or without human insulin and/or the specific insulin receptor inhibitor HNMPA (Fig. 4A). Quantification of PNA+CAR+ cones showed that cone survival was significantly increased in the presence of insulin in the medium culture compared to the control condition (Fig. 4A and 4B). The addition of an insulin receptor inhibitor HNMPA that blocks insulin receptor autophosphorylation, but not insulin growth factor 1 (IGF-1) receptor activation , resulted in a severe loss of PNA+CAR+ cones, not observed with its vehicle (DMSO) (Fig. 4A and 4B). Our results indicate that insulin signaling promotes cone survival in retinal explants confirming previous results in a mouse retinal degeneration model [18, 19].
Next, we induced RD in vivo with subretinal injection of diluted sodium hyaluronate containing (or not) human insulin (2 IU/ml). Quantification on immuno-stained retinal flat-mounts at day 7 (Fig. 4C) revealed that insulin treatment did not alter the numbers of subretinal IBA1+ MPs, but very significantly increased the number of PNA+CAR+ cones compared to PBS controls (Fig. 4D-F). Comparatively, addition of IGF-1 to the detachment inducing gel (at a concentration 100-fold higher than IGF-1 s ED50, which has been shown to reverse hypoalgesia in diabetic mice ) had no effect on the numbers of IBA1+ MPs or PNA+CAR+ cones, quantified on day 7 immuno-stained retinas (Fig. 4G-J).
Taken together, our results showed that insulin and insulin receptor signaling were essential for cone survival ex vivo of adult retinas and that insulin treatment very significantly inhibited RD-induced cone loss despite the unchanged MPs infiltration in vivo. This effect was not due to insulin-induced IGF-1R signaling, which can activate anti-apoptotic IGF-1 receptor signaling [38, 39], as IGF-1 had no comparable effect.
The insulin sensitizer rosiglitazone and metformin prevent RD-induced cone loss
IL-6 and IFN-γ, which we show are increased in RD, can inhibit insulin signaling in type 2 diabetes . This insulin resistance can at least in part be reversed by insulin sensitizers, such as rosaglitazone [40, 41]. To explore whether pharmacological improvement of insulin-signaling could reduce inflammation-induced cone degeneration in RD, we next examined whether rosiglitazone, could prevent cone loss in our mouse model of RD.
Mice received daily intraperitoneal injections of rosiglitazone or vehicle (DMSO 5%) 3 days before and throughout the 7 days of RD. The treatment also did not alter the increased levels of Il-6 and Ifn-γ mRNA levels in whole retinal mRNA and increased Ccl2 in four day RD samples (Fig. 5A). Quantification of IBA1-, PNA-, CAR triple-stained retinal flatmounts (Fig. 5B) also showed that rosiglitazone had no effect on the number of infiltration of subretinal IBA-1+ MPs at day 7 (Fig. 5C). Despite this lack of an anti-inflammatory effect, quantification of PNA+CAR+ cones, revealed that rosiglitazone significantly protected retinas against cone loss at day 7 (Fig. 5D and E). Interestingly, subretinal injection of metformin, a commonly used insulin sensitizer that also exerts an independent anti-inflammatory effect [42, 43], significantly increased cone survival and decreased subretinal MP accumulation in detached retinas compared with PBS controls (Fig. 5G-J).
In summary, our results show that the well-established insulin sensitizers rosiglitazone and metformin significantly curb cone loss in RD. The fact that we observed increased cone survival under rosiglitazone- and insulin- treatment in the absence of an anti-inflammatory effect strongly suggests that restored insulin signaling was the likely mode of action.