Preliminary results

a) Palmitic acid induce circulating monocytes differentiation into inflammatory macrophages

Inflammatory macrophages (iMφs) present in the retina of diabetic eyes mostly derived from circulating monocytes (Mos) and account for most of the cytokine production. We hypothesized that Mos exposure to hyperglycemia and/or hyperlipidemia may induce their differentiation into iMφs.

To test that hypothesis, Mos were isolated from venous blood of healthy volunteer individuals and allowed to differentiate in Mφs for 18h. Mφs were then exposed to either: normoglycemic medium (NG: DMEM with 5 mM glucose plus Ethanol), or hyperglycemic medium (HG:

DMEM with 25 mM glucose plus Ethanol) or palmitic acid (PA: DMEM with 5 mM glucose plus BSA-bounded palmitic acid) during 24h. Cytokines expression were quantified by RT-PCR and multiplex analysis. The expression of VEGF in Mφs was not alter under PA exposure.

Our team (Guillaume Blot) found that HG condition only slightly increased the expression and production of VEGF, CCL2 and IL-1ß compared with NG condition. In contrast, PA-treated Mφs exhibited elevated of pro-inflammatory cytokines, such as CCL2 and IL-1ß.

In this study, we found that only Mφs treated with PA differentiate into inflammatory Mφs.

This data suggests that dyslipidemia, rather than hyperglycemia, may participate to the chronic inflammation in DR.

b) Macrophages induce Müller cell activation after palmitic acid exposure

To investigate the role of inflammatory Mφs in retinal inflammation, we aim to study the interaction between iMφs and Müller glial cells in culture. We exposed hiPSCs-derived Müller cells to PA-treated iMφs supernatants (treated as cited above) for 18 hours.

Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized venous blood from healthy volunteer individuals. PBMCs were incubated in DMEM with 5 mM glucose for 18 hours at 37 ̊C to allow their adherence and differentiation in Mφs.

Mφs were then treated for 24 hours in either normoglycemic medium (NG/EtOH: DMEM with 5 mM glucose plus Ethanol) or hyperglycemic medium with palmitic acid (HG/PA :DMEM with 25 mM glucose plus PA).

hiPSCs-derived Müller cells were then exposed to supernatants obtained from Mφs culture in both conditions.

The expression of genes involved in angiogenesis and inflammatory DR-relevant pathways were then quantified by RT-PCR. Both angiogenesis factors, VEGF and ANGPTL4, and inflammatory factors, IL-1ß, CCL2, TNF-𝛼 and IL6, were significantly increased in hiPSCs-derived Müller cells after exposure to PA-treated iMφs supernatants (Figure 29).

Figure 29: Expression of angiogenesis factors, VEGF and ANGPTL4, and inflammatory factors, CCL2, IL-1ß, IL6, and TNF-𝛼 in hiPSCs-derived Müller cells exposed for 24 hours to supernatants obtained from macrophages previously cultured for 24 hours in either normoglycemic medium (NG SUR-Mos: macrophages cultured DMEM with 5 mM glucose plus Ethanol) or hyperglycemic medium with palmitic acid (Palm SUR-Mos :DMEM with 25 mM glucose plus PA).

A) The mean value of VEGF was 1.458 in NG SUR-Mos versus 6.251 in Palm SUR-Mos (p = 0.0170) B) The mean value of ANGPTL4 was 0.7676 in NG SUR-Mos versus 29.34 in Palm SUR-Mos (p = 0.0001) C) The mean value of CCL2 was 17.54 in NG SUR-Mos versus 56.96 in Palm SUR-Mos (p = 0.0047) D) The mean value of IL-1ß was 1.581 in NG SUR-Mos versus 8.558 in Palm SUR-Mos (p = 0.0003) E) The mean value of IL6 was 69.00 in NG SUR-Mos versus 1836.00 in Palm SUR-Mos (p = 0.0077) F) The mean value of TNF-𝛼 14.59 in NG SUR-Mos versus 98.18 in Palm SUR-Mos (p = 0.0274)

All results were normalized with expression of S26 and expressed as mean ± SEM (n = 6 in each group)

c) Role of IL-1ß and TNF-𝛼 in Müller cell activation To further investigate the interaction of inflammatory macrophages and Müller glial cells in high lipid condition, we aim to determine which cytokines in iMφs supernatants caused Müller cell activation. We study the role of IL-1ß and TNF-𝛼, two main inflammatory cytokines produced by iMφs, on hiPSCs-derived Müller cells.

For this experiment, A2 hiPSCs-derived Müller cells were treated for 18 hours in either:

- Normoglycemic condition (NG: DMEM with 5 mM glucose plus ethanol) - Normoglycemic condition with Palmitate (NG plus BSA-bounded PA) - Hyperglycemic condition (HG: DMEM with 25 mM glucose plus ethanol) - Hyperglycemic condition with Palmitate (HG plus BSA-bouded PA) - Normoglycemic condition with IL-1ß 10 ng/mL

- Normoglycemic condition with TNF-𝛼 10 ng/mL

We evaluated the expression of angiogenesis genes, VEGF and ANGPTL4, as well as inflammatory factors, IL6 and IL-1ß, in hiPSCs-derived Müller cells par quantitative RT-PCR.

The expression of VEGF, ANGPTL4 and IL6 was significantly increased in hiPSCs-derived Müller cells after PA treatment compared with NG or HG alone treatment. The treatment with IL-1ß did not alter the expression of VEGF and ANGPTL4 but had a positive feedback effect on his own expression in Müller cells. Similarly, the treatment with TNF-𝛼 did not alter the expression of VEGF and ANGPTL4 but significantly increase IL6 expression in hiPSCs-derived Müller cells compared with NG or HG treatment (Figure 30).

Figure 30: Expression of angiogenesis factors, VEGF and ANGPTL4, and inflammatory factors, IL6 and IL-1ß in hiPSCs-derived Müller cells exposed for 24 hours either to normoglycemic medium (NG: DMEM with 5 mM glucose plus Ethanol) or normoglycemic medium with palmitic acid (NG-Palm :DMEM with 25 mM glucose plus PA), or to

hyperglycemic medium (HG: DMEM with 25 mM glucose plus Ethanol) or hyperglycemic medium with palmitic acid (HG-Palm :DMEM with 25 mM glucose plus PA), or to IL1- ß, or TNF-a.

A) The mean value of VEGF was 1.028 in NG condition vs 1.685 in NG-Palm condition (p = 0.0009); and was 0.9871 in HG condition vs 1.476 in HG-Palm condition (p = 0.0121); and was 1.08516 in IL1- ß condition; and 1.09685 in TNF-a condition.

B) The mean value of ANGPTL4 was 1.689 in NG condition vs 108.4 in NG-Palm condition (p <0.0001); and was 1.325 in HG condition vs 91.65 in HG-Palm condition (p = <0.0001); and was 2.942 in IL1- ß condition; and 1.861 in TNF-a condition.

C) The mean value of IL6 was 0.9219 in NG condition vs 13.94 in NG-Palm condition (p

<0.0001); and was 1.130 in HG condition vs 11.75 in HG-Palm condition (p = <0.0001); and was 11.84 in IL1- ß condition (p = 0.0004 versus NG condition; p = 0.0005 versus HG condition); and 47.52 in TNF-a condition (p <0.0001 versus NG condition and versus HG condition).

D) The mean value of IL-1ß was 1.236 in NG condition vs 1.091 in NG-Palm condition (p = 0.7682); and was 0.5200 in HG condition vs 0.4921 in HG-Palm condition (p = 0.9173); and was 4.523 in IL1- ß condition (p = 0.0015) versus NG condition; p = 0.0005 versus HG condition); and 0,9833 in TNF-a condition (p = 0.5017 versus NG condition and p = 0.1415 versus HG condition).

All results were normalized with expression of S26 and expressed as mean ± SEM (n = 6 in each group)