Epigallocatechin-3 Gallate Inhibits STAT-1/JAK2/IRF-1/HLA-DR/HLA-B and Reduces CD8 MKG2D Lymphocytes of Alopecia Areata Patients

Background: Alopecia areata (AA) is associated with Interferon- γ (IFN-γ) mediated T-lymphocyte dysfunction and increased circulating Interleukine-17 (IL-17) levels. Epigallocatechin-3-gallate (EGCG) specifically inhibits IFN-γ pathways and unlike Janus Kinase 1 and 2 (JAK1/JAK2) inhibitors (tofacitinib, ruxolitinib), EGCG is safer, more cost-effective, and is a topically active agent. Our objective is to test the mode of action of EGCG in vitro and ex vivo using HaCat, Jurkat cell lines, and peripheral blood mononuclear cells (PBMCs) of AA patients and healthy controls (HCs), respectively. Methods: distribution of T helper cells (Th1, Th17), and cytotoxic cells (CD8) in PBMCs isolated from 30 AA patients and 30 HCs was investigated by flowcytomterty. In vitro treatment of HaCat and Jurkat cells with 40 μm EGCG for 48 h was performed to measure the level of phosphorylation of signal transducer and activator of transcription protein STAT1, and replicated in ex vivo model using PBMCs of AA patients. Results: Interestingly, 40 μm EGCG is capable of completely inhibiting phosphorylation of STAT1 after 48 h in HaCat and Jurkat cells and ex vivo in PBMCs of AA patients. Based on QPCR data, the action of EGCG on p-STAT1 seems to be mediated via downregulation of the expression of JAK2 but not JAK1 leading to the inhibition of human leukocyte antigens (HLA-DR and HLA-B) expression probably via IRF-1. On the other hand, AA patients have significantly increased levels of Th1, Th17, and CD8 cells and the production of IFN-γ and IL-17 by PBMCs in AA patients was significantly higher compared to HC; p = 0.008 and p = 0.006, respectively. Total numbers of CD8+ cells were not significantly different between treated and untreated samples. However, CD8+ cells with positive Natural killer group 2 member D (NKG2D) transmembrane receptor (CD8+ NKG2D+ subset) was significantly reduced when PBMCs were treated with 20 μm EGCG for 48 h. Conclusion: These results suggest that EGCG has a synergistic action that inhibits expression of HLA-DR and HLA-B molecules via the IFN-γ pathway to maintain immune privilege in HF; also it reduces CD8+ NKG2D+ subset.

. Primers used in the q-PCR reactions.

Gene
Forward sequence Reverse Sequence IL-17A  AGATTACTACAACCGATCCACCT  GGGGACAGAGTTCATGTGGTA  STAT1  GCAGGTTCACCAGCTTTATGA  TGAAGATTACGCTTGCTTTTCCT  IRF1  GCAGCTACACAGTTCCAGG  GTCCTCAGGTAATTTCCCTTCCT  IL10  TCAAGGCGCATGTGAACTCC  GATGTCAAACTCACTCATGGCT  FOXP3  CGGACCATCTTCTGGATGAG  TTGTCGGATGATGCCACAG  TGFB1  CTAATGGTGGAAACCCACAACG  TATCGCCAGGAATTGTTGCTG  HLA-DR  ATCATGACAAAGCGCTCCAACTAT  GATGCCCACCAGACCCACAG  HLA-B  CCGGACTCAGAATCTCCTCAG  AAACACAGGTCAGCATGGGAA  GAPDH  GGAGCGAGATCCCTCCAAAAT  GGCTGTTGTCATACTTCTCATGG  β-Actin  TCCCCCAACTTGAGATGTATGAAG  AACTGGTCTCAAGTCAGTGTACAGG  JAK-1  GCGGAGGGATCGACAAATGG  TGGGACATAGCTTAAAGAGGCA  JAK-2  CTCTTTGTCACAACCTCTTTGCC  TTGGAGCATACCAGAGCTTGG  CCL-5 CTCATTGCTACTGCCCTCTGCGCTCCTGC GCTCATCTCCAAAGAGTTGATGTACTC Table S3. Thermal profile used in q-PCR reaction. A 10µ l final volume of real-time PCR reaction, containing 600nM of forward and reverse primers, was run in triplicate. The targeted sequence-specific amplification was detected using SYBER green detector and the thermal profile (Sup table 3). 5 µ g total RNA was reverse transcribed to a first strand cDNA using SuperScript® III First-Strand Synthesis System kit using random hexamer (18080-051, Life Technologies) by following the manufacturer's protocol where cDNA synthesis mix contains 10X RTbuffer+ 20mM MgCl2+ 0.1M DTT+ RnaseOUT+Glyceraldehyde-3phosphate dehydrogenase (GAPDH) was run for each sample as an internal control to normalise any variation in RNA amount, and an NTC control was run to regulate any contamination. The PCR reaction was carried out in 384-well plates using the ABI Prism 7900HT Sequence Detection System (Applied Biosystems).

Optimization of EGCG dosage
EGCG has been used topically at concentrations between 40-660 μΜ without inducing dermal toxicity (Zhao et al., 2015), with, an optimal dose previously used in cell culture of 50-75 µ M with HaCat cells (Zhu et al., 2014) and 100 µ M with epidermal kerantinocytes (Hsu et al., 2003). The first step in this study was to test the range of EGCG dosages that can be tolerated by the HaCat and Jurkat cell lines. 10, 20, 40, 60 and 100 µ M EGCG concentrations were used to treat these cell lines for 24 and 48 h, with cell viability assayed by microscopic evaluation and by staining the dead cells with trypan blue to find the percentage of viable cells in each group.

Cell viability by Trypan blue
The effect of EGCG on cell viability was found to be dose-dependent, regardless of the duration of treatment, with data from 48 h treatment presented here, and data from 24 h treatment found in appendix 3. The viability of HaCat cells compared to untreated samples, reduced slightly when treating the cells with 10 µ M EGCG and continued to drop gradually when increasing the dose of EGCG to 20, 40 and 60 µ M. However, this reduction was mild and not statistically significant. A significant sudden drop in cell viability was observed in samples treated with 100µ M EGCG (p < 0.001), where it declines to about 50% ( Figure S1).
The same trend was seen in Jurkat cells, although a very significant toxic effect (p < 0.001) of EGCG was observed at a lower dose of EGCG (60 µ M) for Jurkat cells, reducing to approximately 50% cell viability, which dropped further to reach about 30% when increasing the dose to 100 µ M. Therefore, it was concluded that 10, 20 and 40 µ M EGCG dosages do not show statistically significant adverse effects on cell viability in either cell line. Slight reduction in viability can be seen in both cell lines after treatment with lower doses of EGCG 10, 20, 40 µ M (not statistically significant) while a significant drop started to be seen at 100 µ M in HaCat cells and 60 µ M in Jurkat cells. The experiment was repeated three times and mean and SD were calculated. Asterisks denote a significant reduction in cell viability, *** p < 0.001.

Microscopic assessment of cell viability
To confirm the viability assay results, microscopic assessment of HaCat and Jurkat cells was performed after 48 h of treatment with EGCG. HaCat cells treated with 10, 20 and 40 µ M EGCG displayed the same morphology as the untreated control group, where the cells proliferated in a compact monolayer in a relatively non-structured pattern. On the other hand, 60 µ M and 100 µ M EGCG treated cells showed marked disruption in the monolayer of cultured cells, and a reduction in cell density with adherent cells displaying a longer, stretched morphology ( Figure S2). The Jurkat cells tend to be round and clump together to form grape-like colonies in untreated samples, and the same morphology can be seen for 10, 20 and 40 µ M EGCG treated samples ( Figure S3). As with the HaCat cells, Jurkat cells cannot tolerate the higher doses of EGCG (60 µ M and 100 µ M) and the cells appeared as discrete entities with small particles floating in the media, which are probably apoptotic bodies when compared to the microscopic images by Ivan et al. (Ivan et al., 2014). These results indicate the adverse effect of EGCG on cell viability, and thus its toxicity at higher dosages.  Cells treated for 48 h with 10, 20, 40, 60 or 100 µ M EGCG or left untreated as a conrol were examined under a light microscope at 20X magnification. The morphology in control versus 10, 20 , 40 µ M EGCG-treated cultures is relatively similar with grape-like colonies; however, 60 and 100 µ M EGCG alters colony morphology forming less compact colonies with more discrete cells .
Based on the viability assay and microscopic findings, we choose 40 µ M of EGCG as an optimal dose that can tolerated by HaCat and Jurkat cells without causing significant cell death. This dose was used in the subsequent experiments to investigate its effect on the expression of key molecules involved in JAK-STAT pathway.