2022 Annual Meeting and Alumni Reunion
Category : Basic and Translational Research Candidate : Seokjoo Lee Poster #: B15
CD11b + Gr-1 + MDSC Prevent IL-6-Induced Treg Dysfunction Partially Through Secretion of IL-10
Seokjoo Lee , Francesca Kahale , Aytan Musayeva , Shudan Wang , Tomas Blanco , Thomas H . Dohlman , Sunil K . Chauhan , Yihe Chen , Reza Dana
Purpose : Regulatory FoxP3 + T cells ( Tregs ) are critical in maintaining corneal allograft tolerance . The highly vascularized inflammatory microenvironment in corneal transplantation adversely affects the suppressive function of Tregs . Emerging evidence indicates the functional crosstalk between CD11b + Gr-1 + myeloidderived suppressor cells ( MDSCs ) and Tregs , but little is known about the impact of CD11b + Gr-1 + MDSCs on the rescue of inflammation-induced Treg dysregulation . The purpose of this study is to determine the rescue effect of CD11b + Gr-1 + MDSCs on IL-6-mediated Treg dysregulation , and to further elucidate the underlying mechanism by which this rescue occurs .
Methods : BALB / c bone marrow cells were cultured in complete RPMI medium in the presence of IL-6 ( 20ng / ml ) and GM-CSF ( 20ng / ml ) for 4 days to differentiate MDSCs . CD11b + Gr-1 + MDSCs and CD11b + cells were obtained using CD11b + Gr-1 + MDSC MACS kits . Tregs were isolated from BALB / c splenocytes using CD4 + CD25 + MACS kits . Tregs ( 2.5x105 ) were co-cultured either with CD11b + Gr-1 + MDSCs or with CD11b + cells at a ratio of 1:1 in the absence or presence of IL-6 ( 40ng / ml ) for 48 hours . IL-10 knockdown ( KD ) CD11b + Gr-1 + MDSCs were generated by IL-10 siRNA ( 10nM ) transfection for 6 hours prior to co-culture with Tregs . FoxP3 expression and IL-10 secretion of Tregs were analyzed by flow cytometry , immunoblotting , and real-time PCR .
Results : Flow cytometric quantification showed Tregs cultured with IL-6 had significantly reduced expression of FoxP3 ( 12.5 ± 0.3 % vs . baseline 15.97 ± 0.6 %, p = 0.0318 ) and modestly decreased secretion of IL-10 ( 5.16 ± 1.2 % vs . baseline 10.7 ± 1.9 %, p = 0.0495 ) as compared to culture without IL-6 . Addition of CD11b + cells to culture partially preserved FoxP3 expression ( 18.2 ± 0.3 %) and IL-10 production ( 32.63 ± 12.3 %) by Tregs in the presence of IL-6 . In contrast , addition of CD11b + Gr-1 + MDSCs to culture led to significantly higher FoxP3 ( 32.93 ± 1.9 %, p = 0.0035 ) and IL-10 ( 60.03 ± 13.3 %, p = 0.0238 ) expression by Tregs than addition of CD11b + cells . Furthermore , Tregs co-cultured with IL-10 KD CD11b + Gr-1 + MDSCs exhibited significantly reduced FoxP3 + Tregs ( 23 ± 1.8 %) and IL-10-secreting FoxP3 + Tregs ( 55.93 ± 4.4 %) as compared to those co-cultured with WT CD11b + Gr-1 + MDSCs ( 32.3 ± 2.4 % and 82.53 ± 3.4 %, respectively , p = 0.0403 and p = 0.0015 , respectively ).
Conclusions : Our data demonstrate that in vitro generated MDSCs are effective in preventing inflammatory cytokine-induced impairment of Treg function , which can be explored as a novel therapeutic approach in ocular autoimmune and alloimmune disorders , including high risk corneal transplantation .