IL 10 regulation of miRNA 155 in activated macrophagesIL-10 regulation of miRNA-155 in activated macrophagesg p gS l i Ch E S A d Mi L d Ali M iSylvia Cheung, Eva So, Andrew Ming-Lum, and Alice MuiSylvia Cheung, Eva So, Andrew Ming Lum, and Alice MuiD t t f S Bi h i t d M l l Bi l U i it f B iti h C l bi V BC C dDepartment of Surgery, Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada

IL-10 does not regulate the transcription of pri-miR-155.Introduction miRNA biogenesis SHIP1 activator is able to inhibit LPS-induced miR-155.IL 10 does not regulate the transcription of pri miR 155.Introduction miRNA biogenesis SHIP1 activator is able to inhibit LPS induced miR 155.

Our lab studies the molecular signalling pathways utilized by A BOu ab stud es t e o ecu a s g a g pat ays ut ed byinterleukin-10 (IL-10) to inhibit macrophage activation The gene

Figure 2. RAW264.7 cells wereA B

interleukin-10 (IL-10) to inhibit macrophage activation. Thej l f IL 10 i t t i i fl t

gene transfected with BIC promotermajor role of IL-10 is to prevent excessive inflammatory reporter (miR-155 host gene) orresponse (which can lead to different disorders including RNA Pol II IkBζ promoter reporter (positive

cancers) by inhibiting synthesis of pro-inflammatory cytokines control). After 24 hours rest, cellscancers) by inhibiting synthesis of pro inflammatory cytokinessuch as TNFα and IL-6 IL-10 is also involved in pri-miRNA were stimulated with LPS +/- IL-10such as TNFα and IL-6. IL-10 is also involved int l t ti t l l t d IL 10 l l b d

for 2 hours. AQX-MN100transplantation tolerance; elevated IL-10 levels are observed

DroshaSHIP1 activator

in patients with successful transplants and administration of Drosha

i ( ) h h l f ( )IL-10 is able to enhance survival in mouse transplantation IL 10 d t ff t th l t f iR 155Figure 5. (A) The chemical structure of a SHIP1 activator, AQX-MN100. (B)

ll d h h l dIL 10 is able to enhance survival in mouse transplantationmodels

IL-10 does not affect the nuclear export of pre-miR-155.nucleus pre-miRNA RAW264.7 cells were pretreated with ethanol or 10 uM AQX-MN100, and

l d h f h d d l (*models. nucleusstimulated with LPS for the indicated times prior to qPCR analysis. (* p <

*** **** )Exportin 5 Figure 3 RAW264 7 cells were

0.05, ***, p < 0.001, **** p < 0.0001).

In this study, we investigate the mechanism of IL-10 function,po t 5

cytoplasmFigure 3. RAW264.7 cells werestimulated with LPS +/- IL-10 for

especially its action on a pro-inflammatory microRNAstimulated with LPS +/- IL-10 forthe indicated times prior to Conclusion and Significanceespecially its action on a pro inflammatory microRNA

(miRNA) miR 155 in macrophages Upregulation of miR 155 pre-miRNAthe indicated times prior tofractionation Levels of pre-miR-

Conclusion and Significance(miRNA), miR-155, in macrophages. Upregulation of miR-155i i t d ith j ti i t l t i i t

pre miRNA fractionation.. Levels of pre-miR-155 in total cellular nuclear andis associated with organ rejection in transplant recipients. 155 in total cellular, nuclear andcytoplasmic fractions were ModelThus, understanding the mechanism by which IL-10 regulates Dicercytoplasmic fractions weredetermined by qPCR (*** p <

Modelg y gmiR-155 will aid in the development of therapeutic to induce

determined by qPCR. ( p <0 001) LPS IL-10miR 155 will aid in the development of therapeutic to induce

tolerance to the transplanted organ miRNA0.001) LPS IL 10

tolerance to the transplanted organ. miRNA

RNA-inducedRNA induced Silencing ComplexIL-10

RISC miRNA

Silencing Complex

RISC: miRNA

SHIP1AKT

miR-155 STAT3l i l i

miR-155 STAT3Translational repression IL-10 inhibits LPS induced miR-155 via SHIP1 and STAT3.miR 155 IL 10 inhibits LPS induced miR 155 via SHIP1 and STAT3.miR-155

A feedback loop is formed involving AKT, miR-155 and SHIP.p g ,This auto-regulatory loop is likely to prevent both excessiveThis auto regulatory loop is likely to prevent both excessivepro-inflammatory AND anti-inflammatory responses duringmRNA degradation pro-inflammatory AND anti-inflammatory responses duringth f i ll f tiMacrophage Inflamed organ gthe course of immune cell function.Macrophage Inflamed organ

Also our work on the SHIP1 activator suggests that AQXAlso, our work on the SHIP1 activator suggests that AQX-i i i l i i fl i d

h b d f d hMN100 can mimic IL-10 in regulating inflammation, and

IL-10 inhibits LPS induction of pri-miR-155 and miR-155 expression in macrophages. thus should prolong graft survival and inducep p p g p g gtransplantation tolerance.transplantation tolerance.

Figure 1. RAW264.7 cellsgwere stimulated with LPS +/-IL-10 for the indicated timesprior to detection of pri-

Figure 4 SCRMB and SHIP1 siRNA transduced cells were treated with Doxp pmiR-155, pre-miR-155 and

Figure 4. SCRMB and SHIP1 siRNA transduced cells were treated with Doxfor 48 ho rs to knockdo n SHIP1 protein Cells ere pretreated ith DMSO

, pmiR-155 levels by qPCR. (* p

for 48 hours to knockdown SHIP1 protein. Cells were pretreated with DMSOSTA 21 STAT3 i hibit f 1 h i t ti l ti ith LPS +/ IL

y q ( p< 0.05, **** p < 0.0001).

or STA-21, a STAT3 inhibitor, for 1 hours prior to stimulation with LPS +/- IL-10 f 2 4 h E i l l f i iR 155 i th 2 h l

, p )10 for 2 or 4 hours. Expression levels of pri-miR-155 in the 2-hour samples

d iR 155 i th 4 h l d b PCR (** < 0 01and miR-155 in the 4-hour samples were measured by qPCR. (** p < 0.01,*** < 0 001 **** < 0 0001)*** p < 0.001, **** p < 0.0001)