Rheumatology Journal Club: RhoA-ROCK in autoimmunity

by Matt McConnell For Presentation on 11/10/2017 Index

• Introduction to RhoA-ROCK signaling literature in autoimmunity: – Downstream of Gna13 (Wu et al., Nat Com, 2017) – Relation to IRF4 and autoimmunity (Biswas et al., JCI, 2010)

• The Paper: – “Cigarette smoke inhibits ROCK2 activation in T cells and modulates IL-22 production” – (Weng et al., Mol Immuno, 2016) INTRODUCTION: RHOA-ROCK AND AUTOIMMUNITY Background: Gna13 and Osteoclastogenesis • Our lab discovered an interesting gene highly expressed during osteoclastogenesis… • But it inhibits osteoclast formation…

(Wu et al., Nat Com, 2017) Background: Gna13 and… Inflammation! • So, if it helps with bone resorption it should help with osteolytic bone disease…

But it also helps with inflammation!

(Wu et al., Nat Com, 2017) Background: Gna13 and Signaling • However, there are two problems with its use as a drug target: – It is a GAP - not drug targetable. – Its activity would need to be increased.

• This is its characterized signaling pathway… – …and RhoA is known to have roles in inflammatory disease…

(Wu et al., Nat Com, 2017) ROCK and RhoA • Since this journal club is primarily going to be about this downstream signaling pathway… • ROCK is short for “Rho-associated Protein Kinase” and it is activated by RhoA activity.

Van de Velde et al. (Prog Neurobio, 2015) ROCK and Autoimmunity

• Another interesting tie-in came from Biswas et al. (JCI, 2010) – They examined ROCK2 through Def6... a model of arthritis - also used in today’s paper (Pernis Lab). ROCK and Th17

• Th17 cells and their IL17A production has been used as a target in autoimmune disease. – ROCK inhibition blocked Th17 cell activity.

(Biswas et al., JCI, 2010) Methods: Def6 Trap Arthritis Model

• They used a Def6 gene trap model for reducing Def6 expression and this model has spontaneous arthritis when crossed with DO11.10 TCR mutant mice per their prior reports.

• They assessed the development of arthritis with the following rubric: – “Joint swelling was monitored by inspection and scored as follows: • 0, no evidence of erythema and swelling; • 1, erythema and mild swelling confined to the wrist or ankle; • 2, erythema and mild swelling extending from the wrist or ankle to the mid- paw; • 3, erythema and moderate swelling extending from the wrist or ankle to the mid-paw; and • 4, erythema and severe swelling encompassing the wrist or ankle, paws, and digits. – Score for all four paws were totaled for each mouse (Brand et al., 2004).” ROCK and Def6 Arthritis

• Their central claim is that ROCK inhibition by Fasudil reduces IL17 and ameliorates autoimmune arthritis.

Relevant RA measurement methods (Biswas et al., JCI, 2010) CIGARETTE SMOKE INHIBITS ROCK2 ACTIVATION IN T-CELLS AND MODULATES IL-22 PRODUCTION Background

• The Pernis Lab focuses on T-cell dysfunction and specifically the IRF4 binding protein Def6. – “[Def6] physically sequesters IRF4 and it also prevents IRF4 phosphorylation by the serine-threonine kinase, ROCK2.”

• IRF4 here is known to enhance Th17 differentiation and is essential for IL17A and IL21 cytokine production. (Brustle et al., 2007; Chen et al., 2008;Huber et al., 2008) Methods

• These works were mainly in vitro experiments using T-cells from their Def6 trapped mice.

• They assessed the cells using: – CD3 and CD28 (re)stimulation – ELISA and qPCR for cytokine production – ROCK kinase activity assays - multiple types. – “Cigarette smoke extract (CSE) was prepared by bubbling the smoke of one cigarette (3R4F, University of Kentucky, Lexington, KY) through 25 ml of PBS for ten minutes.” Fig. 1. Effect of cigarette smoke extract (CSE) on cytokine production by Def6−/−DO11.10CD4+T cells. Fig. 2. Exposure of Def6−/−DO11.10 CD4+T cells to cigarette smoke extract (CSE) inhibits IRF4 phosphorylation and ROCK2 kinase activity. Fig. 2. Exposure of Def6−/−DO11.10 CD4+T cells to cigarette smoke extract (CSE) inhibits IRF4 phosphorylation and ROCK2 kinase activity. Fig. 3. CSE decreases the activity of ARHGEF1 in Def6−/−DO11.10CD4+ T cells. Fig. 4. Effects of cigarette smoke extract (CSE) on cytokine production by nonautoimmune CD4+T cells cultured under either Th0 or Th17 conditions. Fig. 4. Effects of cigarette smoke extract (CSE) on cytokine production by nonautoimmune CD4+T cells cultured under either Th0 or Th17 conditions. Thoughts and Conclusions

• The paper seems to be a little thin on data. – e.g. no in vivo data is presented.

• However, they do show an interesting effect of cigarette smoke in enhancing IL-22 production. – IL-22 is usually increased in RA synovium. (Rutz, et al., 2013; Yang and Zheng, 2014;Xie et al., 2015)

• Coming back to the original idea, however, it seems unlikely that this is a major effector of Gna13’s anti-inflammatory effects… unless this ROCK2 ->-> IL22 effect is dominant over the effects on Th17 differentiation. • Gna13 -> RhoA -> ROCK -> IRF4 -> inflammation

Questions?