Background/Purpose

Aberrant expansion of T_H-17 cells and deregulated production of IL-17 and IL-21 are involved in the pathogenesis of SLE. Production of IL-17 and IL-21 is critically dependent on the transcription factor, IRF4 (Interferon Regulatory Factor 4). Rho-associated protein kinases (ROCKs) can phosphorylate IRF4 and regulate its activity. The finding that ROCK activity is elevated in SLE patients and is associated with human T_H-17 differentiation, coupled with the ability of ROCK inhibitors to ameliorate autoimmunity in murine models of lupus suggest that targeting the ROCK pathway might be a novel therapeutic strategy for the treatment of SLE.  ROCK activation can be inhibited by Y27632 (a nonselective ROCK inhibitor that inhibits both ROCK isoforms, ROCK1 and ROCK2) or by statins (which inhibit ROCKs by interfering with their major upstream activator, RhoA).  Here, we examined the ability of Y27632 and simvastatin to inhibit the production of IL-17 and IL-21 by human T_H-17 cells and SLE T cells.  

Methods

We assessed the capacity of Y27632 (60uM-90uM) and simvastatin (0.2uM) to decrease ROCK activation and IL-17 and IL-21 production by cord blood CD4⁺ T cells cultured under T_H-17-skewing conditions (5ng/mL TGFb, 10ng/mL IL1-b, 20ng/mL IL-6, 50ng/mL IL-23, 5ug/mL anti-IL-4 and 10ug/mL anti-IFN-g). We also assessed the ability of Y27632 and simvastatin to diminish IL-17 and IL-21 production by stimulated SLE CD4⁺T cells.  ROCK activation was determined by an ELISA-based ROCK activity assay.  Plasma levels of IL-17, IL-21, and CCL20 were measured by ELISA. qPCR was used to determine gene expression of IRF4. All patients (N=24) met ACR criteria for SLE.  Demographics and clinical features were as follows: mean age 37 ± 11 years, 96% female, 8% Asian, 21% African American, 21% Caucasian, 50% Hispanic, SLEDAI score 6 ± 4, and 42% with nephritis.

Results

Compared to T_H0 cells, cord blood CD4⁺ T cells cultured under T_H-17-skewing conditions exhibited elevated ROCK activity that was inhibited by both Y27632 and simvastatin. IL-17 production was decreased by 60% in cord blood T_H-17 cells treated with either inhibitor.  IL-21 production was decreased by 83% (90uM Y27632) and 65% (0.2uM simvastatin) in cord blood T_H-17 cells.  Neither Y27632 nor simvastatin decreased IRF4 gene expression suggesting that their effects on IL-17 and IL-21 were not secondary to effects on cell viability.  Both Y27632 and simvastatin decreased IL-17 and IL-21 cytokine production by purified SLE CD4⁺T cells but neither treatment altered IFN-g protein production.  We also confirmed our previous findings that in a subset of SLE patients PBMCs showed elevated ROCK activity compared to PBMCs from healthy controls.  In this new cohort, the majority (79%) of the SLE patients had ROCK values that were at least 2SD above the mean ROCK value for the healthy controls.

Conclusion

These data indicate that the production of IL-17 and IL-21 by SLE T cells can be selectively inhibited by targeting the RhoA-ROCK pathway providing a rationale to inhibit the ROCKs as a means to reverse T cell dysfunction in SLE.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/targeting-the-rhoa-rock-pathway-to-reverse-t-cell-dysfunction-in-sle/