OP0022 RHO EXPRESSION FACILITATES T CELL MIGRATION TO LYMPH NODES IN RESPONSE INFLAMMATION
Authors: E. Malmhäll-Bah, K. M. Andersson, M. C. Erlandsson, M. Brisslert, O. Khan, M. Akula, I. M. Jonsson, M. Bergö, M. I. Bokarewa
Journal: Annals of the Rheumatic Diseases (2021)

Background: Deficiency in geranylgeranyltransferase type I (GGTase-I) results in accumulation of active Rho family proteins RhoA, Rac1 and Cdc42, responsible for cell communication and migration. We reported that mice with GGTase-I deficient macrophages (GLC mice) develop a spontaneous and age-dependent arthritis, reproducing pathology of RA [1]. Objectives: We study how GGTase-I deficiency in Mø changes T cell phenotype to facilitate their translocation to joints and the development of arthritis. Methods: GLC mice were developed on a mixed genetic background (129Ola/Hsd-C57BL/6) by Cre-technology using LysM-promotor to knockout the Pggt1b gene in Mø[2]. CD4 + cells were isolated from spleen and lymph node (LN) of 16 weeks-old mice (GLC n=7, wt n=5) expected to have high prevalence of arthritis. RNA was extracted to measure expression of the Rho proteins and signature genes to characterize differences in Th-subtypes and migration abilities of CD4 + cells between GLC and wt mice. Furthermore, Illumina RNAseq analyzed the transcriptome of LN CD4 + cells. In a separate experiment we treated GLC mice with CTLA4-FP (n=12) or PBS (n=11) for 20 weeks from the age of 5 weeks. Rationale was to disrupt Mø/T cell contact to prevent arthritis. To study Rho-protein dependent phenotype in human RA, we performed RNAseq of sorted CD4 + cells of RA patients. Results: RNAseq showed that CD4 + cells in LN of GLC mice had IFN-γ dependent cytotoxic profile and upregulated numerous pro-inflammatory genes including Eomes , Cxcr3 , Tigit , Tnfsf10 , Il-1rl1 , Stat1 , Jak3 , Irf7 , Irf5 , Ptpn13 . Furthermore, the over-represented genes often depended on the IRF family in their transcription. GLC mice overexpressed Cdc42 and Rac1 in spleen CD4 + compared to wt (p=0.005 and p=0.048 resp.). Spleen GLC CD4 + cells had higher levels of α5β1 and α2β2 integrins, strongly correlating to Cdc42 (r= 0.61 p=0.0027 and r=0.50, p=0.018) and arthritis (r=0.64, p=0.0015 and r=0.69, p=0.0004). Importantly, Cdc42 , Rac1 , and RhoA were higher expressed in LN CD4 + compared to spleen (p=0.016, p=0.031 and p=0.016). In addition, Itgb1 coding for β1 integrin, was upregulated in GLC CD4 + cells of both spleen and LN (p=0.003 p=0.03, resp.), suggesting Rho proteins are important for migration of CD4 + cells to the joint draining LN and for arthritis development. CD4 + cells that migrated to the LN had high proportion of Foxp3 + cells. This also correlated to the expression of Itgb1 (r=0.84, p=0.0012) presenting a plausible mechanism for increased influx of Tregs into joints. Several observations are in favor of this notion. First, GLC mice expressed more Foxp3 in LN compared to spleen CD4 + cells (p=0.016). Second, transcription of Foxp3 in LN CD4 + cells was higher in GLC mice compared to wt (p=0.015). Third, this high Foxp3 coexisted with low transcription of Lef1 (p=0.03), required for Treg immunosuppression. Last, Foxp3 correlated negatively to both Lef1 (r=-0.72, p=0.017), and its cofactor Tcf1 (r=-0.75, p=0.01). CTLA4-FP reduced inflammation in GLC mice evident as lower IFN-γ, IL-6 and TNF-α production (p=0.0002, p&lt;0.0001 and p&lt;0.0001 resp.) and the number of CD25 + CD4 + cells in spleen (p=0.027). In contrast, we observed increased IL-17A production (p=0.056). However, CTLA4-FP treatment did not affect migration of CD4 + cells enriched with Rho-protein into draining LN nor alleviate arthritis. Similar to the GLC mice, CD4 + cells of RA patients with high expression of RhoA, Rac1 and Cdc42 demonstrated enrichment for Th1 signature genes including IFNG , TBX21 , Eomes , IL2RA , IL2RB , IL12RB2 , TNF , IL18RAP (all, adj. p&lt;0.05). Conclusion: This study shows that accumulation of Rho-proteins in CD4 + cells results in pro-inflammatory IFN-γ dependent phenotype in mice and human RA. Accumulation of RhoA, Rac1 and Cdc42 proteins trigger the migration of CD4 + cells into joint draining LN and facilitates arthritis. Inhibiting Mø/T cell contact in GLC mice did not suffice to prevent migration of Rho-protein expressing cells and arthritis References: [1]Khan, O.M., et al. J Clin Invest, 2011. 121 (2): p. 628-39. [2]Akula, M.K., et al. Nat Commun, 2019. 10 (1): p. 3975. Disclosure of Interests: None declared