It is noted that knee swelling was milder and the

It is noted that knee swelling was milder and the intensity of IL-4 staining (MFI) in the vehicle-treated group was lower in the second (Fig. 7a and d) than the first (Figs. 1a and 3f) AIA experiment. The variation in joint swelling between the two studies is most likely due to the models being performed by different investigators, as observed before [50,51]. The exact reason for reduced intensity of IL-4 staining in the second AIA experiment is not unknown, however it is most likely due to the use of different batches, and thus probably different brightness, of the APC anti-IL-4 antibody. The difference in IL-4 staining may also, to some extent, be due to the variation between the severity of the AIA model in the first and second experiment. However, although there was variation in knee swelling and the intensity of IL-4 staining between the two experiments, our results in Fig. 7 confirmed our AIA data in Fig. 1, Fig. 2, Fig. 3 by showing that Cpd43 significantly reduced joint swelling, while enhancing CD4 T cell apoptosis, IL-4 expression and proportion of Tregs. Other reports exist demonstrating that FPR ligands can affect Treg development during the initiation phase of the immune response. For example, administration of lipoxin A4 increases the number of Tregs in LNs [52], and SAA can promote Treg development, however it is not known if this effect is FPR-dependent [53]. Our study, showing that FPR ligation can decrease the expansion of pathogenic T Milnacipran HCl and augment Treg responses even after their initial activation and development, suggests that FPRs have true potential to act as therapeutic targets for the treatment of T cell-mediated diseases. CD4 T cells also play a pivotal role in CIA [3,54]. Here, reduction of CIA severity due to Cpd43 therapy was associated with decreased proliferation and increased apoptosis of CD4 T cells in draining LNs. Reduced T cell expansion due to FPR ligation may be due to the augmented CD4 production of IFNγ in the same lymphoid tissue since IFNγ is known to have anti-proliferative and pro-apoptotic effects on T cells and is inhibitory in CIA [55,56]. These results suggest that, in CIA, FPR ligation by Cpd43 attenuates joint inflammation by reducing the expansion of pathogenic effector CD4 T cells in secondary lymphoid organs. Similar to Cpd43, other FPR ligands have shown therapeutic efficacy in T cell-driven models of RA. Blockade of AnxA1 in adjuvant-induced arthritis after disease onset exacerbated joint inflammation, but the effect of AnxA1 on arthritogenic T cells was not assessed [57]. When given after the onset of disease, BML-111, a lipoxin A4 analog and FPR2 agonist, reduced CIA severity in association with decreased splenocyte proliferation [58]. However, since unpurified splenocytes contain a variety of lymphocytes, including T cells and B cells, capable of undergoing proliferation in response to antigenic stimulation, a specific effect of BML-111 on T cells was not demonstrated. These previous studies, although not demonstrating any specific effects on arthritogenic T cells, support our findings showing the therapeutic effect of Cpd43 in T cell-mediated joint inflammation. Cpd43 increased IL-17A production by splenic CD4 T cells in CIA and AIA, indicating that FPR ligation after the initial T cell activation promotes effector Th17 responses. However, since IL-17A is pathogenic in both of these models [59,60], decreased joint inflammation due to Cpd43 treatment cannot be explained by changes in IL-17A levels. Similarly, although B cells and autoantibodies contribute to the pathogenesis of RA [6], decreased arthritis severity in mice treated with Cpd43 is not due to changes in humoral immunity since B cell responses and autoantibody levels were not affected by FPR ligation. In these studies, Cpd43 could be mediating its effects on arthritogenic T cells via different mechanisms. Firstly, since CD4 cells express FPR2, but not FPR1 [[7], [8], [9]], Cpd43 is likely to have a direct effect on effector and regulatory T cells via FPR2. Indeed, our results showing a reversal of Cpd43-mediated effects on T cells via FPR2 inhibitors confirm that Cpd43 suppresses arthritogenic T cell immunity in an FPR2-dependent manner. In addition, it is possible that Cpd43 affects T cells indirectly. For example, natural killer (NK) cells, which express both FPR1 and FPR2, are activated by FPR ligation and exert a negative effect on pathogenic T cells in disease models such as EAE and CIA [7,[61], [62], [63]]. Therefore, here, Cpd43 could activate NK cells via FPR2 to suppress the disease-causing T cells.