p38α inhibitors demonstrate limited utility in RA despite abundant pre-clinical evidence predicting efficacy. One possible explanation is that p38α has anti-inflammatory functions in addition to its well-defined pro-inflammatory actions. For instance, p38α in macrophages regulates MAPK phosphatases and immunosuppressive cytokines such as IL-10. Genetic deletion of the p38α gene in macrophages increased acute skin edema after toxic exposures like ultraviolet light (12
). These data raise the possibility that p38 deficiency in macrophages could increase the severity of chronic inflammation mediated by innate immunity. If so, then it might explain why inhibition of this kinase results in only a limited benefit in diseases dominated by macrophage cytokines like RA.
Macrophages participate in passive K/BxN arthritis evolution (27
) and in established RA, where anti-TNF and IL-6 therapy demonstrate clinical efficacy (28
). The number of macrophages in rheumatoid synovial biopsies also correlates with joint damage (29
), and depletion of macrophages by therapeutic agents is associated with improvement in RA for some therapeutic agents (30
). Therefore, a therapy that interferes with macrophages de-activation could increase disease severity even though production of some pathogenic cytokines is suppressed.
Previous studies suggested that p38 blockade or deficiency in macrophages suppresses production of the anti-inflammatory cytokine IL-10 and enhances activation of ERK and JNK (12
). Our studies confirmed this observation and led us to explore complex immune-mediated models of inflammation. In vitro experiments focused on chemical p38 inhibition to mimic the situation in human clinical trials, although the results were similar for p38α-deficient macrophages. The only difference observed between genetic and small molecule inhibitors related to IL-6 expression, where genetic deletion appeared to have less effect.
p38α ΔLysM mice demonstrated increased disease severity in a transient model as well as a more persistent month-long chronic model. Cytokine and MMP expression, presumably by from other cell types such as fibroblast like synoviocytes or mast cells, was increased in the joints of p38 deficient mice, as was activation of downstream cytokine signaling molecules like STAT3. Of particular interest, P-ERK levels were also higher in the inflamed joints of p38α ΔLysM mice and correlated with our findings in cultured macrophages. Activation of mast cells and neutrophils are critical initiating events in this model, but the p38α ΔLysM mice have normal p38 expression in these lineages. Thus, the effects observed can be ascribed to macrophages. Similar results in the AIA model in the absence of altered adaptive responses suggest that the pro-inflammatory effect is due to an effect on innate immunity.
The murine data are also consistent with the role of p38 in cultured RA synovial tissue cells, where we confirmed that a p38α inhibitor substantially decreased TNF production from rheumatoid synovial tissue cells (26
). We also noted that IL-6 production was only modestly affected by the inhibitor, while IL-10 production was markedly decreased. These data support the notion that p38 regulates both pro- and anti-inflammatory cytokines in RA cells and that could interfere with clinical efficacy.
As an alternative strategy to targeting downstream kinases like p38, we have advocated shifting emphasis to upstream signaling molecules(6
). This approach has met with success, and it is especially noteworthy that JAK and Syk inhibitors demonstrate efficacy in RA (31
). Our previous studies in the p38 pathway suggest that inhibiting either of its two upstream regulators, namely MKK3 and MKK6, might be more effective than a traditional direct p38 inhibitor. For instance, mice deficient in either MKK have decreased joint inflammation and destruction in the passive K/BxN model and collagen-induced arthritis (22
). MKK3 deficiency also mimics p38 inhibitors in a murine model of allodynia (35
Based on these studies, we evaluated the profile of MKK deficient macrophages in vitro. These studies demonstrated striking dissociation of IL-6 and IL-10 regulation that distinguishes MKK and p38 function. Differential regulation can therefore provide the anti-inflammatory benefit by modulating, rather than blocking, the p38 pathway. We previously showed that p38 and MK2 activities do not necessarily correlate in a linear manner. A threshold level of p38 activation might be necessary for efficient MK2 activation, which is not reached in either mkk3−/−
). Increased activation of other MAPKs like ERK and JNK was also substantially less in the MKK deficient cells.
The potential benefit of targeting MKK3 or MKK6 was supported by studies using bone marrow chimeras. MKK deficient marrow protected normal mice from passive K/BxN arthritis, while normal marrow failed to correct the defect in mkk3−/− or mkk6−/− mice. These data suggest that murine macrophages lacking the MKKs are protective, in contrast to the p38α deficient cells. The chimera study design does not precisely mimic the situation in p38α ΔLysM mice because MKK3 or MKK6 is deleted in all bone marrow cell lineages. However, it provides evidence that myeloid cells deficient in the p38 pathway due to targeting upstream kinases has the opposite effect of blocking p38 in macrophages.
Taken together, the in vitro and in vivo studies suggest that selectively blocking an MKK might be beneficial in inflammatory arthritis by sparing p38-regulated functions like IL-10 and DUSP1 expression. This approach could maintain negative feedback loops that are blocked by p38 inhibitors. Therefore, the data provide a rationale for strategies that inhibit upstream MKKs as a therapeutic approach in RA.