This study is the first to report an association between the responsiveness of the peripheral innate and adaptive immune systems and myocardial dysfunction in patients with RA. We have shown that an 11-cytokine profile differentiated patients with moderate-to-severe LVDD from patients with normal diastolic function. We created an immune response score combining the information from each of the 11 cytokines in the profile to summarize the responsiveness of canonical immune pathways. This score was strongly associated with moderate-to-severe LVDD after adjusting for potential confounders, including age, sex, cardiovascular risk factors, RA characteristics, and immunomodulatory therapies, suggesting it provides unique information above and beyond standard clinical predictors. Neither the differences in ex vivo cytokine production nor the presence of moderate-to-severe LVDD were sufficiently explained by variation in the frequencies of immune cell subsets in the peripheral blood, suggesting that ex vivo functional analysis has the advantage of greater immunological discrimination. We conclude that aberrant systemic immune responsiveness is strongly associated with advanced myocardial dysfunction in patients with RA.
In contrast, the patients with mild LVDD were not significantly different from those with normal LV function. The issue is the extent of continuity along a pathophysiological spectrum from mild to severe LVDD and, ultimately, HF. The immunological findings could represent an epiphenomenon related to established myocardial disease rather than a marker of pre-clinical myocardial injury. However, we favor the alternative hypothesis that the immune response signature is a preclinical predictor of LVDD, for two reasons. First, the significance of “mild” LVDD remains a matter of debate. Whereas severe LVDD is clearly a pre-clinical predictor of future symptomatic HF and mortality, milder LVDD may stabilize or improve in up to 50% of patients (20
). Perhaps only a subset of patients with mild LVDD may, over time, be at substantial risk for functional deterioration.
Second, based on our recently published work and that of others, many of our immunological findings are known to occur early in the course of RA, prior to the development of any systemic disease complications (16
). The responsiveness of cytokines in T cell immune networks was significantly decreased in the group with moderate-to-severe LVDD. Previous studies have demonstrated that in vitro Th1 (i.e., IFN-γ) and Th2 (i.e., IL-4) cytokine release by stimulated PBMC is significantly decreased in patients as compared to control subjects (22
). Moreover, the responsiveness of several cytokines typically produced by myeloid lineages, including monocytes and dendritic cells, or B cells, was significantly increased among the patients with moderate-to-severe LVDD. Previous studies have also established that immune pathways of myeloid lineages are activated in patients with RA, with respect to induction of Toll-like receptor (TLR) signaling (25
) and proinflammatory cytokine production (28
). Immunological profiles similar to ours have previously been associated with disease activity (30
) and severity (32
), suggesting they might predict higher risk of systemic complications. Further, evidence suggests that disease-modifying therapies, including anti-TNF agents, can favorably modulate the aberrant T cell and innate immune responses (29
The immune response score showed considerable overlap between the groups, with over 25% of subjects in both the normal and mild LVDD groups having ‘high’ scores. We speculate that subsets of these patients might be at increased risk for myocardial dysfunction over the course of their disease. Ultimately, the abovementioned issues underscore an important limitation of this study, the cross-sectional design, which precludes assessment of causation. We are planning longitudinal studies of the cohort to address these unanswered questions.
Our findings emphasize that the immune response signature may be most useful in the context of a multivariable risk profile. We showed that the profile of the immune response score, serum IL-6, serum BNP, and glucocorticoid use provided the highest accuracy in identifying individuals with LVDD. Serum IL-6 is an important determinant of myocardial dysfunction and predicts significantly higher risk of incident HF and related mortality in the general population (37
). BNP is a hormone produced by the ventricles in response to pressure or volume overload for the purpose of causing diuresis, natriuresis, and vasodilatation. Serum BNP (or the closely related N-terminal pro-BNP) may be useful in detecting preclinical LVDD, both in the general population and patients with RA (41
). BNP and the N-terminal pro-BNP are associated with clinical disease activity and inflammatory biomarkers in RA, including IL-6, suggesting BNP is also a marker of myocardial inflammation (43
). In our cohort, serum BNP alone had inadequate specificity to be useful as a screening test for myocardial dysfunction (45
), but our findings suggest BNP does add value in the context of a multivariable risk profile. Lastly, therapy with glucocorticoids, particularly high cumulative dose and recent use, is associated with an increased risk of HF (46
This study only included patients with RA, so it is uncertain whether the findings are unique to RA or are similar to what occurs among LVDD/HF patients in the general population. A preliminary analysis of the immune response score in 5 non-RA subjects with moderate-to-severe LVDD as compared to 18 non-RA subjects with normal diastolic function revealed no apparent differences. Though consistent with a unique effect of the immune response signature in RA, considering the small numbers of subjects, the question remains open. Further comparison of the immune response signature between LVDD patients with and without RA might reveal distinctions that could ultimately be informative in understanding the pathogenesis of HF that occurs in these different groups.
Other limitations of this study include the potential effects of multiple testing on our cytokine analyses, but the use of a stringent threshold for statistical significance minimizes this issue. Clinical factors including disease activity and medication effects likely influence the cytokine responses, and this cross-sectional study is unable to disentangle the influence of these variables on the associations of cytokine profiles with LVDD. However, the findings of our multivariate analysis indicate that the immune response signature provides unique information above and beyond standard CV risk factors. The flow cytometry analyses had small sample size and a limited number of markers, so a different flow cytometric approach to biomarker discovery might be considered in future research. Additionally, the cell-based assays are complex and will be challenging to standardize across laboratories in wider usage. However, the successes of cell-based assays including the T-Spot®. TB and QuantiFERON®-TB-Gold tests provide reassurance that this obstacle can be addressed with assay refinement and technological advances. Finally, although only 56% of eligible subjects participated, bias due to the selection of cases with milder disease is unlikely since non-participants were found to have lesser education, which is a known risk factor for more severe RA.
In conclusion, we have reported the discovery of an immune signature based on the responsiveness of ex vivo cytokine production by peripheral blood cells that is associated with advanced myocardial dysfunction in patients with RA. Future studies should evaluate this signature, perhaps in the context of a multivariable risk profile, for predicting deterioration of myocardial function over time. The findings also inform new hypotheses for the role of aberrant systemic immune function in the pathogenesis of myocardial disease in persons with RA.