Background/Purpose: 14-3-3 eta is normally an intracellular protein and only in the disease state is it released into the extracellular space. We have previously presented data describing serum 14-3-3 eta’s diagnostic utility as a marker that complements RF and anti-CCP in early and established RA and is associated with joint damage in RA and PsA. Circulating autoantibodies directed to citrullinated proteins are highly specific for RA, but a significant percentage of patients are or remain sero-negative for anti-CCP. Given that 14-3-3 eta is liberated into the synovial space where PAD enzymes are present, we investigated whether 14-3-3 eta represents a novel citrullination target that could identify anti-CCP negative RA patients.

Methods: Assays to measure the autoantibody levels to either non-citrullinated (non-cit) or citrullinated (cit) 14-3-3 eta were developed using full-length recombinant human 14-3-3 eta. Full-length cit-14-3-3 eta was generated by citrullinating the protein with purified PAD. Reactivity to non-cit and cit-14-3-3 eta was evaluated in 3 anti-CCP positive RA patients to confirm the presence of autoantibodies to cit-14-3-3 eta. To evaluate whether these novel autoantibodies are detectable in anti-CCP negative RA patients and differentially expressed compared to healthy controls, reactivity to both non-cit and cit-14-3-3 eta was measured in 30 anti-CCP negative RA patients and 30 confirmed anti-CCP negative healthy controls.  Mean and median fluroescence intensity [1 MFI = 1 unit (U)] was evaluated and corresponding t-tests and Mann-Whitney U-tests were used to determine differences within and between groups. The area under the ROC curve (AUC) was generated for diagnostic utility estimates and to determine likelihood ratios (LR) for various anti-cit-14-3-3 eta cut-offs.

Results: Compared to non-cit 14-3-3 eta, up to 25X higher reactivity was observed to cit-14-3-3 eta in 2 of the 3 anti-CCP positive RA patients, revealing the novel expression of autoantibodies to the citrullinated form of 14-3-3 eta in RA. Within the anti-CCP negative RA group, significantly higher reactivity was observed to cit versus non-cit-14-3-3 eta at 1943U versus 395U, p=0.01. No significant differences in reactivity were observed within the healthy group. Anti-cit-14-3-3 eta expression was significantly higher in anti-CCP negative RA patients with means (SD) and medians (min-max) of 1943U (3045U) and 306U (68-8982U) compared to 155U (122U) and 100U (45-564U) for healthy controls, p<0.002.  The corresponding ROC AUC for anti-cit-14-3-3 eta differential expression in anti-CCP negative RA patients compared to healthy controls was 0.79 (95% CI 0.68-0.91; p<0.0001). At a cut-off of 320U, the specificity and sensitivity were 90% and 50% delivering an LR positive of 5 increasing to 14 at 439U with a corresponding specificity of 97% and sensitivity of 47%.

Conclusion: Extracellular 14-3-3 eta protein has been described as an RA diagnostic biomarker with prognostic and therapy monitoring applications. 14-3-3 eta also represents a novel citrullination target that is differentially expressed in anti-CCP negative RA patients versus healthy controls, indicating that anti-cit-14-3-3 eta may improve RA diagnosis.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/14-3-3-eta-is-a-novel-citrullination-target-in-rheumatoid-arthritis-that-enhances-diagnostic-utility-in-anti-ccp-negative-patients/