POS1376 SPUTUM CALPROTECTIN LEVELS DO NOT ADD TO ABILITY OF SPUTUM ANTI-CCP AND RF TO PREDICT FUTURE DEVELOPMENT OF RA IN SERUM ANTI-CCP POSITIVE INDIVIDUALS AT-RISK FOR RA

Abstract

Background:

Presence of serum anti-cyclic citrullinated peptide (CCP) antibodies can predict the future development of clinically evident rheumatoid arthritis (RA). Data support that inflammation and autoimmunity in RA likely originates at a mucosal site. Our group previously found that RA-associated antibodies in the lung (i.e., sputum anti-CCP and rheumatoid factor (RF)) are increased in at-risk individuals who later develop RA. Neutrophil extracellular trap (NET) formation can be a source of extracellular citrullinated proteins that are antigenic targets of anti-CCP antibodies. Our group has previously reported enhanced NET formation of sputum neutrophils from the lungs of individuals with RA and at-risk for RA that correlated with sputum anti-CCP antibody levels. Calprotectin is an S100A8/A9 heterodimer released from neutrophils upon NET formation and can be used as a measure of NETosis, but it has not yet been evaluated as a potential predictive biomarker for RA.

Objectives:

The study objective was to evaluate the relationship between sputum calprotectin and transitions from systemic autoimmunity to clinical RA.

Methods:

We included 93 participants who were serum anti-CCP-IgG positive (CCP3, Werfen), without examination evidence of inflammatory arthritis at baseline, had at least one follow-up study visit, and had a baseline sputum sample (i.e., induced sputum by inhalation of hypertonic saline). Twenty-four participants (26%) developed inflammatory arthritis consistent with incident clinical RA within 3 years of follow-up (median 19 months), and 69 participants (74%) did not develop evidence of inflammatory arthritis after at least 12 months of follow-up. Baseline sputum cell-free supernatant and serum were tested by ELISA (Werfen) for calprotectin, anti-CCP-IgG (CCP3), anti-CCP-IgA (CCP3.1 plate with research modification to detect IgA only) and RF-IgM. Sputum antibody positivity was defined as a level above the 95^th percentile in a cohort of 115 healthy controls. Chi-square, non-parametric and cox regression analyses were performed as appropriate.

Results:

Sputum positivity for anti-CCP-IgG, anti-CCP-IgA or RF-IgM strongly predicted which at-risk individuals developed RA (OR=6.0, 95% CI 2.2-16.4, Table 1). Serum anti-CCP-IgG level and serum RF positivity were higher in at-risk individuals who developed RA (Table 1), but the relationship between developing RA and baseline sputum anti-CCP or RF positivity remained significant after adjusting for these covariates (OR=3.6, 95% CI 1.2-11.3, Figure 1A). Sputum calprotectin levels were higher in sputum anti-CCP-IgA (p<0.01), anti-CCP-IgG (p=0.06) and RF-IgM (p=0.02) positive at-risk individuals. However, there was no significant difference in sputum calprotectin levels between at-risk individuals who developed vs. did not develop clinical RA (Table 1, Figure 1B). In at-risk individuals who developed RA, baseline sputum calprotectin levels were indeed lower in those who developed RA in <12 months compared to those who developed RA in 13-36 months (p=0.02, Figure 1C), suggesting that there may be accelerated local clearance of calprotectin with imminent RA onset.

Conclusion:

We confirmed our previous finding that the presence of RA-associated antibodies in the lung, specifically sputum anti-CCP and RF, are significantly higher in at-risk individuals who later develop RA. Although sputum calprotectin levels were higher in sputum anti-CCP and RF positive at-risk individuals, sputum calprotectin levels were not elevated in at-risk individuals who later developed RA, and actually appeared lower in the 12 months preceding RA onset. These data suggest that calprotectin may be associated with mucosal autoantibody generation but not transitions from systemic autoimmunity to clinical RA. Longitudinal studies are needed to determine whether decreasing sputum calprotectin levels result from enhanced local clearance and whether this process plays a role in RA onset. Figure 1Sputum antibody positivity and calprotectin levels in individuals at-risk for RA. Panel A depicts RA-free survival (y-axis) during longitudinal follow-up in months (x-axis) of 93 serum anti-CCP-IgG positive individuals stratified by sputum antibody positivity (red line; N=24) or negativity at baseline (blue line; N=69). Sputum antibody positive=anti-CCP-IgG, anti-CCP-IgA or RF-IgM. P-values calculated based on cox regression and adjusted for serum anti-CCP-IgG level and serum RF-IgM positivity. Panel B depicts sputum calprotectin levels in at-risk individuals who did vs. did not develop RA in follow-up. Panel C depicts sputum calprotectin levels in at-risk individuals who developed RA in 0-12 months vs. 13-36 months after baseline study visit. P-values by Wilcoxon rank sum test.

REFERENCES:

NIL. Table 1Baseline clinical features, serologic biomarkers and sputum biomarkers associated with developing RA in serum anti-CCP-IgG positive individualsDeveloped RA(N=24)Did not develop RA(N=69)p-valueClinical FeaturesAge, in years57 (44-67)63 (63-71)0.437Female20 (83)53 (77)0.578Ever smoker9 (38)23 (33)0.711Chronic lung disease3 (13)5 (7)0.421Serologic Biomarkers≥ 1 shared epitope allele¹12 (50)30 (46)0.747Serum anti-CCP-IgG level, in ELISA units121 (42-863)43 (28-84)0.004Serum RF-IgM+14 (58)18 (26)0.004Serum calprotectin, in ug/mL5 (4-9)5 (3-9)0.651Sputum Biomarkers²Sputum anti-CCP-IgA+8 (33)5 (7)0.004Sputum anti-CCP-IgG+10 (42)10 (15)0.005Sputum RF-IgM+5 (21)6 (9)0.144Sputum anti-CCP-IgA or anti-CCP-IgG+ or RF-IgM+15 (63)15 (22)<0.001Sputum calprotectin, in ug/mL41 (24-122)48 (21-98)0.923All values are median (IQR) or N (%)1. SE testing was only available for 65 of 69 who did not develop RA.2. All met the definition for adequate sputum sample (i.e. <80% squamous epithelial cells on cell differential).

Acknowledgements:

NIL.

Disclosure of Interests:

Timothy Wilson: None declared, Marie Feser: None declared, Kristin Sturm: None declared, Claudia Lugo: None declared, Brian Hattel: None declared, Gary Firestein: None declared, Mark Gillespie: None declared, Troy Torgerson Paid consulting work for Takeda, Pharming healthcare, and Eli Lilly, Jill Norris: None declared, V.Michael Holers: None declared, Kevin Deane Has received ELISA kits from Werfen for research studies., Kristen Demoruelle I have received research grants from Gilead, Boehringer Ingelheim, Pfizer and Bristol Myers Squibb. © The Authors 2025. This abstract is an open access article published in Annals of Rheumatic Diseases under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Neither EULAR nor the publisher make any representation as to the accuracy of the content. The authors are solely responsible for the content in their abstract including accuracy of the facts, statements, results, conclusion, citing resources etc.