Radiographic airway abnormalities in untreated early rheumatoid arthritis are associated with peripheral neutrophil activation

doi:10.21203/rs.3.rs-2362410/v1

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Research Article

Radiographic airway abnormalities in untreated early rheumatoid arthritis are associated with peripheral neutrophil activation

https://doi.org/10.21203/rs.3.rs-2362410/v1

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published 20 Mar, 2023

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Background:

The role of the lung for the initiation and progression of rheumatoid arthritis (RA) is still unclear. Up to 10% of RA patients develop severe treatment resistant lung disease. Understanding early disease mechanisms is of great importance. The objective of this study was to determine whether there is an association between peripheral neutrophil phenotypes and presence of pulmonary abnormalities (PA) on chest high-resolution computed tomography (HRCT) in untreated early RA (ueRA).

Methods:

Clinical data and blood were collected, and HRCT performed at diagnosis on 30 consecutive anti-citrullinated protein antibody (ACPA) and/or rheumatoid factor (RF) positive ueRA patients. HRCTs were evaluated for the presence and extent of RA-associated parenchymal, airway and/or pleural abnormalities. Expression of phenotype markers on neutrophils were determined by flow cytometry. Levels of calprotectin, ACPA and RF were measured using immunoassays.

Results:

The frequency of having any PA was 60%. Airway abnormalities were present in 50%, parenchymal nodules in 43% and interstitial lung abnormalities (ILA) in 10%. Unsupervised multivariate factor analysis showed clustering of any PA with neutrophil activation, parameters of inflammation and RF titres. In univariate analysis, the patients with PA displayed significantly increased CD11b and decreased CD62L expression on neutrophils (1.2-fold, p = 0.014; 0.8-fold, p = 0.012) indicating activation, and significantly increased RF IgM titre and CRP (5.7-fold, p = 0.0025; 2.3-fold, p = 0.0035) as compared to no PA. Titres of RF, but not ACPA, correlated with expression of the neutrophil activation marker CD11b. A stratified analysis demonstrated that airway involvement was the PA subtype with the strongest association with neutrophil activation.

Conclusion:

We report a strong association between radiographic airway findings and activation of circulating neutrophils in early RA supporting a role of innate immunity and the lung in disease onset. Our results also indicate different contributions of RF and ACPA in the RA pathogenesis.

Rheumatoid arthritis

pulmonary abnormalities

HRCT

neutrophil activation

rheumatoid factor

anti-citrullinated protein antibodies

Rheumatoid arthritis (RA) is a systemic inflammatory autoimmune disease primarily affecting joints(1). However, approximately 60% of the patients will develop pulmonary involvement of airways, parenchyma, vasculature and/or pleura(2). Some manifestations are asymptomatic whereas other progress to a life-threatening disease(3). High-resolution computed tomography (HRCT)-detected pulmonary abnormalities (PA) of parenchyma and airways are more prevalent in recent onset RA than in healthy controls (age and sex matched) independently of smoking and may precede the onset of arthritis by many years(4). RA is believed to originate at mucosal sites such as the airways, and several lines of evidence suggest that the break of tolerance and initiation of autoimmunity takes place in the lung as a consequence of repeated environmental stress in individuals with a susceptible genetic background(5). Reported risk factors for pulmonary disease in RA include smoking, presence of autoantibodies and disease duration(2). It is not understood why some RA patients develop pulmonary manifestations and others not, and why some patients suffer from airway disease and others have isolated interstitial involvement.

Approximately 10% of RA patients will develop clinically significant RA-associated interstitial lung disease (RA-ILD) over the course of the disease, but sub-clinical interstitial lung abnormalities (ILA) have been described in up to 44% of the patients(6, 7). Radiologic findings of interstitial lung involvement include reticulations, ground-glass opacities, honeycombing and/or consolidations. Pulmonary nodules (defined as round opacities 3–30 mm) are more prevalent in RA compared to healthy controls (31% versus 7%)(4). Radiologic signs of bronchiolitis are reportedly detected in up to 66% of RA patients(4, 8) and include bronchial wall thickening and air-trapping. Bronchiectasis is more frequently detected in early RA patients compared to healthy controls (17% versus 5%)(4) and the prevalence ranges from 22 to 51% in established RA(9). Obliterative bronchiolitis and ILD are severe extra-articular manifestations of RA with high morbidity and mortality(2). Therapy for these progressive conditions is limited and can at best reduce the rate of decline in lung function. Together, this emphasizes the need for increased knowledge on pulmonary disease mechanisms as well as biomarkers for early and subtype-specific lung diagnoses.

Neutrophils have emerged as a key player in RA based on their capacity to drive inflammation, induce and promote adaptive immune responses and mediate tissue damage(10). Circulating neutrophils in RA display an activated phenotype with increased production of neutrophil extracellular traps (NETs)(11). Anti-citrullinated protein antibodies (ACPAs) and antibodies to immunoglobulins (rheumatic factor; RF) are significant predictors of ILD-development in RA(2, 12). These autoantibodies form immune complexes which are strong inducers of inflammatory responses of neutrophils(10). In a recent study across different RA populations, neutrophil-derived calprotectin was superior to C-reactive protein (CRP) in identifying patients with active disease, and a neutrophil biomarker panel demonstrated good predictive value for extra-articular nodules(13).

Expression of the surface neutrophil glycoprotein CD177 reportedly promote development of some autoimmune diseases but is protective for others(14). Low-density neutrophils have been suggested to represent an immature neutrophil population in RA with enhanced survival and defective TNF signalling possibly leading to lower response to therapy (15). The role of different neutrophil phenotypes in the development of pulmonary disease in RA has not been clarified.

The objective of the present study was to determine if there is an association between neutrophil phenotypes in peripheral blood and presence of pulmonary abnormalities (PA) on chest high-resolution computed tomography (HRCT) in untreated early RA (ueRA).

Study population and design

Thirty consecutive adult patients with recent onset seropositive and disease-modifying anti-rheumatic drug (DMARD)-naïve RA fulfilling the 2010 ACR/EULAR RA classification criteria were included at the time of diagnosis in this cross-sectional study. Seropositivity was defined as a presence of RF (IgA and/or IgM), and/or ACPA. Study activities included medical history, demographics, concomitant medication, smoking habits, patient and physician global assessments of disease activity, swollen and tender joint counts, pulmonary HRCT, radiography of hands- and feet and blood sampling. All study activities were performed within a week, in most cases within 72 hours, from diagnosis before initiation of disease-modifying antirheumatic drugs (DMARDs) including oral prednisolone. Patients with a history of oral glucocorticoid use were eligible if treatment had been discontinued ≥ 2 weeks before inclusion. Use of NSAID was allowed. Symptom duration was defined as time in months since self-reported onset of first disease-associated symptoms. Patients with symptom duration > 36 months were excluded. Additional exclusion criteria included any contraindication for HRCT, current active joint disease other than RA, history of malignancy within five years, immuno-deficiency, active infection requiring anti-infectives within two weeks of inclusion and highly active RA in need of urgent treatment.

Patient data and clinical assessments

Demographic data (age, sex and smoking status) and self-reported history of pulmonary disease (such as chronic bronchitis, asthma, chronic obstructive pulmonary disease and lung cancer) were collected, as well as clinical parameters of disease activity (number of swollen and tender joints of 28 joints, patient global assessment (PGA) of disease activity (VAS 0-100 mm), physician global assessment (PhGA) of disease activity (VAS 0-100 mm), CRP, ESR, presence of joint erosions on radiographs of hands- and feet) and levels of autoantibodies (RF IgM, RF IgA and ACPA) (Table 1). Smoking status was defined as follows: current smoker if ever smoked ≥ 100 cigarettes AND is currently smoking, former smoker if ever smoked ≥ 100 cigarettes AND no smoking for the last 12 months, never smoker if smoked less than 100 cigarettes during a lifetime.

Table 1

Patient demographics and clinical characteristics
Variables	Untreated early RA patients (n = 30)
Age, median (range), years	58 (22–75)
Female sex, n (%)	25 (83%)
Smoking status, n (%) Never smoker Former smoker Current smoker	18 (60%) 6 (20%) 6 (20%)
Self-reported lung disease history, n (%)	4 (13%)
Symptom duration, median (range), months	6 (1–31)
Disease activity, n (%)
SJC28, median (range)	7 (1–17)
TJC28, median (range)	7 (0–16)
ESR (mm), median (range)	19,5 (2–104)
CRP (mg/L), median (range)	7,6 (0,7–105)
DAS28, mean (± SD)	4,9 (± 1,1)
DAS28-CRP, mean (± SD)	4,6 (± 1,0)
CDAI, mean (± SD)	23,6 (± 9,1)
SDAI, mean (± SD)	25,4 (± 10,1)
Joint erosion, n (%)	7 (24%)
RF positive, n (%)^a	22 (73%)
ACPA positive, n (%)^b	23 (77%)
RF and ACPA positive, n (%)^a,b	16 (53%)
RA = Rheumatoid Arthritis; SJC28 = swollen joint count in 28 joints; TJC28 = tender joint count in 28 joints; CRP = C-reactive protein; ESR = Erythrocyte Sedimentation Rate; DAS28 = disease activity score in 28 joints with ESR; CDAI = Clinical disease activity index; SDAI = Simple disease activity index; RF = Rheumatoid factor; ACPA = anti-citrullinated protein antibodies. Symptom duration is defined as time since appearance of patient reported RA symptoms.
^a RF IgM levels > 5 kIE/L and/or RF IgA > 20 kIE/L are considered positive
^b ACPA level > 5 U/mL is considered positive

High-resolution computed tomography (HRCT)

HRCT was performed using a Siemens SOMATOM Force scanner (Siemens Healthineers, Germany) within one week before any treatment was initiated. Volumetric CT scans were achieved in maximal inspiration and expiration using a low dose protocol; 120kV, reference mAs 30, acquisition 192x0.6mm with a pitch of 0.9, rotation time 0.5s applying 4D-Care dose and ADMIRE (strength 3). Images with a slice thickness of 0.75mm were reconstructed with kernels Bf32 and BI64. No contrast was given. All images were evaluated for abnormalities by two experienced thoracic radiologists in consensus. RA-associated HRCT findings were categorized as ILA by the presence of reticulations, ground-glass opacities, honeycombing and/or consolidations; pulmonary nodules by the presence of parenchymal nodule(s) ≥ 3 mm; airway abnormalities by the presence of air trapping and/or bronchial wall thickening (bronchiolitis), and/or bronchiectasis; and pleural abnormalities by the presence of pleural thickening and/or effusion. Pulmonary nodules were quantitively evaluated by means of computed aided detection and semiautomatic nodule segmentation using Syngo. CT Lung CAD, software version VC20G release VC 20 (Siemens Healthcare GmbH, Germany). All patients with parenchymal nodules ≥ 6mm were subjected to follow up according to national guidelines for lung cancer. No patient was diagnosed with cancer based on the initial HRCT performed as part of this study. No evaluation of the vasculature was performed.

Neutrophil characterization

Blood samples were collected in heparin tubes immediately before HRCT (i.e. within one week from diagnosis and before initiation of treatment). All analyses were performed within 6 hours. Neutrophils were separated using density centrifugation (Ficoll-Paque Plus, Cytiva, Sweden), in this paper referred to as low-density granulocytes (LDG) and normal density granulocytes (NDG), and subjected to flow cytometry as previously described(16). Activation of neutrophils was assessed by expression of CD11b and CD62L. 7-aminoactinomycin D (7-AAD) was used to separate dead from live cells. The proportion of LDGs in the peripheral blood mononuclear cell layer was determined based on CD45 expression and side scatter characteristics. The mean fluorescence intensity (MFI) of the surface markers CD62L, CD11b, and CD177 were determined. Gating strategy is presented in Additional file 1, figure S1. All samples were acquired in a FACSVerse equipped with FACSuite Software (BD Biosciences, Franklin Lakes, USA) and analysed with FlowJo Software (TreeStar, Ashland, Oregon, USA). All antibodies used for neutrophil characterization are listed in Additional file 2, table S1.

Autoantibodies and markers of inflammation

Levels of RF were measured using EliA test on Phadia 250 (Thermo Fisher, Waltham, USA). Anti-CCP IgG (ACPA) levels were measured using chemiluminescent microparticle immunoassay on the Alinity i-platform (Abbott Laboratories, Chicago, USA). CRP was analysed using Turbidimetry Alinity and ESR was analysed according to standard lab protocols. Serum calprotectin (S100A8/A9) levels were analysed using particle-enhanced turbidimetric immunoassay (Gentian Diagnostics, Moss, Norway) on an Optilite system (Binding Site, Birmingham, England).

Statistical analysis

Multivariate factor analysis was performed using SIMCA-P software version 17.0.2 (Sartorius Stedim Biotech, Goettingen, Germany). Principal Component analysis (PCA) was used to make an unsupervised analysis and to visualize the overall relationship between neutrophil activation status, neutrophil subtypes, calprotectin, disease activity measures and demographic data. DAS28, DAS28-CRP, CDAI and SDAI were not included since these disease activity measures are calculated form other variables included. Orthogonal partial least squares discriminant analysis (OPLS-DA) was implemented to find differences between groups of patients or subtypes of pulmonary abnormalities. Default settings for the PCA-X and OPLS-DA models were used; data were centred and scalded to unit-variance (UV) in the software to give all variables equal weight. Variable influence on projection (VIP) values were used to discriminate between relevant and less relevant X-variables for each respective OPLS-DA model (Additional file 1, figure S2). The column loading plots presented in Fig. 2A and Fig. 5A consist of variables with VIP-values ≥ 0.95 or VIP-values ≥ 1.0 respectively. Model quality was based on R2 and Q2 parameters and are presented in each figure.

The most relevant X-variables, as indicated by larger bars and smaller error bars in the OPLS-DA column loading plots, were further analysed using univariate statistical analysis. Non-parametric Mann-Whitney U test and Spearman’s correlation test were used for sample sets of continuous variables and Fisher’s exact test was used for categorical variables (GraphPad Prism software version 9.4.0, San Diego, California USA). P-values equal or less than 0.05 were considered significant and *, **, *** indicate p ≤ 0.05, p ≤ 0.01 and p ≤ 0.001 respectively.

Airway abnormalities are the most prevalent HRCT findings in ueRA

Demographic and clinical data are summarized in Table 1. The median age of the RA patients was 58 years, 83% were female, and the median patient-reported symptom duration was 6 months. Four patients reported history of lung disease (all cases being asthma). Among the RA patients, 60% were never smokers and 20% were currently smoking. The mean disease activity was moderate by DAS28 (4.9) but high by CDAI (23.6) composite scores. Seventy-three % of the patients were positive for RF (IgM or IgA), 77% were positive for ACPA, and 53% of the patients were double-positive.

Table 1

The frequency of having any RA-associated PA by HRCT was 60% (18 out of 30 patients) (Table 2). Airway abnormality was the most prevalent HRCT finding in this ueRA cohort (53%). Two patients had bronchiectasis and 14 patients (47%) showed signs of bronchiolitis (bronchial wall thickening and/or air trapping). Parenchymal nodules were found in 13 patients (43%). The mean number of nodules was 1 (range 0–9) and the mean total nodule volume per patient was 48 (range 26–480) mm³. Radiological signs of ILA were uncommon (10%), and all these patients had mild findings of reticulations. No patients showed radiological signs of pleural involvement.

Table 2

RA associated pulmonary abnormalities by HRCT at diagnosis
RA associated pulmonary abnormalities (PA)
Any RA associated PA, n (%)	18 (60%)
Airway abnormalities, n (%) Bronchiectasis Bronchiolitis Bronchial wall thickening Air trapping	15 (50%) 2 (7%) 14 (47%) 4 (13%) 13 (43%)
Parenchymal nodule, n (%)^a	13 (43%)
Interstitial lung abnormalities, n (%) Reticulations Ground glass opacities Honeycombing Consolidations	3 (10%) 3 (10%) 0 0 0
Pleural disease, n (%) Effusion Pleural thickening	0 0 0
^a Parenchymal nodule was defined as ≥ 3 mm

Table 2

Pulmonary abnormalities are associated with a peripheral activated neutrophil phenotype

It is unknown whether peripheral neutrophil phenotypes are related to pulmonary pathology in patients with ueRA. First, unsupervised PCA was used to make unbiased cluster analysis of collected data including; presence of any PA by HRCT, neutrophil activation status (CD11b and CD62L expression), neutrophil subtypes (% LDG and CD177 expression), neutrophil-derived proteolytic enzyme (calprotectin), as well as disease activity measures and demographic data (Fig. 1). Any PA was clustered together with high expression of CD11b (indicating activation) on NDGs, high frequency of LDGs and calprotectin indicating positive association between these variables. This cluster also included RF IgM levels and the clinical parameters of inflammation (CRP, ESR, number of swollen joints (SJC)) and the PhGA. Importantly, history of lung disease or symptom duration did not associate with the PA cluster suggesting that the PA of this study population and neutrophil activation are early events in RA. ACPA levels and current smoker were not clustered with Any PA in this analysis.

Next, an OPLS-DA analysis was performed to identify the variables discriminating the group Any PA from the group No PA. Any PA demonstrated a positive association with RF IgM, high expression of CD11b on NDGs, and CRP, and negative association with high expression of CD62L on NDGs (Fig. 2A). Subsequent univariate analysis confirmed that RA patients with Any PA had significantly increased CD11b expression on NDGs (MFI 6455 vs 5317, p = 0.014) and significantly lower expression of CD62L on NDGs (MFI 10065 vs 12676, p = 0.012), indicating neutrophil activation, as compared to No PA (Fig. 2B-C). The LDG proportions were higher in Any PA compared to No PA, but the proportions were low in both groups (< 4%) except for one outlier in Any PA (Additional file 1, figure S3A).

Pulmonary abnormalities are associated with levels of RF but not levels of ACPA

A significant increase in levels of RF (75.4 vs 8.6, p = 0.0025), but not ACPA (336.1 vs 162.5 p = 0.17), was found in patients with Any PA as compared to No PA (Fig. 2D-E). Sensitivity analysis of autoantibody levels and smoking status did not indicate that the difference was caused only by smoking (open circles in Fig. 2D-E). In addition, patients with Any PA had higher levels of CRP (11.5 vs 5.0, p = 0.0035) compared to No PA patients, while there was no significant difference in ESR between groups (Fig. 2F-G). The Any PA and No PA groups did not differ in disease activity measures CDAI, SDAI, SCJ28, TCJ28 and DAS28, nor with respect to smoking status (Additional file 2, table S2). Furthermore, there were no significant differences in calprotectin serum levels in patients with versus without PA, nor in patients with versus without joint erosions (Additional file 1, figure S3 B-C). Thus, presence of PA in this study population was associated with CRP, high RF IgM titres and neutrophil activation.

The fact that both RF IgM titres and markers of neutrophil activation in this ueRA cohort were associated with the presence of PA prompted us to investigate if there is a correlation between these two factors. Indeed, there was a significant positive correlation of RF IgM titres and CD11b expression on NDGs (r = 0.37, p = 0.05) (Fig. 3A). However, no significant correlation was found using loss of CD62L as a marker of activation (r=-0.19, p = 0.33) (Fig. 3B). No correlation was found between levels of ACPA IgG and mean fluorescence intensity of CD11b nor CD62L on NDGs (Additional file 1, figure S4).

Airway abnormality is the main subtype behind the association of PA, neutrophil activation and RF titres

The overlap between the radiographic entities is illustrated by a Venn diagram in Fig. 4A. The vast majority (15/18) of patients with PA had airway abnormalities. Three patients with pulmonary nodules did not have any other PA. All patients with ILA also had airway abnormalities by HRCT. The small size of the ILA group precluded any further analysis with respect to this radiographic finding in this study.

To further elucidate which specific radiographic finding is the major driver for the association of Any PA with neutrophil activation, we performed an OPLS-DA analysis in which the three non-overlapping sub-groups; Airway abnormalities (including patients with coexisting nodules and/or ILA), Pulmonary nodules only (excluding patients with coexisting airway abnormalities) and No PA. The OPLS-DA score-plot demonstrated a separation between patients with Airway abnormalities and patients with No PA, indicating that these groups are different form each other with respect to neutrophil activation status, neutrophil subtypes, calprotectin, disease activity and demographic data. On the other hand, patients with Pulmonary nodules only clustered together with No PA indicating similarities between the groups (Fig. 4B).

Discriminant analysis of the two groups Airway abnormalities and No PA using OPLS revealed that Airway abnormalities was associated with the expression of CD11b on NDGs and RF IgM, and No PA with the expression of CD62L on NDGs i.e. less neutrophil activation (Fig. 5A). Subsequent univariate analysis demonstrated a significant increase in CD11b (1.3-fold, p = 0.014) (Fig. 5B) and decrease in CD62L (0.6-fold, p = 0.003) (Fig. 5C) expression on NDGs in patients with airway abnormalities as compared to no pulmonary abnormalities. Furthermore, the group Airway abnormalities had significantly higher levels of RF IgM (8.8-fold, p = 0.0002) (Fig. 5D), but not ACPA, titres (p = 0.16) (Fig. 5E) compared to No PA. Again, sensitivity analysis of smoking status did not indicate that current smoking could explain the differences in RF titres. As in the non-stratified analysis, CRP levels were significantly higher (2.8-fold, p = 0.013), but not ESR (p = 0.14) (Fig. 5F-G), in Airway abnormalities as compared to No PA. Taken together, the stratified analysis indicates that airway abnormality is the major subtype behind the association of PA with high levels of RF IgM and neutrophil activation.

Lung disease remains one of the most therapeutically challenging extraarticular manifestations in RA and cause increased morbidity and premature mortality(17). This is the first study that investigates, and provides evidence for, an association of peripheral neutrophil activation and pulmonary abnormalities, in particular signs of airway involvement, in ueRA. Our data supports a role for innate immunity and the lung in RA disease pathogenesis and confirms earlier reported fact that circulating neutrophils are more activated in RA(13). Furthermore, our data highlights the importance of RF in disease pathogenesis, and it is tempting to speculate that RFs might participate in priming of neutrophils.

Neutrophils are key modulators of innate and adaptive immune responses during acute inflammation and infections, as well as of the resolution of inflammation of affected tissues(10). In the joints in RA, neutrophils accumulate and become activated by immune complexes such as RFs and evidence suggest degranulation and NETosis by inflammatory neutrophils lead to activation of stromal and immune cells, ACPA formation and cartilage destruction (11). Interestingly, all patients in the present study had active arthritis but only the individuals with arthritis and PA had sign of peripheral neutrophil activation.

Increasing evidence support that inflammatory events and immune activation, including NETosis and ACPA formation, at mucosal sites such as in the airways are key steps in the initiation of RA (5, 18). Our findings that radiographic signs of airway inflammation are associated with neutrophil activation and systemic inflammation in untreated RA patients at time of diagnosis support this hypothesis. Whether this initial inflammatory activity, predominantly of airways, in early RA will resolve by treatment, or progress further to lung disease of different types is not answered by the present data.

The significant correlation of RF titres and the expression of a neutrophil activation marker is interesting. Neutrophils have been reported to be activated by both RF and ACPAs in vitro(19, 20). Our data, suggest that RF might be more important for neutrophil activation and for airway inflammation than ACPAs in early RA. The latter agrees with findings from the Swedish LURA study, where the frequencies of airway abnormalities were equal in ACPA + and ACPA- patients(12). Furthermore, in agreement with earlier reports, the PCA of our cohort data did not cluster current smokers with Airway abnormalities (12) but support close association between smoking, arthralgia (tender joint count) and ACPAs(21).

The increased activation of neutrophils in patients with PA was found in normal density granulocytes but not in LDGs. The proportion of LDGs in this ueRA cohort was low compared to systemic lupus erythematosus (16). Whether there is an association of frequency and/or activation of LDGs and lung disease in RA needs to be evaluated in further studies. According to Bach et al, neutrophil-derived calprotectin was superior to CRP in identifying patients with active disease (13). However, the levels of calprotectin were not different in patients with PA as compared to no PA in our study and we could not confirm the reported association of calprotectin and joint erosion which was reported for established RA(13).

The frequency of PA found in association with RA varies greatly between studies and most studies have been performed on established RA with years of treatment at inclusion and the potential impact of different DMARDs on lung disease development has still not been clarified. A key strength of our study is that HRCT assessments and neutrophil analyses were collected on patients at the time of RA diagnosis, using the same HRCT equipment and laboratory facility, and before initiation of any DMARD or glucocorticoid therapy. Neutrophil analysis was performed fresh on the same day (same time point for all samples) limiting influence of auto-activation in vitro. In the present cohort, most of the patients had RA-associated PA on HRCT, and the prevalence of the different subtypes are in overall agreement with the Swedish LURA study(4). Another strength of our study is that an unsupervised approach was used to evaluate the multidimensional patient data. This strategy resulted in an improved separation of the different radiographic entities. A limitation of our study is the relatively small sample size, which resulted in low numbers of patients with ILA and limited the possibilities for further analysis on this group.

We report an association between the presence of PA on HRCT and increased activation status of circulating neutrophils at diagnosis in patients with ueRA. Furthermore, our data demonstrate a significant correlation between higher levels of RF, but not levels of ACPA, and airway abnormalities suggesting that these autoantibodies mediate different disease associated processes. Taken together, our data supports a role for innate immunity and the lung in RA disease pathogenesis. Further studies are needed to elucidate the role of neutrophils in the development of RA-associated progressive lung disease.

ACPA - anti-citrullinated protein antibodies

CDAI – clinical disease activity index

CRP – C-reactive protein

DAS28 – disease activity score, 28 joints

DMARD – disease modifying anti-rheumatic drug

ESR – erythrocyte sedimentation rate

HRCT – High-resolution computed tomography

EULAR – European league against rheumatism

ILA – interstitial lung abnormality

LDG – low density granulocyte

NGD – normal density granulocyte

NET – neutrophil extracellular traps

OPLS-DA – orthogonal partial least squares discriminant analysis

PA – pulmonary abnormality

PBMC – peripheral blood mononuclear cell

PCA – principal component analysis

PGA – patient global assessment of disease activity

PhGA – physicians’ global assessment of disease activity

RA – rheumatoid arthritis

RF - rheumatoid factor

SJC-28 – swollen joint count of 28 joints

SDAI – simple disease activity index

TJC-28 – tender joint count of 28 joints

ueRA – untreated early rheumatoid arthritis

VIP – variable influence on projection

Ethical approval and consent to participate

The study was conducted in compliance with the Declaration of Helsinki. All patients gave informed consent, and the study protocol was approved by the Swedish Ethical Review Authority (2019-05725).

Consent for publication

Not applicable

Availability of data and materials

The datasets supporting the conclusions of this article are included within the article and additional files.

Competing interests

The authors have declared no conflicts of interest.

Funding

This work was supported by grants from the Swedish Research Council [2020-06193], the Rune and Ulla Amlöv foundation, the Local Research and Development Council Västra Götaland [VGFOUREG-932185, 940809], the Gothenburg Society of Medicine [GLS-935160, 961518], the Swedish Rheumatism Association [R-969281], and the Swedish state under the agreement between the Swedish government and the county councils, the ALF-agreement [ALFGBG-942903, 943046].

Authors’ contributions

AKHE had full access to all of the data in the study and takes responsibility for the integrity of the data analysis. Conceptualization, study design - AKHE, AR, IG, ACL, JV, ÅJ, Recruitment of patients - AKHE, MJ, DG, KSt, KSv, Assessment of HRCT - JV, ÅJ, Clinical assessments and data - AKHE, MJ, DG, KSt, KSv, Laboratory data acquisition – GL, TS, Analysis and interpretation of data - All authors, Figure design - TS, Manuscript drafting- AKHE, CKM, TS, Review and editing – All authors, Approved manuscript for publication - All authors.

Acknowledgments

We are grateful to all patients participating in the study and to the study nurses at Rheumatology, Sahlgrenska University Hospital for excellent work.

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Additional file 3 is not available with this version

No competing interests reported.

Additionalfile1.docx
Additional file 1: Supplementary figure 1. Gating strategy of flowcytometry data analysis, Supplementary figure 2. VIP data, Supplementary figure 3. A) Univariate analysis of % LDG in Any PA vs No PA, B) calprotectin levels in Any PA vs No PA and C) calprotectin levels in joint erosions vs No joint erosions, Supplementary figure 4. Correlation analysis of ACPA levels and neutrophil activation markers CD11b (A) and CD62L (B)
Additionalfile2.docx
Additional file 2: Supplementary table 1. Cluster of differentiation markers used for neutrophil characterization. Supplementary table 2. Demographics and clinical data in patients with versus without RA-Associated PA

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published 20 Mar, 2023

Read the published version in Arthritis Research & Therapy →

Editorial decision: Major revision
24 Jan, 2023
Reviews received at journal
19 Jan, 2023
Reviewers agreed at journal
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Reviewers invited by journal
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Radiographic airway abnormalities in untreated early rheumatoid arthritis are associated with peripheral neutrophil activation

Status:

Journal Publication

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Abstract

Background:

Methods:

Results:

Conclusion:

Figures

Background

Patients And Methods

Study population and design

Patient data and clinical assessments

High-resolution computed tomography (HRCT)

Neutrophil characterization

Autoantibodies and markers of inflammation

Statistical analysis

Results

Airway abnormalities are the most prevalent HRCT findings in ueRA

Pulmonary abnormalities are associated with a peripheral activated neutrophil phenotype

Pulmonary abnormalities are associated with levels of RF but not levels of ACPA

Discussion

Conclusions

Abbreviations

Declarations

References

Additional file

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1