Angiogenic T Cells: potential biomarkers for the early diagnosis of interstitial lung disease in autoimmune diseases?

Background: We explored, for the first time, the contribution of angiogenic T cells (TAng) in interstitial lung disease associated to autoimmune disease (AD-ILD+) as potential biomarkers of the disease, evaluating their role in the underlying vasculopathy and lung fibrosis. Additionally, the relationship of TAng with clinical manifestations and cellular and molecular endothelial dysfunctionrelated biomarkers was assessed. (2) Methods: We included 57 AD-ILD+ patients (21 with rheumatoid arthritis (RA)-ILD+, 21 with systemic sclerosis (SSc)-ILD+ and 15 with other AD-ILD+) and three comparative groups: 45 AD-ILD-- patients (25 RA-ILD-- and 20 SSc-ILD--); 21 idiopathic pulmonary fibrosis (IPF) patients; 21 healthy controls (HC). TAng were considered as CD3+CD184+CD31+ by flow cytometry. (3) Results: A similar TAng frequency was found between AD-ILD+ and IPF, being in both cases lower than that observed in AD-ILD- and HC. A lower TAng frequency was associated with negative Scl-70 status and lower FEV1/FVC ratio in SSc-ILD+, as well as with men in RA-ILD+ and non-specific interstitial pneumonia radiological pattern in other AD-ILD+. No relationship between TAng and endothelial progenitor cells, endothelial cells and vascular endothelial growth factor gene expression and protein levels was disclosed. (4) Conclusions: Our findings suggest TAng as potential biomarkers for the early diagnosis of ILD in AD.Funding: V.P.-C. and S.R.-M. are supported by funds of RETICS Program [RD16/0012/0009, Instituto de Salud Carlos III (ISCIII), co-funded by European Regional Development Fund (ERDF); FG is supported by funds of the RICORS Program (RD21/0002/0025) from ISCIII, co-funded by the European Union; OG is staff personnel of Xunta de Galicia (Servizo Galego de Saude (SERGAS) through a research-staff stabilization contract (ISCIII/SERGAS) and his work is funded by ISCIII and ERDF [RD16/0012/0014 (RIER) and PI17/00409]. He is the beneficiary of project funds from the Research Executive Agency of the European Union in the framework of MSCA-RISE Action of the H2020 Programme, project 734899—Olive-Net. RL-M is a recipient of a Miguel Servet type II Program fellowship from ISCIII, co-funded by the European Social Fund, ‘Investing in your future’ (CPII21/00004)

Endothelial Progenitor Cells: Relevant Players in the Vasculopathy and Lung Fibrosis Associated with the Presence of Interstitial Lung Disease in Systemic Sclerosis Patients

Endothelial progenitor cells (EPC), which are key effectors in the physiologic vascular network, have been described as relevant players in autoimmune diseases. We previously showed that EPC frequency may help to identify the presence of interstitial lung disease (ILD) in rheumatoid arthritis patients. Given that ILD constitutes the main cause of mortality in systemic sclerosis (SSc) patients, we aimed to determine the EPC contribution to the pathogenic processes of vasculopathy and lung fibrosis in SSc-ILD+. EPC quantification was performed by flow cytometry on blood from 83 individuals: 21 SSc-ILD+ patients and subjects from comparative groups (20 SSc-ILD- and 21 idiopathic pulmonary fibrosis (IPF) patients and 21 healthy controls (HC)). EPC were considered as CD34+, CD45low, CD309+, and CD133+. A significant increase in EPC frequency was found in SSc-ILD+ patients when compared to HC (p < 0.001). SSc-ILD+ patients exhibited a higher EPC frequency than SSc-ILD- patients (p = 0.012), whereas it was markedly reduced compared to IPF patients (p < 0.001). EPC frequency was higher in males (p = 0.04) and negatively correlated to SSc duration (p = 0.04) in SSc-ILD+ patients. Our results indicate a role of EPC in the processes of vasculopathy and lung fibrosis in SSc-ILD+. EPC frequency may be considered as a biomarker of ILD in SSc patients.V.P.-C. is supported by a pre-doctoral grant from IDIVAL [PREVAL 18/01]. S.R.-M. is supported by funds from the RETICS Program [RD16/0012/0009, Instituto de Salud Carlos III (ISCIII), co-funded by the European Regional Development Fund (ERDF)]. B.A.-M. is a recipient of a ‘López Albo’ Post-Residency Programme funded by Servicio Cántabro de Salud. L.L.-G. is supported by funds from INNVAL20/06 (IDIVAL). R.P.-F. is supported by funds from the START project [FOREUM18/34]. O.G. is staff personnel of Xunta de Galicia (Servizo Galego de Saude (SERGAS) through a research-staff stabilization contract (ISCIII/SERGAS), and his work is funded by ISCIII and the ERDF [grants RD16/0012/0014 (RIER) and PI17/00409]. He is a beneficiary of project funds from the Research Executive Agency (REA) of the European Union in the framework of MSCA-RISE Action of the H2020 Programme, project 734899—Olive-Net. R.L.-M. is a recipient of a Miguel Servet type I fellowship [ISCIII, co-funded by the European Social Fund, ‘Investing in your future’, CP16/00033]

Endothelial Progenitor Cells as a Potential Biomarker in Interstitial Lung Disease Associated with Rheumatoid Arthritis

Interstitial lung disease (ILD) increases morbidity and mortality in patients with rheumatoid arthritis (RA). Although the pathogenesis of ILD associated with RA (RA-ILD+) remains poorly defined, vascular tissue is crucial in lung physiology. In this context, endothelial progenitor cells (EPC) are involved in endothelial tissue repair. However, little is known about their implication in RA-ILD+. Accordingly, we aimed to investigate the potential role of EPC related to endothelial damage in RA-ILD+. EPC quantification in peripheral blood from 80 individuals (20 RA-ILD+ patients, 25 RA-ILD? patients, 21 idiopathic pulmonary fibrosis (IPF) patients, and 14 healthy controls) was performed by flow cytometry. EPC were considered as CD34+, CD45low, CD309+ and CD133+. A significant increase in EPC frequency in RA-ILD+ patients, as well as in RA-ILD? and IPF patients, was found when compared with controls (p < 0.001, p = 0.02 and p < 0.001, respectively). RA-ILD+ patients exhibited a higher EPC frequency than the RA-ILD? ones (p = 0.003), but lower than IPF patients (p < 0.001). Our results suggest that EPC increase may represent a reparative compensatory mechanism in patients with RA-ILD+. The degree of EPC frequency may help to identify the presence of ILD in RA patients and to discriminate RA-ILD+ from IPFThis work was partially supported by the European Regional Development Fund (ERDF) and ‘Fondo de Investigación Sanitaria’ [PI18/00043] from Instituto de Salud Carlos III (ISCIII), Health Ministry, Spain. VP-C is supported by a pre-doctoral grant from IDIVAL [PREVAL 18/01]. SR-M is supported by funds of RETICS Program [RD16/0012/0009, ISCIII, co-funded by ERDF]. BA-M is a recipient of a ‘López Albo’ Post-Residency Programme funded by Servicio Cántabro de Salud. LL-G is supported by funds of ISCIII, co-funded by ERDF [PI18/00042]. OG is beneficiary of a grant funded by Xunta de Galicia, Consellería de Educación, Universidade Formación Profesional and Consellería de Economía, Emprego e Industria (GAIN), GPC IN607B2019/10. RL-M is a recipient of a Miguel Servet type I fellowship [ISCIII, co-funded by European Social Fund—ESF, CP16/00033]

Elevated VCAM-1, MCP-1 and ADMA serum levels related to pulmonary fibrosis of interstitial lung disease associated with rheumatoid arthritis

Introduction: Early diagnosis of interstitial lung disease (ILD) associated with rheumatoid arthritis (RA) constitutes a challenge for the clinicians. Pulmonary vasculopathy is relevant in the development of interstitial lung disease. Accordingly, we aimed to explore the role of vascular cell adhesion molecule-1 (VCAM-1), monocyte chemoattractant protein-1 (MCP-1) and asymmetric dimethylarginine (ADMA), key molecules in the vasculopathy, as potential biomarkers of pulmonary fibrosis in RA-ILD+. Methods: We included 21 RA-ILD+ patients and two comparative groups: 25 RA-ILD- patients and 21 idiopathic pulmonary fibrosis (IPF) patients. Serum levels of the molecules were determined by ELISA, and mRNA expression was quantified by qPCR. Results: VCAM-1, MCP-1 and ADMA serum levels were increased in RA-ILD+ patients in relation to RA-ILD- and IPF patients. Additionally, RA-ILD+ patients exhibited increased CCL2 (gene encoding MCP-1) and decreased PRMT1 (gene related to ADMA synthesis) mRNA expression in relation to RA-ILD- patients. A lower expression of VCAM1, CCL2, and PRMT1 was observed in RA-ILD+ patients when compared with those with IPF. Furthermore, MCP-1 serum levels and PRMT1 mRNA expression were positively correlated with RA duration, and ADMA serum levels were positively associated with C-reactive protein in RA-ILD+ patients. Conclusion: Our study suggests that VCAM-1, MCP-1 and ADMA could be considered as useful biomarkers to identify ILD in RA patients, as well as to discriminate RA-ILD+ from IPF, contributing to the early diagnosis of RA-ILD+.Funding: VP-C is supported by funds of PI18/00042 from Instituto de Salud Carlos III (ISCIII), co-funded by European Regional Development Fund (ERDF). SR-M is supported by funds of RETICS Program (RD16/0012/0009) from ISCIII, co-funded by ERDF; FG is supported by funds of the RICORS Program (RD21/ 0002/0025) from ISCIII, co-funded by the European Union; OG is staff personnel of Xunta de Galicia (Servizo Galego de Saude (SERGAS) through a research-staff stabilization contract (ISCIII/SERGAS) and his work is funded by ISCIII and ERDF [RD16/0012/0014 (RIER) and PI17/00409]. He is beneficiary of project funds from the Research Executive Agency of the European Union in the framework of MSCA-RISE Action of the H2020 Programme, project 734899—Olive-Net. RL-M is a recipient of a Miguel Servet type II Program fellowship from ISCIII, co-funded by the European Social Fund, ‘Investing in your future’ (CPII21/00004)

Role of VEGF polymorphisms in the susceptibility and severity of interstitial lung disease

The search for biomarkers that can help to establish an early diagnosis and prognosis of interstitial lung disease (ILD) is of potential interest. VEGF polymorphisms have been implicated in the development of several lung disorders. Consequently, we assessed, for the first time, the role of VEGF polymorphisms in the susceptibility and severity of ILD. A total of 436 Caucasian ILD patients (244 with idiopathic interstitial pneumonias (IIPs) and 192 with non-IIP) and 536 ethnically-matched healthy controls were genotyped for VEGF rs833061, rs1570360, rs2010963, rs3025020, and rs3025039 polymorphisms by TaqMan assays. Pulmonary function tests were collected from all the patients. VEGF serum levels were determined by ELISA in a subgroup of patients. No VEGF genotype, allele, carrier, or haplotype differences were found between ILD patients and controls as well as between IIP and non-IIP patients. However, an association of rs1570360 with IIP in women and also with lung function in IIP patients was found. None of the VEGF polymorphisms were associated with VEGF levels. In conclusion, our results suggest that VEGF does not seem to play a relevant role in ILD, although rs1570360 may influence the severity of ILD in women and a worse outcome in IIP patients.Funding: This research was partially supported by a grant from Spanish Society of Pulmonology and Thoracic Surgery (SEPAR 474-2017). S.R.-M. was supported by funds of the RETICS Program (RD16/0012/0009) from the “Instituto de Salud Carlos III” (ISCIII), co-funded by the European Regional Development Fund. V.P.-C. was supported by a pre-doctoral grant from IDIVAL (PREVAL 18/01). B.A.-M. was recipient of a “López Albo” post-residency program funded by Servicio Cántabro de Salud. L.L.-G. was supported by funds from IDIVAL (INNVAL 20/06). O.G. was beneficiary of a grant funded by Xunta de Galicia, Consellería de Educación, Universidade e Formación Profesional and Consellería de Economía, Emprego e Industria (GAIN), GPC IN607B2019/10. R.L.-M. was a recipient of a Miguel Servet type I program fellowship from the ISCIII, co-funded by the ESF, “Investing in your future” (grant CP16/00033)

Omentin: a biomarker of cardiovascular risk in individuals with axial spondyloarthritis

ABSTRACT: Cardiovascular (CV) disease is the main cause of mortality in axial spondyloarthritis (axSpA). CV risk is enhanced by dysregulation of adipokines. Low omentin levels were associated with metabolic dysfunction and CV disease in conditions different from axSpA. Accordingly, we evaluated the genetic and functional implication of omentin in CV risk and subclinical atherosclerosis in a cohort of 385 axSpA patients. Subclinical atherosclerosis was evaluated by carotid ultrasound. Omentin rs12409609, in linkage disequilibrium with a polymorphism associated with CV risk, was genotyped in 385 patients and 84 controls. Serum omentin levels were also determined. omentin mRNA expression was assessed in a subgroup of individuals. Serum and mRNA omentin levels were lower in axSpA compared to controls. Low serum omentin levels were related to male sex, obesity, inflammatory bowel disease (IBD) and high atherogenic index. rs12409609 minor allele was associated with low omentin mRNA expression in axSpA. No association was observed with subclinical atherosclerosis at the genetic or functional level. In conclusion, in our study low omentin serum levels were associated with CV risk factors in axSpA. Furthermore, rs12409609 minor allele may be downregulating the expression of omentin. These data support a role of omentin as a CV risk biomarker in axSpA.We wish to thank all the patients and controls that participated in this study. This work was supported by funds of a NEXT-VAL grant (NVAL17/10) (Instituto de Investigación Sanitaria IDIVAL) awarded to FG. SR-M is supported by funds of the RETICS Program (RD16/0012/0009) from the ‘Instituto de Salud Carlos III´ (ISCIII), co-funded by the European Regional Development Fund (ERDF). VP-C is supported by a pre-doctoral grant from IDIVAL (PREVAL 18/01). VM is supported by funds of a Miguel Servet type I programme (grant CP16/00033) (ISCIII, co-funded by the European Social Fund (ESF)). LL-G is supported by funds of PI18/00042 (ISCIII, co-funded by ERDF). RL-M is a recipient of a Miguel Servet type I programme fellowship from the ISCIII, co-funded by the ESF (grant CP16/00033)

Role of the IL33 and IL1RL1 pathway in the pathogenesis of Immunoglobulin A vasculitis

Cytokines signalling pathway genes are crucial factors of the genetic network underlying the pathogenesis of Immunoglobulin-A vasculitis (IgAV), an inflammatory vascular condition. An influence of the interleukin (IL)33- IL1 receptor like (IL1RL)1 signalling pathway on the increased risk of several immune-mediated diseases has been described. Accordingly, we assessed whether the IL33-IL1RL1 pathway represents a novel genetic risk factor for IgAV. Three tag polymorphisms within IL33 (rs3939286, rs7025417 and rs7044343) and three within IL1RL1 (rs2310173, rs13015714 and rs2058660), that also were previously associated with several inflammatory diseases, were genotyped in 380 Caucasian IgAV patients and 845 matched healthy controls. No genotypes or alleles differences were observed between IgAV patients and controls when IL33 and IL1RL1 variants were analysed independently. Likewise, no statistically significant differences were found in IL33 or IL1RL1 genotype and allele frequencies when IgAV patients were stratified according to the age at disease onset or to the presence/absence of gastrointestinal (GI) or renal manifestations. Similar results were disclosed when IL33 and IL1RL1 haplotypes were compared between IgAV patients and controls and between IgAV patients stratified according to the clinical characteristics mentioned above. Our results suggest that the IL33-IL1RL1 signalling pathway does not contribute to the genetic network underlying IgAV.Acknowledgements: We are indebted to the patients and healthy controls for their essential collaboration to this study. We also thank the National DNA Bank Repository (Salamanca) for supplying part of the control samples. This study was supported by European Union FEDER funds and `Fondo de Investigaciones Sanitarias´ (Grant PI18/00042) from ‘Instituto de Salud Carlos III’ (ISCIII, Health Ministry, Spain). DP-P is a recipient of a Río Hortega programme fellowship from the ISCIII, co-funded by the European Social Fund (ESF, `Investing in your future´) (Grant Number CM20/00006). SR-M is supported by funds of the RETICS Program (RD16/0012/0009) (ISCIII, cofunded by the European Regional Development Fund (ERDF)). VP-C is supported by a pre-doctoral grant from IDIVAL (PREVAL 18/01). BA-M is a recipient of a `López Albo´ Post-Residency Programme funded by Servicio Cántabro de Salud. LL-G is supported by funds from IDIVAL (INNVAL20/06). OG is staff personnel of Xunta de Galicia (Servizo Galego de Saude (SERGAS)) through a research-staff stabilization contract (ISCIII/SERGAS) and his work is funded by ISCIII and the European Union FEDER fund (Grant Numbers RD16/0012/0014 (RIER) and PI17/00409). He is beneficiary of project funds from the Research Executive Agency (REA) of the European Union in the framework of MSCA-RISE Action of the H2020 Programme, project 734899—Olive-Net. RL-M is a recipient of a Miguel Servet type I programme fellowship from the ISCIII, co-funded by ESF (`Investing in your future´) (Grant Number CP16/00033)

HLA association with the susceptibility to anti-synthetase syndrome

Objective: To investigate the human leukocyte antigen (HLA) association with anti-synthetase syndrome (ASSD). Methods: We conducted the largest immunogenetic HLA-DRB1 and HLA-B study to date in a homogeneous cohort of 168 Caucasian patients with ASSD and 486 ethnically matched healthy controls by sequencing-based-typing. Results: A statistically significant increase of HLA-DRB1*03:01 and HLA-B*08:01 alleles in patients with ASSD compared to healthy controls was disclosed (26.2% versus 12.2%, P=1.56E-09, odds ratio-OR [95% confidence interval-CI]=2.54 [1.84-3.50] and 21.4% versus 5.5%, P=18.95E-18, OR [95% CI]=4.73 [3.18-7.05]; respectively). Additionally, HLA-DRB1*07:01 allele was significantly decreased in patients with ASSD compared to controls (9.2% versus 17.5%, P=0.0003, OR [95% CI]=0.48 [0.31-0.72]). Moreover, a statistically significant increase of HLA-DRB1*03:01 allele in anti-Jo-1 positive compared to anti-Jo-1 negative patients with ASSD was observed (31.8% versus 15.5%, P=0.001, OR [95% CI]=2.54 [1.39-4.81]). Similar findings were observed when HLA carrier frequencies were assessed. The HLA-DRB1*03:01 association with anti-Jo-1 was unrelated to smoking history. No HLA differences in patients with ASSD stratified according to the presence/absence of the most representative non-anti-Jo-1 anti-synthetase autoantibodies (anti-PL-12 and anti-PL-7), arthritis, myositis or interstitial lung disease were observed. Conclusions: Our results support the association of the HLA complex with the susceptibility to ASSD

Role of MUC1 rs4072037 polymorphism and serum KL-6 levels in patients with antisynthetase syndrome

Mucin 1/Krebs von den Lungen-6 (KL-6) is proposed as a serum biomarker of several interstitial lung diseases (ILDs), including connective tissue disorders associated with ILD. However, it has not been studied in a large cohort of Caucasian antisynthetase syndrome (ASSD) patients. Consequently, we assessed the role of MUC1 rs4072037 and serum KL-6 levels as a potential biomarker of ASSD susceptibility and for the differential diagnosis between patients with ILD associated with ASSD (ASSD-ILD?+) and idiopathic pulmonary fibrosis (IPF). 168 ASSD patients (149 ASSD-ILD?+), 174 IPF patients and 523 healthy controls were genotyped for MUC1 rs4072037 T?>?C. Serum KL-6 levels were determined in a subgroup of individuals. A significant increase of MUC1 rs4072037 CC genotype and C allele frequencies was observed in ASSD patients compared to healthy controls. Likewise, MUC1 rs4072037 TC and CC genotypes and C allele frequencies were significantly different between ASSD-ILD+ and IPF patients. Additionally, serum KL-6 levels were significantly higher in ASSD patients compared to healthy controls. Nevertheless, no differences in serum KL-6 levels were found between ASSD-ILD+ and IPF patients. Our results suggest that the presence of MUC1 rs4072037 C allele increases the risk of ASSD and it could be a useful genetic biomarker for the differential diagnosis between ASSD-ILD+ and IPF patients

Vaspin in atherosclerotic disease and cardiovascular risk in axial spondyloarthritis: a genetic and serological study

Background: Vaspin is a novel anti-inflammatory adipokine associated with cardiovascular (CV) disease and inflammation in chronic inflammatory conditions different from axial spondyloarthritis (axSpA). Given the high incidence of CV disease (mainly due to accelerated atherosclerosis) exhibited by axSpA patients, we wondered if vaspin could also be a key molecule in this process. However, data on the role of vaspin regarding atherosclerotic disease in the context of axSpA is scarce. For this reason, we aimed to evaluate the implication of vaspin, at the genetic and serological level, in subclinical atherosclerosis and CV risk in axSpA. Methods: This study included 510 patients diagnosed with axSpA. Carotid ultrasound (US) was performed to evaluate the presence of subclinical atherosclerosis. Three vaspin gene variants (rs2236242, rs7159023, and rs35262691) were genotyped by TaqMan probes. Serum vaspin levels were assessed by enzyme-linked immunosorbent assay. Statistical analysis was performed using STATA® v.11.1. Results: Serum vaspin levels were significantly higher in female patients than in males and also in obese patients when compared to those with normal weight (p < 0.05). At the genetic level, we disclosed that the minor allele of rs2236242 (A) was associated with lower serum vaspin levels in axSpA, while the rs7159023 minor allele (A) was linked to higher serum levels (p < 0.05). When the three polymorphisms assessed were combined conforming haplotypes, we disclosed that the TGC haplotype related to high serum levels of vaspin (p = 0.01). However, no statistically significant association was observed between vaspin and markers of subclinical atherosclerosis, both at the genetic and serological level. Conclusions: Our results revealed that vaspin is linked to CV risk factors that may influence on the atherosclerotic process in axSpA. Additionally, we disclosed that serum vaspin concentration is genetically modulated in a large cohort of patients with axSpA.This work was partially supported by funds of a NEXT-VAL grant (NVAL17/10) (Instituto de Investigación Sanitaria IDIVAL) awarded to FG. RL-M is a recipient of a Miguel Servet type I programme fellowship (grant CP16/00033) from the “Instituto de Salud Carlos III” (ISCIII) and co-funded by the European Social Fund, ESF). SR-M is supported by funds of the RETICS Program (RD16/0012/0009) from ISCIII and co-funded by the European Regional Development Fund. VP-C is supported by a pre-doctoral grant from IDIVAL (PREVAL18/01). LL-G is supported by funds of a Miguel Servet type I programme fellowship from ISCIII (grant CP16/00033, co-funded by the ESF). OG is beneficiary of a grant funded by Xunta de Galicia, Consellería de Educación, Universidade e Formación Profesional and Consellería de Economía, Emprego e Industria (GAIN), GPC IN607B2019/10