Identification of early risk factors for anti-citrullinated-protein-antibody positive rheumatoid arthritis-a prospective cohort study.

OBJECTIVE: Individuals positive for anti-cyclic-peptide-antibodies (anti-CCP) and musculoskeletal complaints (MSK-C) are at risk for developing rheumatoid arthritis (RA). In this study we aimed to investigate factors involved in arthritis progression. METHODS: Anti-CCP2-positive individuals with MSK-C referred to a rheumatologist were recruited. Individuals lacked arthritis at clinical and ultrasound examination and were followed for ≥three years or until clinical arthritis diagnosis. Blood samples from inclusion were analyzed for; nine anti-citrullinated-protein-antibody (ACPA) reactivities (citrullinated α-1-enolase, fibrinogen, filaggrin, histone, vimentin and tenascin peptides); 92 inflammation-associated proteins; and HLA-shared epitope alleles. Cox regression was applied to the data to identify independent predictors in a model. RESULTS: 267 individuals were included with median follow up of 49 months (IQR: 22-60). 101 (38%) developed arthritis after median 14 months (IQR: 6-27). The analysis identified that presence of at least one ACPA reactivity (HR 8.0, 95% CI 2.9-22), ultrasound detected tenosynovitis (HR 3.4, 95% CI 2.0-6.0), IL6 levels (HR 1.5, 95% CI 1.2-1.8) and IL15-Rα levels (HR 0.6, 95% CI 0.4-0.9) are significant independent predictors for arthritis progression in a prediction model (Harrell's C 0.76 [SE 0.02], AUC 0.82 [95% CI 0.76-0.89], cross-validated AUC 0.70 [95% CI 0.56-0.85]). CONCLUSION: We propose a high-Risk-RA phase characterized by presence of ACPA reactivity, tenosynovitis, IL6, and IL15-Rα and suggest that these factors need to be further investigated for their biological effects and clinical values, to identify individuals at particular low risk and high risk for arthritis progression.

Salivary Microbiota and Host-Inflammatory Responses in Periodontitis Affected Individuals With and Without Rheumatoid Arthritis.

Objectives: Periodontitis and rheumatoid arthritis (RA) are two widespread chronic inflammatory diseases with a previously suggested association. The objective of the current study was to compare the oral microbial composition and host´s inflammatory mediator profile of saliva samples obtained from subjects with periodontitis, with and without RA, as well as to predict biomarkers, of bacterial pathogens and/or inflammatory mediators, for classification of samples associated with periodontitis and RA. Methods: Salivary samples were obtained from 53 patients with periodontitis and RA and 48 non-RA with chronic periodontitis. The microbial composition was identified using 16S rRNA gene sequencing and compared across periodontitis patients with and without RA. Levels of inflammatory mediators were determined using a multiplex bead assay, compared between the groups and correlated to the microbial profile. The achieved data was analysed using PCoA, DESeq2 and two machine learning algorithms, OPLS-DA and sPLS-DA. Results: Differential abundance DESeq2 analyses showed that the four most highly enriched (log2 FC >20) amplicon sequence variants (ASVs) in the non-RA periodontitis group included Alloprevotella sp., Prevotella sp., Haemophilus sp., and Actinomyces sp. whereas Granulicatella sp., Veillonella sp., Megasphaera sp., and Fusobacterium nucleatum were the most highly enriched ASVs (log2 FC >20) in the RA group. OPLS-DA with log2 FC analyses demonstrated that the top ASVs with the highest importance included Vampirovibrio sp. having a positive correlation with non-RA group, and seven ASVs belonging to Sphingomonas insulae, Sphingobium sp., Novosphingobium aromaticivorans, Delftia acidovorans, Aquabacterium spp. and Sphingomonas echinoides with a positive correlation with RA group. Among the detected inflammatory mediators in saliva samples, TWEAK/TNFSF12, IL-35, IFN-α2, pentraxin-3, gp130/sIL6Rb, sIL-6Ra, IL-19 and sTNF-R1 were found to be significantly increased in patients with periodontitis and RA compared to non-RA group with periodontitis. Moreover, correlations between ASVs and inflammatory mediators using sPLS-DA analysis revealed that TWEAK/TNFSF12, pentraxin-3 and IL-19 were positively correlated with the ASVs Sphingobium sp., Acidovorax delafieldii, Novosphingobium sp., and Aquabacterium sp. Conclusion: Our results suggest that the combination of microbes and host inflammatory mediators could be more efficient to be used as a predictable biomarker associated with periodontitis and RA, as compared to microbes and inflammatory mediators alone.

Periodontal Health and Oral Microbiota in Patients with Rheumatoid Arthritis.

This study aimed to investigate the periodontal health of patients with established rheumatoid arthritis (RA) in relation to oral microbiota, systemic and oral inflammatory mediators, and RA disease activity. Forty patients underwent full-mouth dental/periodontal and rheumatological examination, including collection of blood, saliva, gingival crevicular fluid (GCF) and subgingival plaque. Composition of plaque and saliva microbiota were analysed using 16S rRNA sequencing and levels of inflammatory mediators by multiplex-immunoassay. The majority of the patients (75%) had moderate or severe periodontitis and the rest had no/mild periodontitis. Anti-citrullinated protein antibody (ACPA) positivity was significantly more frequent in the moderate/severe periodontitis (86%) compared to the no/mild group (50%). No significance between groups was observed for RA disease duration or activity, or type of medication. Levels of sCD30/TNFRSF8, IFN-α2, IL-19, IL-26, MMP-1, gp130/sIL-6Rß, and sTNF-R1 were significantly higher in serum or GCF, and April/TNFSF13 was significantly higher in serum and saliva samples in moderate/severe periodontitis. The microbial composition in plaque also differed significantly between the two groups. In conclusion, the majority of RA patients had moderate/severe periodontitis and that this severe form of the disease was significantly associated with ACPA positivity, an altered subgingival microbial profile, and increased levels of systemic and oral inflammatory mediators.

Predictors of the first cardiovascular event in patients with systemic lupus erythematosus - a prospective cohort study.

INTRODUCTION: Cardiovascular disease (CVD) is a major cause of premature mortality among Systemic lupus erythematosus (SLE) patients. Many studies have measured and evaluated risk factors for premature subclinical atherosclerosis, but few studies are prospective and few have evaluated risk factors for hard endpoints, i.e. clinically important cardiovascular events (CVE). We investigated the impact of traditional and lupus associated risk factors for the first ever CVE in a longitudinal cohort of SLE patients. METHODS: A total of 182 SLE patients (mean age 43.9 years) selected to be free of CVE were included. Cardiovascular and autoimmune biomarkers were measured on samples collected after overnight fasting at baseline. Clinical information was collected at baseline and at follow up. End point was the first ever CVE (ischemic heart, cerebrovascular or peripheral vascular disease or death due to CVD). Impact of baseline characteristics/biomarkers on the risk of having a first CVE was evaluated with Cox regression. RESULTS: Follow up was 99.5% after a mean time of 8.3 years. Twenty-four patients (13%) had a first CVE. In age-adjusted Cox regression, any positive antiphospholipid antibody (aPL), elevated markers of endothelial activation (von Willebrand factor (vWf), soluble vascular cellular adhesion molecule-1 (sVCAM-1)) and fibrinogen predicted CVEs. Of SLE manifestations, arthritis, pleuritis and previous venous occlusion were positively associated with future CVEs while thrombocytopenia was negatively associated. Among traditional risk factors only age and smoking were significant predictors. In a multivariable Cox regression model age, any positive aPL, vWf and absence of thrombocytopenia were all predictors of the first CVE. CONCLUSIONS: In addition to age, positive aPL, biomarkers indicating increased endothelial cell activity/damage, and absence of thrombocytopenia were independent predictors of CVEs in this prospective study. Our results indicate that activation of the endothelium and the coagulation system are important features in SLE related CVD. Furthermore, we observed that the risk of CVEs seems to differ between subgroups of SLE patients.

Decreased binding of annexin v to endothelial cells: a potential mechanism in atherothrombosis of patients with systemic lupus erythematosus.

OBJECTIVE: The cause of the exceedingly high risk of atherothrombosis in systemic lupus erythematosus (SLE) is not clear but antiphospholipid antibodies (aPL) and potentially antithrombotic annexin V have been implicated. METHODS AND RESULTS: Twenty-six women (52+/-8.2 years) with SLE and a history of cardiovascular disease (CVD) (SLE cases) were compared with 26 women with SLE but no CVD (SLE controls) and 26 healthy women (population controls). Common carotid intima-media thickness (IMT) was determined by B-mode ultrasound as a surrogate measure of atherosclerosis. Annexin V binding to human umbilical vein endothelial cells (HUVECs) as determined by flow cytometry after 24-hour culture with plasma was decreased when plasma from SLE cases was used (SLE cases versus population controls: P=0.002; SLE cases versus SLE controls P=0.02). Antibodies against cardiolipin were among IgG antibodies causing decreased binding. There was a positive association between annexin V binding and IMT (R=0.73; P<0.001) among SLE cases. Immunohistochemical analysis revealed presence of annexin V in all human atherosclerotic plaques tested, especially at sites prone to rupture. CONCLUSIONS: Decreased annexin V binding to endothelium caused by antibodies may represent a novel mechanism of atherothrombosis. We hypothesize that even though annexin V may promote plaque growth at some disease stages, it may also stabilize plaque.

The A-1087IL-10 allele is associated with cardiovascular disease in SLE.

The risk of cardiovascular disease (CVD) in SLE patients is very high. It is therefore surprising that IL-10 has been discussed both as pathogenic in SLE and as an atheroprotective cytokine. In contrast, TNF is believed to be atherogenic and we recently reported that raised activity in the TNF-system is implicated in SLE-related CVD. Twenty-six (aged 52 +/- 8 years) female patients with SLE and a history of CVD (myocardial infarction, angina, stroke or claudication) were compared with 26 age-matched SLE patients without CVD (SLE controls) or 26 age-matched population controls. The -1087IL-10 gene polymorphism was determined by PCR with restriction endonuclease mapping. Serum IL-10 and TNF-levels were determined by ELISA. The A allele frequency of -1087IL-10 gene in SLE/CVD was higher than in SLE controls (0.62 versus 0.42, p < 0.05). Ten (38%) of 26 SLE/CVD exhibited IL-10 AA genotype compared with five (19%) of 26 SLE controls. Serum IL-10 and TNF-levels were raised in SLE/CVD compared with SLE controls or population controls (p < 0.001). Furthermore, in SLE/CVD, a significantly reduced IL-10:TNF ratio was observed in patients with IL-10 AA genotype compared with AG or GG genotype (0.56 versus 0.77 versus 1.24, p < 0.05). In SLE controls and population controls, individuals with IL-10 GG genotype tended to have higher IL-10:TNF ratio. In conclusion, the A-1087IL-10 allele which has been reported to cause a lower capacity for IL-10 production could contribute to CVD in SLE. Furthermore, the IL-10 AA genotype is associated with reduced ratio of atheroprotective to atherogenic cytokines in SLE patients with CVD.

Platelet-activating factor-acetylhydrolase and other novel risk and protective factors for cardiovascular disease in systemic lupus erythematosus.

OBJECTIVE: There is an important inflammatory component to atherosclerosis and cardiovascular disease (CVD). It is therefore interesting that the risk of CVD is high in inflammatory diseases such as systemic lupus erythematosus (SLE). In this study, we investigated nontraditional risk factors for the development of CVD in patients with SLE. METHODS: Twenty-six women (mean age 52 years) with SLE and a history of CVD were compared with 26 age-matched women with SLE and no clinical manifestations of CVD (SLE controls) and 26 age-matched healthy women (population controls). Serum levels of several novel nontraditional risk and protective factors were determined: heat-shock protein (HSP)-related factors (Hsp60, Hsp70, anti-human Hsp60, anti-human Hsp70, and anti-mycobacterial Hsp65), platelet-activating factor-acetylhydrolase (PAF-AH) activity, secretory phospholipase A(2) GIIA (sPLA(2)), and anti-endothelial cell antibody (AECA). The intima-media thickness and the presence of plaques in the common carotid arteries were determined by B-mode ultrasound as a surrogate measure of atherosclerosis. RESULTS: Levels of PAF-AH, but not HSP-related factors, AECA, or sPLA(2), were significantly increased in SLE cases. Only PAF-AH discriminated between SLE cases and SLE controls (P = 0.005). PAF-AH was significantly associated with low-density lipoprotein (LDL) cholesterol and total cholesterol in the SLE cases (r = 0.50, P = 0.0093 and r = 0.54, P = 0.0045), but not in either control group. CONCLUSION: The increased levels of PAF-AH in SLE cases and the association between PAF-AH and LDL cholesterol adds support to the notion that PAF-AH may promote atherothrombosis in SLE. The role of HSPs in CVD is complex, since anti-Hsp65 appears to be associated with the presence of CVD, whereas Hsp70 might protect against it. In this cross-sectional study, levels of HSP-related factors, AECA, and sPLA(2) were not associated with CVD in SLE.

Elevated triglycerides and low levels of high-density lipoprotein as markers of disease activity in association with up-regulation of the tumor necrosis factor alpha/tumor necrosis factor receptor system in systemic lupus erythematosus.

OBJECTIVE: To investigate how blood lipid levels are related to disease activity, clinical characteristics, and serum levels of tumor necrosis factor alpha (TNF alpha) and its soluble type 1 and 2 receptors, sTNFR1 and sTNFR2, in systemic lupus erythematosus (SLE). METHODS: Fasting blood samples were obtained from an unselected cohort of SLE patients at Karolinska Hospital (n = 208, mean +/- SD age 45.7 +/- 14.2 years). Disease activity was estimated using the SLE Disease Activity Measure (SLAM). Levels of circulating TNF alpha, sTNFR1, and sTNFR2 were determined by enzyme-linked immunosorbent assay. Blood lipid levels obtained after overnight fasting were analyzed by routine chemistry. RESULTS: Triglyceride (TG) levels were associated with the SLAM score (r = 0.48, P < 0.0001) and with the activities of TNF alpha (r = 0.29, P = 0.0001), sTNFR1 (r = 0.38, P < 0.0001), and sTNFR2 (r = 0.40, P < 0.0001). High-density lipoprotein (HDL) levels were negatively associated with the SLAM score (r = -0.27, P = 0.0003) and with the activities of TNF alpha (r = -0.15, P = 0.04) and sTNFR2 (r = -0.19, P = 0.01). High levels of TGs, total cholesterol, TNF alpha, sTNFR1, and sTNFR2 all showed close correlations with the presence of nephritis and arterial disease (P < 0.05). In multiple logistic regression models, the TNF alpha activity and TG levels were independent determinants (P = 0.003 for both) of active disease (SLAM score > or =7). CONCLUSION: Dyslipoproteinemia with high TG/low HDL levels correlates with disease activity in SLE, and enhanced activity in the TNFalpha/sTNFR system seems to be an important underlying factor. Both dyslipoproteinemia and enhanced activity of the TNF alpha system are closely related to cardiovascular and renal manifestations in SLE, and thus both may serve as markers of more severe disease.

Oxidised LDL modulates immune-activation by an IL-12 dependent mechanism.

Oxidised low density lipoprotein (oxLDL) is one factor that may cause the immune reaction in the artery wall characteristic of atherosclerosis. OxLDL can promote immune activation as determined by enhanced secretion of IFN-gamma and TNF by immune competent cells. We previously demonstrated that Platelet-activating factor (PAF)-like lipids and/or lysophosphatidylcholine (LPC) in OxLDL contribute significantly to this immune activation, but these factors may also inhibit immune activation, at higher concentrations. We here demonstrate that IL-12 induces enhanced IFN-gamma secretion in peripheral blood mononuclear cells (PBMC), with no addition of a specific antigen, as determined by ELISPOT. Antibodies to IL-12 and to MHC class II inhibited both IL-12- and oxLDL-induced IFN-gamma secretion. OxLDL induced IL-12 production in PBMC. In the presence of IL-10, a T helper 2 cytokine, oxLDL induced a decreased IFN-gamma secretion, indicating that the local cytokine-milieu may determine the immunological properties of oxLDL. IL-10 could also be induced by OxLDL. Mononuclear leukocytes were prepared directly from human atherosclerotic plaques obtained at carotid operations. OxLDL had the capacity to induce IL-12, IL-10 and TNF from plaque cells using ELISPOT. Taken together, our data indicate that oxLDL can modulate immune reactivity in atherosclerosis by a nonspecific mechanism. OxLDL can be inhibitory, especially at higher concentrations. However, oxLDL can also promote immune activation by functioning as an adjuvant, potentiating and/or modulating immune-reactions via IL-12 and other cytokines including IL-10. This suggests that a specific T cell epitope in oxLDL is not necessary for oxLDL-induced T cell activation.