Lorena Álvarez-Rodríguez

Aging is associated with circulating cytokine dysregulation

PURPOSE Aging is accompanied by a progressive increase in pro-inflammatory cytokine status. However, little is known about the development of age-dependent modifications in other circulating cytokines. The aim of this study was to investigate in vivo the influence of age on circulating cytokine production in healthy subjects (HC). METHODS Circulating cytokines were measured by CBA and ELISA in 73 HC. Intracellular cytokine production was assessed in CD3+ and CD14+ cells by flow cytometry. Production of cytokines in cell culture supernatants was also studied after polyclonal stimulation. RESULTS Subjects were divided into three different groups according to age: 28 young HC (<30 years, 26.2 ± 2.4), 24 middle age HC (30-60 years, 44.7 ± 8.4) and 21 elderly HC (>60 years, 70.6 ± 7.9). Age was positively correlated with the circulating levels of IL-12p70, IL-1β, TNFα, IL-6, and IL-10. Age had a negative correlation with circulating levels of IL-17. Besides, age was positively correlated with spontaneous intracellular expression of proinflammatory cytokines in circulating monocytes. No correlation was found with other intracellular cytokine expression or with the production of cytokines in cell culture supernatants after in vitro stimulation. Gender had a marginal effect on the circulating cytokine profile. CONCLUSION Aging has a significant impact on the production of circulating cytokines in healthy individuals. The circulating cytokine milieu may contribute to the development of age-restricted conditions.

Age and low levels of circulating vitamin D are associated with impaired innate immune function

This study investigated in vivo the influence of age and vitamin D status on innate immune function in HC. Serum 25OHD was measured in 71 HC. TLR expression on various subpopulations of PBMCs, as well as TLR function by stimulating PBMCs with specific ligands, was assessed by flow cytometry. Circulating cathelicidin levels were determined by ELISA. Serum 25OHD levels decreased with age, and there was a significant inverse correlation between 25OHD levels and age. There was a negative correlation between serum 25OHD levels and MFI expression of TLR7 on B cells, T cells, and monocytes. TLR7 function, addressed by in vitro stimulation with a specific agonist, was significantly correlated with serum 25OHD levels, and this was especially a result of the results in HC older than 60 years. MFI expression of TLR5 on T cells and TLR2 on monocytes was also negatively correlated with serum 25OHD levels. TLR1 (monocytes) and TLR2 (monocytes) expression was positively correlated with age. Furthermore, TLR4 and TLR8 function was negatively correlated with age. Circulating cathelicidin levels decreased with age and were positively correlated with 25OHD levels. Aging is accompanied by changes in expression and function of several TLRs. Serum 25OHD levels decrease with age and are also associated with a change in expression and defective function of certain TLRs, especially those involved in viral response.

Circulating cytokines in active polymyalgia rheumatica

Objective: To characterise the circulating cytokine profile and the cellular source of circulating cytokines in polymyalgia rheumatica (PMR). Methods: The study included 34 patients with active untreated PMR and 17 age-matched healthy controls (HC). Circulating cytokines were measured by cytometric bead array and ELISA. Intracellular cytokines were assessed in CD3+ and CD14+ cells by flow cytometry. Cytokines in cell culture supernatants were also determined after polyclonal stimulation of patients’ peripheral blood mononuclear cells. Results: Circulating levels of interleukin-6 (IL6) were significantly higher in subjects with active PMR than in HC. Corticosteroid (CS) treatment was followed by a decrease in the level of IL6. Intracellular cytokine staining showed that circulating monocytes did not produce higher amounts of proinflammatory cytokines in patients with PMR than in HC. There was a discordance between serum levels and cytokine-producing monocyte and T cells, and it was not possible to demonstrate a Th1 bias in the peripheral compartment. Conclusions: Active PMR is characterised by increased serum levels of IL6, but not of other proinflammatory cytokines, that are rapidly suppressed by CS treatment. As circulating monocytes do not show increased production of proinflammatory cytokines, IL6 may be mainly produced in the inflamed tissue. A study of the circulating cytokine profile and its cellular source may provide a clue to new therapeutic options.

Interleukin-1RN gene polymorphisms in elderly patients with rheumatic inflammatory chronic conditions: association of IL-1RN*2/2 genotype with polymyalgia rheumatica.

The objective of this study was to investigate whether there is an association between IL1RN polymorphism and disease susceptibility for three age-related inflammatory conditions: polymyalgia rheumatica (PMR), giant cell arteritis (GCA), and elderly-onset rheumatoid arthritis (EORA). A tandem-repeat polymorphism within IL1RN intron 2 was analyzed in 139 PMR, 69 GCA, and 156 RA patients (75 with EORA) as well as in 437 healthy subjects, together with the in vitro production of IL-1beta. Our results showed that the IL1RN*2/2 genotype was more frequent in PMR patients compared with controls (p = 0.032, odds ratio = 1.785, 95% confidence interval = 1.047-3.044) and GCA patients (p = 0.008, odds ratio = 4.661, 95% confidence interval = 1.352-16.065). We found no difference in the distribution of genotypes between PMR and EORA or between EORA and controls. However, the frequency of the IL-1RN*2/2 genotype had a tendency to be higher in patients with EORA compared with young onset RA. The presence of IL1RN*1 or IL1RN*2 allele was not associated with severity of the disease in PMR and GCA patients and did not influence the production of IL-1beta. In conclusion, the IL1RN*2 polymorphism in a homozygous state was associated with an increased susceptibility to PMR and may give some clues for a differential therapy with GCA.

Toll-like receptor 4 gene polymorphism and giant cell arteritis susceptibility: a cumulative meta-analysis.

OBJECTIVE Toll-like receptor (TLR) 4 (+896 A/G) gene polymorphism has been reported to be associated with susceptibility to giant cell arteritis (GCA) with inconsistent results. To provide a more definitive conclusion, a cumulative meta-analysis of the association of TLR4 (+896 A/G) polymorphism with GCA susceptibility combining previous studies was performed. METHODS The cumulative meta-analysis included 3 case-control studies which provided a total of 437 patients and 1023 controls. Meta-analysis was conducted by fitting random effects models and checked for heterogeneity and publication bias. Combined odds ratio (OR) and associated 95% confidence intervals (CI) were obtained by using a DerSimonian and Laird random effects model. RESULTS Three studies, all from a South European ancestry, were identified through a literature search and included in this meta-analysis. The cumulative meta-analysis showed that the pooled random effects OR was non significant (OR: 1.46, 95% CI: 0.95-2.25; p = 0.082). There is some evidence suggestive of moderate-high heterogeneity between the three studies, I2 34.2% (95% CI: 0-55.2). CONCLUSION This cumulative meta-analysis does not demonstrate an association of TLR4 (+896 A/G) gene polymorphism with susceptibility to GCA. Further studies using larger samples and in populations from different geographic origins are needed to determine the exact role of TLR4 gene polymorphisms in GCA.

Lack of association between Toll-like receptor 4 gene polymorphisms and giant cell arteritis

OBJECTIVE Coding variants in Toll-like receptor 4 (TLR4) have been reported to be associated with inflammatory diseases. The aim of this study was to determine whether two of these polymorphisms (+896 A/G and +1196 C/T) are associated with susceptibility and clinical features of GCA. We also attempted to correlate the functional consequences of these polymorphisms. METHODS A total of 72 patients with GCA and 126 age-matched controls were genotyped using allele-specific PCR and restriction fragment length polymorphism analysis. TLR4 expression was studied on peripheral blood mononuclear cells by flow cytometry and TLR4 function was assessed by stimulating monocytes in vitro with a specific ligand. RESULTS There was no significant difference in allele frequency or genotype of TLR4 (+896 A/G and +1196 C/T) between GCA patients and controls. The clinical characteristics of these patients were unrelated to the presence of these polymorphisms. Furthermore, we did not observe an association with TLR4 expression or a distinct phenotype of TLR4 response with the +896 A/G and +1196 C/T genotypes. CONCLUSION Our results do not support the association of these TLR4 variants with GCA. Studies including a larger number of patients and patient populations from different geographical origin are needed.

Anti-carbamylated protein antibodies in patients with ageing associated inflammatory chronic disorders

to future research, highlighting the power of routinely captured data. As routinely captured data are more frequently utilized to address clinical questions in everyday practice, the strengths and limitations of such datasets will become more apparent. Funding: No specific funding was received from any funding bodies in the public, commercial or not-for-profit sectors to carry out the work described in this article.

Phagocyte dysfunction in polymyalgia rheumatica and other age-related, chronic, inflammatory conditions

This study was conducted to evaluate phagocyte function in patients with age‐related chronic inflammatory conditions. It included 95 patients with PMR, 17 with GCA, 40 with EORA, and 25 age‐matched HCs. Serum IL‐8 was determined with a bead array. The chemotactic capacity, phagocytic ability, and oxidative burst activity of circulating leukocytes were determined with flow cytometry kits. Patients with active chronic inflammatory diseases showed a significant increase in circulating levels of IL‐8 that remained elevated in patients with PMR or EORA, despite treatment. No correlation was found between circulating IL‐8 and the migratoty capacity of neutrophils. Neutrophils from patients with active EORA without stimulus and after fMLP stimuli showed a higher capacity to migrate than those of the HCs (P=0.033). The phagocytic activity of granulocytes in the patients with GCA was significantly higher than in the HCs and the patients with PMR or EORA (P<0.05). The percentage and MFI of phagocytes that produce ROIs when stimulated with Escherichia coli was significantly reduced in neutrophils and monocytes from the patients with age‐restricted inflammatory conditions. We concluded that the effector functions of phagocytes, determined to be chemotaxis, phagocytosis, and oxidative burst, are deregulated in age‐restricted inflammatory disorders and may have a pathogenic role.

Toll-like receptor 9 gene polymorphisms in polymyalgia rheumatica and giant cell arteritis

Ageing is accompanied by qualitative and quantitative changes in the immune system, which are grouped by the term immunosenescence (1) and by the appearance of age-restricted disorders, such as polymyalgia rheumatica (PMR) and giant cell arteritis (GCA). The pathogenesis of both conditions remains unknown, but recent evidence supports the involvement of innate immunity (2, 3). There are several factors that support the study of Tolllike receptor 9 (TLR9) in PMR and GCA. First, an increased expression of TLR9 in circulating B cells in GCA patients with active disease was found (2). Second, it has been hypothesized that the initial events in the vessel wall of patients with GCA are mediated by the transition of immature adventitial dendritic cells (DCs) to the mature state through TLR activation (3). Third, functional defects in the TLR9 pathway have been implicated in chronic granulomatous conditions (3). Furthermore, some TLR9 polymorphisms are functionally relevant and related to the development of other chronic inflammatory conditions (4, 5). Our aim was to determine whether two polymorphisms in TLR9 (T–1486C and T–1237C) are associated with susceptibility and clinical features in PMR and GCA and to search for the functional consequences of these polymorphisms. The present study included 268 PMR patients, 97 GCA patients, and 128 age-matched healthy controls (HC) (Table 1) who all provided signed informed consent. The regional ethics committee approved the study. Genotyping of the TLR9 [ 1486 T > C (rs187084) and 1237 T > C (rs5743836)] polymorphisms was performed using polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) as described previously (6). Intracellular expression of TLR9 was assessed on T cells, B cells, and monocytes by flow cytometry as described previously (2). The study population was found to be in Hardy– Weinberg equilibrium for both TLR9 gene polymorphisms. The allele, genotype, and haplotype analysis of TLR9 T–1486C and T–1237C revealed that neither of them were significantly associated with disease susceptibility (Supplementary Tables 1–3). We observed no significant difference between PMR and GCA patients (Supplementary Table 4). Furthermore, no significant association between the TLR9 gene polymorphisms and the intensity of the acute phase response or the presence of ischaemic manifestations and polymyalgic syndrome was seen. No significant association between the TLR9 gene polymorphisms and the presence of at least one relapse/recurrence, number of

Evaluation of Toll-like-receptor gene family variants as prognostic biomarkers in rheumatoid arthritis

Rheumatoid arthritis (RA) is a systemic autoimmune disease whose main feature is persistent joint inflammation. Toll-like receptors (TLRs) play critical roles in the activation of innate and adaptive immune responses, and influence the activity of NFκB, a key player in chronic inflammation. We aimed at investigating the association of TLR allelic variants with susceptibility and severity of RA through a systematic, high-throughput, analysis of TLR genes. All coding exons and flanking regions of nine members of the TLR family (TLR1-9) were analyzed in 66 patients with RA and 30 healthy controls by next generation sequencing. We focussed on three single allelic variants, N248S in TLR1, Q11L in TLR7 and M1V in TLR8 based on the allelic frequencies in both patient and control populations, the predicted impact on protein function and the novelty in RA research. Analysis of these selected variants in a larger cohort of 402 patients with RA and in 208 controls revealed no association with susceptibility. However, the M1V allele was associated with a lower need for disease-modifying antirheumatic drugs (DMARDs) (p=0.008) and biologic treatments (p=0.021). Functional studies showed that the M1V variant leads to a reduced production of inflammatory cytokines, IL-1β, IL-6 and TNFα, in response to TLR8 agonists. Thus, the presence of this variant confers a significant protective effect on disease severity. These results show for the first time the association between the M1V variant of TLR8 and reduced disease severity in RA, which could have prognostic value for these patients.