Iraide Alloza

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

Atherosclerosis is the main risk factor for cardiovascular disease (CVD), which is the leading cause of mortality worldwide. Atherosclerosis is initiated by endothelium activation and, followed by a cascade of events (accumulation of lipids, fibrous elements, and calcification), triggers the vessel narrowing and activation of inflammatory pathways. The resultant atheroma plaque, along with these processes, results in cardiovascular complications. This review focuses on the different stages of atherosclerosis development, ranging from endothelial dysfunction to plaque rupture. In addition, the post-transcriptional regulation and modulation of atheroma plaque by microRNAs and lncRNAs, the role of microbiota, and the importance of sex as a crucial risk factor in atherosclerosis are covered here in order to provide a global view of the disease.

A recent genome-wide association study reported five loci for which there was strong, but sub-genome-wide significant evidence for association with multiple sclerosis risk. The aim of this study was to evaluate the role of these potential risk loci in a large and independent data set of ≈ 20,000 subjects. We tested five single nucleotide polymorphisms rs228614 (MANBA), rs630923 (CXCR5), rs2744148 (SOX8), rs180515 (RPS6KB1), and rs6062314 (ZBTB46) for association with multiple sclerosis risk in a total of 8499 cases with multiple sclerosis, 8765 unrelated control subjects and 958 trios of European descent. In addition, we assessed the overall evidence for association by combining these newly generated data with the results from the original genome-wide association study by meta-analysis. All five tested single nucleotide polymorphisms showed consistent and statistically significant evidence for association with multiple sclerosis in our validation data sets (rs228614: odds ratio = 0.91, P = 2.4 × 10(-6); rs630923: odds ratio = 0.89, P = 1.2 × 10(-4); rs2744148: odds ratio = 1.14, P = 1.8 × 10(-6); rs180515: odds ratio = 1.12, P = 5.2 × 10(-7); rs6062314: odds ratio = 0.90, P = 4.3 × 10(-3)). Combining our data with results from the previous genome-wide association study by meta-analysis, the evidence for association was strengthened further, surpassing the threshold for genome-wide significance (P < 5 × 10(-8)) in each case. Our study provides compelling evidence that these five loci are genuine multiple sclerosis susceptibility loci. These results may eventually lead to a better understanding of the underlying disease pathophysiology.

Several studies have highlighted the association of the 12q13.3-12q14.1 region with coeliac disease, type 1 diabetes, rheumatoid arthritis and multiple sclerosis (MS); however, the causal variants underlying diseases are still unclear. The authors sought to identify the functional variant of this region associated with MS.Tag-single nucleotide polymorphism (SNP) analysis of the associated region encoding 15 genes was performed in 2876 MS patients and 2910 healthy Caucasian controls together with expression regulation analyses.rs6581155, which tagged 18 variants within a region where 9 genes map, was sufficient to model the association. This SNP was in total linkage disequilibrium (LD) with other polymorphisms that associated with the expression levels of FAM119B, AVIL, TSFM, TSPAN31 and CYP27B1 genes in different expression quantitative trait loci studies. Functional annotations from Encyclopedia of DNA Elements (ENCODE) showed that six out of these rs6581155-tagged-SNPs were located in regions with regulatory potential and only one of them, rs10877013, exhibited allele-dependent (ratio A/G=9.5-fold) and orientation-dependent (forward/reverse=2.7-fold) enhancer activity as determined by luciferase reporter assays. This enhancer is located in a region where a long-range chromatin interaction among the promoters and promoter-enhancer of several genes has been described, possibly affecting their expression simultaneously.This study determines a functional variant which alters the enhancer activity of a regulatory element in the locus affecting the expression of several genes and explains the association of the 12q13.3-12q14.1 region with MS.

Cytokine and cytokine receptor genes, including IL2RA, IL7R and IL12A, are known risk factors for multiple sclerosis (MS). Excitotoxic oligodendroglial death mediated by glutamate receptors contributes to demyelinating reactions. In the present study, we screened 368 single-nucleotide polymorphisms (SNPs) in 55 genes or gene clusters coding for cytokines, cytokine receptors, suppressors of cytokine signaling (SOCS), complement factors and glutamate receptors for association with MS in a Spanish-Basque resident population. Top-scoring SNPs were found within or nearby the genes coding for SOCS-1 (P=0.0005), interleukin-28 receptor, alpha chain (P=0.0008), oncostatin M receptor (P=0.002) and interleukin-22 receptor, alpha 2 (IL22RA2; P=0.003). The SOCS1 rs243324 variant was validated as risk factor for MS in a separate cohort of 3919 MS patients and 4003 controls (combined Cochran-Mantel-Haenszel P=0.00006; odds ratio (OR)=1.13; 95% confidence interval (CI)=1.07-1.20). In addition, the T allele of rs243324 was consistently increased in relapsing-remitting/secondary progressive versus primary-progressive MS patients, in each of the six data sets used in this study (P(CMH)=0.0096; OR=1.24; 95% CI 1.05-1.46). The association with SOCS1 appears independent from the chr16MS risk locus CLEC16A.

Several variants in strong linkage disequilibrium (LD) at the SP140 locus have been associated with multiple sclerosis (MS), Crohn's disease (CD) and chronic lymphocytic leukemia (CLL). To determine the causal polymorphism, we have integrated high-density data sets of expression quantitative trait loci (eQTL), using GEUVADIS RNA sequences and 1000 Genomes genotypes, with MS-risk variants of the high-density Immunochip array performed by the International Multiple Sclerosis Genetic Consortium (IMSGC). The variants most associated with MS were also correlated with a decreased expression of the full-length RNA isoform of SP140 and an increase of an isoform lacking exon 7. By exon splicing assay, we have demonstrated that the rs28445040 variant was the causal factor for skipping of exon 7. Western blots of peripheral blood mononuclear cells from MS patients showed a significant allele-dependent reduction of the SP140 protein expression. To confirm the association of this functional variant with MS and to compare it with the best-associated variant previously reported by GWAS (rs10201872), a case-control study including 4384 MS patients and 3197 controls was performed. Both variants, in strong LD (r(2) = 0.93), were found similarly associated with MS [P-values, odds ratios: 1.9E-9, OR = 1.35 (1.22-1.49) and 4.9E-10, OR = 1.37 (1.24-1.51), respectively]. In conclusion, our data uncover the causal variant for the SP140 locus and the molecular mechanism associated with MS risk. In addition, this study and others previously reported strongly suggest that this functional variant may be shared with other immune-mediated diseases as CD and CLL.

Cardiovascular disease, the leading cause of mortality worldwide, is primarily caused by atherosclerosis, which is characterized by lipid and inflammatory cell accumulation in blood vessels and carotid intima thickening. Although disease management has improved significantly, new therapeutic strategies focused on accelerating atherosclerosis regression must be developed. Atherosclerosis models mimicking in vivo-like conditions provide essential information for research and new advances toward clinical application. New nanotechnology-based therapeutic opportunities have emerged with apoA-I nanoparticles (recombinant/reconstituted high-density lipoproteins, rHDL) as ideal carriers to deliver molecules and the discovery that microRNAs participate in atherosclerosis establishment and progression. Here, a therapeutic strategy to improve cholesterol efflux is developed based on a two-step administration of rHDL consisting of a first dose of antagomiR-33a-loaded rHDLs to induce adenosine triphosphate-binding cassette transporters A1 overexpression, followed by a second dose of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine rHDLs, which efficiently remove cholesterol from foam cells. A triple-cell 2D-atheroma plaque model reflecting the cellular complexity of atherosclerosis is used to improve efficiency of the nanoparticles in promoting cholesterol efflux. The results show that sequential administration of rHDL potentiates cholesterol efflux indicating that this approach may be used in vivo to more efficiently target atherosclerotic lesions and improve prognosis of the disease.

Multiple sclerosis (MS) shares some risk genes with other disorders hallmarked by an autoimmune pathogenesis, most notably IL2RA and CLEC16A. We analyzed 10 single-nucleotide polymorphisms (SNPs) in nine risk genes, which recently emerged from a series of non-MS genome-wide association studies (GWAS), in a Spanish cohort comprising 2895 MS patients and 2942 controls. We identified two SNPs associated with MS. The first SNP, rs6859219, located in ANKRD55 (Chr5), was recently discovered in a meta-analysis of GWAS on rheumatoid arthritis (RA), and emerged from this study with genome-wide significance (odds ratio (OR)=1.35; P=2.3 × 10−9). The second SNP, rs12785878, is located near DHCR7 (Chr11), a genetic determinant of vitamin D insufficiency, and showed a size effect in MS similar to that recently observed in Type 1 diabetes (T1D; OR=1.10; P=0.009). ANKRD55 is a gene of unknown function, and is flanked proximally by the IL6ST–IL31RA gene cluster. However, rs6859219 did not show correlation with a series of haplotype-tagging SNPs covering IL6ST–IL31RA, analyzed in a subset of our dataset (D′< 0.31; r2< 0.011). Our results expand the number of risk genes shared between MS, RA and T1D.

Objectives The mechanism by which atheroma plaque becomes unstable is not completely understood to date but analysis of differentially expressed genes in stable versus unstable plaques may provide clues. This will be crucial toward disclosing the mechanistic basis of plaque instability, and may help to identify prognostic biomarkers for ischaemic events. The objective of our study was to identify differences in expression levels of 59 selected genes between symptomatic patients (unstable plaques) and asymptomatic patients (stable plaques). Methods 80 carotid plaques obtained by carotid endarterectomy and classified as symptomatic (>70% stenosis) or asymptomatic (>80% stenosis) were used in this study. The expression levels of 59 genes were quantified by qPCR on RNA extracted from the carotid plaques obtained by endarterectomy and analyzed by means of various bioinformatic tools. Results Several genes associated with autophagy pathways displayed differential expression levels between asymptomatic and symptomatic (i.e. MAP1LC3B, RAB24, EVA1A). In particular, mRNA levels of MAP1LC3B, an autophagic marker, showed a 5−fold decrease in symptomatic samples, which was confirmed in protein blots. Immune system−related factors and endoplasmic reticulum-associated markers (i.e. ERP27, ITPR1, ERO1LB, TIMP1, IL12B) emerged as differently expressed genes between asymptomatic and symptomatic patients. Conclusions Carotid atherosclerotic plaques in which MAP1LC3B is underexpressed would not be able to benefit from MAP1LC3B−associated autophagy. This may lead to accumulation of dead cells at lesion site with subsequent plaque destabilization leading to cerebrovascular events. Identified biomarkers and network interactions may represent novel targets for development of treatments against plaque destabilization and thus for the prevention of cerebrovascular events.

A recent large-scale study in multiple sclerosis (MS) using the ImmunoChip platform reported on 11 loci that showed suggestive genetic association with MS. Additional data in sufficiently sized and independent data sets are needed to assess whether these loci represent genuine MS risk factors.The lead SNPs of all 11 loci were genotyped in 10 796 MS cases and 10 793 controls from Germany, Spain, France, the Netherlands, Austria and Russia, that were independent from the previously reported cohorts. Association analyses were performed using logistic regression based on an additive model. Summary effect size estimates were calculated using fixed-effect meta-analysis.Seven of the 11 tested SNPs showed significant association with MS susceptibility in the 21 589 individuals analysed here. Meta-analysis across our and previously published MS case-control data (total sample size n=101 683) revealed novel genome-wide significant association with MS susceptibility (p<5×10(-8)) for all seven variants. This included SNPs in or near LOC100506457 (rs1534422, p=4.03×10(-12)), CD28 (rs6435203, p=1.35×10(-9)), LPP (rs4686953, p=3.35×10(-8)), ETS1 (rs3809006, p=7.74×10(-9)), DLEU1 (rs806349, p=8.14×10(-12)), LPIN3 (rs6072343, p=7.16×10(-12)) and IFNGR2 (rs9808753, p=4.40×10(-10)). Cis expression quantitative locus effects were observed in silico for rs6435203 on CD28 and for rs9808753 on several immunologically relevant genes in the IFNGR2 locus.This study adds seven loci to the list of genuine MS genetic risk factors and further extends the list of established loci shared across autoimmune diseases.

Human endogenous retroviruses (HERVs) are repetitive sequences derived from ancestral germ-line infections by exogenous retroviruses and different HERV families have been integrated in the genome. HERV-Fc1 in chromosome X has been previously associated with multiple sclerosis (MS) in Northern European populations. Additionally, HERV-Fc1 RNA levels of expression have been found increased in plasma of MS patients with active disease. Considering the North-South latitude gradient in MS prevalence, we aimed to evaluate the role of HERV-Fc1on MS risk in three independent Spanish cohorts.A single nucleotide polymorphism near HERV-Fc1, rs391745, was genotyped by Taqman chemistry in a total of 2473 MS patients and 3031 ethnically matched controls, consecutively recruited from: Northern (569 patients and 980 controls), Central (883 patients and 692 controls) and Southern (1021 patients and 1359 controls) Spain. Our results were pooled in a meta-analysis with previously published data.Significant associations of the HERV-Fc1 polymorphism with MS were observed in two Spanish cohorts and the combined meta-analysis with previous data yielded a significant association [rs391745 C-allele carriers: pM-H = 0.0005; ORM-H (95% CI) = 1.27 (1.11-1.45)]. Concordantly to previous findings, when the analysis was restricted to relapsing remitting and secondary progressive MS samples, a slight enhancement in the strength of the association was observed [pM-H = 0.0003, ORM-H (95% CI) = 1.32 (1.14-1.53)].Association of the HERV-Fc1 polymorphism rs391745 with bout-onset MS susceptibility was confirmed in Southern European cohorts.

Vascular smooth muscle cells (VSMCs) are central players in carotid atherosclerosis plaque development. Although the precise mechanisms involved in plaque destabilization are not completely understood, it is known that VSMC proliferation and migration participate in plaque stabilization. In this study, we analyzed expression patterns of genes involved in carotid atherosclerosis development (e.g., transcription factors of regulation of SMC genes) of VSMCs located inside or outside the plaque lesion that may give clues about changes in phenotypic plasticity during atherosclerosis. VSMCs were isolated from 39 carotid plaques extracted from symptomatic and asymptomatic patients by endarterectomy. Specific biomarker expression, related with VSMC phenotype, was analyzed by qPCR, western immunoblot, and confocal microscopy. MYH11, CNN1, SRF, MKL2, and CALD1 were significantly underexpressed in VSMCs from plaques compared with VSMCs from a macroscopically intact (MIT) region, while SPP1, KLF4, MAPLC3B, CD68, and LGALS3 were found significantly upregulated in plaque VSMCs versus MIT VSMCs. The gene expression pattern of arterial VSMCs from a healthy donor treated with 7-ketocholesterol showed high similarity with the expression pattern of carotid plaque VSMCs. Our results indicate that VSMCs isolated from plaque show a typical SMC dedifferentiated phenotype with macrophage-like features compared with VSMCs isolated from a MIT region of the carotid artery. Additionally, MYH11, KLF5, and SPP1 expression patterns were found to be associated with symptomatology of human carotid atherosclerosis.

Interleukin-12 (IL-12) and the more recently discovered IL-23 and IL-27 constitute a unique family of structurally related, heterodimeric cytokines that regulate cell-mediated immune responses and T helper 1 (Th1)-type inflammatory reactions. Not surprisingly, the potentiality of treating conditions such as multiple sclerosis (MS) and rheumatoid arthritis (RA) through pharmacological interference with IL-12 pathways has received widespread attention. In this review we have examined over 50 substances with reported IL-12 inhibitory effects. We demonstrate that a majority of these belong to a limited number of major functional classes, each of which targets discrete events in the IL-12 biological pathway. Thus, most IL-12 inhibitory substances appear to work either through inhibition of transcription factor NF-kappa B activation, up-regulation of intracellular cAMP, blockage of posttranslational processing or interference with signal transduction pathways. In addition, cyclophilin-binding drugs, and generic inhibitors of nuclear histone deacetylases, and of ion channels, pumps and antiporters are emerging as potential leads to novel targets for interference with IL-12 production. Many inhibitors of NF-kappa B and of IL-12 signal transduction have been proven effective in limiting or preventing disease in experimental autoimmune encephalomyelitis (EAE) models of MS. The sharing of the p40 subunit, the IL-12R beta 1 and components of the signal transduction pathways between IL-12 and IL-23 raises the question as to whether the beneficial effects of various drugs previously ascribed to inhibition of IL-12 may, in fact, have been due to concurrent blockage of both cytokines, or of IL-23, rather than IL-12. Moreover, the homodimeric beta(2)-form of IL-12, though originally considered to display only antagonistic effects, is now emerging as a pronounced agonist in a variety of inflammatory processes. Reassessment of IL-12 inhibitory compounds is therefore needed to scrutinize their effects on IL-12 alpha beta, beta(2) and IL-23 formation. This is likely to open exciting perspectives to the identification of drugs that target these cytokines either indiscriminately or selectively. The functional diversity of presently available inhibitors should facilitate an unprecedented flexibility in designing future trials for the treatment of IL-12- and IL-23-mediated disorders.

Celecoxib (CE) is a nonsteroidal anti-inflammatory drug (NSAID) that is a specific inhibitor of cyclooxygenase 2 (COX2). It is indicated for a variety of chronic inflammatory conditions, including rheumatoid arthritis. Over the last few years, adverse cardiovascular effects and increased risk for heart attacks have been associated with this drug. In addition, evidence is emerging for COX2-independent molecular targets. CE has been shown to induce apoptosis in various cancer cells lines through a COX2-independent mechanism that seems to involve inactivation of protein kinase Akt and inhibition of endoplasmic reticulum (ER) Ca2+ ATPase. In this study, we show that both CE and an analog devoid of COX2 inhibitory activity [1-(4-sulfamoyl phenyl)-3-trifluoromethyl-5-(4-trifluoromethylphenyl)pyrazole, CEA] inhibit the secretion of the dimeric interleukin-12 (IL-12) alphabeta and beta2 forms with identical IC50 values of 20 and 30 microM, respectively, whereas no such effect was seen with rofecoxib. Reverse transcription-polymerase chain reaction analysis showed that this inhibition was not due to a blockage of transcription of the alpha- and beta-chain expression cassettes. Secretion of the beta monomer form was less strongly inhibited, suggestive for a mechanism primarily targeting dimer assembly in the ER. Analysis of intracellular fractions revealed that both CE and CEA increased the association of IL-12 with calreticulin, an endoplasmic reticulum-resident chaperone involved in the retention of misfolded cargo proteins while blocking interaction with ERp44. Our findings reveal a previously undescribed effect of celecoxib on oligomer protein folding and assembly in the endoplasmic reticulum and ensuing secretion and suggest that celecoxib-driven alteration of the secretome may be involved in some of its clinical side effects.

Abstract The C allele of a single nucleotide polymorphism (SNP), rs6897932, located in the interleukin‐7 receptor alpha chain ( IL7RA ) was recently found to be associated with multiple sclerosis and Type I diabetes. We analysed 13 SNPs in the IL7RA gene in a combined cohort of patients with chronic inflammatory arthropathies (rheumatoid arthritis and juvenile idiopathic arthritis; 368 patients and 532 unaffected subjects). No significant associations with disease were found with the exception of the non‐synonymous SNP rs6897932. This SNP showed modest enrichment of the TT genotype in arthritic patients compared with controls [ P = 0.02; OR 1.72 (95% CI 1.08–2.75)]. Our data are suggestive for a role of rs6897932 in predisposition to chronic inflammatory arthropathies.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system with presumed autoimmune origin, triggered by genetic and environmental risk factors. A recent genome-wide association study conducted on MS identified new biallelic markers outside the HLA (human leucocyte antigen) region involved in disease susceptibility: rs1109670 (DDEF2); rs1458175 (PDZRN4); rs1529316 and rs2049306 (CSMD1); rs16914086 (TBC1D2); rs1755289 (SH3GL2); rs1841770 (ZIC1); rs651477 (EN1); rs7607490 (TRIB2); rs397020 (C20orf46); rs908821 (SLC25A36); rs7672826 (MGC45800) and rs9523762 (GPC5). We aimed at replicating these top association signals in a Spanish cohort of 2863 MS patients and 2930 sex- and age-matched controls. Only rs9523762 mapping in the GPC5 gene was significantly associated (G allele, P=1.6 × 10(-5); odds ratio (95% confidence interval)=1.23 (1.12-1.36)), supporting a role for this proteoglycan in MS predisposition. The independent replication of association signals to validate data generated by genome-wide association scans is a first step in the effort to improve patient care.

A recent meta-analysis of genome-wide association screens coupled to a replication exercise in a combined US/UK collection led to the identification of 4 single nucleotide polymorphisms (SNPs) in three gene loci, i.e. TNFRSF1A, CD6 and IRF8, as novel risk factors for multiple sclerosis with genome-wide level of significance. In the present study, using a combined all-Spain collection of 2515 MS patients and 2942 healthy controls, we demonstrate significant association of rs17824933 in CD6 (P(CMH)=0.004; OR=1.14; 95% CI 1.04-1.24) and of rs1860545 in TNFRSF1A (P(CMH)=0.001; OR=1.15; 95% CI 1.06-1.25) with MS, while the low-frequency coding non-synonymous SNP rs4149584 in TNFRSF1A displayed a trend for association (P(CMH)=0.062; OR=1.27; 95% CI 0.99-1.63). This data reinforce a generic role for CD6 and TNFRSF1A in susceptibility to MS, extending to populations of southern European ancestry.

Background and objectives: Ten genes previously showing different evidence of association with multiple sclerosis have been selected to validate. Methods: Eleven polymorphisms were genotyped with the iPLEX™ Sequenom in a well-powered collection of Spanish origin including 2863 multiple sclerosis cases and 2930 controls. Results: Replication extended to the following polymorphisms: PKN2 (rs305217), GTF2B (rs7538427), EPHA4 (rs1517440), YTHDF3 (rs12115114), ANKFN1 (rs17758761) and PTPRM (rs4798571), which did not reach the threshold of significance in a follow-up of the first genome-wide association study (GWAS) conducted in multiple sclerosis; TMEM39A (rs1132200), which appeared as a newly identified susceptibility gene in the same study; a gene previously reaching GWAS significance in Italy, CBLB (rs9657904); IL12B (rs6887695, rs10045431), a susceptibility gene shared by diverse autoimmune diseases and, finally, another gene showing inconclusive association with multiple sclerosis, CNR1 (rs1049353). Conclusions: Pooled analysis corroborated the effect on MS predisposition of three genes: TMEM39A [rs1132200: p M-H =0.001; OR M-H (95% CI)= 0.84 (0.75–0.93)], IL12B [rs6887695: p M-H =0.03; OR M-H (95% CI)= 1.09 (1.01–1.17)] and CBLB [rs9657904: p M-H =0.01; OR M-H (95% CI)= 0.89 (0.81–0.97)].

To determine whether polymorphisms in the interferon-gamma (IFNgamma)/interleukin-26 (IL-26; formerly, AK155) gene cluster contribute to sex-based differential susceptibility to rheumatoid arthritis (RA).Four microsatellite markers, located in a 118-kb interval that contains both the IFNgamma and IL-26 genes on chromosome 12q15, were typed in 251 patients with RA and 198 unrelated healthy controls (all of whom lived in Northern Ireland) by means of polymerase chain reaction-based fragment analysis.Marker D12S2510, which is located 3 kb 3' from the IL-26 gene, was significantly associated with RA in women (corrected P [P(corr)] = 0.008, 2 degrees of freedom [2 df]) but not in men (P = 0.99, 2 df). A 3-marker haplotype, IFNGCA*13;D12S2510*8;D12S2511*9, was inferred that showed significant underrepresentation in women with RA (odds ratio 0.50, 95% confidence interval 0.32-0.78; P = 0.002, P(corr) = 0.03) but not in men with RA.Our results demonstrate that common polymorphisms in the IFNgamma/IL-26 gene region may contribute to sex bias in susceptibility to RA, by distorting the propensity of female carriers versus male carriers to contract this disease. These results conform to our recent observations of a role for this gene cluster in sex-based differential susceptibility to another Th1-type inflammatory disease, multiple sclerosis.

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system white matter characterized by inflammation, demyelination and axonal damage. The cytotoxic T lymphocyte antigen-4 (CTLA-4) protein plays a key role in the down-regulation of T cell activation. We analysed the CTLA4 +49A/G and CT60 polymorphisms in a cohort of 120 MS trio families recruited from the Flanders region in Belgium. Both polymorphisms were genotyped by polymerase chain reaction-restriction fragment length polymorphism (RFLP). The +49 G-allele was significantly more transmitted to affected probands (P = 0.005). No transmission distortion was observed for the CT60 polymorphism. Haplotype analysis revealed significant overtransmission of the +49 A/G*G-CT60*G haplotype (P = 0.0025), and undertransmission of the +49 A/G*A-CT60*G haplotype (P = 0.015). The CTLA4 gene has been the focus of intense investigation in MS. Of 15 recently published papers, only six reported significant associations of various CTLA4 polymorphisms with MS, with the remainder being negative. Ours is the first report investigating the CT60 polymorphism in MS. Our data highlight a need for further scrutiny of the CTLA4 gene in MS.

CD6 has recently been identified and validated as risk gene for multiple sclerosis (MS), based on the association of a single nucleotide polymorphism (SNP), rs17824933, located in intron 1. CD6 is a cell surface scavenger receptor involved in T-cell activation and proliferation, as well as in thymocyte differentiation. In this study, we performed a haptag SNP screen of the CD6 gene locus using a total of thirteen tagging SNPs, of which three were non-synonymous SNPs, and replicated the recently reported GWAS SNP rs650258 in a Spanish-Basque collection of 814 controls and 823 cases. Validation of the six most strongly associated SNPs was performed in an independent collection of 2265 MS patients and 2600 healthy controls. We identified association of haplotypes composed of two non-synonymous SNPs [rs11230563 (R225W) and rs2074225 (A257V)] in the 2(nd) SRCR domain with susceptibility to MS (P max(T) permutation = 1×10(-4)). The effect of these haplotypes on CD6 surface expression and cytokine secretion was also tested. The analysis showed significantly different CD6 expression patterns in the distinct cell subsets, i.e. - CD4(+) naïve cells, P = 0.0001; CD8(+) naïve cells, P<0.0001; CD4(+) and CD8(+) central memory cells, P = 0.01 and 0.05, respectively; and natural killer T (NKT) cells, P = 0.02; with the protective haplotype (RA) showing higher expression of CD6. However, no significant changes were observed in natural killer (NK) cells, effector memory and terminally differentiated effector memory T cells. Our findings reveal that this new MS-associated CD6 risk haplotype significantly modifies expression of CD6 on CD4(+) and CD8(+) T cells.

Carotid artery atherosclerosis is a risk factor to develop cerebrovascular disease. Atheroma plaque can become instable and provoke a cerebrovascular event or else remain stable as asymptomatic type. The exact mechanism involved in plaque destabilization is not known but includes among other events smooth muscle cell (SMC) differentiation. The goal of this study was to perform thorough analysis of gene expression differences in SMCs isolated from carotid symptomatic versus asymptomatic plaques. Comparative transcriptomics analysis of SMCs based on RNAseq technology identified 67 significant differentially expressed genes and 143 significant differentially expressed isoforms in symptomatic SMCs compared with asymptomatic. 37 of top-scoring genes were further validated by digital PCR. Enrichment and network analysis shows that the gene expression pattern of SMCs from stable asymptomatic plaques is suggestive for an osteogenic phenotype, while that of SMCs from unstable symptomatic plaque correlates with a senescence-like phenotype. Osteogenic-like phenotype SMCs may positively affect carotid atheroma plaque through participation in plaque stabilization via bone formation processes. On the other hand, plaques containing senescence-like phenotype SMCs may be more prone to rupture. Our results substantiate an important role of SMCs in carotid atheroma plaque disruption.

An intronic variant in ANKRD55, rs6859219, is a genetic risk factor for multiple sclerosis, but the biological reasons underlying this association are unknown. We characterized the expression of ANKRD55 in human PBMCs and cell lines. Three ANKRD55 transcript variants (Ensembl isoforms 001, 005, and 007) could be detected in PBMCs and CD4(+) T cells but were virtually absent in CD8(+), CD14(+), CD19(+), and CD56(+) cells. Rs6859219 was significantly associated with ANKRD55 transcript levels in PBMCs and CD4(+) T cells and, thus, coincides with a cis-expression quantitative trait locus. The processed noncoding transcript 007 was the most highly expressed variant in CD4(+) T cells, followed by 001 and 005, respectively, but it was not detected in Jurkat, U937, and SH-SY5Y cell lines. Homozygotes for the risk allele produced more than four times more transcript copies than did those for the protective allele. ANKRD55 protein isoforms 005 and 001 were predominantly located in the nucleus of CD4(+) T cells and Jurkat and U937 cells. ANKRD55 was produced by primary cultures of murine hippocampal neurons and microglia, as well as by the murine microglial cell line BV2, and it was induced by inflammatory stimuli. ANKRD55 protein was increased in the murine mouse model of experimental autoimmune encephalomyelitis. Flow cytometric analysis of CNS-infiltrating mononuclear cells showed that CD4(+) T cells and monocytes expressed ANKRD55 in experimental autoimmune encephalomyelitis mice, with the higher fluorescence intensity found in CD4(+) cells. A low percentage of microglia also expressed ANKRD55. Together, these data support an important role for ANKRD55 in multiple sclerosis and neuroinflammation.

Summary Cytokine gene polymorphisms are known to influence susceptibility and disease course of many autoimmune diseases. Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system white matter characterized by inflammation, demyelination and axonal damage. We analysed both the well‐known intronic variable number of tandem repeat (VNTR) and +33 C/T single‐nucleotide polymorphisms (SNP) in the IL‐4 gene, as well as the functional Q551R SNP in the IL4‐R gene in a cohort of three distinct populations comprising sporadic cases and controls from the northern Spanish Basque Country and Northern Ireland, as well as family trios from Belgium. The IL‐4 +33 TT genotype was decreased in primary progressive (PP) versus relapsing‐remitting (RR) patients in the Northern Irish population (OR = 0.14; 95% CI = 0.018–1.09). Two‐marker haplotype distribution of the VNTR and +33 C/T SNP in PP patients differed from that seen in RR patients in Northern Ireland ( P = 0.03). The R allele of the Q551R SNP was significantly under‐transmitted in the Belgian trio families ( P = 0.003), although this effect was not seen in the Northern Irish and Basque data sets. We did not identify IL‐4–IL4‐R gene–gene interaction in determining susceptibility or clinical parameters of MS. Disease or genetic heterogeneity or both may be responsible for the observed lack of reproduction in different populations. Our data reinforce recent findings for a role of IL4‐R in susceptibility to MS.

Inflammation in carotid atherosclerotic plaque is linked to plaque rupture and cerebrovascular accidents. The balance between pro- and anti-inflammatory mediators governs development of the plaque, and may mediate enhancement of lesion broadening or, on the contrary, delay progression. In addition to macrophages and endothelial cells, smooth muscle cells (SMCs), which are the dominant cell subset in advanced plaques, are crucial players in carotid atherosclerosis development given their ability to differentiate into distinct phenotypes in reponse to specific signals received from the environment of the lesion. Carotid atheroma SMCs actively contribute to the inflammation in the lesion because of their acquired capacity to produce inflammatory mediators. We review the successive stages of carotid atheroma plaque formation via fatty streak early-stage toward more advanced rupture-prone lesions and document involvement of cytokines and chemokines and their cellular sources and targets in plaque progression and rupture.

The quality control mechanisms directing the folding of cytokines in the endoplasmic reticulum (ER) are poorly understood. We have investigated ER chaperone usage by the cytokine interferon-γ (IFN-γ). ATP-depletion or inhibition of N-glycosylation was found to cause IFN-γ to accumulate into detergent-insoluble aggregates in the ER. Six chaperones, GRP94, GRP78, ERp72, PDI, CaBP1/P5 and CRT were found to associate with IFN-γ during its steady state folding. Interaction of the five first chaperones with IFN-γ was regulated co-ordinately by ATP. These chaperones were recently reported to be part of a multi-chaperone complex involved in the folding of complex, multi-subunit proteins. Our data suggest that also proteins with a relatively simple quaternary structure such as cytokines may fold in association with this complex. In addition, we identified calreticulin as the major chaperone interacting with IFN-γ, and the related class II cytokine interleukin-10, during heat-shock in vivo. IFN-γ was maintained in a folding-competent form by calreticulin during heat-shock and released during subsequent recovery at 37 °C. This interaction was observed in both recombinant (CHO-F11) and natural producer cells (Jurkat, NK-92MI) of IFN-γ. Since cytokines such as IFN-γ and IL-10 are frequently produced in the course of inflammatory conditions associated with fever, the thermo-protective effect of calreticulin may constitute a previously unrecognized component of the cellular cytokine production machinery, of likely relevance in sustaining cytokine folding and secretion in pathophysiological conditions.

Interleukin-12 (IL-12), p80, and IL-23 are structurally related cytokines sharing a p40 subunit. We have recently demonstrated that celecoxib and its COX-2-independent analogue 4-trifluoromethyl-celecoxib (TFM-C) inhibit secretion but not transcription of IL-12 (p35/p40) and p80 (p40/p40). This is associated with a mechanism involving altered cytokine-chaperone interaction in the endoplasmic reticulum (ER). In the present study, we found that celecoxib and TFM-C also block secretion of IL-23 (p40/p19 heterodimers). Given the putative ER-centric mode of these compounds, we performed a comprehensive RT-PCR analysis of 23 ER-resident chaperones/foldases and associated co-factors. This revealed that TFM-C induced 1.5-3-fold transcriptional up-regulation of calreticulin, GRP78, GRP94, GRP170, ERp72, ERp57, ERdj4, and ERp29. However, more significantly, a 7-fold up-regulation of homocysteine-inducible ER protein (HERP) was observed. HERP is part of a high molecular mass protein complex involved in ER-associated protein degradation (ERAD). Using co-immunoprecipitation assays, we show that TFM-C induces protein interaction of p80 and IL-23 with HERP. Both HERP siRNA knockdown and HERP overexpression coupled to cycloheximide chase assays revealed that HERP is necessary for degradation of intracellularly retained p80 by TFM-C. Thus, our data suggest that targeting cytokine folding in the ER by small molecule drugs could be therapeutically exploited to alleviate inappropriate inflammation in autoimmune conditions.

Celecoxib, a highly specific cyclooxygenase-2 (COX-2) inhibitor has been reported to have COX-2-independent immunomodulatory effects. However, celecoxib itself has only mild suppressive effects on arthritis. Recently, we reported that a 4-trifluoromethyl analogue of celecoxib (TFM-C) with 205-fold lower COX-2-inhibitory activity inhibits secretion of IL-12 family cytokines through a COX-2-independent mechanism that involves Ca2+-mediated intracellular retention of the IL-12 polypeptide chains. In this study, we explored the capacity of TFM-C as a new therapeutic agent for arthritis. To induce collagen-induced arthritis (CIA), DBA1/J mice were immunized with bovine type II collagen (CII) in Freund's adjuvant. Collagen antibody-induced arthritis (CAIA) was induced in C57BL/6 mice by injecting anti-CII antibodies. Mice received 10 μg/g of TFM-C or celecoxib every other day. The effects of TFM-C on clinical and histopathological severities were assessed. The serum levels of CII-specific antibodies were measured by ELISA. The effects of TFM-C on mast cell activation, cytokine producing capacity by macophages, and neutrophil recruitment were also evaluated. TFM-C inhibited the severity of CIA and CAIA more strongly than celecoxib. TFM-C treatments had little effect on CII-specific antibody levels in serum. TFM-C suppressed the activation of mast cells in arthritic joints. TFM-C also suppressed the production of inflammatory cytokines by macrophages and leukocyte influx in thioglycollate-induced peritonitis. These results indicate that TFM-C may serve as an effective new disease-modifying drug for treatment of arthritis, such as rheumatoid arthritis.

Cardiovascular disease (CVD), the leading cause of mortality worldwide is primarily caused by atherosclerosis, which is promoted by the accumulation of low-density lipoproteins into the intima of large arteries. Multiple nanoparticles mimicking natural HDL (rHDL) have been designed to remove cholesterol excess in CVD therapy. The goal of this investigation was to assess the cholesterol efflux efficiency of rHDLs with different lipid compositions, mimicking different maturation stages of high-density lipoproteins (HDLs) occurring in vivo.the cholesterol efflux activity of soybean PC (Soy-PC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), DPPC:Chol:1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (LysoPC) and DPPC:18:2 cholesteryl ester (CE):LysoPC rHDLs was determined in several cell models to investigate the contribution of lipid composition to the effectiveness of cholesterol removal.DPPC rHDLs are the most efficient particles, inducing cholesterol efflux in all cellular models and in all conditions the effect was potentiated when the ABCA1 transporter was upregulated.DPPC rHDLs, which resemble nascent HDL, are the most effective particles in inducing cholesterol efflux due to the higher physical binding affinity of cholesterol to the saturated long-chain-length phospholipids and the favored cholesterol transfer from a highly positively curved bilayer, to an accepting planar bilayer such as DPPC rHDLs. The physicochemical characteristics of rHDLs should be taken into consideration to design more efficient nanoparticles to promote cholesterol efflux.

Intronic single-nucleotide polymorphisms (SNPs) in the ANKRD55 gene are associated with the risk for multiple sclerosis (MS) and rheumatoid arthritis by genome-wide association studies (GWAS). The risk alleles have been linked to higher expression levels of ANKRD55 and the neighboring IL6ST (gp130) gene in CD4 + T lymphocytes of healthy controls. The biological function of ANKRD55, its role in the immune system, and cellular sources of expression other than lymphocytes remain uncharacterized. Here, we show that monocytes gain capacity to express ANKRD55 during differentiation in immature monocyte-derived dendritic cells (moDCs) in the presence of interleukin (IL)-4/granulocyte-macrophage colony-stimulating factor (GM-CSF). ANKRD55 expression levels are further enhanced by retinoic acid agonist AM580 but downregulated following maturation with interferon (IFN)-γ and lipopolysaccharide (LPS). ANKRD55 was detected in the nucleus of moDC in nuclear speckles. We also analyzed the adjacent IL6ST , IL31RA , and SLC38A9 genes. Of note, in healthy controls, MS risk SNP genotype influenced ANKRD55 and IL6ST expression in immature moDC in opposite directions to that in CD4 + T cells. This effect was stronger for a partially correlated SNP, rs13186299, that is located, similar to the main MS risk SNPs, in an ANKRD55 intron. Upon analysis in MS patients, the main GWAS MS risk SNP rs7731626 was associated with ANKRD55 expression levels in CD4 + T cells. MoDC-specific ANKRD55 and IL6ST mRNA levels showed significant differences according to the clinical form of the disease, but, in contrast to healthy controls, were not influenced by genotype. We also measured serum sgp130 levels, which were found to be higher in homozygotes of the protective allele of rs7731626. Our study characterizes ANKRD55 expression in moDC and indicates monocyte-to-dendritic cell (Mo–DC) differentiation as a process potentially influenced by MS risk SNPs.

Introduction: SNPs associated with genome-wide risk for multiple sclerosis (MS) modulate expression of ankyrin repeat domain protein 55 (ANKRD55). The function of ANKRD55 is not well understood. A role for ANKRD55 in ciliar transport in multiciliated cells has been reported. To gain deeper insight in how ANKRD55 may modulate neuro-inflammatory parameters, we identified the ANKRD55 interactomes from human neuroblastoma, astrocytic, microglial and monocytic cell lines. Methods: Cell lines were transfected with synthetic ANKRD55 RNA in conjunction with nanoparticles. ANKRD55 interactomes were determined by affinity purification coupled to mass spectrometry (AP-MS) and analyzed bioinformatically. Results were validated and interpreted using confocal immunofluorescence microscopy, RNAseq transcriptomics, and a visible immunoprecipitation assay (VIP). Results: Shared among the interactomes were two 14-3-3 isoforms. Unique to the microglial interactome were eight proteins belonging to the intraflagellar transport complex B (IFT-B). The IFT-B complex is known to mediate anterograde protein trafficking from the base to the tip of cilia. The dimer IFT46-IFT56 was identified as the minimum entity of IFT-B needed to support interaction with ANKRD55. To verify whether ANKRD55 is a ciliar transport protein, we induced ciliogenesis by serum starvation. Primary ARL13B+ cilia could be induced in the astrocytic and neuroblastoma, but not microglial, cell lines. By confocal microscopy, ANKRD55 was not detectable in these cilia but was enriched at the basal body. In the microglial cell line, ANKRD55 and IFT-B components were enriched at the centrosome. In two human primary myeloid cell models, monocyte-derived microglia (MoMG) and monocyte-derived dendritic cells (MoDC), we were able to recapitulate the co-localization of ANKRD55 and IFT81 at the centrosome. Discussion: Our work shows that an ANKRD55-IFT-B-like complex is assembled in microglial cells. Together with the finding that ANKRD55 was not detected in primary cilia, the results suggest that ANKRD55 is associated with an IFT-B pathway that can operate independent of ciliogenesis.

Celecoxib is a selective cyclooxygenase-2 (COX2) inhibitor. We have previously shown that celecoxib inhibits experimental autoimmune encephalomyelitis (EAE) in COX-2-deficient mice, suggestive for a mode of action involving COX2-independent pathways. In the present study, we tested the effect of a trifluoromethyl analogue of celecoxib (TFM-C) with 205-fold lower COX-2 inhibitory activity in two models of neuroinflammation, i.e. cerebellar organotypic cultures challenged with LPS and the EAE mouse model for multiple sclerosis. TFM-C inhibited secretion of IL-1β, IL-12 and IL-17, enhanced that of TNF-α and RANTES, reduced neuronal axonal damage and protected from oxidative stress in the organotypic model. TFM-C blocked TNF-α release in microglial cells through a process involving intracellular retention, but induced TNF-α secretion in primary astrocyte cultures. Finally, we demonstrate that TFM-C and celecoxib ameliorated EAE with equal potency. This coincided with reduced secretion of IL-17 and IFN-γ by MOG-reactive T-cells and of IL-23 and inflammatory cytokines by bone marrow-derived dendritic cells. Our study reveals that non-coxib analogues of celecoxib may have translational value in the treatment of neuro-inflammatory conditions.

Multiple sclerosis (MS) is a prevalent neurological disease of complex etiology. Here, we describe the characterization of a multi-incident MS family that nominated a rare missense variant (p.G420D) in plasminogen (PLG) as a putative genetic risk factor for MS. Genotyping of PLG p.G420D (rs139071351) in 2160 MS patients, and 886 controls from Canada, identified 10 additional probands, two sporadic patients and one control with the variant. Segregation in families harboring the rs139071351 variant, identified p.G420D in 26 out of 30 family members diagnosed with MS, 14 unaffected parents, and 12 out of 30 family members not diagnosed with disease. Despite considerably reduced penetrance, linkage analysis supports cosegregation of PLG p.G420D and disease. Genotyping of PLG p.G420D in 14446 patients, and 8797 controls from Canada, France, Spain, Germany, Belgium, and Austria failed to identify significant association with disease (P = 0.117), despite an overall higher prevalence in patients (OR = 1.32; 95% CI = 0.93-1.87). To assess whether additional rare variants have an effect on MS risk, we sequenced PLG in 293 probands, and genotyped all rare variants in cases and controls. This analysis identified nine rare missense variants, and although three of them were exclusively observed in MS patients, segregation does not support pathogenicity. PLG is a plausible biological candidate for MS owing to its involvement in immune system response, blood-brain barrier permeability, and myelin degradation. Moreover, components of its activation cascade have been shown to present increased activity or expression in MS patients compared to controls; further studies are needed to clarify whether PLG is involved in MS susceptibility.

The ankyrin repeat domain-55 (ANKRD55) gene contains intronic single nucleotide polymorphisms (SNPs) associated with risk to contract multiple sclerosis, rheumatoid arthritis or other autoimmune disorders. Risk alleles of these SNPs are associated with higher levels of ANKRD55 in CD4+ T cells. The biological function of ANKRD55 is unknown, but given that ankyrin repeat domains constitute one of the most common protein-protein interaction platforms in nature, it is likely to function in complex with other proteins. Thus, identification of its protein interactomes may provide clues. We identified ANKRD55 interactomes via recombinant overexpression in HEK293 or HeLa cells and mass spectrometry. One hundred forty-eight specifically interacting proteins were found in total protein extracts and 22 in extracts of sucrose gradient-purified nuclei. Bioinformatic analysis suggested that the ANKRD55-protein partners from total protein extracts were related to nucleotide and ATP binding, enriched in nuclear transport terms and associated with cell cycle and RNA, lipid and amino acid metabolism. The enrichment analysis of the ANKRD55-protein partners from nuclear extracts is related to sumoylation, RNA binding, processes associated with cell cycle, RNA transport, nucleotide and ATP binding. The interaction between overexpressed ANKRD55 isoform 001 and endogenous RPS3, the cohesins SMC1A and SMC3, CLTC, PRKDC, VIM, β-tubulin isoforms, and 14-3-3 isoforms were validated by western blot, reverse immunoprecipitaton and/or confocal microscopy. We also identified three phosphorylation sites in ANKRD55, with S436 exhibiting the highest score as likely 14-3-3 binding phosphosite. Our study suggests that ANKRD55 may exert function(s) in the formation or architecture of multiple protein complexes, and is regulated by (de)phosphorylation reactions. Based on interactome and subcellular localization analysis, ANKRD55 is likely transported into the nucleus by the classical nuclear import pathway and is involved in mitosis, probably via effects associated with mitotic spindle dynamics.

Autophagy has emerged in recent years as a critical cellular survival mechanism for cell homeostasis and may play a protective role in atherosclerosis. We aimed to review here the role autophagy plays in different cell types present in carotid atherosclerotic plaques and that may be associated with the development of unstable carotid atheroma plaque.We performed a thorough literature exploration in this area of research covering the three main cell types present in carotid atheroma plaques.Reviewed reports indicate that the role of autophagy in stable or unstable carotid atherosclerotic plaques depends on the different cell types and phenotypes, the stage and morphology of the plaque and the specific autophagy factor/s involved.Although defective autophagy could be one of the causes for carotid atheroma plaques to become unstable, it is important to take into account that autophagic players can act differentially in different cell types and different stages of the developed plaque.This review provides an overview of the role of autophagy in the main cell types in carotid atherosclerosis (i.e. macrophages, endothelial cells and smooth muscle cells).

HSP70 chaperones mediate protein folding by ATP-dependent interaction with short linear peptide segments that are exposed on unfolded proteins. The mode of action of the <i>Escherichia coli</i> homolog DnaK is representative of all HSP70 chaperones, including the endoplasmic reticulum variant BiP/GRP78. DnaK has been shown to be effective in assisting refolding of a wide variety of prokaryotic and eukaryotic proteins, including the α-helical homodimeric secretory cytokine interferon-γ (IFN-γ). We screened solid-phase peptide libraries from human and mouse IFN-γ to identify DnaK-binding sites. Conserved DnaK-binding sites were identified in the N-terminal half of helix B and in the C-terminal half of helix C, both of which are located at the IFN-γ dimer interface. Soluble peptides derived from helices B and C bound DnaK with high affinity in competition assays. No DnaK-binding sites were found in the loops connecting the α-helices. The helix C DnaK-binding site appears to be conserved in most members of the superfamily of interleukin (IL)-10-related cytokines that comprises, apart from IL-10 and IFN-γ, a series of recently discovered small secretory proteins, including IL-19, IL-20, IL-22/IL-TIF, IL-24/MDA-7 (melanomadifferentiation-associated gene), IL-26/AK155, and a number of viral IL-10 homologs. These cytokines belong to a relatively small group of homodimeric proteins with highly interdigitated interfaces that exhibit the strongly hydrophobic character of the interior core of a single-chain folded domain. We propose that binding of DnaK to helix C in the superfamily of IL-10-related cytokines may constitute the hallmark of a novel conserved regulatory mechanism in which HSP70-like chaperones assist in the formation of a hydrophobic dimeric "folding" interface.

Affinity capture methods are widely used for isolation and analysis of protein complexes. Short peptide tags fused to the protein of interest normally facilitate straightforward purification and detection of interacting proteins. We investigated the suitability of applying C-terminally hexahistidine-tagged interleukin-12 (IL-12) alpha- and beta-chains as "bait" proteins for cocapturing novel binding partners using heterologous recombinant human embryonic kidney-293 (HEK-293) cell lines. The beta-chain, but not the alpha-chain, extracted from cell lysates was capable of binding to the Ni(2+)-nitrilotriacetic acid affinity resin under nondenaturing conditions. Retention of the alpha-chain on this matrix was dependent on treatment of cell lysates with high concentrations of chaotropes such as urea. Since under these conditions any noncovalent protein associations are destroyed, prior cross-linking of proteins interacting with the alpha-chain in intact cells was required. The use of the thiol-cleavable cross-linker 3,3'-dithiobis(succinimidyl proprionate) facilitated dissociation of alpha-chain-binding proteins by means of dithiothreitol following purification. Using this approach we were able to demonstrate a strong interaction between the endoplasmic reticulum chaperone calreticulin (CRT) and the IL-12 alpha-chain that was confirmed in a reciprocal anti-CRT immunoprecipitation assay. The assay presented here provides a simple approach to exposing concealed hexahistidine tags while retaining native noncovalent protein interactions and should be generally applicable in a range of pull-down or affinity capture methods aiming at analysis of protein complexes.

The human IL22RA2 gene co-produces three protein isoforms in dendritic cells (IL-22 binding protein-1, IL-22BPi2 and IL-22BPi3). Two of these, IL-22BPi2 and IL-22BPi3, are capable of neutralizing the biological activity of IL-22. The function of IL-22BPi1, which differs from IL-22BPi2 through an in-frame 32-amino acid insertion provided by an alternatively spliced exon, remains unknown. We demonstrate that IL-22BPi1 is secreted detectably, but at much lower levels than IL-22BPi2, and unlike IL-22BPi2 and IL-22BPi3, is largely retained in the endoplasmic reticulum (ER). As opposed to IL-22BPi2 and IL-22BPi3, IL-22BPi1 is incapable of neutralizing or binding to IL-22 measured in bioassay or assembly-induced IL-22 co-folding assay. We performed interactome analysis in transfected human cell lines, to disclose the mechanism underlying the poor secretion of IL-22BPi1 and identified GRP78, GRP94, GRP170 and calnexin as main interactors. Structure-function analysis revealed that, like IL-22BPi2, IL-22BPi1 binds to the substrate-binding domain of GRP78 as well as to the middle domain of GRP94. Ectopic expression of wild-type GRP78 enhanced, and ATPase-defective GRP94 mutant decreased, secretion of both IL-22BPi1 and iL-22BPi2, while neither of both affected IL-22BPi3 secretion. Thus, IL-22BPi1 is a bona fide client protein of the ER chaperones GRP78 and GRP94 similar to IL-22BPi. However, in contrast to IL-22BPi2 and IL-22BPi3, IL-22BPi1 activates an unfolded protein response resulting in increased protein levels of GRP78 and GRP94. Cloning of the IL22RA2 alternatively spliced exon into an unrelated cytokine, IL-2, bestowed similar characteristics on the resulting protein. Upon silencing of IL22RA2 expression in natural producer cells, i.e. human immature monocyte-derived dendritic cells, GRP78 levels were significantly reduced, suggesting that native IL22RA2 expression naturally contributes to upregulating levels of GRP78 in these cells. The IL22RA2 alternatively spliced exon was reported to be recruited through a single mutation in the proto-splice site of a Long Terminal Repeat retrotransposon sequence in the ape lineage. Our work suggests that positive selection of IL-22BPi1 was not driven by IL-22 antagonism as in the case of IL-22BPi2 and IL-22BPi3, but by capacity for induction of an UPR response.

Single nucleotide polymorphisms (SNPs) near SOCS1 are associated with multiple sclerosis (MS), but the most important SNPs in the area and mechanisms by which they influence the disease are unknown.A haplotype-tagging association study was performed covering 60.5kbp around SOCS1, and the index SNP was validated in a total of 2292 individuals. mRNA expression of SOCS1 and nearby genes was measured in MS patients with different disease courses and healthy controls. SOCS1 protein expression was studied by flow cytometry in a separate cohort of patients and controls. Differentiation and maturation of monocyte-derived dendritic cells (moDCs) were also studied.One SNP, rs423674, reached genome-wide significance. No genotype-specific mRNA expression differences were seen, but, by flow cytometry, significant interactions were observed between genotypes for rs423674 and disease activity (relapse or remission) in B cells and regulatory T cells. Furthermore, homozygotes for the risk allele (GG) showed higher levels of CD1a and CD86 than carriers of the protective allele (GT) in immature moDCs and a greater increase of HLA-DR+ cell percentage than GT heterozygotes upon maturation.rs423674, or its genetic proxies, may influence MS risk by modulating SOCS1 expression in a cell-specific manner and by influencing dendritic cell function.

Recently, we reported an association between a SNP in IL28RA and MS. Here, we performed a fine-mapping of the IL28RA locus by genotyping 10 haplotype-tagging SNPs in a Basque-Spanish population. In addition, based on shared genetic risk loci between autoimmune diseases, a psoriasis-associated SNP located at this locus, rs4649203, was genotyped in four independent populations, comprising a total of 2582 cases and 2614 controls. We did not find any consistent association between IL28RA and MS in these populations, suggesting that, although it may play a role in other autoimmune diseases, this gene is unlikely of general relevance to MS pathogenesis.

Interleukin‐12 (IL‐12) is a heterodimeric cytokine composed of two subunits, p35 and p40. The disulfide‐linked homodimer (p40) 2 has been shown to be a potent IL‐12 antagonist. In the present study, the p40 subunit was refolded from Escherichia coli inclusion bodies. Formation of (p40) 2 was greatly increased in a redox buffer containing reduced and oxidized glutathione, but was not significantly affected by the cosolvents urea, GdnHCl or Chaps. While protein disulfide isomerase (PDI), GroEL/ES or DnaK/J/GrpE suppressed aggregation during refolding of p40, only DnaK/J/GrpE and PDI enhanced p40 dimerization. Oxidative assembly of p40 into (p40) 2 by PDI, but not suppression of aggregation, was strongly dependent on inclusion of BSA in the refolding buffer. It is concluded that both chaperone‐like and disulfide isomerase effects are essential for correct folding of p40 into dimers.

Annals of NeurologyVolume 52, Issue 4 p. 524-525 Letters Interleukin-12 p40 polymorphism and susceptibility to multiple sclerosis Iraide Alloza MSc, Iraide Alloza MSc Cytokine Biology and Genetics Programme, McClay Research Centre, School of Pharmacy, Belfast, United KingdomSearch for more papers by this authorShirley Heggarty PhD, Shirley Heggarty PhD Department of Medical Genetics, Queen's University of Belfast, Belfast, United KingdomSearch for more papers by this authorAn Goris MSc, An Goris MSc Rega Institute for Medical Research, University of Leuven, Leuven, BelgiumSearch for more papers by this authorColin A. Graham PhD, Colin A. Graham PhD Department of Medical Genetics, Queen's University of Belfast, Belfast, United KingdomSearch for more papers by this authorBénédicte Dubois MD, PhD, Bénédicte Dubois MD, PhD Department of Neurology, University Hospital Gasthuisberg, University of Leuven, Leuven, BelgiumSearch for more papers by this authorGavin McDonnell MD, Gavin McDonnell MD Department of Neurology, Royal Victoria Hospital, Belfast, United KingdomSearch for more papers by this authorStanley A. Hawkins FRCP, Stanley A. Hawkins FRCP Department of Neurology, Royal Victoria Hospital, Belfast, United KingdomSearch for more papers by this authorHerwig Carton MD, PhD, Herwig Carton MD, PhD Department of Neurology, University Hospital Gasthuisberg, University of Leuven, Leuven, BelgiumSearch for more papers by this authorKoen Vandenbroeck PhD, Koen Vandenbroeck PhD Cytokine Biology and Genetics Programme, McClay Research Centre, School of Pharmacy, Belfast, United KingdomSearch for more papers by this author Iraide Alloza MSc, Iraide Alloza MSc Cytokine Biology and Genetics Programme, McClay Research Centre, School of Pharmacy, Belfast, United KingdomSearch for more papers by this authorShirley Heggarty PhD, Shirley Heggarty PhD Department of Medical Genetics, Queen's University of Belfast, Belfast, United KingdomSearch for more papers by this authorAn Goris MSc, An Goris MSc Rega Institute for Medical Research, University of Leuven, Leuven, BelgiumSearch for more papers by this authorColin A. Graham PhD, Colin A. Graham PhD Department of Medical Genetics, Queen's University of Belfast, Belfast, United KingdomSearch for more papers by this authorBénédicte Dubois MD, PhD, Bénédicte Dubois MD, PhD Department of Neurology, University Hospital Gasthuisberg, University of Leuven, Leuven, BelgiumSearch for more papers by this authorGavin McDonnell MD, Gavin McDonnell MD Department of Neurology, Royal Victoria Hospital, Belfast, United KingdomSearch for more papers by this authorStanley A. Hawkins FRCP, Stanley A. Hawkins FRCP Department of Neurology, Royal Victoria Hospital, Belfast, United KingdomSearch for more papers by this authorHerwig Carton MD, PhD, Herwig Carton MD, PhD Department of Neurology, University Hospital Gasthuisberg, University of Leuven, Leuven, BelgiumSearch for more papers by this authorKoen Vandenbroeck PhD, Koen Vandenbroeck PhD Cytokine Biology and Genetics Programme, McClay Research Centre, School of Pharmacy, Belfast, United KingdomSearch for more papers by this author First published: 24 September 2002 https://doi.org/10.1002/ana.10348Citations: 14Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. References 1 van Veen T, Crusius JBA, Schrijver HM, et al. Interleukin-12p40 genotype plays a role in the susceptibility to multiple sclerosis. Ann Neurol 2001; 50: 275. 2 Hall MA, McGlinn E, Coakley G, et al. Genetic polymorphism of IL-12 p40 gene in immune-mediated disease. Genes Immun 2000; 1: 219–224. 3 Morahan G, Huang D, Ymer SI, et al. Linkage disequilibrium of a type 1 diabetes susceptibility locus with a regulatory IL12B allele. Nat Genet 2001; 27: 218–221. 4 Thomson G. Mapping disease genes–family-based association studies. Am J Hum Genet 1995; 57: 487–498. 5 Pericak-Vance MA, Rimmler JB, Saunders AM, et al. A meta-analysis of genome screens in multiple sclerosis. Mult Scler 2001; 7: 3–11. Citing Literature Volume52, Issue4October 2002Pages 524-525 ReferencesRelatedInformation

Carotid atherosclerotic plaque rupture can lead to cerebrovascular accident (CVA). By comparing RNA-Seq data from vascular smooth muscle cells (VSMC) extracted from carotid atheroma surgically excised from a group of asymptomatic and symptomatic subjects, we identified more than 700 genomic variants associated with symptomatology (p &lt; 0.05). From these, twelve single nucleotide polymorphisms (SNPs) were selected for further validation. Comparing genotypes of a hospital-based cohort of asymptomatic with symptomatic patients, an exonic SNP in the BIRC6 (BRUCE/Apollon) gene, rs35286811, emerged as significantly associated with CVA symptomatology (p = 0.002; OR = 2.24). Moreover, BIRC6 mRNA levels were significantly higher in symptomatic than asymptomatic subjects upon measurement by qPCR in excised carotid atherosclerotic tissue (p &lt; 0.0001), and significantly higher in carriers of the rs35286811 risk allele (p &lt; 0.0001). rs35286811 is a proxy of a GWAS SNP reported to be associated with red cell distribution width (RDW); RDW was increased in symptomatic patients (p &lt; 0.03), but was not influenced by the rs35286811 genotype in our cohort. BIRC6 is a negative regulator of both apoptosis and autophagy. This work introduces BIRC6 as a novel genetic risk factor for stroke, and identifies autophagy as a genetically regulated mechanism of carotid plaque vulnerability.

Vascular smooth muscle cells (VSMCs) provide vital contractile force within blood vessel walls, yet can also propagate cardiovascular pathologies through proliferative and pro-inflammatory activities. Such phenotypes are driven, in part, by the diverse effects of long non-coding RNAs (lncRNAs) on gene expression. However, lncRNA characterisation in VSMCs in pathological states is hampered by incomplete lncRNA representation in reference annotation. We aimed to improve lncRNA representation in such contexts by assembling non-reference transcripts in RNA sequencing datasets describing VSMCs stimulated in vitro with cytokines, growth factors, or mechanical stress, as well as those isolated from atherosclerotic plaques. All transcripts were then subjected to a rigorous lncRNA prediction pipeline. We substantially improved coverage of lncRNAs responding to pro-mitogenic stimuli, with non-reference lncRNAs contributing 21–32% for each dataset. We also demonstrate non-reference lncRNAs were biased towards enriched expression within VSMCs, and transcription from enhancer sites, suggesting particular relevance to VSMC processes, and the regulation of neighbouring protein-coding genes. Both VSMC-enriched and enhancer-transcribed lncRNAs were large components of lncRNAs responding to pathological stimuli, yet without novel transcript discovery 33–46% of these lncRNAs would remain hidden. Our comprehensive VSMC lncRNA repertoire allows proper prioritisation of candidates for characterisation and exemplifies a strategy to broaden our knowledge of lncRNA across a range of disease states.

Chaperones facilitate proper folding of peptides and bind to misfolded proteins as occurring during periods of cell stress. Complexes of peptides with chaperones induce peptide-directed immunity. Here we analyzed the interaction of (pre)proinsulin with the best characterized chaperone of the hsp70 family, bacterial DnaK.Of a set of overlapping 13-mer peptides of human preproinsulin high affinity binding to DnaK was found for the signal peptide and one further region in each proinsulin domain (A- and B-chain, C-peptide). Among the latter, peptides covering most of the B-chain region B11-23 exhibited strongest binding, which was in the range of known high-affinity DnaK ligands, dissociation equilibrium constant (K'd) of 2.2 ± 0.4 μM. The B-chain region B11-23 is located at the interface between two insulin molecules and not accessible in insulin oligomers. Indeed, native insulin oligomers showed very low DnaK affinity (K'd 67.8 ± 20.8 μM) whereas a proinsulin molecule modified to prevent oligomerization showed good binding affinity (K'd 11.3 ± 7.8 μM).Intact insulin only weakly interacts with the hsp70 chaperone DnaK whereas monomeric proinsulin and peptides from 3 distinct proinsulin regions show substantial chaperone binding. Strongest binding was seen for the B-chain peptide B 11-23. Interestingly, peptide B11-23 represents a dominant autoantigen in type 1 diabetes.

The metalloantibiotic bacitracin is a known inhibitor of protein disulfide isomerase (PDI). The disulfide-linked interleukin-12 (IL-12) alphabeta-heterodimer and beta2-homodimer forms are crucial mediators of cell-mediated immune responses and inflammatory reactions. Bacitracin was found to potently block secretion of both the alphabeta- and beta2-dimer forms of IL-12, while it did not affect secretion of the beta-monomer. This inhibition coincided with a reduction in the intracellular amount of PDI found in complex with the beta-chain during intracellular transit. Bacitracin did not affect mRNA levels of the alphabeta- and beta-chain. Similar to bacitracin, N-acetylcysteine blocked alphabeta- and beta2-secretion as well as PDI-beta-chain complex formation. Thus, blocking PDI or shifting the endoplasmic reticulum towards a more reduced status disrupts the oxidative folding pathway or assembly of IL-12 dimer forms. The assembly stage of cytokines in the endoplasmic reticulum may represent a novel target for pharmacological intervention.

Of the three interleukin-22 binding protein (IL-22BP) isoforms produced by the human IL22RA2 gene, IL-22BPi2 and IL-22BPi3 are capable of neutralizing IL-22. The longest isoform, IL-22BPi1, does not bind IL-22, is poorly secreted, and its retention within the endoplasmic reticulum (ER) is associated with induction of an unfolded protein response (UPR). Therapeutic modulation of IL-22BPi2 and IL-22BPi3 production may be beneficial in IL-22-dependent disorders. Recently, we identified the ER chaperones GRP94 and cyclophilin B in the interactomes of both IL-22BPi1 and IL-22BPi2. In this study, we investigated whether secretion of the IL-22BP isoforms could be modulated by pharmacological targeting of GRP94 and cyclophilin B, either by means of geldanamycin, that binds to the ADP/ATP pocket shared by HSP90 paralogs, or by cyclosporin A, which causes depletion of ER cyclophilin B levels through secretion. We found that geldanamycin and its analogs did not influence secretion of IL-22BPi2 or IL-22BPi3, but significantly enhanced intracellular and secreted levels of IL-22BPi1. The secreted protein was heterogeneously glycosylated, with both high-mannose and complex-type glycoforms present. In addition, cyclosporine A augmented the secretion of IL-22BPi1 and reduced that of IL-22BPi2 and IL-22BPi3. Our data indicate that the ATPase activity of GRP94 and cyclophilin B are instrumental in ER sequestration and degradation of IL-22BPi1, and that blocking these factors mobilizes IL-22BPi1 toward the secretory route.

Lopez de Lapuente, A., A. Feliu, N. Ugidos, M. Mecha, J. Mena, I. Astobiza, J. Riera, F. Carillo-Salinas, M. Comabella, X. Montalban, I. Alloza, C. Guaza, and K. Vandenbroeck. 2016. Novel insights into the multiple sclerosis risk gene ANKRD55. J. Immunol. 196: [4553–4565][1]. The eighth author

The IL22RA2 locus is associated with risk for multiple sclerosis (MS) but causative variants are yet to be determined. In a single nucleotide polymorphism (SNP) screen of this locus in a Basque population, rs28385692, a rare coding variant substituting Leu for Pro at position 16 emerged significantly (p = 0.02). This variant is located in the signal peptide (SP) shared by the three secreted protein isoforms produced by IL22RA2 (IL-22 binding protein-1(IL-22BPi1), IL-22BPi2 and IL-22BPi3). Genotyping was extended to a Europe-wide case-control dataset and yielded high significance in the full dataset (p = 3.17 × 10−4). Importantly, logistic regression analyses conditioning on the main known MS-associated SNP at this locus, rs17066096, revealed that this association was independent from the primary association signal in the full case-control dataset. In silico analysis predicted both disruption of the alpha helix of the H-region of the SP and decreased hydrophobicity of this region, ultimately affecting the SP cleavage site. We tested the effect of the p.Leu16Pro variant on the secretion of IL-22BPi1, IL-22BPi2 and IL-22BPi3 and observed that the Pro16 risk allele significantly lowers secretion levels of each of the isoforms to around 50%–60% in comparison to the Leu16 reference allele. Thus, our study suggests that genetically coded decreased levels of IL-22BP isoforms are associated with augmented risk for MS.

The main aim of our study was to fine-map the genetic association of a region around the SOCS1 gene with multiple sclerosis (MS) and to study the functional implication of the most associated genetic variants. For that, we genotyped 22 haplotype tagging SNPs, covering a 60 kb region around the gene, in 574 MS patients and 647 healthy controls. 14 SNPs showed a significant association, some of them reaching p values of 10−7. The most strongly associated SNPs lie in intergenic regions 20–40 kb upstream of SOCS1. qPCR of mRNA from whole peripheral blood mononuclear cells (PBMCs) obtained from MS patients and healthy controls who had been genotyped for the most associated SNP, rs423674, and the index SNP from a preliminary screening, rs243324, revealed that SOCS1 was significantly higher expressed in relapsing remitting MS (RRMS) patients during relapse and in primary progressive MS (PPMS) patients than in controls (p = 0.0015). We did not see genotype related differential expression of SOCS1 mRNA in whole PBMCs. Flow cytometry analysis of SOCS1 protein expression on PBMC subsets showed that the risk allele of rs423674 was associated with lower SOCS1 main fluorescence intensity (MFI) in regulatory T cells (Tregs), both in the relapse and in the remission stages of RRMS. Moreover, Tregs from homozygotes for the risk allele tended to downregulate SOCS1 in the relapse stage more than carriers of the protective allele. In contrast, in B lymphocytes, CD4 and CD8 T lymphocytes and NK cells, homozygotes for the risk alleles upregulated SOCS1 in the relapse stage, while carriers of the risk allele tended to downregulate it. Further, immature monocyte-derived dendritic cells (moDC) from homozygotes for the risk allele of rs423674 showed a higher significant expression of the differentiation marker CD1a than heterozygotes, and, after exposure to bacterian lypopolysaccharide (LPS) or myelin basic protein (MBP), mature moDC tended to have a higher expression of maturation markers CD80, CD86 and HLA-DR than heterozygotes, suggesting that the risk allele of rs423674 might be associated with increased differentiation and maturation of moDC. This effect is likely independent from SOCS1, because the correlation between the expression of SOCS1 and maturation marker tended to be inverse. Our results suggest that the association of rs423674 with MS might be mediated by its relation with SOCS1 expression in immune cells and by its association with DC differentiation and maturation.

IL22RA2 (IL-22 binding protein, IL-22BP) is a soluble high-affinity antagonist of the cytokine IL-22, and has emerged recently with genome-wide significance as a risk locus for multiple sclerosis (MS). We performed a single nucleotide polymorphism (SNP) screen to identify putative causative variants, and identified a non-synonymous (NS) SNP significantly associated with MS in a multi-center screen including over 16,000 MS patients and controls. In order to evaluate if this SNP affected IL22RA2 protein processing and secretion, a site-directed mutagenesis was carried out, and both constitutive CMV-promoter-based and Tet-Express inducible vectors expressing C-terminally FLAG-tagged wild-type and mutant proteins were generated. Vectors were transfected in HEK293 cells and the intracellular and secreted protein fractions were analyzed by western blot. This showed that this NS SNP modulates secretion levels of the protein, with lower levels observed for the mutant than for the wild-type protein. As part of this work we performed a characterization of the N-glycosylation status of the recombinant protein and demonstrate that it contains uniquely high-mannose chains (43 kD) which are converted into complex sugar chains in the secreted form augmending its Mr to 50 kD. We have also investigated transcription, secretion and N-glycosylation of IL22RA2 produced by monocyte-derived dendritic cells and have characterized in detail the intracellular and secreted forms of the natural protein. We will present both a detailed overview of the genetic study and of the characterization of recombinant and natural IL22RA2.

Heterodimer formation of IL-12 and IL-23 is known to take place in the ER. Peptidyl-prolyl cis–trans isomerases B and C (PPIB/C; cyclophilins B and C) locate to the endoplasmic reticulum (ER). PPIB interacts with multiple ER residents including calnexin, calreticulin, GRP94, BiP, Erp72. Cyclosporine A (CsA) induces secretion of PPIB/C thus strongly reducing the intracellular concentration of both cyclophilins. Intracellular removal of PPIB/C can also be attained by siRNA knockdown. We used both approaches to assess the role of PPIB/C on assembly and secretion of the heterodimeric cytokines IL-12 and IL-23 in a recombinant HEK293 cell line permanently expressing the p40 subunit, and transiently transfected with either the p19 or p35 subunits. Removal of PPIB/C reduced secretion of IL-12 and blocked that of IL-23. This did not coincide with altered levels of mRNA suggestive for a ER-centered effect. Pull down of the p40 subunit via a C-terminal his-tag revealed association with PPIB but not PPIC. These observations were validated in monocyte-derived dendritic cells stimulated with IFN-gamma and zymosan, inducers of both IL-12 and IL-23. Knock-down of PPIB/C is known to enhance the oxidation status of the ER. Our data indicates that PPIB is involved in assembly of IL-12 and IL-23 heterodimer assembly either via its peptidyl-prolyl cis–trans isomerase activity and/or via facilitating disulfide-bond formation. This data show also that the known suppressive effect of CsA on IL-12/IL-23 secretion form natural producer cells may be due to both transcriptional repression and interference with ER assembly.

IL23A, or p19, forms part of heterodimeric cytokine IL-23, and is known to be upregulated under conditions of endoplasmic reticulum (ER) stress in lymphocytes and antigen presenting cells. The purpose of our study was first to verify whether ER stress induction is confined to the p19 subunit only, or whether other subunits of the IL12-/IL-23 family (p35, EBI3, p40, p28) are also upregulated. Second, we wondered also whether the ER-stress-induced upregulation of p19 is restricted to cells of immune lineage or reflects a more generalized pathway that can be activated also in non-myeloid cancer cell lines. We used QPCR to assess kinetics and extent of upregulation of the 5 subunits in the lung epithelial cell line A549 under ER stress conditions, assessed p19 upregulation in HEK293, Hela, A549, Du145 MCF-7, PC3, HT29 and U937 cell lines by ER stress, and analysed the subcellular location of p19 in ER-stressed A549, Du145 and U937 cells. Thapsigargin-induced upregulation of p19 mRNA but not of the other subunits was observed independent of cell type, and was not reflected in enhanced levels of IL-23 secretion measured by ELISA. Immunofluorescence microscopy revealed perinuclear location of p19 in Du145, A549 and U937 cells. Sucrose gradient centrifugation to isolate nuclei of ER-stressed A549 cells revealed that a fraction of p19 was associated with the nucleus. ER stress-induced p19 upregulation is seen in cancer cell and may reflect a novel biological function of p19 independent from its role as composing subunit of the cytokine IL-23.

Introduction: Celecoxib, a highly specific cyclooxygenase-2 (COX-2) inhibitor has been reported to have COX-2independent immunomodulatory effects. However, celecoxib itself has only mild suppressive effects on arthritis. Recently, we reported that a 4-trifluoromethyl analogue of celecoxib (TFM-C) with 205-fold lower COX-2-inhibitory activity inhibits secretion of IL-12 family cytokines through a COX-2-independent mechanism that involves Ca 2 + -mediated intracellular retention of the IL-12 polypeptide chains. In this study, we explored the capacity of TFM-C as a new therapeutic agent for arthritis. Methods: To induce collagen-induced arthritis (CIA), DBA1/J mice were immunized with bovine type II collagen (CII) in Freund’s adjuvant. Collagen antibody-induced arthritis (CAIA) was induced in C57BL/6 mice by injecting anti-CII antibodies. Mice received 10 μg/g of TFM-C or celecoxib every other day. The effects of TFM-C on clinical and histopathological severities were assessed. The serum levels of CII-specific antibodies were measured by ELISA. The effects of TFM-C on mast cell activation, cytokine producing capacity by macophages, and neutrophil recruitment were also evaluated. Results: TFM-C inhibited the severity of CIA and CAIA more strongly than celecoxib. TFM-C treatments had little effect on CII-specific antibody levels in serum. TFM-C suppressed the activation of mast cells in arthritic joints. TFMC also suppressed the production of inflammatory cytokines by macrophages and leukocyte influx in thioglycollateinduced peritonitis. Conclusion: These results indicate that TFM-C may serve as an effective new disease-modifying drug for treatment of arthritis, such as rheumatoid arthritis.

Event Abstract Back to Event Confirmation of Human Endogenous Retrovirus HERV-Fc1 role in multiple sclerosis risk J Varade1, B De La Hera1, I Alloza2, 3, A Alcina4, R Álvarez-Lafuente5, K Vandenbroeck2, 3, F Matesanz4 and E Urcelay1* 1 Immunology Department, Hospital Clínico San Carlos. Instituto de Investigación Sanitaria del Hospital Clínico San Carlos (IdISSC), Spain 2 Neurogenomiks Laboratory, Dept. of Neuroscience, University of the Basque Country UPV/EHU,, Spain 3 IKERBASQUE, Basque Foundation for Science,, Spain 4 Inst. Parasitología y Biomedicina “López Neyra”, CSIC, Spain 5 Multiple Sclerosis Unit, Hospital Clínico San Carlos. Instituto de Investigación Sanitaria del Hospital Clínico San Carlos (IdISSC), Spain Background: Multiple sclerosis (MS) is a chronic demyelinating disease with an aetiology not completely ascertained, albeit the prevailing hypothesis contemplates the triggering effect of environmental factors in genetically susceptible individuals. The genetics of MS has undergone a spectacular advance propelled by the genome wide association studies (GWAS) recently performed. However, this robust approach accounts only for part of the estimated genetic load, and where the so called missing heritability lies is intriguing. Provided GWAS do not scrutinize repetitive regions of the genome, their role in MS predisposition gains interest. Part of these repetitive sequences corresponds to human endogenous retroviruses (HERVs), which are proviral loci inherited under a Mendelian pattern. We aimed to validate the previously reported association of HERV-Fc1 with MS susceptibility. Methods: The rs391745 polymorphism located upstream of the HERV-Fc1 locus in chromosome X was genotyped by TaqMan assays in independent cohorts from Central (714 patients and 692 controls), South (880 patients and 1359 controls) and North (368 patients and 980 controls) Spain. Results: North and Central Spanish cohorts including non primary progressive MS patients and showing association results without heterogeneity were combined to achieve a higher statistical power. A significant association with MS risk was evidenced [pM-H=0.02; OR (95% CI)=1.27 (1.03-1.55)], replicating previously reported Danish and Norwegian results. Meta-analysis of all available data yielded a strong effect [p=0.0003; OR (95% CI)=1.32 (1.32-1.53)]. Conclusion: Our work supports the association of HERV-Fc1 in MS pathogenesis, identifying a chromosome-X genetic marker that could lay behind genetic differences in MS clinical forms. Acknowledgements Angel García and Carmen Martínez provided expert technical assistance. Belen de la Hera is recipient of a PhD scholarship from ‘Fondo de Investigaciones Sanitarias’ (FI11/00560), Jezabel Varade is recipient of a contract from “Ministerio de Economía y Competitividad” (PTA2011-6137-1) and Elena Urcelay works for the Fundación para la Investigación Biomédica-Hospital Clínico San Carlos. Fondo de Investigaciones Sanitarias FEDER-FIS [PI08/1636 and PI10/1985] and Fundación Alicia Koplowitz. Keywords: Multiple Sclerosis, genetic marker, Herv-FC1, inmunogenetics, neuroimmunology Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Immune-mediated disease pathogenesis Citation: Varade J, De La Hera B, Alloza I, Alcina A, Álvarez-Lafuente R, Vandenbroeck K, Matesanz F and Urcelay E (2013). Confirmation of Human Endogenous Retrovirus HERV-Fc1 role in multiple sclerosis risk. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00309 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 19 Mar 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. E Urcelay, Immunology Department, Hospital Clínico San Carlos. Instituto de Investigación Sanitaria del Hospital Clínico San Carlos (IdISSC), Madrid, Spain, eurcelay.hcsc@salud.madrid.org Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers J Varade B De La Hera I Alloza A Alcina R Álvarez-Lafuente K Vandenbroeck F Matesanz E Urcelay Google J Varade B De La Hera I Alloza A Alcina R Álvarez-Lafuente K Vandenbroeck F Matesanz E Urcelay Google Scholar J Varade B De La Hera I Alloza A Alcina R Álvarez-Lafuente K Vandenbroeck F Matesanz E Urcelay PubMed J Varade B De La Hera I Alloza A Alcina R Álvarez-Lafuente K Vandenbroeck F Matesanz E Urcelay Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. 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