SERS analysis of cancer cell-secreted purines reveals a unique paracrine crosstalk in MTAP-deficient tumors.

The tumor microenvironment (TME) is a dynamic pseudoorgan that shapes the development and progression of cancers. It is a complex ecosystem shaped by interactions between tumor and stromal cells. Although the traditional focus has been on the paracrine communication mediated by protein messengers, recent attention has turned to the metabolic secretome in tumors. Metabolic enzymes, together with exchanged substrates and products, have emerged as potential biomarkers and therapeutic targets. However, traditional techniques for profiling secreted metabolites in complex cellular contexts are limited. Surface-enhanced Raman scattering (SERS) has emerged as a promising alternative due to its nontargeted nature and simplicity of operation. Although SERS has demonstrated its potential for detecting metabolites in biological settings, its application in deciphering metabolic interactions within multicellular systems like the TME remains underexplored. In this study, we introduce a SERS-based strategy to investigate the secreted purine metabolites of tumor cells lacking methylthioadenosine phosphorylase (MTAP), a common genetic event associated with poor prognosis in various cancers. Our SERS analysis reveals that MTAP-deficient cancer cells selectively produce methylthioadenosine (MTA), which is taken up and metabolized by fibroblasts. Fibroblasts exposed to MTA exhibit: i) molecular reprogramming compatible with cancer aggressiveness, ii) a significant production of purine derivatives that could be readily recycled by cancer cells, and iii) the capacity to secrete purine derivatives that induce macrophage polarization. Our study supports the potential of SERS for cancer metabolism research and reveals an unprecedented paracrine crosstalk that explains TME reprogramming in MTAP-deleted cancers.

Corrigendum: mTORC1-dependent AMD1 regulation sustains polyamine metabolism in prostate cancer.

This corrects the article DOI: 10.1038/nature22964.

METTL1 promotes tumorigenesis through tRNA-derived fragment biogenesis in prostate cancer.

Newly growing evidence highlights the essential role that epitranscriptomic marks play in the development of many cancers; however, little is known about the role and implications of altered epitranscriptome deposition in prostate cancer. Here, we show that the transfer RNA N7-methylguanosine (m7G) transferase METTL1 is highly expressed in primary and advanced prostate tumours. Mechanistically, we find that METTL1 depletion causes the loss of m7G tRNA methylation and promotes the biogenesis of a novel class of small non-coding RNAs derived from 5'tRNA fragments. 5'tRNA-derived small RNAs steer translation control to favour the synthesis of key regulators of tumour growth suppression, interferon pathway, and immune effectors. Knockdown of Mettl1 in prostate cancer preclinical models increases intratumoural infiltration of pro-inflammatory immune cells and enhances responses to immunotherapy. Collectively, our findings reveal a therapeutically actionable role of METTL1-directed m7G tRNA methylation in cancer cell translation control and tumour biology.

mTORC1-dependent AMD1 regulation sustains polyamine metabolism in prostate cancer.

Activation of the PTEN-PI3K-mTORC1 pathway consolidates metabolic programs that sustain cancer cell growth and proliferation. Here we show that mechanistic target of rapamycin complex 1 (mTORC1) regulates polyamine dynamics, a metabolic route that is essential for oncogenicity. By using integrative metabolomics in a mouse model and human biopsies of prostate cancer, we identify alterations in tumours affecting the production of decarboxylated S-adenosylmethionine (dcSAM) and polyamine synthesis. Mechanistically, this metabolic rewiring stems from mTORC1-dependent regulation of S-adenosylmethionine decarboxylase 1 (AMD1) stability. This novel molecular regulation is validated in mouse and human cancer specimens. AMD1 is upregulated in human prostate cancer with activated mTORC1. Conversely, samples from a clinical trial with the mTORC1 inhibitor everolimus exhibit a predominant decrease in AMD1 immunoreactivity that is associated with a decrease in proliferation, in line with the requirement of dcSAM production for oncogenicity. These findings provide fundamental information about the complex regulatory landscape controlled by mTORC1 to integrate and translate growth signals into an oncogenic metabolic program.

1H NMR-Based Urine Metabolomics Reveals Signs of Enhanced Carbon and Nitrogen Recycling in Prostate Cancer.

Prostate cancer is the second most common tumor and the fifth cause of cancer-related death among men worldwide. PC cells exhibit profound signaling and metabolic reprogramming that account for the acquisition of aggressive features. Although the metabolic understanding of this disease has increased in recent years, the analysis of such alterations through noninvasive methodologies in biofluids remains limited. Here, we used NMR-based metabolomics on a large cohort of urine samples (more than 650) from PC and benign prostate hyperplasia (BPH) patients to investigate the molecular basis of this disease. Multivariate analysis failed to distinguish between the two classes, highlighting the modest impact of prostate alterations on urine composition and the multifactorial nature of PC. However, univariate analysis of urine metabolites unveiled significant changes, discriminating PC from BPH. Metabolites with altered abundance in urine from PC patients revealed changes in pathways related to cancer biology, including glycolysis and the urea cycle. We found out that metabolites from such pathways were diminished in the urine from PC individuals, strongly supporting the notion that PC reduces nitrogen and carbon waste in order to maximize their usage in anabolic processes that support cancer cell growth.

Pharmacological Targeting of the ER-Resident Chaperones GRP94 or Cyclophilin B Induces Secretion of IL-22 Binding Protein Isoform-1 (IL-22BPi1).

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.

Novel Insights into the Multiple Sclerosis Risk Gene ANKRD55.

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.

A functional variant that affects exon-skipping and protein expression of SP140 as genetic mechanism predisposing to multiple sclerosis.

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.

Genome-wide significant association with seven novel multiple sclerosis risk loci.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Cell-specific effects in different immune subsets associated with SOCS1 genotypes in multiple sclerosis.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: rs423674, or its genetic proxies, may influence MS risk by modulating SOCS1 expression in a cell-specific manner and by influencing dendritic cell function.

The PP2A regulator IER5L supports prostate cancer progression.

Prostate cancer exhibits high prevalence and accounts for a high number of cancer-related deaths. The discovery and characterization of molecular determinants of aggressive prostate cancer represents an active area of research. The Immediate Early Response (IER) family of genes, which regulate Protein Phosphatase 2A (PP2A) activity, has emerged among the factors that influence cancer biology. Here, we show that the less studied member of this family, Immediate Early Response 5 like (IER5L), is upregulated in aggressive prostate cancer. Interestingly, the upregulation of IER5L expression exhibits a robust association with metastatic disease in prostate and is recapitulated in other cancer types. In line with this observation, IER5L silencing reduces foci formation, migration and invasion ability in a variety of human and murine prostate cancer cell lines. In vivo, using zebrafish and immunocompromised mouse models, we demonstrate that IER5L-silencing reduces prostate cancer tumor growth, dissemination, and metastasis. Mechanistically, we characterize the transcriptomic and proteomic landscapes of IER5L-silenced cells. This approach allowed us to identify DNA replication and monomeric G protein regulators as downstream programs of IER5L through a pathway that is consistent with the regulation of PP2A. In sum, we report the alteration of IER5L in prostate cancer and beyond and provide biological and molecular evidence of its contribution to tumor aggressiveness.

Molecular method for the characterization of Coxiella burnetii from clinical and environmental samples: variability of genotypes in Spain.

BACKGROUND: Coxiella burnetii is a highly clonal microorganism which is difficult to culture, requiring BSL3 conditions for its propagation. This leads to a scarce availability of isolates worldwide. On the other hand, published methods of characterization have delineated up to 8 different genomic groups and 36 genotypes. However, all these methodologies, with the exception of one that exhibited limited discriminatory power (3 genotypes), rely on performing between 10 and 20 PCR amplifications or sequencing long fragments of DNA, which make their direct application to clinical samples impracticable and leads to a scarce accessibility of data on the circulation of C. burnetii genotypes. RESULTS: To assess the variability of this organism in Spain, we have developed a novel method that consists of a multiplex (8 targets) PCR and hybridization with specific probes that reproduce the previous classification of this organism into 8 genomic groups, and up to 16 genotypes. It allows for a direct characterization from clinical and environmental samples in a single run, which will help in the study of the different genotypes circulating in wild and domestic cycles as well as from sporadic human cases and outbreaks. The method has been validated with reference isolates. A high variability of C. burnetii has been found in Spain among 90 samples tested, detecting 10 different genotypes, being those adaA negative associated with acute Q fever cases presenting as fever of intermediate duration with liver involvement and with chronic cases. Genotypes infecting humans are also found in sheep, goats, rats, wild boar and ticks, and the only genotype found in cattle has never been found among our clinical samples. CONCLUSIONS: This newly developed methodology has permitted to demonstrate that C. burnetii is highly variable in Spain. With the data presented here, cattle seem not to participate in the transmission of C. burnetii to humans in the samples studied, while sheep, goats, wild boar, rats and ticks share genotypes with the human population.

Genotyping of Coxiella burnetii from domestic ruminants in northern Spain.

BACKGROUND: Information on the genotypic diversity of Coxiella burnetii isolates from infected domestic ruminants in Spain is limited. The aim of this study was to identify the C. burnetii genotypes infecting livestock in Northern Spain and compare them to other European genotypes. A commercial real-time PCR targeting the IS1111a insertion element was used to detect the presence of C. burnetii DNA in domestic ruminants from Spain. Genotypes were determined by a 6-loci Multiple Locus Variable number tandem repeat analysis (MLVA) panel and Multispacer Sequence Typing (MST). RESULTS: A total of 45 samples from 4 goat herds (placentas, N = 4), 12 dairy cattle herds (vaginal mucus, individual milk, bulk tank milk, aerosols, N = 20) and 5 sheep flocks (placenta, vaginal swabs, faeces, air samples, dust, N = 21) were included in the study. Samples from goats and sheep were obtained from herds which had suffered abortions suspected to be caused by C. burnetii, whereas cattle samples were obtained from animals with reproductive problems compatible with C. burnetii infection, or consisted of bulk tank milk (BTM) samples from a Q fever surveillance programme. C. burnetii genotypes identified in ruminants from Spain were compared to those detected in other countries. Three MLVA genotypes were found in 4 goat farms, 7 MLVA genotypes were identified in 12 cattle herds and 4 MLVA genotypes were identified in 5 sheep flocks. Clustering of the MLVA genotypes using the minimum spanning tree method showed a high degree of genetic similarity between most MLVA genotypes. Overall 11 different MLVA genotypes were obtained corresponding to 4 different MST genotypes: MST genotype 13, identified in goat, sheep and cattle from Spain; MST genotype 18, only identified in goats; and, MST genotypes 8 and 20, identified in small ruminants and cattle, respectively. All these genotypes had been previously identified in animal and human clinical samples from several European countries, but some of the MLVA genotypes are described here for the first time. CONCLUSIONS: Genotyping revealed a substantial genetic diversity among domestic ruminants from Northern Spain.

Four-year evaluation of the effect of vaccination against Coxiella burnetii on reduction of animal infection and environmental contamination in a naturally infected dairy sheep flock.

Vaccination is considered one of the best options for controlling Coxiella burnetii infection in livestock. The efficacy of a phase I vaccine was investigated over 4 years in a sheep flock with confirmed C. burnetii infection. Shedding was not detected in ewes and yearlings in the last 2 years, but C. burnetii still persisted in the environment.

Identification of Androgen Receptor Metabolic Correlome Reveals the Repression of Ceramide Kinase by Androgens.

Prostate cancer (PCa) is one of the most prevalent cancers in men. Androgen receptor signaling plays a major role in this disease, and androgen deprivation therapy is a common therapeutic strategy in recurrent disease. Sphingolipid metabolism plays a central role in cell death, survival, and therapy resistance in cancer. Ceramide kinase (CERK) catalyzes the phosphorylation of ceramide to ceramide 1-phosphate, which regulates various cellular functions including cell growth and migration. Here we show that activated androgen receptor (AR) is a repressor of CERK expression. We undertook a bioinformatics strategy using PCa transcriptomics datasets to ascertain the metabolic alterations associated with AR activity. CERK was among the most prominent negatively correlated genes in our analysis. Interestingly, we demonstrated through various experimental approaches that activated AR reduces the mRNA expression of CERK: (i) expression of CERK is predominant in cell lines with low or negative AR activity; (ii) AR agonist and antagonist repress and induce CERK mRNA expression, respectively; (iii) orchiectomy in wildtype mice or mice with PCa (harboring prostate-specific Pten deletion) results in elevated Cerk mRNA levels in prostate tissue. Mechanistically, we found that AR represses CERK through interaction with its regulatory elements and that the transcriptional repressor EZH2 contributes to this process. In summary, we identify a repressive mode of AR that influences the expression of CERK in PCa.

Seroepidemiological study of Q fever in domestic ruminants in semi-extensive grazing systems.

BACKGROUND: Q fever, a worldwide zoonotic disease caused by Coxiella burnetii, is endemic in northern Spain where it has been reported as responsible for large series of human pneumonia cases and domestic ruminants' reproductive disorders. To investigate pathogen exposure among domestic ruminants in semi-extensive grazing systems in northern Spain, a serosurvey was carried out in 1,379 sheep (42 flocks), 626 beef cattle (46 herds) and 115 goats (11 herds). Serum antibodies were analysed by ELISA and positive samples were retested by Complement Fixation test (CFT) to detect recent infections. RESULTS: ELISA anti-C. burnetii antibody prevalence was slightly higher in sheep (11.8 +/- 2.0%) than in goats (8.7 +/- 5.9%) and beef cattle (6.7 +/- 2.0%). Herd prevalence was 74% for ovine, 45% for goat and 43% for bovine. Twenty-one percent of sheep flocks, 27% of goat and 14% of cattle herds had a C. burnetii seroprevalence >or= 20%. Only 15 out of 214 ELISA-positive animals reacted positive by CFT. Age-associated seroprevalence differed between ruminant species with a general increasing pattern with age. No evidence of correlation between abortion history and seroprevalence rates was observed despite the known abortifacient nature of C. burnetii in domestic ruminants. CONCLUSIONS: Results reported herein showed that sheep had the highest contact rate with C. burnetii in the region but also that cattle and goats should not be neglected as part of the domestic cycle of C. burnetii. This work reports basic epidemiologic patterns of C. burnetii in semi-extensive grazed domestic ruminants which, together with the relevant role of C. burnetii as a zoonotic and abortifacient agent, makes these results to concern both Public and Animal Health Authorities.

Targeting PML in triple negative breast cancer elicits growth suppression and senescence.

Oncogene addiction postulates that the survival and growth of certain tumor cells is dependent upon the activity of one oncogene, despite their multiple genetic and epigenetic abnormalities. This phenomenon provides a foundation for molecular targeted therapy and a rationale for oncogene-based stratification. We have previously reported that the Promyelocytic Leukemia protein (PML) is upregulated in triple negative breast cancer (TNBC) and it regulates cancer-initiating cell function, thus suggesting that this protein can be therapeutically targeted in combination with PML-based stratification. However, the effects of PML perturbation on the bulk of tumor cells remained poorly understood. Here we demonstrate that TNBC cells are addicted to the expression of this nuclear protein. PML inhibition led to a remarkable growth arrest combined with features of senescence in vitro and in vivo. Mechanistically, the growth arrest and senescence were associated to a decrease in MYC and PIM1 kinase levels, with the subsequent accumulation of CDKN1B (p27), a trigger of senescence. In line with this notion, we found that PML is associated to the promoter regions of MYC and PIM1, consistent with their direct correlation in breast cancer specimens. Altogether, our results provide a feasible explanation for the functional similarities of MYC, PIM1, and PML in TNBC and encourage further study of PML targeting strategies for the treatment of this breast cancer subtype.

Genomic and Functional Regulation of TRIB1 Contributes to Prostate Cancer Pathogenesis.

Prostate cancer is the most frequent malignancy in European men and the second worldwide. One of the major oncogenic events in this disease includes amplification of the transcription factor cMYC. Amplification of this oncogene in chromosome 8q24 occurs concomitantly with the copy number increase in a subset of neighboring genes and regulatory elements, but their contribution to disease pathogenesis is poorly understood. Here we show that TRIB1 is among the most robustly upregulated coding genes within the 8q24 amplicon in prostate cancer. Moreover, we demonstrate that TRIB1 amplification and overexpression are frequent in this tumor type. Importantly, we find that, parallel to its amplification, TRIB1 transcription is controlled by cMYC. Mouse modeling and functional analysis revealed that aberrant TRIB1 expression is causal to prostate cancer pathogenesis. In sum, we provide unprecedented evidence for the regulation and function of TRIB1 in prostate cancer.

The Rare IL22RA2 Signal Peptide Coding Variant rs28385692 Decreases Secretion of IL-22BP Isoform-1, -2 and -3 and Is Associated with Risk for Multiple Sclerosis.

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.