Targeted resequencing of the 13q13 spondyloarthritis-linked locus identifies a rare variant in FREM2 possibly associated with familial spondyloarthritis.

OBJECTIVES: The strong heritability of spondyloarthritis remains poorly explained, despite several large-scale association studies. A recent linkage analysis identified a new region linked to SpA on 13q13. Here we searched for variants potentially explaining this linkage signal by deep-sequencing of the region. METHODS: Re-sequencing of the 1.4 Mb target interval was performed in 92 subjects from the 43 best-linked multicases families (71 spondyloarthritis and 21 unaffected relatives), using hybridization capture-based protocol (Illumina Nextera®). Variants of interest were then genotyped by TaqMan and high resolution melting to check their co-segregation with disease in the same families and to test their association with spondyloarthritis in an independent cohort of 1,091 unrelated cases and 399 controls. Expression of FREM2 was assessed by immunostaining. RESULTS: Of the 7,563 variants identified, 24 were non-synonymous coding single-nucleotide variants. Two of them were located in the FREM2 gene on a haplotype co-segregating with the disease, including one common variant (R1840W, minor allele frequency=0.11) and one rare variant (R727H, minor allele frequency=0.0001). In the case-control analysis, there was no significant association between R1840W and spondyloarthritis (P-value=0.21), whereas R727H was not detected in any of the genotyped individuals. Immunostaining experiments revealed that FREM2 is expressed in synovial membrane, cartilage and colon. CONCLUSIONS: Targeted re-sequencing of a spondyloarthritis-linked region allowed us to identify a rare non-synonymous coding variant in FREM2, co-segregating with spondyloarthritis in a large family. This gene is expressed in several tissues relevant to spondyloarthritis pathogenesis, supporting its putative implication in spondyloarthritis.

Interleukin 27 is a novel cytokine with anti-inflammatory effects against spondyloarthritis through the suppression of Th17 responses.

Introduction: Spondylarthritis (SpA) development in HLA-B27/human ß2-microglobulin transgenic rat (B27-rat) is correlated with altered conventional dendritic cell (cDC) function that promotes an inflammatory pattern of CD4+T cells, including a biased expansion of pro-inflammatory Th17 population and imbalance of regulatory T cells cytokine profile. Transcriptomic analysis revealed that cDCs from B27-rats under express IL-27, an anti-inflammatory cytokine which induces the differentiation of IL-10+ regulatory T cells and inhibits Th17 cells. Methods: Here, we first investigated whether in vitro addition of exogenous IL-27 could reverse the inflammatory pattern observed in CD4+ T cells. Next, we performed preclinical assay using IL-27 to investigate whether in vivo treatment could prevent SpA development in B27-rats. Results: in vitro addition of IL-27 to cocultures of cDCs and CD4+ T cell subsets from B27-rats reduced IL-17 and enhanced IL-10 production by T cells. Likewise, IL-27 inhibited the production of IL-17 by CD4+ T cells from SpA patients. Interestingly, in vivo treatment with recombinant IL-27 starting before SpA onset, inhibited SpA development in B27-rats through the suppression of IL-17/TNF producing CD4+ T cells. Discussion: Overall, our results reveal a potent inhibitory effect of IL-27 and highlight this cytokine as a promising new therapeutic target in SpA, especially for SpA patients non responders to currently approved biotherapies.

A condensate-hardening drug blocks RSV replication in vivo.

Biomolecular condensates have emerged as an important subcellular organizing principle1. Replication of many viruses, including human respiratory syncytial virus (RSV), occurs in virus-induced compartments called inclusion bodies (IBs) or viroplasm2,3. IBs of negative-strand RNA viruses were recently shown to be biomolecular condensates that form through phase separation4,5. Here we report that the steroidal alkaloid cyclopamine and its chemical analogue A3E inhibit RSV replication by disorganizing and hardening IB condensates. The actions of cyclopamine and A3E were blocked by a point mutation in the RSV transcription factor M2-1. IB disorganization occurred within minutes, which suggests that these molecules directly act on the liquid properties of the IBs. A3E and cyclopamine inhibit RSV in the lungs of infected mice and are condensate-targeting drug-like small molecules that have in vivo activity. Our data show that condensate-hardening drugs may enable the pharmacological modulation of not only many previously undruggable targets in viral replication but also transcription factors at cancer-driving super-enhancers6.

Multimodal imaging as optical biopsy system for gastritis diagnosis in humans, and input of the mouse model.

BACKGROUND: Gastric inflammation is a major risk factor for gastric cancer. Current endoscopic methods are not able to efficiently detect and characterize gastric inflammation, leading to a sub-optimal patients' care. New non-invasive methods are needed. Reflectance mucosal light analysis is of particular interest in this context. The aim of our study was to analyze reflectance light and specific autofluorescence signals, both in humans and in a mouse model of gastritis. METHODS: We recruited patients undergoing gastroendoscopic procedure during which reflectance was analysed with a multispectral camera. In parallel, the gastritis mouse model of Helicobacter pylori infection was used to investigate reflectance from ex vivo gastric samples using a spectrometer. In both cases, autofluorescence signals were measured using a confocal microscope. FINDINGS: In gastritis patients, reflectance modifications were significant in near-infrared spectrum, with a decrease between 610 and 725 nm and an increase between 750 and 840 nm. Autofluorescence was also modified, showing variations around 550 nm of emission. In H. pylori infected mice developing gastric inflammatory lesions, we observed significant reflectance modifications 18 months after infection, with increased intensity between 617 and 672 nm. Autofluorescence was significantly modified after 1, 3 and 6 months around 550 and 630 nm. Both in human and in mouse, these reflectance data can be considered as biomarkers and accurately predicted inflammatory state. INTERPRETATION: In this pilot study, using a practical measuring device, we identified in humans, modification of reflectance spectra in the visible spectrum and for the first time in near-infrared, associated with inflammatory gastric states. Furthermore, both in the mouse model and humans, we also observed modifications of autofluorescence associated with gastric inflammation. These innovative data pave the way to deeper validation studies on larger cohorts, for further development of an optical biopsy system to detect gastritis and finally to better surveil this important gastric cancer risk factor. FUNDING: The project was funded by the ANR EMMIE (ANR-15-CE17-0015) and the French Gastroenterology Society (SNFGE).

Multispectral imaging detects gastritis consistently in mouse model and in humans.

Gastritis constitutes the initial step of the gastric carcinogenesis process. Gastritis diagnosis is based on histological examination of biopsies. Non-invasive real-time methods to detect mucosal inflammation are needed. Tissue optical properties modify reemitted light, i.e. the proportion of light that is emitted by a tissue after stimulation by a light flux. Analysis of light reemitted by gastric tissue could predict the inflammatory state. The aim of our study was to investigate a potential association between reemitted light and gastric tissue inflammation. We used two models and three multispectral analysis methods available on the marketplace. We used a mouse model of Helicobacter pylori infection and included patients undergoing gastric endoscopy. In mice, the reemitted light was measured using a spectrometer and a multispectral camera. We also exposed patient's gastric mucosa to specific wavelengths and analyzed reemitted light. In both mouse model and humans, modifications of reemitted light were observed around 560 nm, 600 nm and 640 nm, associated with the presence of gastritis lesions. These results pave the way for the development of improved endoscopes in order to detect real-time gastritis without the need of biopsies. This would allow a better prevention of gastric cancer alongside with cost efficient endoscopies.

A TLR2-Activating Fraction From Mycobacterium abscessus Rough Variant Demonstrates Vaccine and Diagnostic Potential.

Mycobacterium abscessus is a prevalent pathogenic mycobacterium in cystic fibrosis (CF) patients and one of the most highly drug resistant mycobacterial species to antimicrobial agents. It possesses the property to transition from a smooth (S) to a rough (R) morphotype, thereby influencing the host innate immune response. This transition from the S to the R morphotype takes place in patients with an exacerbation of the disease and a persistence of M. abscessus. We have previously shown that the exacerbation of the Toll-like receptor 2 (TLR2)-mediated inflammatory response, following this S to R transition, is essentially due to overproduction of bacilli cell envelope surface compounds, which we were able to extract by mechanical treatment and isolation by solvent partition in a fraction called interphase. Here, we set up a purification procedure guided by bioactivity to isolate a fraction from the R variant of M. abscessus cells which exhibits a high TLR2 stimulating activity, referred to as TLR2-enriched fraction (TLR2eF). As expected, TLR2eF was found to contain several lipoproteins and proteins known to be stimuli for TLR2. Vaccination with TLR2eF showed no protection toward an M. abscessus aerosol challenge, but provided mild protection in ΔF508 mice and their FVB littermates when intravenously challenged by M. abscessus. Interestingly however, antibodies against TLR2eF compounds were detected during disease in CF patients. In conclusion, we show the potential for compounds in TLR2eF as vaccine and diagnostic candidates, in order to enhance diagnosis, prevent and/or treat M. abscessus-related infections.

HLA-B27 Subtypes Predisposing to Ankylosing Spondylitis Accumulate in an Endoplasmic Reticulum-Derived Compartment Apart From the Peptide-Loading Complex.

OBJECTIVE: It was previously shown that HLA-B27 subtypes predisposing to spondyloarthritis (SpA), i.e., B*27:02, B*27:05, and B*27:07, displayed an increased propensity to form intracellular oligomers and to accumulate at a high density in cytoplasmic vesicles, as compared to the non-SpA-associated HLA-B*07:02 and HLA-B*27:06. This study was undertaken to characterize the nature and content of HLA-B-containing vesicles and to further examine their relevance to SpA predisposition. METHODS: Vesicles containing HLA-B proteins were detected in transfected HeLa cells and in cells from SpA patients or HLA-B27/human ß2 -microglobulin (hß2 m)-transgenic rats, by microscopy. The nature and content of HLA-B-containing vesicles were characterized in colocalization experiments with appropriate markers. RESULTS: The SpA-associated HLA-B*27:04 subtype accumulated at higher levels (P < 10-5 ) in cytoplasmic vesicles compared to HLA-B*27:06, from which it differs only by 2 substitutions, reinforcing the correlation between vesicle formation and SpA predisposition. Colocalization studies showed that those vesicles contained misfolded HLA-B heavy chain along with ß2 m and endoplasmic reticulum (ER) chaperones (calnexin, calreticulin, BiP, glucose-regulated protein 94-kd) and belonged to the ER but were distinct from the peptide-loading complex (PLC). Similar vesicles were observed in immune cells from HLA-B27+ SpA patients, in greater abundance than in healthy controls (P < 0.01), and in dendritic cells from HLA-B27/hß2 m transgenic rats, correlating with SpA susceptibility. CONCLUSION: Accumulation of misfolded HLA-B heavy chain along with ß2 m and ER chaperones into ER-derived vesicles distinct from the PLC is a characteristic feature of HLA-B27 subtypes predisposing to SpA. This phenomenon could contribute to HLA-B27 pathogenicity, via a noncanonical mechanism.

Interaction of metabolic and respiratory acidosis with α and ß-adrenoceptor stimulation in rat myocardium.

BACKGROUND: The effects of acute respiratory versus metabolic acidosis on the myocardium and their consequences on adrenoceptor stimulation remain poorly described. We compared the effects of metabolic and respiratory acidosis on inotropy and lusitropy in rat myocardium and their effects on the responses to α- and ß-adrenoceptor stimulations. METHODS: The effects of acute respiratory and metabolic acidosis (pH 7.10) and their interactions with α and ß-adrenoceptor stimulations were studied in isolated rat left ventricular papillary muscle (n=8 per group). Intracellular pH was measured using confocal microscopy and a pH-sensitive fluorophore in isolated rat cardiomyocytes. Data are mean percentages of baseline±SD. RESULTS: Respiratory acidosis induced more pronounced negative inotropic effects than metabolic acidosis did both in isotonic (45±3 versus 63±6%, P<0.001) and isometric (44±5 versus 64±3%, P<0.001) conditions concomitant with a greater decrease in intracellular pH (6.85±0.07 versus 7.12±0.07, P<0.001). The response to α-adrenergic stimulation was not modified by respiratory or metabolic acidosis. The inotropic response to ß-adrenergic stimulation was impaired only in metabolic acidosis (137±12 versus 200±33%, P<0.001), but this effect was not observed with administration of forskolin or dibutiryl-cyclic adenosine monophosphate. This effect might be explained by a change in transmembrane pH gradient only observed with metabolic acidosis. The lusitropic response to ß-adrenergic stimulation was not modified by respiratory or metabolic acidosis. CONCLUSION: Acute metabolic and respiratory acidosis induce different myocardial effects related to different decreases in intracellular pH. Only metabolic acidosis impairs the positive inotropic effect of ß-adrenergic stimulation.

Live-cell imaging reveals multiple interactions between Epstein-Barr virus nuclear antigen 1 and cellular chromatin during interphase and mitosis.

Epstein-Barr virus (EBV) establishes a life-long latent infection in humans. In proliferating latently infected cells, EBV genomes persist as multiple episomes that undergo one DNA replication event per cell cycle and remain attached to the mitotic chromosomes. EBV nuclear antigen 1 (EBNA-1) binding to the episome and cellular genome is essential to ensure proper episome replication and segregation. However, the nature and regulation of EBNA-1 interaction with chromatin has not been clearly elucidated. This activity has been suggested to involve EBNA-1 binding to DNA, duplex RNA, and/or proteins. EBNA-1 binding protein 2 (EBP2), a nucleolar protein, has been proposed to act as a docking protein for EBNA-1 on mitotic chromosomes. However, there is no direct evidence thus far for EBP2 being associated with EBNA-1 during mitosis. By combining video microscopy and Förster resonance energy transfer (FRET) microscopy, we demonstrate here for the first time that EBNA-1 and EBP2 interact in the nucleoplasm, as well as in the nucleoli during interphase. However, in strong contrast to the current proposed model, we were unable to observe any interaction between EBNA-1 and EBP2 on mitotic chromosomes. We also performed a yeast double-hybrid screening, followed by a FRET analysis, that led us to identify HMGB2 (high-mobility group box 2), a well-known chromatin component, as a new partner for EBNA-1 on chromatin during interphase and mitosis. Although the depletion of HMGB2 partly altered EBNA-1 association with chromatin in HeLa cells during interphase and mitosis, it did not significantly impact the maintenance of EBV episomes in Raji cells.

The traffic of proteins between nucleolar organizer regions and prenucleolar bodies governs the assembly of the nucleolus at exit of mitosis.

The building of nuclear bodies after mitosis is a coordinated event crucial for nuclear organization and function. The nucleolus is assembled during early G(1) phase. Here, two periods (early G1a and early G1b) have been defined. During these periods, the nucleolar compartments (DFC, GC) corresponding to different steps of ribosome biogenesis are progressively assembled. In telophase, rDNA transcription is first activated and PNBs (reservoirs of nucleolar processing proteins) are formed. The traffic of the processing proteins between incipient nucleoli and PNBs was analyzed using photoactivation. We demonstrate that the DFC protein fibrillarin passes from one incipient nucleolus to other nucleoli but not to PNBs, and that the GC proteins, B23/NPM and Nop52, shuttle between PNBs and incipient nucleoli. This difference in traffic suggests a way of regulating assembly first of DFC and then of GC. The time of residency of GC proteins is high in incipient nucleoli compared to interphase nuclei, it decreases in LMB-treated early G1a cells impairing the assembly of GC. Because the assembly of the nucleolus and that of the Cajal body at the exit from mitosis are both sensitive to CRM1 activity, we discuss the fact that assembly of GC and/or its interaction with DFC in early G1a depends on shuttling between PNBs and NORs in a manner dependent on Cajal body assembly.

In nucleoli, the steady state of nucleolar proteins is leptomycin B-sensitive.

BACKGROUND INFORMATION: The nucleolus is a dynamic structure. It has been demonstrated that nucleolar proteins rapidly associate with and dissociate from nucleolar components in continuous exchanges with the nucleoplasm using GFP (green fluorescent protein)-tagged proteins. However, how the exchanges within one nucleolus and between nucleoli within the nuclear volume occurred is still poorly understood. RESULTS: The movement of PAGFP (photoactivatable GFP)-tagged proteins that become visible after photoactivation can be followed. In the present study, we establish the protocol allowing quantification of the traffic of PAGFP-tagged nucleolar proteins in nuclei containing two nucleoli. The traffic in the activated area, at the periphery of the activated area and to the neighbouring nucleolus is measured. Protein B23 is rapidly replaced in the activated area, and at the periphery of the activated area the steady state suggests intranucleolar recycling of B23; this recycling is LMB (leptomycin B)-sensitive. The pool of activated B23 is equally distributed in the volume of the two nucleoli within 2 min. The three-dimensional distribution of the proteins Nop52 and fibrillarin is less rapid than that of B23 but is also LMB-sensitive. In contrast, traffic of fibrillarin from the nucleoli to the CB (Cajal body) was not modified by LMB. CONCLUSIONS: We propose that the steady state of nucleolar proteins in nucleoli depends on the affinity of the proteins for their partners and on intranucleolar recycling. This steady state can be impaired by LMB but not the uptake in the neighbouring nucleolus or the CB.