Immunodeficiency, auto-inflammation and amylopectinosis in humans with inherited HOIL-1 and LUBAC deficiency

We report the clinical description and molecular dissection of a new fatal human inherited disorder characterized by chronic auto-inflammation, invasive bacterial infections and muscular amylopectinosis. Patients from two kindreds carried biallelic loss-of-expression and loss-of-function mutations in HOIL1, a component the linear ubiquitination chain assembly complex (LUBAC). These mutations resulted in impairment of LUBAC stability. NF-κB activation in response to interleukin-1β (IL-1β) was compromised in the patients’ fibroblasts. By contrast, the patients’ mononuclear leukocytes, particularly monocytes, were hyperresponsive to IL-1β. The consequences of human HOIL-1 and LUBAC deficiencies for IL-1β responses thus differed between cell types, consistent with the unique association of auto-inflammation and immunodeficiency in these patients. These data suggest that LUBAC regulates NF-κB-dependent IL-1β responses differently in different cell types

Arginine butyrate: a therapeutic candidate for Duchenne muscular dystrophy.

International audienceAs a strategy to treat Duchenne muscular dystrophy, we used arginine butyrate, which combines two pharmacological activities: nitric oxide pathway activation, and histone deacetylase inhibition. Continuous intraperitoneal administration to dystrophin-deficient mdx mice resulted in a near 2-fold increase in utrophin (protein homologous to dystrophin) in skeletal muscle, heart, and brain, accompanied by an improvement of the dystrophic phenotype in both adult and newborn mice (45 and 70% decrease in creatine kinase level, respectively; 14% increase in tidal volume, 30% decrease in necrotic area in limb and 23% increase in isometric force). Intermittent administration, as performed in clinical trials, was then used to reduce the frequency of injections and to improve safety. This also enhanced utrophin level around 2-fold (EC50=284 mg/ml) and alleviated the dystrophic phenotype (inverted grid and grip test performance near to wild-type values, creatine kinase level decreased by 50%). Skin biopsies were used to monitor treatment efficacy, instead of invasive muscle biopsies, and this could be done a few days after the start of treatment. A 2-fold increase in utrophin expression was also shown in cultured human myotubes. In vivo and in vitro experiments demonstrated that the drug combination acts synergistically. Together, these data constitute a proof of principle of the beneficial effects of arginine butyrate on muscular dystrophy

Immunodeficiency, autoinflammation and amylopectinosis in humans with inherited HOIL-1 and LUBAC deficiency

International audienceWe report the clinical description and molecular dissection of a new fatal human inherited disorder characterized by chronic autoinflammation, invasive bacterial infections and muscular amylopectinosis. Patients from two kindreds carried biallelic loss-of-expression and loss-of-function mutations in HOIL1 (RBCK1), a component of the linear ubiquitination chain assembly complex (LUBAC). These mutations resulted in impairment of LUBAC stability. NF-κB activation in response to interleukin 1β (IL-1β) was compromised in the patients' fibroblasts. By contrast, the patients' mononuclear leukocytes, particularly monocytes, were hyper-responsive to IL-1β. The consequences of human HOIL-1 and LUBAC deficiencies for IL-1β responses thus differed between cell types, consistent with the unique association of autoinflammation and immunodeficiency in these patients. These data suggest that LUBAC regulates NF-κB–dependent IL-1β responses differently in different cell types

Schematic representation of MALT1 isoforms and variants thereof.

<p>The two reported human MALT1 isoforms A (NP_006776.1) and B (NP_776216.1) are depicted. The drawings show the different domains, the auto-cleavage sites (red arrows), the catalytic cysteine residue (black), the reported ubiquitination site (green) and shows the regions required for binding BCL10 (grey) and TRAF6 (blue). The truncated variants of the two isoforms, used or discussed in the present work, are represented in the shaded boxes at the bottom, with predicted molecular weights. The 11-amino acid deletion of isoform B is depicted by a red triangle. Ig, Immunoglobulin-like domain.</p

Functional impact of C-terminal cleavage on MALT1 isoforms A and B.

<p><b>(A)</b> NF-κB luciferase reporter gene assay in HEK293 cells transfected with either, MALT1A WT, MALT1A-781, MALT1B WT, or MALT1B-770 in the absence or presence of CARD11-L244P. Luciferase activity was recorded after 24h. Data show the mean of triplicate determinations from 7 independent experiments. Statistical significance was calculated using the Student T-test. Western blot analysis, performed in parallel to control for protein expression, is shown below with samples from a representative experiment. An anti-tubulin immunoblot is provided as loading control. <b>(B)</b> CBM reconstitution was performed using MALT1A, MALT1B as well as their constitutively cleaved variants, together with the MALT1 protease substrate A20. Immunoblot analyses for MALT1 show the respective C-terminal auto-cleavage bands (white arrow heads). For A20, they show cleaved fragments as previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169026#pone.0169026.ref009" target="_blank">9</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169026#pone.0169026.ref022" target="_blank">22</a>]. A20p37 is a proteasome sensitive degradation product of A20 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169026#pone.0169026.ref009" target="_blank">9</a>]. Its weak detection when MALT1A WT is used is consistent with high proteolytic activity resulting in early processing of A20 and disappearance of A20p37 by the time of harvest. A non-specific (NS) band detected by the anti-A20 antibody is provided as loading control.</p

MALT1 auto-cleavage at R149 is induced by TRAF6.

<p><b>(A)</b> MALT1A was expressed in HEK293 cells, either alone, or together with CARD11-L244P, or as part of a CBM reconstitution assay (transfection ratio 1:1:1:1 using a control plasmid), or a TM reconstitution assay (TRAF6 + MALT1 (transfection ratio 1:1:2 with “2” referring to a control plasmid), or a CBMT reconstitution assay (transfection ratio 1:1:1:1). Western Blot analyses with anti-FLAG and anti-MALT1 (MT1/410) antibodies are shown, displaying MALT1 auto-cleaved fragments (C-terminal, white arrow head; N-terminal depicted as p16/p76, grey arrow heads). Loading controls with an anti-tubulin antibody are also provided. <b>(B)</b> The CBMT reconstitution assay was performed as in (A) using either TRAF6-WT, or TRAF6-C70A, or TRAF6 289–522. Anti-FLAG Western Blot analyses are shown, displaying MALT1 auto-cleaved fragments (C-terminal, white arrow heads; N-terminal p16, grey arrow heads) as well as MALT1 mono-ubiquitinated species (black arrow heads). An anti-cleaved BCL10 immunoblot providing evidence for proteolytic activity of MALT1 is also shown together with a non-specific (NS) band detected by the BCL10 antibody, as loading control.</p

Pulmonary hypertension in patients with idiopathic pulmonary fibrosis and combined pulmonary fibrosis and emphysema: hemodynamic severity

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Post-translational modifications of MALT1 in human lymphocytes.

<p><b>(A)</b> OCI-Ly3 cells were grown for 4 days in the presence or absence of 50 μM z-VRPR-fmk. Cells were harvested for MALT1 immunoprecipitation and lysate analysis using an anti-MALT1 antibody (H-300), as described in the Methods section. The MALT1 faster and slower migrating species are indicated with white and black arrow heads, respectively. The BCL10 antibody recognizing more specifically the uncleaved form of BCL10 (ep606y) was reported already [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169026#pone.0169026.ref038" target="_blank">38</a>]. Densitometry of MALT1and BCL10 signal intensities was measured in 3 independent experiments and is shown as Mean ± SD. Cells were counted at the end of each experiment and counts are represented as Mean ± SD (N = 3). Statistical significance was calculated using the Student T-test. <b>(B)</b> Jurkat cells were stimulated with PMA (10 ng/ml) anti-CD28 (1 μM) and anti-CD3 (1 μM) in the presence of 5 μM MG-132, for various times before full cell extraction and analysis of MALT1 (MT1/410 antibody) and BCL10 (antibodies specified on the figure) by immunoblotting. The MALT1 faster and slower migrating species are indicated with a white and a black arrow head, respectively. <b>(C)</b> Primary human CD3 T cells were stimulated with PMA (10 ng/ml) and Ionomycin (1 μM) in the presence of 5 μM MG-132, for various times before full cell extraction and analysis of MALT1 (MT1/410 antibody). The MALT1 faster and slower migrating species are indicated with a white and a black arrow head, respectively. Anti-tubulin immunoblots are provided as loading controls.</p