MALT1 Protease Activity Is Required for Innate and Adaptive Immune Responses

<div><p>CARMA-BCL10-MALT1 signalosomes play important roles in antigen receptor signaling and other pathways. Previous studies have suggested that as part of this complex, MALT1 functions as both a scaffolding protein to activate NF-κB through recruitment of ubiquitin ligases, and as a protease to cleave and inactivate downstream inhibitory signaling proteins. However, our understanding of the relative importance of these two distinct MALT1 activities has been hampered by a lack of selective MALT1 protease inhibitors with suitable pharmacologic properties. To fully investigate the role of MALT1 protease activity, we generated mice homozygous for a protease-dead mutation in MALT1. We found that some, but not all, MALT1 functions in immune cells were dependent upon its protease activity. Protease-dead mice had defects in the generation of splenic marginal zone and peritoneal B1 B cells. CD4⁺ and CD8⁺ T cells displayed decreased T cell receptor-stimulated proliferation and IL-2 production while B cell receptor-stimulated proliferation was partially dependent on protease activity. In dendritic cells, stimulation of cytokine production through the Dectin-1, Dectin-2, and Mincle C-type lectin receptors was also found to be partially dependent upon protease activity. <i>In vivo</i>, protease-dead mice had reduced basal immunoglobulin levels, and showed defective responses to immunization with T-dependent and T-independent antigens. Surprisingly, despite these decreased responses, MALT1 protease-dead mice, but not MALT1 null mice, developed mixed inflammatory cell infiltrates in multiple organs, suggesting MALT1 protease activity plays a role in immune homeostasis. These findings highlight the importance of MALT1 protease activity in multiple immune cell types, and in integrating immune responses <i>in vivo</i>.</p></div

MALT1 protease-dead mice develop inflammatory cell infiltrates in multiple organs.

<p>H&E-stained histological sections of tissues from MALT1 <i>Wt</i> (left panels A, C, E, G; n = 3) and <i>Malt1</i>^PD/PD (right panels B, D, F H; n = 6) mice. Results from <i>Malt1</i>^-/- (n = 3) and <i>Malt1</i>^+/PD (n = 2) mice were identical to <i>Wt</i> mice. (A, B) Lung at 100X magnification. Airways of <i>Wt</i> and <i>Malt1</i>^PD/PD mice, including the B, bronchiolar (B) and terminal airways (TA) were relatively clear of inflammatory infiltrates. Distinctive findings were present in the lungs of <i>Malt1</i>^PD/PD mice, which featured pulmonary veins (V) surrounded by a prominent mixed mononuclear inflammatory cell infiltrate predominated by lymphocytes and histiocytic cells (inset, 400X magnification). (C, D) Limbic region of eye (150X magnification). Cornea from <i>Wt</i> eye demonstrates typical anatomical features of a thin corneal epithelium (CE) overlying the substantia propria (SP). Occasional linear nuclei in the SP are part of the normal fibroblast populations. Sclera (S), pigmented iris (I) and lens (L) are normal in <i>Malt1</i>^PD/PD mice. Cornea from <i>Malt1</i>^PD/PD mice consistently had neutrophilic inflammatory cell infiltrates (arrows) of varying severity in the substantia propria of the cornea. This particular <i>Malt1</i>^PD/PD mouse also had a florid neutrophilic infiltrate towards the central portion of the cornea (inset, 150X magnification). (E, F) Non-glandular stomach (50X magnification). Normal non-glandular stomach was comprised of stratified squamous epithelium (Ep) with a small amount of keratinized stratum corneum (Sc) and only occasional inflammatory cells evident in the submucosa (Sub). Non-glandular stomach from <i>Malt1</i>^PD/PD mice demonstrated marked hyperkeratosis of the stratum corneum and prominent hyperplasia of the epithelium. A mixed inflammatory infiltrate was commonly present in the mural wall, with this particular mouse having a moderate neutrophilic infiltrate in the submucosa (arrow). (G, H) Glandular stomach (50X magnification). The fundic mucosa (Muc) of the <i>Wt</i> mouse is regular, even, and is formed by undulating gastric pits. The relatively acellular tunica adventitia (Ta) is the thin tissue layer (arrow) sandwiched between the thicker tunica muscularis (Tm) and the adipose tissue (Ad) / pancreas (Pan). Reactive hyperplasia of the glandular mucosa was present in <i>Malt1</i>^PD/PD mice, and characterized by prominent thickening of the mucosa and loss of regular and even mucosal architecture. Mixed inflammatory cell infiltrates were increased in the mural wall of stomachs from <i>Malt1</i>^PD/PD mice, with this particular mouse demonstrating an intense inflammatory infiltrate of the tunica adventicia that is predominated by populations of lymphocytes and histiocytic cells.</p

Mixed inflammatory infiltrates of T cells, B cells, and macrophages in MALT1 protease-dead mice.

<p>IHC photomicrographs (80X magnification, insets 400X magnification) of inflammatory cell infiltrates surrounding vessels (V) of the lung and tunica adventicia of glandular stomach in <i>Malt1</i>^PD/PD mice, using antibodies against: (A, D) CD3 to detect T cells; (B, E) CD45 to detect B-cell; and (C, F) F4/80 to detect macrophage. Non-selective background staining was higher with the F4/80 antibody, as evident with the pale staining of erythrocytes within the vessel lumen (V, Fig 8C). Therefore, macrophage identification was made by considering the higher staining intensity and morphology of cells stained (inset, Fig 8C). IHC photomicrographs of inflammatory cell infiltrates in lung of <i>Malt1</i>^PD/PD mice (8A, C) demonstrates a mixed population of T-cells, B-cells and macrophages comprising the perivascular cuffs of pulmonary vessels. Similar to the lung infiltrates of <i>Malt1</i>^PD/PD, inflammatory infiltrates in deeper glandular portions of stomach had mixed population of T cells, B cells and macrophages in the tunica adventicia (Fig 8D, F).</p

MALT1 protease activity is required for maximal T-dependent and T-independent antibody responses <i>in vivo</i>.

<p>(A) <i>Wt</i>, <i>Malt1</i>^-/-, and <i>Malt1</i>^PD/PD mice (n = 6/group) were immunized with the T-dependent antigen KLH, and serum anti-KLH antibody titers were measured by ELISA on days 7, 14, 21 and 28 after immunization. (B) <i>Wt</i>, <i>Malt1</i>^-/-, and <i>Malt1</i>^PD/PD mice (n = 4-5/group) were immunized with the T-independent antigen TNP-Ficoll, and serum anti-TNP antibody titers were measured by ELISA on days 7, 14, 21 and 28 after immunization. Data represents Ig levels in individual animals, and averages are indicated by horizontal lines. Significance was determined relative to the <i>Wt</i> groups, with *<i>p</i> < 0.05, **<i>p</i> < 0.01, †<i>p</i> < 0.001.</p

The development of MZ and B1 B cells populations is dependent upon MALT1 protease activity.

<p>Representative flow cytometry analysis of lymphocyte populations in MALT1 <i>Wt</i>, <i>Malt1</i>^-/-, and <i>Malt1</i>^PD/PD mice. (A) CD4⁺CD8a⁺, CD4⁺CD8a^-, and CD4^-CD8a⁺ T cell populations in the thymus. (B) CD4⁺CD8a⁺, CD4⁺CD8a^-, and CD4^-CD8a⁺ T cell populations in the spleen. (C) Mature follicular (IgD^hiIgM^lo), transitional T1 (IgD^loIgM^hi), and T2 (IgD^hiIgM^hi) B cell populations in the spleen. (D, F) MZ B cell (CD21^-CD23⁺) population in the spleen. (E, G) B1 B cell (IgM^hiCD5^lo) population in peritoneal fluid. Bar graphs represent average cell numbers for 3–4 mice/group. Significance was determined relative to the <i>Wt</i> groups, with *<i>p</i> < 0.05, **<i>p</i> < 0.01. Error bars represent +/- SEM.</p

MALT1 protease activity is required for TCR- but not BCR-mediated lymphocyte activation.

<p>(A) CD4⁺ T cells and (B) CD8⁺ T cells from the spleens of <i>Wt</i>, <i>Malt1</i>^-/-, and <i>Malt1</i>^PD/PD mice were activated by co-stimulation with anti-CD3 and anti-CD28 beads, and proliferation was measured after 96 h by ³H-thymidine incorporation, and IL-2 production after 24 h by MSD. The total IL-2 produced by CD4⁺ and CD8⁺ T cells from <i>Wt</i> mice was approximately 2–3 ng/ml and 100–400 pg/ml, respectively. Where indicated, T cells were treated with 126 μM of the MALT1 inhibitor Z-VRPR-FMK (VRPR) for 45 min prior to stimulation. (C) B220⁺ B cells from the spleens of <i>Wt</i>, <i>Malt1</i>^-/-, and <i>Malt1</i>^PD/PD mice were stimulated with 20 μg/ml anti-IgM or 20 μg/ml LPS and proliferation was measured after 96 h by ³H-thymidine incorporation. Bar graphs from Fig 3A-C represent the average response expressed as a percent of the stimulated <i>Wt</i> control (LPS treatment for B220⁺ cells) for 4–6 mice/group, and are representative of 3 or more studies. (D) Splenic B220⁺ B cells from <i>Wt</i> and <i>Malt1</i>^PD/PD mice were labeled with CFSE prior to stimulation with anti-IgM and proliferation was analyzed by FACS three days post stimulation. <i>Wt</i> EC50 = 2.6 μg/ml; <i>Malt1</i>^PD/PD EC50 = 6.4 μg/ml. Each data point represents the average response of five mice expressed as a percent of <i>Wt</i> proliferation at the highest dose. (E) Splenic B220+ B cells were either unstimulated or (F) stimulated with α-IgM (20 μg/ml) for three days and viability was analyzed by FACS. Each bar is an average from five mice. Significance was determined relative to the stimulated <i>Wt</i> groups, with *<i>p</i> < 0.05, **<i>p</i> < 0.01, †<i>p</i> < 0.001, ‡<i>p</i> < 0.0001. Error bars represent +/- SEM.</p

Decreased basal serum Ig levels in MALT1 null and protease-dead mice.

<p>ELISA analysis of IgA, IgG1, IgG2a, IgG2b, IgG3, and IgM levels in non-immunized sera from <i>Wt</i>, <i>Malt1</i>^-/-, and <i>Malt1</i>^PD/PD mice (n = 6/group). Data represents Ig levels in individual animals, and averages are indicated by horizontal lines. Significance was determined relative to the <i>Wt</i> groups, *<i>p</i> < 0.05, **<i>p</i> < 0.01, †<i>p</i> < 0.001, ‡<i>p</i> < 0.0001.</p

CLR stimulation of cytokine production by BMDCs involves MALT1 protease activity.

<p>BMDCs from <i>Wt</i>, <i>Malt1</i>^-/-, and <i>Malt1</i>^PD/PD mice were stimulated with (A) curdlan to activate Dectin-1, (B) anti-Dectin-2 antibody to activate Dectin-2, and (C) TDB to activate Mincle. IFN-γ, IL-1β, IL-10, IL-12 p70, IL-6, KC (IL-8 homologue), and TNF-α levels were measured after 20 h by MSD, and results are shown only for cytokines for which significant expression was detected. Maximum cytokine concentration ranges detected were: curdlan, 1–3 ng/ml IL-1β, 4–20 ng/ml IL-6, 4–9 ng/ml KC, 0.5–1 ng/ml IL-10, 0.2–0.4 ng/ml IL-12p70, and 40–100 ng/ml TNF-α; anti-Dectin-2, 0.1–0.3 ng/ml IL-6, 0.5–2 ng/ml KC, and 3–15 ng/ml TNF-α; TDB, 0.3–0.6 ng/ml KC, and 0.3–2 ng/ml TNF-α. Where indicated, cells were treated 126 μM Z-VRPR-FMK (VRPR) for 45 min prior to stimulation. All bar graphs represent the average response expressed as a percent of the stimulated <i>Wt</i> control for 6 mice/group, and are representative of 3 or more studies. Significance was determined relative to the stimulated <i>Wt</i> groups, with *<i>p</i> < 0.05, **<i>p</i> < 0.01, †<i>p</i> < 0.001, ‡<i>p</i> < 0.0001. Error bars represent +/- SEM.</p

A C472A mutation in MALT1 inactivates protease activity without changing protein expression.

<p>(A) Homologous recombination of the <i>Wt Malt1</i> locus (<i>I</i>) in C57BL/6-derived embryonic stem cells was used to introduce a catalytically inactive C472A mutation into exon 12 of the <i>Malt1</i> gene (for clarity, the genomic locus is not draw to scale). The resulting neo cassette-containing mice (<i>II</i>) were crossed to a Flp recombinase-expressing deleter mouse strain to generate animals carrying the protease-dead allele (<i>III</i>) used in these studies. These mice were in turn crossed to a Cre recombinase-expressing deleter strain to excise exon 12 and generate a null allele (<i>IV</i>). Close triangles, FRT sites; open triangles, <i>loxP</i> sites. (B) Purified total B cells from the spleens of <i>Wt</i>, <i>Malt1</i>^-/-, and <i>Malt1</i>^PD/PD mice were treated with (+) or without (-) PMA plus ionomycin for 1 h and assessed by Western blotting for expression of MALT1, CYLD, Bcl10, and proteolytically cleaved CYLD and Bcl10. For clarity, the images were cropped. Uncropped western blots are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127083#pone.0127083.s001" target="_blank">S1 Fig</a> (Complete western blots confirming MALT1 (C472A) expression and loss of protease activity).</p

Discovery of Small Molecule RIP1 Kinase Inhibitors for the Treatment of Pathologies Associated with Necroptosis

Potent inhibitors of RIP1 kinase from three distinct series, 1-aminoisoquinolines, pyrrolo­[2,3-b]­pyridines, and furo­[2,3-d]­pyrimidines, all of the type II class recognizing a DLG-out inactive conformation, were identified from screening of our in-house kinase focused sets. An exemplar from the furo­[2,3-d]­pyrimidine series showed a dose proportional response in protection from hypothermia in a mouse model of TNFα induced lethal shock