Published: January 7th, 2019
MALT1 regulates innate immunity but how this occurs remains ill-defined. We used the selective MALT1 paracaspase inhibitor MLT-827 to unravel the contribution of MALT1 to innate signaling downstream of Toll-like or C-type lectin-like receptors, demonstrating that MALT1 regulates the production of myeloid cytokines, and downstream of C-type lectin-like receptors, selectively.
Besides its function in lymphoid cells, which has been addressed by numerous studies, the paracaspase MALT1 also plays an important role in innate cells downstream of pattern recognition receptors. Best studied are the Dectin-1 and Dectin-2 members of the C-type lectin-like receptor family that induce a SYK- and CARD9-dependent signaling cascade leading to NF-κB activation, in a MALT1-dependent manner. By contrast, Toll-like receptors (TLR), such as TLR-4, propagate NF-κB activation but signal via an MYD88/IRAK-dependent cascade. Nonetheless, whether MALT1 might contribute to TLR-4 signaling has remained unclear. Recent evidence with MLT-827, a potent and selective inhibitor of MALT1 paracaspase activity, indicates that TNF- production downstream of TLR-4 in human myeloid cells is independent of MALT1, as opposed to TNF- production downstream of Dectin-1, which is MALT1 dependent. Here, we addressed the selective involvement of MALT1 in pattern recognition sensing further, using a variety of human and mouse cellular preparations, and stimulation of Dectin-1, MINCLE or TLR-4 pathways. We also provided additional insights by exploring cytokines beyond TNF-, and by comparing MLT-827 to a SYK inhibitor (Cpd11) and to an IKK inhibitor (AFN700). Collectively, the data provided further evidence for the MALT1-dependency of C-type lectin-like receptor —signaling by contrast to TLR-signaling.
The paracaspase activity of MALT1 (Mucosa-associated lymphoid tissue lymphoma translocation protein 1) was revealed in 20081,2. Since then, a number of studies have reported its critical contribution to antigen receptor responses in lymphocytes. Genetic models in the mouse as well as pharmacology data support a key role in T cells, in T-cell dependent autoimmunity and in B-cell lymphoma settings3,4. In lymphocytes, MALT1 paracaspase activation occurs upon assembly of a CARD11-BCL10-MALT1 complex5, which is triggered by antigen-r....
Experiments were conducted according to the guidelines and standards of the Novartis Human Research Ethics Committee.
1. Preparation of Peripheral Blood Mononuclear Cells (PBMCs) from Human Buffy Coats
NOTE: We received buffy coats from healthy volunteers one day after collection, in 50 mL bags. They were provided under informed consent and collected through the Interregionale Blutspende Schweizeriches Rotes Kreuz. We handled them using the p.......
In myeloid cells, MALT1 relays activation signals downstream of several C-type lectin-like receptors, such as Dectin-1, Dectin-2 and MINCLE6. These pathways rely on (hem)ITAM motif-containing receptors (e.g., Dectin-1) or ITAM motif-containing co-receptors (e.g., FcRγ, for Dectin-2 and MINCLE) that recruit and activate the SYK kinase (Figure 1). This leads to activation of a protein kinase C isoform, namely PKC.......
In this work, we used simple experimental settings to study signaling pathways in human and mouse innate cells, and interrogate their dependency on MALT1 proteolytic function. Expanding on previous work11, our study showed that MALT1 paracaspase activity controls C-type lectin-like receptor induced cytokine production, including TNF-α. In contrast, TLR-4-induced TNF-α was independent of MALT1 in both species. Collectively, these data corroborated the key and selective contribution of the.......
|100 µm nylon cell strainer
|14 ml Falcon tube
|15 mL Falcon tube
|50 mL Falcon tube
|6 well plates
|96 well flat-bottom plate, with low evaporation lid
|96 well V-bottom plate
|Ammonium Chloride - NH4Cl
|Assay diluent RD1-W ELISA
|Cell culture microplate, 384 well, black
|Ethylenediaminetetraacetic acid disodium salt dihydrate
|gentleMACS C tubes
|MACS Miltenyi Biotec
|MACS Miltenyi Biotec
|Heat-inactivated Fetal bovine serum
|HTRF hu IL-23
|HTRF hu TNF-α
|HTRF reconstitution buffer
|50mM Phosphate buffer, pH 7.0, 0.8M KF, 0.2% BSA
|LPS used in human samples
|LPS used in murine samples
|155 mM NH4Cl, 10 mM KHCO3, 1 mM EDTA, pH 7.4
|Microplate, 384 well white
|Monocytes enrichment kit
|Nalgene Mr. Frosty Cryo 1 °C Freezing Container
|cooling device (containing Propanol-2)
|PBS 1x pH 7.4 [-] CaCl2 [-] MgCl2
|Potassium bicarbonate - KHCO3
|Resazurin solution for viability assessment
|Tris-based buffer containing tripropylamine
|Recovery cell culture freezing medium
|Roswell Park Memorial Institute Medium (RPMI) with Glutamax
|+ 10% FBS for iMoDCs
+ 10% FBS + 1 mM Sodium Pyruvate + 100 U/mL Pen/Strep + 5 µM β-mercaptoethanol for human PBMCs and monocytes
+ 10% FBS + Pen/Strep + 5 µM β-mercaptoethanol for murine splenocytes
|PBS pH 7.4 + 2% FBS + 1 mM EDTA pH 8.0
|UltraPure 0.5 M EDTA pH 8.0
|V-PLEX Proinflammatory Panel 1 Human Kit
|electrochemiluminescent multiplex assay (IL-1β, TNF-α, IL-6, IL-8)
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