Myeloid Innate Signaling Pathway Regulation by MALT1 Paracaspase Activity

Summary

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.

Abstract

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.

Introduction

The paracaspase activity of MALT1 (Mucosa-associated lymphoid tissue lymphoma translocation protein 1) was revealed in 2008^1,2. 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 settings^3,4. In lymphocytes, MALT1 paracaspase activation occurs upon assembly of a CARD11-BCL10-MALT1 complex⁵, which is triggered by antigen-r....

Protocol

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.......

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
100 µm nylon cell strainer	Sigma	CLS431752
14 ml Falcon tube	BD Falcon	352057
15 mL Falcon tube	Falcon	352090
50 mL Falcon tube	Falcon	352070
6 well plates	Costar	3516
96 well flat-bottom plate, with low evaporation lid	Costar	3595
96 well V-bottom plate	Costar	734-1798
Ammonium Chloride - NH4Cl	Sigma	A9434
Assay diluent RD1-W ELISA	R&D	895038	Assay diluent
Cell culture microplate, 384 well, black	Greiner	781986
Depleted Zymosan	Invivogen	tlrl-dzn	now: tlrl-zyd
Dimethyl sulfoxide	Sigma	D2650	DMSO
EDTA-Na2	Sigma	E5134	Ethylenediaminetetraacetic acid disodium salt dihydrate
ELISA muTNF-α	R&D	SMTA00
Ficoll-Paque Plus	GE Healthcare	17-1440-03
gentleMACS C tubes	MACS Miltenyi Biotec	130-096-334
gentleMACS dissociator	MACS Miltenyi Biotec	130-093-235
GM-CSF	Novartis	-
Heat-inactivated Fetal bovine serum	Gibco	10082	FBS
HTRF hu IL-23	CisBio	62HIL23PEG
HTRF hu TNF-α	CisBio	62TNFPEC
HTRF reconstitution buffer	CisBio	62RB3RDE	50mM Phosphate buffer, pH 7.0, 0.8M KF, 0.2% BSA
IFN-γ	R&D	L4516
IL-4	Novartis	-
Isoflurane	Abbott	Forene
Lipopolysaccharides (LPS)	Sigma	L4391	LPS used in human samples
Lipopolysaccharides	Sigma	L4516	LPS used in murine samples
Lysis buffer	Self-made	-	155 mM NH4Cl, 10 mM KHCO3, 1 mM EDTA, pH 7.4
Magnet	Stemcell	18001
Microplate, 384 well white	Greiner	784075
Monocytes enrichment kit	Stemcell	19059
Nalgene Mr. Frosty Cryo 1 °C Freezing Container	Nalgene	5100-0001	cooling device (containing Propanol-2)
PBS 1x pH 7.4 [-] CaCl2 [-] MgCl2	Gibco	10010	Phosphate-buffered saline
Penicillin/Streptomycin	Gibco	15140	Pen/Strep
Potassium bicarbonate - KHCO3	Sigma	P9144
PrestoBlue	Invitrogen	A13262	Resazurin solution for viability assessment
Propanol-2	Merck	1.09634
Read buffer	MesoScale Discovery	R92TC-3	Tris-based buffer containing tripropylamine
Recovery cell culture freezing medium	Gibco	12648-010	freezing medium
Roswell Park Memorial Institute Medium (RPMI) with Glutamax	Gibco	61870	+ 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
Separation buffer	Self-made	-	PBS pH 7.4 + 2% FBS + 1 mM EDTA pH 8.0
Sodium Pyruvate	Gibco	11360
Trehalose-6,6-dibehenate	Invivogen	tlrl-tdb	TDB
Tween 20	Sigma	P7949	Polysorbate 20
UltraPure 0.5 M EDTA pH 8.0	Invitrogen	15675	Ethylenediaminetetraacetic acid
Viewseal sealer	Greiner BioOne	676070
V-PLEX Proinflammatory Panel 1 Human Kit	MesoScale Discovery	K15049D	electrochemiluminescent multiplex assay (IL-1β, TNF-α, IL-6, IL-8)
β-Mercaptoethanol	Gibco	31350