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-receptor proximal signaling downstream of the T- or B- cell receptor. There is also ample evidence that a similar CARD9-BCL10-MALT1 complex is important for propagating signals downstream of C-type lectin-like receptors (CLLR), e.g., Dectin-1, Dectin-2 and MINCLE in myeloid cells^6,7. Dectin-1 has been particularly well studied because this pathway is critical for host defense against fungal infections^8,9. Implication of MALT1 in Toll-like receptor (TLR) pathways, however, has remained controversial¹⁰. Recent evidence in human myeloid cells ruled out a direct role for MALT1 paracaspase activity in the regulation of TNF- production downstream of TLR-4¹¹.

In the present work, we used various experimental settings and stimulatory conditions in human and mouse myeloid cells to probe innate signaling pathways, relying on specific pharmacological tool inhibitors and measurement of cytokine production.

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 procedure below, at room temperature unless specified otherwise.

1. Prepare a sterile and clean pair of scissors and a 1 L beaker with a plastic bag (under a lamina flow).
2. Transfer the buffy coat into the beaker and carefully open it with the scissors.
3. Using a 25 mL pipette, add 100 mL of phosphate-buffered saline buffer/ethylenediaminetetraacetic acid (PBS/EDTA: PBS 1x pH 7.4 containing no CaCl₂ and no MgCl₂, supplemented with 2 mM EDTA pH 8.0).
   NOTE: Using the same pipette and after slowly pipetting the solution up and down, dispense 25 mL of the diluted buffy coat into 6 conical centrifuge tubes of 50 mL pre-filled with 15 mL of polysaccharide-based density gradient.
4. Centrifuge 20 min at 800 x g with moderate acceleration (set at 4 out of 9) and without brake to allow for separation of cells based on their densities.
   NOTE: After centrifugation, three layers will be visible; a pellet containing red blood cells and granulocytes, an upper layer made of plasma, and in between a white ring containing peripheral blood mononuclear cells (PBMCs).
5. Harvest the PBMC ring using a 10 mL pipette and transfer to new 50 mL tubes.
   NOTE: Two to three 50 mL tubes are normally required per buffy coat. At this stage, some plasma likely contaminates the collected PBMCs, which should have no impact on the subsequent enrichment steps.
6. Top up to 50 mL every tube using PBS/EDTA and proceed to three successive washes with decreasing centrifugation time and speed (15 min at 520 x g, 10 min at 330 x g, 8 min at 150 x g).
   NOTE: At each step, the supernatant is poured off in a liquid waste container and the cell pellet is resuspended in 50 mL of PBS/EDTA (pellets may be pooled after the first wash).
7. After the final wash, resuspend the pellet in 25 mL of an ice-cold lysis buffer (see Table of Materials) to lyse the red blood cells by osmotic pressure.
8. Incubate until the solution becomes clear (≤5 min at room temperature).
9. Stop the reaction by adding 25 mL of separation buffer (PBS 1x pH 7.4 containing no CaCl₂ and no MgCl₂, supplemented with 2% heat-inactivated fetal bovine serum (FBS) and 1 mM EDTA pH 8.0).
10. Wash one more time at 150 x g for 8 min.
    NOTE: At this stage PBMCs can be used as a bulk population (see step 3: PBMCs and monocytes treatments and stimulatory conditions) or processed for monocyte enrichment (see step 2: Preparation of monocytes from PBMCs), or frozen down for subsequent use (see step 5: Monocytes and PBMCs freezing/thawing procedures).

2. Preparation of Monocytes from PBMCs

1. Resuspend the PBMCs obtained at step 1.10 in separation buffer and count them to reach 5 x 10⁷cells/mL.
2. Transfer the cell suspension into a 14 mL conical centrifuge tube with cap.
3. Add 50 µL of monocyte enrichment antibody cocktail per mL of cell suspension, vortex, and incubate 10 min at 4 °C.
4. Add 50 µL of monocyte enrichment beads per mL of cells.
   NOTE: The beads must be thoroughly vortexed to ensure homogeneity of the suspension.
5. After adding the beads, shortly vortex the cell suspension and incubate 5 min at 4 °C.
6. Rinse the upper part of the tube with separation buffer until the tube is filled up to 10 mL.
7. Slowly mix the solution by pipetting up and down.
8. Place the tube without cap into a separation magnet.
9. Incubate for 2.5 min at room temperature.
10. Pour slowly into a 15 mL conical centrifuge tube.
11. At this stage, use monocytes directly (see step 3: PBMCs and monocytes treatments and stimulatory conditions) or differentiate them into immature Monocyte-derived Dendritic Cells (iMoDCs) (see step 4), or freeze them down for subsequent use (see step 5).

3. PBMCs and Monocytes Treatments and Stimulatory Conditions

1. Count cells and dilute them in culture medium (Roswell Park Memorial Institute medium (RPMI) 10% FBS + 1 mM Sodium Pyruvate + 100 U/mL Penicillin Streptomycin (Pen/Strep) + 5 µM β-mercaptoethanol), down to 1.25 x 10⁴ cells/well.
2. Distribute 30 µL of cell suspension per well of a 384-well plate.
3. Add 15 µL of 4x concentrated compound solutions and pre-incubate for 1 h at 37 °C, 5% CO₂.
4. Add lipopolysaccharide (LPS) to a final concentration of 10 ng/mL, or depleted zymosan (DZ) to a final concentration of 100 µg/mL, or keep in plain medium.
5. Incubate overnight at 37 °C, 5% CO₂.
6. Take 10 µL of the supernatant to measure secreted TNF-a levels (see step 7: Cytokines and viability measurements).

4. Monocytes Differentiation into iMoDCs and Stimulatory Conditions

1. Count monocytes (enriched at step 2) and centrifuge the cell suspension at 520 x g for 5 min.
2. Pipette off the supernatant and add culture medium (RPMI+ 10% FBS to obtain a final cell suspension of 0.4 x 10⁶ cells/mL.
3. Add 80 ng/mL recombinant human IL-4 + 100 ng/mL GM-CSF.
4. Dispense 5 mL of cell suspension per well in a 6-well plate.
5. Incubate for 7 days at 37 °C, 5% CO₂.
6. At day 7, harvest cells by pipetting gently to avoid their activation.
7. Centrifuge at 520 x g for 5 min.
8. Vacuum aspirate and resuspend in 50 mL of culture medium without growth factors.
9. Centrifuge at 520 x g for 5 min and resuspend at 1 x 10⁶ cells/mL in culture medium.
10. Dispense 100 µL of cell suspension (10⁵ cells) per well of a 96-well flat bottom plate.
11. Pre-incubate for 1 h at 37 °C after adding 50 µL of 4x concentrated compound solutions, prepared as described at step 8: Compounds preparation.
12. Add 50 µL of stimuli (4x concentrated), prepared as described at step 9.
13. Incubate 24 h at 37 °C.
14. Mix each well and transfer cells and supernatant (SN) into a 96-well V-bottom plate.
15. Spin down at 475 x g for 5 min.
16. Transfer supernatant into a new 96-well flat bottom plate, seal and freeze at -20 °C until further use.

5. Monocytes and PBMCs Freezing/Thawing Procedures

1. Freezing
   1. Spin down PBMC or monocyte cell preparations at 520 x g for 5 min.
   2. Vacuum aspirate the supernatant and resuspend cells in freezing medium at 1 x 10⁷ cells/mL.
   3. Dispense 1 mL of cell suspension into cryotubes, transfer the tubes to a specific cooling device (see Materials table) and place it at -80 °C.
2. Thawing
   1. Thaw the cryotube and quickly transfer its content into a 15 mL conical centrifuge tube containing 9 mL of culture medium.
   2. Centrifuge at 520 x g for 5 min.
   3. Vacuum aspirate supernatant and resuspend cell pellet in 5 mL of culture medium. Cells are now ready for further experimental processing.

6. Mouse Spleen Cells Preparation and Treatment

NOTE: We conducted animal sacrifices according to the guidelines and standards of the Novartis Animal Welfare Organization. Studies were approved by the Ethics Committee of the regional governmental authority (Kantonales Veterinäramt der Stadt Basel). We sacrificed animals by isoflurane over-exposure, with all efforts made to minimize suffering.

1. Harvest the spleen and dissociate tissue using a tube equipped with a mechanical tissue grinder device and filled with 5 mL of cold RPMI medium.
2. Use the spleen program of the dissociation machine to grind organs.
3. Filter the cells through a 100 mm nylon cell strainer.
4. Transfer the suspension into 50 mL tubes and centrifuge at 4 °C for 10 min at 320 x g.
5. Vacuum aspirate the supernatant, resuspend the cell pellet in 3 mL of ice-cold lysis buffer, and incubate for ≤2 min on ice.
6. Stop lysis by adding 7 mL of RPMI medium.
7. Filter again through a 100 µm nylon cell strainer.
8. Spin down cell suspension at 330 x g for 10 min at 4 °C.
9. Vacuum aspirate supernatant and resuspend cells at 11 x 10⁶ cells/mL in complete medium (RPMI supplemented with 10% FBS, 100 U/mL Pen/Step and 5 µM β-Mercaptoethanol).
10. Plate 1 x 10⁶ cells/well (90 µL) in a 96-well plate (flat bottom).
11. Add 5 µL of 20x concentrated compound solution previously diluted in RPMI medium as described in step 8.3: Serial dilution for murine spleen cells.
12. Incubate for 30 min at 37 °C, 5% CO₂.
13. Add 5 µL of 20x concentrated DZ (final concentration 30 µg/mL) or 20x concentrated LPS + IFN-g (TLR-4) (final concentration 1 µM LPS and 10 ng/mL IFN-g).
14. Incubate overnight at 37 °C, 5% CO₂.
15. Centrifuge at 330 x g for 10 min.
16. Transfer supernatants in new plates, seal and freeze at -20 °C until further use.

7. Cytokines and Viability Measurements

1. Human TNF-a measurement by HTRF (Homogenous Time Resolved Fluorescence)
   NOTE: The protocol followed the supplier’s recommendations, briefly summarized below.
   1. Mix 1 volume of reconstituted reagent (anti TNF-a-cryptate and anti-TNF-a-XL665) with 19 volumes of reconstitution buffer (50 mM phosphate buffer pH 7.0, 0.8 M potassium fluoride (KF), 0.2% bovine serum albumin (BSA)).
   2. Mix the two ready-to-use antibody solutions 1:1 just prior to dispensing the reagents.
   3. Dispense 10 µL of supernatants from step 3.6 into white 384-well plates.
   4. Dispense 10 µL of the antibody mix.
   5. Cover the plate with a sealer and incubate at 4 °C overnight.
   6. Read plate on a microplate reader (50–200 flash).
2. Human IL-23 measurement by HTRF (Homogenous Time Resolved Fluorescence)
   NOTE: The protocol followed the supplier’s recommendations, briefly summarized below.
   1. Mix one volume of reconstituted reagent (anti IL-23-cryptate-antibody and anti-IL-23 D2-antibody) with 19 volumes of detection buffer #3.
   2. Mix the two ready-to-use antibody solutions 1:1 just prior to dispensing the reagents.
   3. Dispense 10 µL of supernatants from step 3.6 into white 384-well plates.
   4. Dispense 10 µL of the antibody mix.
   5. Cover the plate with a sealer and incubate at 4 °C overnight.
   6. Read plate on a microplate reader (50–200 nm flash).
3. Human IL-6, IL-8, IL-1β and TNF-α measurements by electrochemiluminescence
   NOTE: All samples were diluted at 1/150 in diluent 2 (first dilution: 10 µL in 150 µL, then 20 µL in 180 µL). The protocol followed the supplier’s recommendations:
   1. Dilute samples and standard in diluent 2.
   2. Proceed to dilution of the standard in diluent 2 using a 1/4 serial fold dilution.
   3. Wash plates three times with wash buffer.
   4. Dispense 50 µL of samples or standard per well.
   5. Incubate for 2 h at room temperature under agitation.
   6. Wash the plate four times with PBS + 0.05% Polysorbate 20.
   7. Add 25 µL of detection antibody (60 µL of each antibody for 3 mL final) in diluent 3.
   8. Incubate for 2 h at room temperature under agitation.
   9. Wash the plate four times with PBS + 0.05% Polysorbate 20.
   10. Avoiding bubbles, add 150 µL per well of read buffer (Tris-based buffer containing tripropylamine, diluted 2x in ddH₂0) as a co-reactant for light generation in electrochemiluminescence immunoassays.
   11. Read the plate (without delay) on a multiplex plate reader.
4. Mouse TNF-a measurement by ELISA following the supplier’s protocol
   1. Dilute supernatant 1:1 in assay diluent (ready to use protein-containing buffer).
      1. Prepare reagents, samples, and standard dilutions as described in the kit. Add 50 μL of assay diluent to each well.
      2. Add 50 μL of standard, control, or sample per well.
   2. Mix by gently tapping the plate frame for 1 min.
   3. Cover with the provided adhesive strip and incubate for 2 h at room temperature.
   4. Aspirate each well and wash with 400 μL/well (repeat this step five times in total).
   5. After the final wash, remove any remaining wash buffer by aspirating.
   6. Invert the plate and blot against clean paper towels.
   7. Add 100 μL of mouse TNF-α conjugate to each well. Cover with a new adhesive strip.
   8. Incubate for 2 h at room temperature.
   9. Repeat the aspiration/wash as in step 7.4.4.
   10. Add 100 μL of substrate solution to each well and incubate for 30 min at room temperature protected from light.
   11. Add 100 μL of diluted hydrochloric acid solution (stop solution) to each well. Gently tap the plate to ensure thorough mixing.
   12. Measure the optical density at 450 nm (with the correction wavelength set at 560 nm) using a microplate reader (to be done within 30 min).
5. Cell viability
   1. After removing supernatants from PBMC or monocyte preparations, assess cell viability using a ready to use resazurin solution (oxidation-reduction indicator) added directly to the cell suspension to 10% final concentration.
   2. Incubate for 1 to 2 h at 37 °C, 5% CO₂.
   3. Read fluorescence at 590 nm (Excitation 540 nm) using a microplate reader.

8. Compound Preparation

1. Serial dilution for iMoDCs
   1. Dilute the MLT-827 stock solution (10 mM in dimethyl sulfoxide (DMSO) into medium to reach 8 µM in one go (4x concentrated).
   2. Perform a six-step 1:5 serial dilution, using medium + 0.08% DMSO. Use the same medium + 0.08% DMSO solution for the vehicle (no compound) condition.
2. Single dose testing for iMoDCs
   1. Dilute the MLT-827, AFN700 and Cpd11 stock solutions into medium to reach 4 µM in one go (4x concentrated).
3. Serial dilution for murine spleen cells
   1. Dilute a 10 µM MLT-827 solution (obtained from a 10 mM stock solution following a one go dilution in medium) to 0.01 µM, with a DMSO end concentration of 0.1%.
   2. For the dilution steps, take 2 µL of each dilution, add 38 µL RPMI, and pipet 5 µL into the well.
      NOTE: All treatments are performed in triplicate.

9. Stimuli Preparation

1. Depleted zymosan (DZ)
   1. Add 2 mL of sterile endotoxin-free water to 10 mg of DZ.
   2. Vortex to homogenize the stock solution, vortex also prior each use.
   3. Aliquot solution and store aliquots at -20 °C.
2. Trehalose-6,6-dibehenate (TDB)
   1. Add 100 µL of DMSO to 1 mg TDB, heat at 60 °C in a water bath for 15–30 s.
   2. Vortex and immediately add 900 µL of sterile PBS, vortex again.
   3. Heat for 10–15 min at 60 °C and homogenize by vortexing prior each use.
   4. Keep the solution at 4 °C.
   5. Perform serial dilutions and single dose preparation as for the MLT-827 compound.
      NOTE: Because TDB needs to be prepared in DMSO, a final 1% DMSO concentration is present during cell stimulation.

Results

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 PKCδ, whic...

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

List of materials used in this article

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