Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1β in Human Monocyte-derived Dendritic Cells

Summary

Dendritic cells (DCs) secrete IL-1β in response to TLR8 recognition of synthetic purine, R848, followed by NLRP3 inflammasome activation with nigericin, therefore, IL-1β can be used to measure NLRP3 inflammasome activity. Intracellular cytokine staining, immunoblotting, and ELISA are used to accurately measure NLRP3 inflammasome priming and activation via IL-1β expression.

Abstract

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue remodeling and repair, and virologic control^1,2 . Interleukin-1β is an essential element of the innate immune response and contributes to eliminate invading pathogens while preventing the establishment of persistent infection^1-5.

Inflammasomes are the key signaling platform for the activation of interleukin 1 converting enzyme (ICE or Caspase-1). The NLRP3 inflammasome requires at least two signals in DCs to cause IL-1β secretion⁶. Pro-IL-1β protein expression is limited in resting cells; therefore a priming signal is required for IL-1β transcription and protein expression. A second signal sensed by NLRP3 results in the formation of the multi-protein NLRP3 inflammasome. The ability of dendritic cells to respond to the signals required for IL-1β secretion can be tested using a synthetic purine, R848, which is sensed by TLR8 in human monocyte derived dendritic cells (moDCs) to prime cells, followed by activation of the NLRP3 inflammasome with the bacterial toxin and potassium ionophore, nigericin.

Monocyte derived DCs are easily produced in culture and provide significantly more cells than purified human myeloid DCs. The method presented here differs from other inflammasome assays in that it uses in vitro human, instead of mouse derived, DCs thus allowing for the study of the inflammasome in human disease and infection.

Introduction

Activation of innate immune system is required to steer adaptive immune responses during infection, disease, and vaccination⁷. Dendritic cells are the most potent antigen presenting cells of the innate immune system; they are specialized for uptake of antigens, migration to lymph nodes, and activation of naïve CD4⁺ and cytolytic CD8⁺ T-cells^8-10. To enable rapid pathogen detection the innate immune system utilizes numerous germline encoded pattern recognition receptors (PRR) that recognize conserved pathogen derived motifs or host derived markers of cell stress and damage. Toll like receptors (TLRs) are membrane bound pattern recognition receptors that recognize certain extracellular phagocytized pathogen associated molecular patterns (PAMPs) and danger associated molecular patterns (DAMPs). By contrast nod like receptors (NLRs) are cytosolic and respond to a diverse range of PAMPs and DAMPs. Nod like receptors represent a second line of defense against pathogens that evade cell surface and endocytic PRRs. The interaction of pathogen derived, or “danger” associated, factors with TLR and NLR ligands leads to a state of DC maturation resulting in increased DC interaction with other immune cells and promotion of T cell and natural killer cell activation¹¹.

Interleukin-1β is a crucial component of the host defense against infection. Upon recognition of a microorganism, the highly proinflammatory cytokine, IL-1β, is secreted and functions as a chemo attractant and activator of innate and adaptive immune cells. In vivo IL-1β is largely responsible for the acute phase response including fever and inflammatory cytokine synthesis¹².

Most NLRs contain a C terminal leucine rich repeat domain that is thought to function in ligand sensing, a central nucleotide binding domain (NACHT) that is important for NLRP3 oligomerization, and an N terminal effector domain (PYD in NLRP3) that mediates signal transduction to downstream targets through protein protein interactions. The NLRP3 protein defines the most intensely studied inflammasome complex. This protein is a member of the NLR family and has the ability to form a multi molecular protein complex composed of NLRP3, the adaptor protein PYCARD (also known as ASC), and ICE. Upon inflammasome activation PYCARD binds to NLRP3 N terminal domains and recruits ICE via caspase activation and recruitment domain (CARD) domains. Interleukin-1 converting enzyme is initially generated as a zymogen containing a CARD motif at its N-terminus. Inflammasome formation results in bringing two ICE molecules sufficiently close to induce their autocatalytic activation. The inflammasome complex is necessary for activating ICE thus allowing it to convert cytoplasmic pro-IL-1β to mature cytokine.

Successful secretion of IL-1β in DCs requires sensing of two different and independent danger signals. First, TLR sensing of PAMPs, DAMPs, or cytokine signaling (TNFα or IL-1β) causes an upregulation of cytoplasmic pro-IL-1β protein expression. A second, often different, signal is required for inflammasome complex formation upstream of ICE maturation. A few inflammasome stimulating signals include bacterial membrane pore forming toxins (such as nigericin), lysosomal disrupting crystals (such as monosodium urate crystals, MSU), and extracellular ATP. The upstream mechanism leading to NLRP3 inflammasome activation by these diverse activators is unclear. Studies investigating signaling upstream of inflammasome formation proposes that intracellular events, such as induction of hypokalemia or reactive oxygen species (ROS) indirectly activate the inflammasome^13-28.

Amongst the different viral activators of the NLRP3 inflammasome is influenza, which provides both the primary and secondary signal required for IL-1β secretion^3,29-33 . Using mouse NLRP3 knockout models it was found that IL-1β secretion in DCs is NLRP3 dependent³². Additionally, NLRP3 knockout mice attracted fewer leukocytes to the site of infection and experienced higher mortality^2,5. Two recent papers suggest a mechanism for NLRP3 inflammasome activation during Influenza virus infection; first, priming through recognition of viral RNA by TLR7 or TLR8 (depending on TLR expression of the responding cell) or through sensing of commensal bacteria by other TLRs to induce cytoplasmic pro-IL-1β expression, followed by a second signal, activation of NLRP3 inflammasome formation by viral ion channel protein M2 on the trans Golgi network^33,34. In the latter step, triggering of the NLRP3 inflammasome is accomplished by disturbance of the intracellular ionic milieu leading to ROS production, which is, simply, sensed by NLPR3 as a signal to form the inflammasome. However, the precise mechanism of inflammasome activation upstream of ICE activity during Influenza infection still remains unclear.

This work describes a technique valuable for studying the NLRP3 inflammasome in human moDCs that can be used as a foundation for further investigation of the pathway underlying DC based IL-1β secretion in response to TLR8 ligation with R848 followed by activation of the inflammasome by a well known activator of NLRP3, nigericin. Variations of this method can be used with other cell types including, but not limited to: monocytes, macrophages, other DC subsets, and epithelial cells.

Protocol

Ethics Statement: Research samples are obtained and stored for research with donors’ consent. All samples should be coded or anonymized prior to use. This protocol follows the guidelines of our Institutional Review Board.

1. Differentiation of Human Peripheral Blood Monocytes into Monocyte Derived Dendritic Cells.

Note: Human buffy coats serve as the source of human peripheral blood cells (PBMCs) and were obtained from the New York Blood Center (New York, NY). Blood donors are healthy volunteers. The 5 day procedure begins with the plating of human peripheral blood mononuclear cells (PBMCs) onto tissue culture flasks^35,36. Notable differences from the published protocols are the following:

1. Use 225 cm² non pyrogenic polystyrene tissue culture flasks with a filter cap to adhere a total of 2x10⁸ PBMCs per flask instead of 10 cm polystyrene tissue culture plates (58 cm²) (step 1).
2. Prepare media with 5% pooled human serum (PHS; 30 ml) in 500 ml RPMI-1640 + L-glutamine, 5 ml 1 M HEPES buffer, and 1.4 ml 50 mg/ml Gentamicin (5% PHS media) followed by filtration through a 20 µM Filter. Add a total of 50 ml 5% PHS media per 225 cm² tissue culture flask.
3. Wash adhered cells three times with 25 ml fresh RPMI-1640. Shake vigorously for 5 sec during each wash and aspirate non adherent cells (step 4).
4. Add 190 µl of 400 IU/µl IL-4 and 380 µl of 100 IU/µl GM-CSF per flask (step 3) on day 0, 2, and 4. Day 0 is defined as the day PBMCs are initially plated in Step 1 (Step 8).
5. Harvest day 5 moDCs at a concentration of 1x10⁶ cells/ml (step 15).
6. Use moDCs immediately in their resting state. Steps 17-22 are never performed. Note: Samples should be processed as soon as possible after collection for best results.
7. Aliquot moDCs at a concentration of 2x10⁵ cells/well (200 µl/well of the 1x10⁶ cells/ml) in a 96 well round bottomed plate (western blot, ELISA, FACs) or onto a poly-L-lysine treated chamberslide (microscopy) for experimentation. Note: There are at least 4 conditions: Completely unstimulated (negative control), priming only, activation only, and priming followed by activation. Conditions can be expanded to include diluent controls for R848 and nigericin if desired. If the downstream assay is ICS, duplicates are necessary for isotype controls.

2. Priming the Inflammasome - Signal 1

1. Reconstitute lyophilized R848 in DMSO according to the manufacturer's instructions. Dilute working stock at RT with RPMI-1640.
2. Add R848 at a 10 µM final concentration to appropriate wells for 18 hr. Place cells in an incubator at 37 °C and 5% CO₂.

3. Activating the NLRP3 Inflammasome - Signal 2

1. Reconstitute lyophilized nigericin in ethanol according to the manufacturer's instructions. Dilute working stock at RT with RPMI-1640 before adding to the appropriate conditions.
2. Add nigericin at a 20 µM final concentration and return to the incubator at 37 °C and 5% CO₂ for 6 hr. No washing steps occur between priming and during activation. Note: Secretion of IL-1β is increased by the presence of calcium ionophores, brefeldin A, monensin, dinitrophenol, or carbonyl cyanide chlorophenylhydrazone^37,38. Therefore, addition of these secretory inhibitors are not recommended when analyzing inflammasome priming or activity since it will alter secretion of IL-1β.

4. IL-1ß Sample Collection

1. Centrifuge the plate with cells and supernatants at 974 x g for 3 min.
2. Without disturbing the cell pellet, aspirate the supernatant and transfer to a separate round bottomed plate in order to measure cytokine secretion from the supernatants. Note: Supernatants can be temporarily stored at -20 °C or -80 °C for long term storage.
3. Wash the cellular pellets three times with 200 µl of 1x PBS at 974 x g for 3 min to remove any extracellular IL-1β from the cellular samples. Note: When washing cell pellets from a 96 well plate, remove the wash by swift plate inversion into a collection bin so as to not disturb the sample.

5. Measuring IL-1ß From Cellular and Supernatant Samples

1. Intracellular Cytokine Staining (ICS): The ICS protocol is described below^39,40. Note: do not suspend the cells if the downstream assay is microscopy. All washes and aspirations should be done without disturbing the cell layer/pellet (depending on the downstream assay).
   1. Add 100 µl of 5% PHS media into appropriate wells. Add appropriate volume (approximately 1 µl/2x10⁵ cells, or 1 µl/well) of fluorescently labeled α-CD11c and α-CD14 (phenotype markers; clone B-ly6 and MφP9, respectively) or isotype control to the cells for 10 min at RT in the dark.
   2. Wash three times with 1x PBS at 974 x g for 3 min.
   3. Fix the cells by adding 100 µl of 4% PFA for 20 min at RT in the dark.
   4. Pause Point: Wash with 100 µl 1x PBS at 974 x g for 3 min and store at 4 °C O/N in 1x PBS.
   5. Add 100 µl of permeabilization buffer to cells for 30 min. Note: permeabilization buffer is composed of 1x PBS with 0.3% Triton X-100, and 1% bovine serum albumin (BSA) for permeabilization. Do not disturb adherent cells when downstream assay is microscopy.
   6. Add appropriate volume (approximately 62 ng antibody/2x10⁵ cells, approximately or 2.5 µl/well) of α-IL-1β-FITC (clone 8516) or isotype control for 2 hr in an incubator at 37 °C.
   7. Wash cells three times with 200 µl of permeabilization buffer at 974 x g for 3 min in the dark.
   8. Optional: Stain with DAPI, add mountant, and place coverslip gently on top of a glass slide. Allow mountant to cure O/N before capturing microscopy images.
   9. Acquire the data by FACs or microscopy. Note: Compare the isotype for each condition to the appropriate staining when analyzing by FACs or microscopy.
      1. FACs: Acquire one sample at a time. Set the FSC/SSC gate on the live cells, followed by gating on CD11c⁺CD14^- cells before analyzing the moDC population pro-IL-1β staining.
      2. Microscopy: Set the exposure time using the positive staining sample - R848 treated. Use DAPI⁺pro-IL-1β⁺ and DAPI⁺pro-IL-1β^- to determine percentage of pro-IL-1β+ expressing moDCs.
2. SDS-Page: Immunoblotting is performed to detect pro-IL-1β. Note: The technique described below combines standard immunoblotting technique, gradient 4-20% polyacrylamide gel, and fluorescent or chemiluminescent detection^41,42 .
   1. Lyse cells directly in 10 µl denaturing lysis buffer (5 µl β-mercaptoethanol/950 µl Laemmli sample buffer followed by diluting 1:1 with cell lysis buffer). Transfer lysate to 1.5 ml Eppendorf tubes.
   2. Heat lysate on a dry plate for 10 min at 100 °C.
   3. Spin lysate at 20,800 x g on a minicentrifuge for 1 min to concentrate the liquid volume. Pause point: samples can be frozen at -20 °C for short term storage.
   4. Load the total volume onto the polyacrylamide gel. Run 140 V through the gel box for approximately 1 hr, or until the dye front runs off the bottom of the gel.
   5. Transfer the protein from the polyacrylamide gel onto a PVDF Immobilon-FL membrane for 90 min at 100 V. Block the membrane with 5% BSA in Tris Buffered Saline/ 0.1% Tween-20 (TBS-T) for 1 hr. Note: PVDF Immobilon-FL Is best for use with fluorescent detection. A membrane with a different composition is recommended for other detection techniques to increase the signal to background ratio.
   6. Dilute primary and secondary antibodies in 10 ml of 5% BSA/TBS-T. Perform primary antibody incubations at 4 °C O/N while shaking and secondary antibody at RT for 1 hr while shaking.
   7. Wash the membrane 3 times with TBS-T for 5 min while shaking between primary and secondary antibody incubations and again between secondary incubations and imaging. Note: Bands can be detected using fluorescent or chemiluminescent detection. Follow the manufacturer’s instructions for detection.
3. ELISA: Samples that are frozen for storage need to equilibrate to RT before analysis. Spin down samples in a centrifuge to consolidate supernatant condensation. Follow the manufacturer’s instructions for measurement of IL-1β. Note: In this protocol IL-1β is specifically measured; however, simultaneous measurement of TNFα, IL-6, and the immunomodulatory cytokine IL-10 ensure the appropriate conditions were primed.

Results

These techniques measure TLR8 priming with R848. Intracellular cytokine staining for pro-IL-1β allows for microscopy and FACs readouts from CD14^-CD11c⁺ moDCs. Both techniques can be quantified relative to a non primed, or resting, cell control as well as an isotype control (Figures 1 and 2). Percent of pro-IL-1β⁺ staining cells is multiplied by the geometric median of this population to provide the median fluorescent intensity (MFI). The ...

Discussion

Inflammatory cytokines are integral in steering the innate and adaptive immune response to fight viral infection. Secreted IL-1β has been shown to increase during Influenza infection^3,43,44 . The precise mechanisms by which these cytokines are processed in response to viral recognition in human dendritic cells are not fully understood. Myeloid DC isolation kits are expensive and time consuming. Isolation kits and FAC sorting may unintentionally stress or activat...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
IL-4	R&D
GM-CSF	Genzyme	NDC 58468-0180-2	We acquire this item through our local pharmacy with a prescription
RPMI 1640 with L-glutamine	Cellgro	10-040-CV
Peripheral blood mononuclear cells	New York Blood Center		PBMCs were isolated from the blood of healthy donors
12 well tissue culture plates	Sigma-Aldrich	3516
96 well round bottom tissue culture plates	Sigma-Aldrich	3799
α-IL-1β-FITC	R&D	IC201F
FITC isotype control	Miltenyi Biotec	130-092-213
α-β-Tubulin	Santa Cruz	SC-9014
α-IL-1β	R&D	mab201
PVDF Immobilon-FL membrane	Millipore	IPFL00010
gradient 4-12% polycrylamide gel	Bio Rad	161-1159
Laemmli sample buffer	Bio Rad	161-0737
BSA	Equitech Bio Inc		30% solution sterile/filtered
PFA	Electron Microscopy Sciences	15710	16% solution
Human inflammatory cytokine bead array kit	BD	551811
Nigericin	Invivogen	tlrl-nig
R848	3M Corp.
α-CD14	BD	340436
β-mercaptoethanol	Sigma-Aldrich	M6250-10ML
TBS			On site stock room
Tween-20	Sigma-Aldrich	P2287-100mL
Nunc EasYFlask 225 cm2, Filter Cap, 70 ml working volume, 30/Cs	Thermo Scientific	159933
20 μM Sterile Disposable Filter Units	Thermo Scientific	569-0020
HEPES	Invitrogen	15630080
Goat α-mouse IRDye 800CW	Licor	926-32210
Donkey α-rabbit IRDye 680RD	Licor	926-68073
Spectra multicolor broad range protein ladder	Thermo Scientific	26634
Tris Glycine SDS 10x	Bio Rad	1610732
Tris Glycine 10x	Bio Rad	161-0734
Methanol - 4 L	Fisher Scientific	A433P-4
8 chamber polystyrene vessel tissue culture treated glass slide	BD Falcon	354108
Poly-L-Lysine	Sigma	P4707