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). B…

Representative 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 (…

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

Name of Reagent/ Equipment	Company	Catalog Number	Comments/Description
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
α-CD11c	BD	555392
β-mercaptoethanol	Sigma-Aldrich	M6250-10ML
TBS			On site stock room
Tween-20	Sigma-Aldrich	P2287-100mL
Nunc EasYFlask 225cm2, Filter Cap, 70mL working volume, 30/Cs	Thermo Scientific	159933
20 μM Sterile Disposable Filter Units	Thermo Scientific	569-0020
Gentamicin	Invitrogen	15750060
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 – 4L	Fisher Scientific	A433P-4
Prolong Gold antifade Reagent with DAPI	Life Technologies	P-36931
8 chamber polystyrene vessel tissue culture treated glass slide	BD Falcon	354108
Poly-L-Lysine	Sigma	P4707