Background
Nucleotide-binding oligomerization (NOD) -like receptors (NLRs) are cytosolic pattern recognition receptors responsible for detecting pathogen- or danger-associated molecular patterns (PAMPs or DAMPs), and are a critical surveillance system for innate immunity. Most NLRs share common structural characteristics: a C-terminal leucine-rich repeat domain that recognizes PAMPs or DAMPs, a central NOD domain, and a variable N-terminal effector domain [
1]. They are categorized into five families based on their N-terminal domains. NLRP3 (NALP3) has a pyrin N-terminal domain which binds with the adaptor protein, ASC, to recruit pro-caspase-1 (p45), forming a multi-protein complex termed the inflammasome. Upon inflammasome activation, active caspase-1 (p20 or p10) is cleaved and pro-inflammatory cytokines such as interleukin (IL) -1β and -18, which play an important role in host defense against infection, are subsequently released.
Cigarette smoking has been known to increase susceptibility to infection likely from dysregulation of immune function [
2], but the precise underlying molecular mechanisms remain unclear. A previous study showed that cigarette smoke alone does not induce secretion of IL-1β, an inflammasome cytokine, in human monocyte THP1 cells [
3]. On the other hand, NLRP3 appears to be required for bronchoalveolar secretion of IL-1β in response to cigarette smoke in an in vivo murine model [
4]. However, it is not known whether cigarette smoke affects NLRP3 cellular concentrations or how it interacts with other pathogens to affect cellular protein levels. This question is important, as the identification of an effect, and underlying mechanism, could provide us a therapeutic target to control dysregulated immune function in smokers.
The aim of our study was to investigate the molecular basis for effects of cigarette smoke extract (CSE) on the NLRP3 inflammasome, a component also activated by lipopolysaccharide (LPS). We investigated if CSE interacts with LPS and modulates NLRP3 inflammasome activity and cytokine release. To this end, we utilized human monocyte THP1 cells and primary human peripheral blood macrophages, and C57BL/6 mice to assess the in vivo effects of cigarette smoke.
Methods
Antibodies and reagents
Antibodies against ubiquitin and IL-1β were obtained from Cell Signaling Technology (Danvers, MA). Leupeptin, ATP, and antibodies against β-actin and GAPDH were acquired from Sigma-Aldrich (St. Louis, MO). NLRP6 antibodies were purchased from Abcam (Cambridge, MA). NLRP3 antibodies were from Adipogen (San Diego, CA). Antibody against ASC was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Protein A/G agarose beads, pcDNA3.1D TOPO cloning kits, LPS, and antibodies targeting the V5 tag were purchased from Thermo Fisher Scientific (Waltham, MA). IL-18 antibodies were acquired from MBL International (Woburn, MA). Caspase-1 antibodies were obtained from R&D Systems (Minneapolis, MN). Cycloheximide (CHX) was purchased from Enzo Life Sciences (Farmingdale, NY). MG-132 was purchased from Ubiquitin-Proteasome Biotechnologies (Aurora, CO). CSE in a vacuum sealed bottle was purchased from Murty Pharmaceuticals (Lexington, KY).
Cell culture
Human monocyte THP1 cells were purchased from Sigma-Aldrich. Human peripheral blood macrophages and human macrophage cell culture medium were purchased from Celprogen (Torrance, CA). The clonal primary macrophages were derived from human peripheral blood, and confirmed positive for Mcl-1, CD4, CD14, CD206, CD11b/CR3, CD2, and CD19 expression per the company. RPMI 1640 medium was purchased from Thermo Fisher Scientific. Fetal bovine serum (FBS) was purchased from Gemini (Sacramento, CA). THP1 cells were cultured in RPMI 1640 medium supplemented with 10% FBS. Human peripheral blood macrophages were cultured in human macrophage cell culture medium supplemented with 10% FBS. For the half-life experiments, CHX was used at a concentration of 40 μg/mL in fresh medium without FBS supplement, avoiding the possible breakdown of CHX when it is mixed with an alkaline substance (i.e., nicotine from CSE). For analysis of secreted proteins, the medium was removed from treated cells with the same total cell number and medium volume per well and precipitated with trichloroacetic acid (TCA). The precipitated pellet was then mixed with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer and analyzed by immunoblotting.
qRT-PCR
RNA was isolated from THP1 cells using the RNeasy Mini Kit from Qiagen (Valencia, CA) per the protocol supplied in the kit. The concentration of each RNA sample was measured, followed by conversion to cDNA using the High-Capacity RNA-to-cDNA kit from Thermo Fisher Scientific. Real-time PCR was carried out in a C1000 Thermal Cycler from Bio-Rad (Hercules, CA) using SYBR Select Master Mix from Thermo Fisher Scientific per the included protocol. The primers used were NLRP3 (5′-ATGAGTGCTGCTTCGACATC-3′, 5′-TTGTCACTCAGGTCCAGCTC-3′), and GAPDH (5′-ATCATCCCTGCCTCTACTGC-3′, 5′-GTCAGGTCCACCACTGACAC-3′).
Immunoprecipitation and immunoblotting
Cells were collected in lysis buffer (0.25% Triton X-100 in PBS and 1:1000 protease inhibitor mixture) and sonicated for 12 s, followed by centrifugation at 16,100 × g for 10 min. The cell lysate was then incubated and rotated with 5 μL of anti-ubiquitin antibody at room temperature for 1 h. Each sample was then incubated with 30 μL of protein A/G agarose beads and rotated overnight at 4 °C. After incubation overnight, the beads were spun down at 0.1 × g for 3 min and then washed with lysis buffer a total of 3 times. SDS-PAGE sample loading buffer was added to the beads and they were boiled for 5 min before immunoblot analysis. Immunoblotting was carried out as follows; Equal amounts of protein in sample loading buffer were separated by gel electrophoresis, and transferred onto nitrocellulose membranes [
5]. Restore PLUS Western Blot Stripping Buffer from Thermo Fisher Scientific (Waltham, MA) was used to reprobe membranes to detect multiple proteins.
Cloning and mutagenesis
Human
NLRP3 cDNA was cloned into a pcDNA3.1D/V5-His vector provided in the pcDNA3.1D TOPO cloning kit. Site directed mutagenesis of
NLRP3 was performed using the QuikChange II XL kit from Agilent Technologies (Santa Clara, CA) as previously described [
6].
Transfection
2.5 × 106 Human peripheral blood macrophages were suspended in 100 μL of 20 mM HEPES in PBS and were mixed with 4 μg of either WT NLRP3-V5 or K689R NLRP3-V5 plasmid DNA in a cuvette. The cells were nucleofected using the Y-010 protocol on an Amaxa Nucleofector II machine (Basel, Switzerland). After transfection, 1 mL of 10% FBS human macrophage cell culture medium was added to each cuvette. The samples were then transferred to 6-well plates containing 1 mL of 10% FBS human macrophage cell culture medium, for a total of 2 mL of culture medium. The cells were grown until they reached approximately 50% confluency (~48–72 h) before half-life experiments were initiated.
Animal study
Male C57BL/6 mice ranging from 8 to 12 weeks of age were purchased from the Jackson Laboratory (Bar Harbor, ME) and exposed to 4 non-filtered cigarettes (University of Kentucky research cigarettes, Lot number 1R5F), 5 days per week, for a total of 6 months. The mice were deposited in a smoking chamber which allows the restrained mice to get direct cigarette smoke exposure towards their nose [
7]. A mouse was exposed to 8.32 mg total particulate matter per day via the targeted delivery system [
8]. Age-matched littermates were used as controls and were exposed to filtered air. After 6 months, the mice were sacrificed by administration of CO
2. Following euthanasia, the lungs were immediately extracted and frozen in liquid nitrogen for storage at -80 °C. The lungs were then homogenized in lysis buffer (1% Triton X-100 in PBS and 1:1000 protease inhibitor mixture), before analysis via SDS-PAGE and immunoblotting. The protocol described was approved by the University of Pittsburgh Institutional Animal Care and Use Committee (Protocol #: 12101008).
Statistical analysis
A Mann-Whitney U test or a Kruskal-Wallis equality of populations rank test were used to compare experimental groups. We employed non-parametric methods as our sample sizes were relatively small to check a normal sample distribution. All analyses were performed two-tailed, using Stata Statistical Software: Release 13.0 (StataCorp. 2013. College Station, TX: StataCorp LP).
Discussion
Our study reveals a unique observation that CSE decreases NLRP3 protein levels, mediated by increased ubiquitin proteasomal processing. Further, we demonstrate that CSE suppresses NLRP3 levels even in the presence of endotoxin thereby preventing the release of IL-1β and IL-18, critical cytokines for antimicrobial host defense. Our findings provide potential mechanistic insights for smoking-related immunosuppression, and the results may uncover unique opportunities to develop therapeutic strategies to modulate cytokine signaling. For example, small molecules that stabilize NLRP3 protein levels (e.g. targeting of NLRP3 deubiquitinating enzymes) might be one opportunity that emerges from the results of these and other studies.
Cigarette smoke has been known to dysregulate both innate and adaptive immune function, making smokers more susceptible to infection with worse outcomes [
2,
10]. Specifically, smokers have increased susceptibility to bacterial pneumonia, tuberculosis, periodontitis and surgical infections [
2]. The function of neutrophils and macrophages in smokers is defective, and they secrete lower levels of IL-6 and tumor necrosis factor (TNF) [
11], which are crucial for early response to pathogens [
12].
In addition, IL-1β and IL-18 are also known to play an important role in host defense. IL-1β activates the release of TNF and IL-6, and induces Th17 cell differentiation for cellular adaptive responses [
13]. IL-18 is essential for the induction of IFN-γ and regulation of Th1 responses [
14]. Both cytokines, IL-1β and IL-18, are synthesized as premature forms in cells, and cleaved by caspase-1 (p20 or p10) to be bioactive. Caspase-1 is activated by multi-protein complexes, inflammasomes, consisting of three components: a sensor NLR, adaptor ASC, and effector pro-caspase-1 (p45). The most studied is the NLRP3 inflammasome, which is associated with immune responses that limit microbial invasion, thereby protecting hosts [
15]. A previous study showed that cigarette smoke decreases caspase-1 activity when THP1 cells are stimulated with asbestos [
16]. However, it is not known whether cigarette smoke directly affects NLRP3 protein mass. Our study shows that CSE decreases the level of NLRP3 protein via increased degradation, most likely increased ubiquitin-mediated proteasomal processing. The CSE-induced degradation of NLRP3 was observed despite addition of LPS, a known inhibitor of NLRP3 ubiquitination that stabilizes the NLRP3 protein [
6]. Release of IL-1β and IL-18 was also decreased after CSE exposure, likely from a decreased amount of activated NLRP3 inflammasome complex, as evidenced by reduced levels of active caspase-1 (Fig.
4b).
The ubiquitin proteasome system mediates disposal of the majority of proteins in cells. In lung epithelial cells, cigarette smoke increases total cellular poly-ubiquitinated proteins [
17], which is consistent with our findings (Fig.
2a). CSE also induces the degradation of proteins involved with cell death and proliferation [
18,
19]. Our studies indicate that CSE also suppresses immune function by modifying the activity of the ubiquitin proteasome system.
Previous studies suggest that the downstream products of NLRP3 inflammasomes such as IL-1β or IL-18 are associated with the pathophysiology of smoke-driven chronic obstructive pulmonary disease (COPD) although direct evidence to link NLRP3 protein with the disease is sparse. It is possible that the response to cigarette smoke could differ by cell type, model system, or kinetics. The level of IL-1β and/or IL-18 was increased in the lungs, lavage fluid, or sputum of COPD subjects or animals exposed to cigarette smoke [
20‐
23]. However, cigarette smoke alone does not secrete IL-1β in THP1 cells [
3], and we found that the release of IL-1β and IL-18 is reduced after CSE exposure in THP1 cells and monocyte-derived macrophages. Pulmonary cells such as lung epithelial cells or alveolar macrophages could have different responses to cigarette smoke exposure in terms of cellular NLRP3 protein levels, while monocytes or monocyte-derived macrophages have decreased NLRP3 protein levels with cigarette smoke leading to immunosuppression. Indeed, we did not observe significant changes in NLRP3 protein levels in A549 alveolar epithelial cells and Beas-2B bronchial epithelial cells after CSE exposure (data not shown). Another possibility is that increased IL-1β and IL-18 in COPD is mainly derived from a pathway other than NLRP3 inflammasome activation. Although IL-18 knockout mice exhibit reduced pulmonary inflammation and emphysema compared to wild-type mice after cigarette smoke [
21,
24], pulmonary inflammation occurred independent of NLRP3/caspase-1 axis after four weeks of cigarette smoke exposure [
25]. To clarify these points, further studies are necessary.
Cigarettes smoke is a mixture of more than 4500 chemical compounds [
26]. Cigarette smokers inhale and absorb many toxic compounds both in vaporous and particulate phases by burning cigarettes. Immune modulation by cigarette smoke results from the sum of all CSE compounds over time rather than a single compound [
27]. We used CSE stored in a vacuum sealed bottle at -80 °C and thereby minimizing the potential confounding effect that might occur by dissipating reactive intermediates. It is unclear to what extent the cigarette smoke in the airway is absorbed into systemic circulation in a human body. However, the dose range of CSE in our study seems appropriate to study immunologic effects based on previous studies [
28‐
30], although the degree of systemic absorption may differ individually [
31].
Acknowledgements
Not applicable