Toll-like receptor 4-interacting SPA4 peptide suppresses the NLRP3 inflammasome in response to LPS and ATP stimuli

Anti-inflammatory activity of TLR4-interacting SPA4 peptide via suppression of TLR4-priming of the NLRP3-inflammasome.

Abstract

Inflammation is induced because of interplay among multiple signaling pathways and molecules during infectious and noninfectious tissue injuries. Crosstalk between Toll-like receptor-4 signaling and the neuronal apoptosis inhibitor protein, major histocompatibility class 2 transcription activator, incompatibility locus protein from Podospora anserina, and telomerase-associated protein (NACHT), leucine-rich repeat (LRR), and pyrin domain-containing protein 3 (NLRP3) inflammasome against pathogen- or damage-associated molecular patterns can cause exaggerated inflammation. We previously established that the Toll-like receptor-4-interacting SPA4 peptide suppresses gram-negative bacterial lipopolysaccharide (Toll-like receptor-4 ligand)-induced nuclear factor-κB and inflammatory response. In the present study, we hypothesized that the SPA4 peptide exerts its anti-inflammatory effects by suppressing the crosstalk between Toll-like receptor-4 signaling and the NLRP3 inflammasome. We evaluated binding of the lipopolysaccharide-ligand to cell-surface Toll-like receptor-4 in the presence or absence of adenosine triphosphate (an NLRP3 inflammasome inducer) by flow cytometry. The expression and activity of NLRP3 inflammasome-related parameters were studied in cells challenged with lipopolysaccharide and adenosine triphosphate using molecular and immunologic methods. The cells were challenged with lipopolysaccharide and treated with SPA4 peptide before (pre-adenosine triphosphate) or after (post-adenosine triphosphate) secondary challenge with adenosine triphosphate. Our data demonstrate that the Toll-like receptor-4-interacting SPA4 peptide does not affect the binding of lipopolysaccharide to Toll-like receptor-4 in the presence or absence of adenosine triphosphate. We also found that the SPA4 peptide inhibits mRNA and cellular protein levels of pro-interleukin-1β and NLRP3, formation of the NLRP3 inflammasome, caspase activity, and release of interleukin-1β. Furthermore, the SPA4 peptide treatment reduced the secreted levels of interleukin-1β from cells overexpressing Toll-like receptor-4 compared with cells expressing the dominant-negative form of Toll-like receptor-4. Together our results suggest that the SPA4 peptide exerts its anti-inflammatory activity by suppressing Toll-like receptor-4-priming of the NLRP3 inflammasome

Introduction

The host innate immune system induces inflammatory responses because of activation of several PRRs present on the cell membrane and in the cytoplasm. These receptors sense PAMPs and DAMPs and initiate a network of inflammatory signaling pathways, which, in turn, release various proinflammatory cytokines, chemokines, and chemical mediators [1]. An exaggerated inflammatory response causes acute tissue injury, which leads to organ damage or chronic inflammatory pathologic conditions [2, 3].

In an infectious or noninfectious inflammatory condition, the PAMPs and DAMPs can activate the TLR and intracellular Nod-like receptor signaling pathways [4, 5]. During an endotoxic shock-induced lung injury, LPS (PAMP) and subsequent stimulation with ATP (DAMP; released from necrotic cells at the site of injury) can result in crosstalk between TLR4 and NLRP3 (a member of Nod-like receptor family), formation of the NLRP3 inflammasome, and production of IL-1β [4]. IL-1β is a major proinflammatory cytokine that is produced in large amounts during injury. The activation and secretion of mature IL-1β occur in 2 phases. The first priming phase includes activation of TLR4-NF-κB signaling by LPS. In the second activation step with ATP, the NLRP3 inflammasomes are formed [4, 6]. The NLRP3 inflammasome formation involves the association of the pyrin domain of the NLRP3 protein with an ASC containing a caspase activation and recruitment domain. The ASC then recruits procaspase-1 through its caspase activation and recruitment domain, leading to cleavage of procaspase-1 into caspase-1 and maturation of pro-IL-1β into IL-1β [4, 7, 8]. Thus, signaling via TLR4 and the NLRP3 inflammasome is needed for an enhanced release of IL-1β and the exaggerated inflammation observed in acute injurious and inflammatory conditions, such as endotoxic shock, lung injury, and acute respiratory distress syndrome [9], and chronic conditions of arthritis [10], diabetes [11], atherosclerosis [12], and cryopyrin-associated autoinflammatory syndrome [13].

A TLR4-interacting SPA4 peptide, amino acids GDFRYSDGTPVNYTNWYRGE, was recently identified [14]. Our results demonstrated that the SPA4 peptide binds to TLR4 and suppresses NF-κB and the inflammatory response against gram-negative bacterial LPS (a TLR4 ligand) in cell systems [14–16] and in a mouse model of endotoxic shock-induced lung inflammation [16]. Results from cells transfected with the dominant-negative construct of MYD88 demonstrated that the SPA4 peptide suppresses TLR4–MYD88-dependent NF-κB activity and TNF-α cytokine [16]. In this study, we hypothesized that the SPA4 peptide would interfere with the crosstalk between TLR4 signaling and the NLRP3 inflammasome to suppress an exaggerated inflammatory response in cells challenged with LPS and ATP. Thus, we investigated the effect of the SPA4 peptide on the steady-state mRNA and protein levels of pro-IL-1β and NLRP3, formation of the NLRP3 inflammasome, activation of caspase, and release of IL-1β in an established dendritic cell line and primary mouse alveolar macrophages.

Our results demonstrate that the SPA4 peptide does not interfere with LPS binding to TLR4 in the presence or absence of the NLRP3 inflammasome inducer ATP. Furthermore, upon treatment of LPS- and ATP-challenged cells with SPA4 peptide, the mRNA and cellular protein levels of NLRP3 and pro-IL-1β, NLRP3 inflammasome formation, caspase activity, and secreted levels of IL-1β are significantly reduced. Together, our results indicate that the suppression of LPS–TLR4 signaling by SPA4 peptide [14–16] further reduces the NLRP3 inflammasome and alleviates inflammatory response.

MATERIALS AND METHODS

SPA4 peptide

SPA4 peptide (amino acid sequence GDFRYSDGTPVNYTNWYRGE) was synthesized at GenScript Piscataway Township, NJ, USA). Mass spectrometry and HPLC confirmed the purity and composition of the batches of the peptide. The endotoxin concentration of reconstituted peptide solutions was measured by Limulus amebocyte lysate assay (Charles River Laboratories, Wilmington, MA, USA) per the manufacturer’s instructions.

Cell culture systems

Murine bone-marrow-derived JAWSII dendritic cells.

Murine bone-marrow-derived JAWSII dendritic cells (American Type Culture Collection, Manassas, VA, USA) were maintained in α-MEM (Cellgro; Mediatech, Manassas, VA, USA) supplemented with 20% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 50 µg/ml gentamicin (Life Technologies, Grand Island, NY, USA), and 5 ng/ml recombinant murine granulocyte macrophage colony-stimulating factor (PeproTech, Rocky Hill, NJ, USA).

Primary mouse alveolar macrophages.

Primary mouse alveolar macrophages were harvested from 5–6-wk-old, female C57BL6/J mice (The Jackson Laboratory, Bar Harbor, ME, USA) using a standard protocol [17]. Studies in mice were approved by the Institutional Animal Care and Use and Biosafety Committees at the University of Oklahoma Health Sciences Center (Oklahoma City, OK, USA). An angiocatheter was placed in the trachea and BALF was collected using ice-cold, endotoxin-, calcium-, and magnesium-free Dulbecco’s phosphate-buffered saline [18]. BALF was centrifuged at 400 g for 10 min at 4°C. Pelleted cells were suspended into RPMI 1640 medium containing 5% heat-inactivated FBS, 5 mM HEPES with 10 µg/ml gentamicin, 50 U/ml penicillin, and 50 µg/ml streptomycin, and incubated at 37°C for 2 h. Nonadherent cells were removed, and adherent cells were washed before conducting the experiments. The morphology and phenotype of a representative preparation of cells were assessed by Wright-Giemsa staining and flow cytometry after staining with fluorochrome-conjugated cell surface marker (CD45, CD11b, CD68)-specific antibodies. Adherent cells were CD45⁺CD11b⁺CD68⁺ (27.5%) and CD45⁺CD11b⁺CD68⁻ (60.3%).

Transient transfection with mouse TLR4 plasmid DNA constructs.

Mouse bone marrow-derived JAWSII dendritic cells (1 × 10⁶ cells) were transfected with 2 µg of each plasmid construct encoding WTTLR4 or TLR4DN (Pro at position 712 substituted with His), using TransIT-TKO transfection reagent (Mirus Bio, Madison, WI, USA) per the previously published method [16, 19]. The plasmid DNA constructs were obtained from Dr. Lynn Hajjar (University of Washington, Seattle, WA, USA). Viability and morphology of the cells were assessed by the trypan blue exclusion method and Wright-Giemsa staining, respectively.

Binding of LPS (TLR4-ligand) to the cells

About 100,000 dendritic cells were incubated with 1 µg/ml (final concentration) of BODIPY-labeled Escherichia coli O55:B5-derived LPS (BODIPY LPS, Life Technologies) with or without 10 µM SPA4 peptide at 37°C and run on a flow cytometer. Any shift in the cell-associated FL1 (green) fluorescence was determined as a measure of LPS binding to the cells. Unchallenged, untreated cells or cells incubated with BODIPY LPS in the presence of a 10-fold excess amount of plain E. coli O55:B5-derived LPS (Calbiochem; EMD Millipore, Billerica, MA, USA) were included as controls. Extracellular ATP is a well-characterized endogenous DAMP. Although ATP triggers the NLRP3 inflammasome by binding to the purinergic receptor P2X7 and through K⁺ efflux [4], we examined any effect of 2.5 mM ATP (Sigma-Aldrich, St. Louis, MO, USA) on LPS binding to the cells.

Challenges with LPS and ATP and treatment with SPA4 peptide

Although LPS is a known PAMP and an inducer of TLR4 signaling, ATP is subsequently released as one of the DAMPs from necrotic cells at the site of tissue injury and triggers the formation of NLRP3 inflammasome [20, 21]. We challenged the genetically transfected or untransfected dendritic cells or alveolar macrophages with E. coli O111:B4-derived LPS before a second challenge with ATP and treated the cells with SPA4 peptide. In a pre-ATP treatment model, the LPS (100 ng/ml)-challenged cells were treated with SPA4 peptide (10 µM) at 2.5 h before the addition of ATP (2.5 mM) at 3.5 h. In a post-ATP treatment model, the LPS-challenged cells were treated with ATP at 3.5 h and SPA4 peptide at 4 h.

In a separate group, the LPS (100 ng/ml)-challenged cells were treated with SPA4 peptide alone (10 µM) at 4 h. The LPS- or ATP-challenged cells, unchallenged and untreated, or SPA4 peptide-treated cells were included as controls. Additional controls included an NLRP3 inflammasome inhibitor, glyburide (200 µM; InvivoGen, Carlsbad, CA, USA) [22], and a pan-caspase inhibitor, Z-VAD-FMK (10 and 50 µM, InvivoGen). The cell-free supernatants and cells from all treatment groups were harvested at 5 h.

Expression of IL-1β and NLRP3 mRNA

LPS-TLR4-NF-κB activity was recently recognized as being associated with the transcription of IL-1β [23] and NLRP3 [24]. Thus, we evaluated whether SPA4 peptide could reduce mRNA expression of IL-1β and NLRP3. Approximately 1 × 10⁶ JAWSII dendritic cells were seeded per well. The next day, the cells were challenged with LPS and ATP and were treated with SPA4 peptide, as described above. Total RNA was extracted from cell lysates using the RNeasy kit (Qiagen, Germantown, MD, USA). The reverse-transcriptase reaction was performed for 1 h at 42°C using 2 μg of total RNA, 1 μg of oligo(dT), 200 U of Moloney murine leukemia virus reverse-transcriptase enzyme, 500 μM deoxynucleotide triphosphates mix, and 25 U of RNase inhibitor (Promega, Fitchburg, WI, USA). The cDNA equivalent of 30 ng of total RNA was then used to carry out 50 cycles of PCR. Each cycle consisted of incubation at 95°C for 15 s, 58°C for 30 s, and 72°C for 30 s (Bio-Rad Laboratories, Hercules, CA, USA). Each reaction was set up in triplicate wells in a total volume of 20 μl, which contained 3 μl cDNA, 2 μl of each forward and reverse primer, and 10 μl RT² SYBR Green qPCR master mix (Qiagen). Primer sequences for the target (IL-1β, NLRP3) [25, 26] and housekeeping β-actin [27] genes were as follows: IL-1β = 5′-TGAGCTGAAAGCTCTCCACC-3′, 5′-CTGATGTACCAGTTGGGGAA-3′; NLRP3 = 5′-CACTTGGATCTAGCCACATC-3′, 5′-AGCTCCAGCTTAAGGGAACTC-3′; and β-actin = 5′-CCGGAAATCGTGCGTGACATTAAG-3′, 5′-TGATCTCCTTCTGCATCCTGTCGG-3′

The primers were synthesized at Integrated DNA Technologies (Coralville, IA, USA). The primers of IL-1β and NLRP3 were derived from the mature IL-1β region (positions 491–510 on the IL-1β nucleotide sequence; accession NM_008631) and pyrin domain (positions 371–388 on the NLRP3 nucleotide sequence; accession NM_145827), respectively. The quantitative values of the genes of interest were normalized with those of the β-actin housekeeping gene. Fold changes over the control values were calculated using the ΔΔCt method for relative quantification (Qiagen).

Formation of ASC specks

Extracellular ATP triggers the oligomerization of NLRP3 and ASC, which cause the formation of “specks” in LPS-challenged cells [7]. The specks are about 1–2 µm in diameter and can be observed with immunocytochemistry [28]. Approximately 50,000 primary mouse alveolar macrophages were challenged with LPS (100 ng/ml) and ATP (2.5 mM) and were treated with SPA4 peptide (10 μM), as described above. Glyburide (200 µM), an NLRP3 inflammasome inhibitor, was added at 2.5 h in pre-ATP treatment model [22]. The cells were fixed in 3.5% paraformaldehyde and permeabilized in DMEM containing 10% FBS, 0.05% saponin, and 10 mM HEPES. Nonspecific sites were blocked with 1% BSA. Cells were incubated with 1:200 diluted rabbit anti-ASC antibody (Adipogen International, San Diego, CA, USA; raised against a synthetic peptide corresponding to the N-terminal region of human ASC) overnight at 4°C. Subsequently, cells were washed and incubated with 10 µg/ml Alexa Fluor 488-conjugated secondary anti-rabbit antibody (Invitrogen). Finally, Hoechst dye 33258 (1 µg/ml) was added for nuclear staining, and the slides were mounted with Vectashield (Vector Laboratories, Burlingame, CA, USA). All images were acquired at ×20 objective using the fluorescence microscope. Fluorescence micrographs were taken from 5 different fields selected at random. A blinded observer counted the numbers of specks per total number of cells.

Colocalization of ASC and NLRP3 proteins within specks

Next, we assessed the colocalization of ASC and NLRP3 proteins within the specks as an indicator of NLRP3 inflammasome formation by immunocytochemistry. Approximately 50,000 primary mouse alveolar macrophages were challenged with LPS and ATP and were treated with SPA4 peptide, as described above. The cells were fixed in 3.5% paraformaldehyde and permeabilized in DMEM containing 10% FBS, 0.05% saponin, and 10 mM HEPES. Nonspecific sites were blocked with 1% BSA. Cells were incubated with 1:200 diluted rabbit anti-ASC antibody and 1:50 diluted mouse anti-NLRP3 antibody raised against recombinant mouse NLRP3 (pyrin domain/aa 1–93; Adipogen) overnight at 4°C. Subsequently, cells were washed and incubated with 10 µg/ml Alexa Fluor 488-conjugated anti-rabbit secondary antibody for ASC (green) and Alexa Fluor 568-conjugated anti-mouse secondary antibody for NLRP3 (red; Invitrogen). Finally, Hoechst dye 33258 (1 µg/ml; blue) was added for nuclear staining, and the slides were mounted with Vectashield. All the images were acquired at ×63 oil immersion objective under Zeiss (Carl Zeiss Inc., Thornwood, NY, USA) confocal microscope and processed using Zeiss LSM Image Examiner or ZEN 2011 programs. The merged confocal images were observed for specks and colocalization of NLRP3 and ASC proteins within the specks.

Measurement of caspase activity

Approximately 1 × 10⁶ dendritic cells were challenged with LPS and ATP and treated with SPA4 peptide, as described above. A pan-caspase inhibitor, Z-VAD-FMK (10 and 50 µM) was added alone or in combination with SPA4 peptide. Cells were lysed with 200 µl of ice-cold radioimmunoprecipitation buffer containing 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5% sodium deoxycholate, 1 mM EDTA, phosphatase inhibitors (200 µM sodium orthovanadate, 10 µM sodium fluoride), protease inhibitor cocktail (inhibits broad categories of Ser, Cys, and metalloproteases; Roche Diagnostics, Indianapolis, IN, USA), and detergents (0.1% SDS and 1% Igepal CA-630; Sigma-Aldrich). About 200 μg of cell lysate protein was suspended in a reaction buffer containing 25 mM HEPES (pH 7.4) and 2 mM dithiothreitol and was incubated with 200 μM acetyl-Tyr, Val, Ala, Asp, and 7-amido 4-methylcoumarin (a fluorogenic substrate; Enzo Life Sciences, Farmingdale, NY, USA) to assess caspase activity. Fluorescence readings were then taken at 360-nm excitation and 460-nm emission wavelengths (BioTek Instruments, Winooski, VT, USA). Recombinant mouse caspase-1 protein (BioVision, Milpitas, CA, USA) was included as a positive control. The background fluorescence readings (fluorogenic substrate suspended in the reaction buffer) were subtracted from the readings of the experimental wells.

Expression of pro-IL-1β, NLRP3, and active caspase-1

To study the expression of cellular pro-IL-1β and NLRP3, we suspended 20 µg of total cell lysate protein in 2× SDS sample buffer and boiled it for 5 min. The cell lysate proteins were separated on Novex 4–20% Tris-glycine SDS-PAGE gradient gel (Invitrogen) and transferred onto a 0.2-μm nitrocellulose membrane. The nonspecific sites were blocked with 7% of nonfat milk for 1 h. The membrane was then washed thrice with TBST (0.1% Tween 20) and immunoblotted overnight with 1:1000 diluted IL-1β (Cell Signaling Technology, Beverly, MA, USA) or NLRP3 (Adipogen) antibodies. The membrane was washed and incubated with 1:1000 diluted anti-rabbit or anti-mouse HRP-conjugated antibody (Cell Signaling). The immune complexes were visualized using West Femto chemiluminescent substrate reagent (Pierce Biotechnology, Rockford, IL, USA) on an Omega imaging system (UltraLum, Claremont, CA, USA). The membrane was then incubated with stripping buffer containing 10% SDS, 0.5 M Tris-HCl (pH 6.8), and 100 mM β-mercaptoethanol at 55°C for 45 min, washed, and incubated with a 1:2000 diluted antibody for actin to assess protein loading (Sigma-Aldrich). The next day, the membrane was washed and incubated with 1:1000 diluted anti-rabbit HRP-conjugated antibody (Cell Signaling).

For immunoblotting of active caspase-1, secreted proteins were precipitated from the cell-free supernatants of alveolar macrophages using the methanol–chloroform protein-precipitation method [29]. In brief, methanol, cell-free supernatant, and chloroform were mixed together in a ratio of (1:1:0.25), vortexed vigorously, and centrifuged at 13,000 g for 5 min at 4°C. Three phases were obtained after centrifugation. The upper phase was removed, and 1 volume of methanol was added to the remaining 2 bottom phases. The mixture was again vortexed and centrifuged. The supernatant was discarded without disturbing the pellet. The pellet was dried at 55°C for 10 min on a dri-block heater (Techne, Burlington, NJ, USA). The pellet was suspended in 40 µl of 1× SDS sample buffer, boiled, and run on a Novex 4–20% Tris-Gly SDS-PAGE gradient gel (Invitrogen). The separated proteins were immunoblotted overnight with 1:200 diluted caspase-1 p10 antibody (Santa Cruz Biotechnology, Dallas, TX, USA). Active caspase-1 contains 2 heterodimers; each heterodimer is composed of 1 subunit of 20 kDa and 1 unit of 10 kDa [30]. The membrane was washed and incubated with 1:1000 diluted anti-rabbit HRP-conjugated antibody. The immune complexes were visualized using West Femto chemiluminescent substrate reagent.

Levels of cellular pro-ILβ-1 and secreted IL-1β

The levels of pro-IL-1β and secreted IL-1β were measured using a commercially available ELISA kit (BioLegend, San Diego, CA, USA).

Statistics

Statistical significance was analyzed using ANOVA to test the differences between the multiple groups (Prism Software, GraphPad Software, La Jolla, CA, USA). P values are shown within the figures.

RESULTS

SPA4 peptide does not affect LPS binding to the TLR4-expressing dendritic cells in the presence or absence of ATP

Previously published results from our laboratory [14] demonstrated the binding between SPA4 peptide and TLR4 protein in complex with MD2 in a computationally docked model and using an immunochemical assay. Earlier, we published that the SPA4 peptide does not bind to LPS [15]. Here, we determined the effect of the SPA4 peptide on the binding of LPS to the TLR4-expressing cells in the presence or absence of ATP. An increase in MFI, percentage of fluorescent cells, and a positive shift in the flow cytometric histogram indicated the binding of BODIPY LPS to the TLR4-expressing JAWSII cells (Fig. 1). Addition of ATP did not affect LPS binding because no change was noted in MFI, percentage of cells, or the histogram pattern (Fig. 1). As expected, a decrease in MFI and percentage of fluorescent cells and a negative shift in the flow cytometric histogram indicated reduced binding of BODIPY LPS in the presence of a 10-fold excess amount of unlabeled LPS. Together, our results demonstrated that the SPA4 peptide does not affect the binding of LPS to TLR4.

Figure 1. SPA4 peptide does not affect the binding of LPS to dendritic cells.

The binding of BODIPY LPS was assessed with or without ATP in the reaction assay tubes. (A) A flow cytometric dot plot of cells was first plotted to gate out any cell debris using forward and side scatter on x and y axes. Cells with moderate or high forward and side scatters were gated in region P. (B) Flow cytometric histogram charts were then plotted on the FL1 channel for the cells gated in region P. A significant positive shift was seen in the histogram plot of cells challenged with BODIPY LPS (brown line) compared with control (black line). No further shift was observed when BODIPY LPS-challenged cells were treated with the SPA4 peptide (red line). As expected, a negative shift was observed when BODIPY LPS-challenged cells were incubated with a 10-fold excess of unlabeled LPS (blue line). A similar pattern was observed when the BODIPY LPS binding was assessed in combination with ATP. (C) The percent cells and MFI of cells gated in the M and R regions are shown in tabulated form. The results are from 1 representative experiment of 3 experiments performed separately.

SPA4 peptide treatment inhibits the steady state levels of IL-1β and NLRP3 mRNA

The maturation and secretion of active IL-1β requires 2 steps. The first step involves priming with LPS via TLR4, resulting in de novo synthesis of pro-IL-1β and an increase in the expression of NLRP3 protein [6, 31]. Our previously published results showed that the SPA4 peptide suppresses LPS–TLR4–NF-κB activity in therapeutic models [14–16]. Because NF-κB induces a number of inflammatory genes, including IL-1β and NLRP3 [23, 24], we studied the effect of the SPA4 peptide on mRNA expression of IL-1β and NLRP3 (Fig. 2). As expected, LPS significantly induced the mRNA expressions of IL-1β (average 841-fold) and NLRP3 (average 1.7-fold) compared with the unchallenged, untreated control (Fig. 2). The addition of SPA4 peptide reduced the LPS-induced expression of the IL-1β gene to one-fourth (Fig. 2A) and the NLRP3 mRNA expression to one-third (Fig. 2B) of the ones in the LPS-challenged cells.

Figure 2. SPA4 peptide suppresses mRNA levels of IL-1β and NLRP3 in LPS-primed dendritic cells.

The LPS-challenged dendritic cells were treated with SPA4 peptide (10 μM) at 4 h. The cells were harvested at 5 h. Expression of (A) IL-1β and (B) NLRP3 mRNA was determined by quantitative real-time PCR. The results were normalized with mRNA levels of β-actin and fold changes were calculated compared with the unchallenged, untreated control. Bars represent means ± sem of the fold changes noted in 3 experiments performed in triplicates separately.

In the second step, endogenous DAMP, such as ATP, induced the NLRP3 inflammasome, leading to the release of an exaggerated amount of IL-1β (summarized in Fig. 3) [7, 32, 33]. Here, we addressed whether the TLR4-immunomodulatory activity of SPA4 peptide would carry the effect further in reducing the NLRP3 inflammasome and IL-1β in response to endogenous DAMPs released during severe injury. As expected, the LPS and ATP challenges increased the mRNA of IL-1β and NLRP3 from basal levels (Fig. 4A and B). The ATP challenge alone did not have an effect on the level of IL-1β mRNA but slightly decreased the level of NLRP3 mRNA. The SPA4 peptide treatment of LPS- and ATP-challenged cells reduced the levels of both IL-1β and NLRP3 mRNA to about one-third and one-half of those in unchallenged, untreated cells (Fig. 4A and B), respectively.

Figure 3. A drawing demonstrating the association between the TLR4 and NLR pathways.

The inflammatory response (or maturation of IL-1β) involving TLR4 and NLR is a 2-step process: 1) Priming step (left half of the circle)—LPS binds to TLR4 and activates downstream NF-κB and transcription and translation of NLRP3 and pro-IL-1β; and 2) Activation step (right half of the circle)—ATP induces NLRP3 inflammasome assembly through interaction between NLRP3 and ASC. Upon activation of the NLRP3 inflammasome, procaspase-1 undergoes self-proteolytic cleavage and releases caspase-1, which in turn converts pro-IL-1β into its active form IL-1β. In this study, we included 2 models of SPA4 peptide treatment: 1) pre-ATP treatment (left)—SPA4 peptide was added to LPS-primed cells at 2.5 h, followed by ATP addition at 3.5 h; and 2) post-ATP treatment (right)—SPA4 peptide was added after 30 min of ATP addition at 4 h. Cell-free medium supernatants and cell lysates were harvested at 5 h in all treatment models.

Figure 4. SPA4 peptide reduces mRNA levels of IL-1β and NLRP3 in pre-ATP and post-ATP treatment models.

Expression of (A) IL-1β and (B) NLRP3 mRNA was determined by quantitative real-time PCR. The results were normalized with mRNA expression level of the β-actin housekeeping gene. Bars represent means ± sem of pooled results from 3 experiments performed in triplicates separately on different occasions.

Cellular levels of pro-IL-1β and NLRP3 proteins are reduced in SPA4 peptide-treated cells

The NLRP3 and pro-IL-1β proteins serve as the substrates for the NLRP3 inflammasome and caspase-1, respectively. The LPS challenge induced synthesis of pro-IL-1β. The expression of pro-IL-1β was slightly reduced in cells challenged with LPS and ATP, indicating the conversion of pro-IL-1β into active IL-1β (Fig. 5A). Treatment with the SPA4 peptide suppressed the expression of pro-IL-1β in cells challenged with LPS or LPS in combination with ATP. The amounts of pro-IL-1β measured by ELISA reflected the changes observed with immunoblotting (Fig. 5B). A similar expression pattern was observed for NLRP3 protein in cell lysates (Fig. 5C).

Figure 5. SPA4 peptide reduces the cellular protein pool of pro-IL-1β and NLRP3 in dendritic cells.

Twenty micrograms of cell lysate protein was separated on SDS-PAGE gel, transferred on the nitrocellulose membrane, and immunoblotted with (A) pro-IL-1β or (C) NLRP3-specific antibodies. The membrane was then stripped, and immunoblotted with an actin-specific antibody. The densitometry was performed on the immune complexes. Arbitrary densitometric units for pro-IL-1β or NLRP3 proteins were normalized with those of actin and plotted as a bar chart. Immunoblots are from 1 representative experiment of 3 experiments. (B) The intracellular protein pool of pro-IL-1β (picogram per microgram of total cellular protein) in cell lysate of dendritic cells was detected by ELISA. Bars represent means ± sem of pooled results from 3 experiments performed in triplicates separately on different occasions.

NLRP3 inflammasome or speck formation is suppressed in SPA4 peptide-treated cells

The activation of the NLRP3 inflammasome is reflected by coassembly of NLRP3 and ASC proteins, into a large protein complex called a speck [7]. The addition of ATP to LPS-primed cells causes assembly of the NLRP3 inflammasome, producing specks (Figs. 6 and 7). Endogenous NLRP3 and ASC proteins were distributed across the cytoplasm in the primary mouse alveolar macrophages, which were unchallenged and untreated or treated with SPA4 peptide alone (Figs. 6 and 7). As expected, a substantial increase in the number of specks was observed in cells challenged with LPS and ATP. The LPS or ATP challenges, on their own, did not induce specks in the cells. The SPA4 peptide significantly suppressed the number of specks to about one-third that of cells challenged with LPS and ATP (Fig. 6). We did not observe any conspicuous changes in the MFI of specks between the groups (data not shown).

Figure 6. SPA4 peptide reduces the assembly of the NLRP3 inflammasome.

LPS-primed primary mouse alveolar macrophages were challenged with ATP and treated with SPA4 peptide as described in Fig. 3. (A) Cells were fixed and immunostained for ASC. The formation of ASC specks was analyzed by fluorescence microscopy. Fluoromicrographs were taken from each treatment well in 3 separate experiments. A speck in a representative fluoromicrograph of LPS- and ATP-challenged cell is enlarged for better visualization (white arrowhead). Glyburide was included as a positive control and as an inhibitor of inflammasome formation. The fluoromicrographs were taken using ×20 objective. (B) The number of specks in fluoromicrographs were counted, divided by the total number of cells, and multiplied by 100 to calculate the percentage of cells exhibiting ASC specks. Bars represent means ± sem of pooled results from 3 experiments performed separately on different occasions.

Figure 7. Colocalization of ASC and NLRP3 within the specks in primary mouse alveolar macrophages.

(A) Cross-reactivity of NLRP3 and ASC primary antibodies. Twenty micrograms of cell lysate protein was separated on SDS-PAGE gel, transferred on the nitrocellulose membrane, and immunoblotted with ASC- and NLRP3-specific antibodies. Lane 1: lysate protein from unchallenged, untreated cells; and Lane 2: lysate protein from cells challenged with LPS and ATP. (B) LPS-primed primary mouse alveolar macrophages were challenged with ATP and treated with SPA4 peptide as described in Fig. 3. Cells were fixed and immunostained for ASC (green) and NLRP3 (red), and counterstained with nuclear dye (blue). Specks are shown as circles. Confocal images of stained cells were taken using ×63 oil-immersion objective. Colocalization of ASC and NLRP3 proteins was observed in yellow. Results are from 1 experiment representative of 3 separate experiments. Glyburide was included as an inhibitor of NLRP3 inflammasome formation.

We assessed whether NLRP3 colocalized with ASC within these specks. The possibility of cross-reactivity of ASC- and NLRP3-specific antibodies with respective antigens was assessed by Western immunoblotting. The ASC-specific antibody recognized an immunoreactive band of 25 kDa, and NLRP3-specifc antibody recognized a major immunoreactive protein of ∼110 kDa (Fig. 7A). We observed colocalization of ASC and NLRP3 proteins within the specks in all treatment groups (Fig. 7B). Treatment with SPA4 peptide suppressed the speck formation altogether (Figs. 6 and 7B).

SPA4 peptide treatment reduces caspase activity and release of IL-1β

The NLRP3 inflammasome recruits procaspase-1, activating caspase-1, which later cleaves pro-IL-1β into biologically active IL-1β. When LPS-primed dendritic cells were challenged with ATP, a substantial increase was observed in caspase activity and IL-1β production, compared with LPS or ATP alone or at basal levels (Figs. 8 and 9). Corresponding to the suppression of the NLRP3 inflammasome, we observed a significant reduction in caspase activity in SPA4 peptide-treated cells. The results were consistent in both pre-ATP and post-ATP treatment models. Z-VAD-FMK is known to suppress caspase activity [34]. When cells were treated with the SPA4 peptide in combination with Z-VAD-FMK, there was no additive effect on caspase activity (Fig. 8). Likewise, SPA4 peptide reduced the secreted levels of IL-1β in pre-ATP and post-ATP models. However, the addition of Z-VAD-FMK to SPA4 peptide-treated cells further decreased the secreted levels of IL-1β in the pre-ATP model (Fig. 9A). Such was not the case in the post-ATP model (Fig. 9B).

Figure 8. SPA4 peptide treatment reduces caspase activity.

The caspase activity was measured in 200 µg of cellular protein after incubating with N-acetyl-Tyr-Val-Ala-Asp-7-amino-4-methylcoumarin (fluorogenic substrate) for 2 h. Fluorescence readings were taken at 380 nm excitation and 460 nm emission wavelengths. The background readings of substrate solution without any cell protein were subtracted from the readings of each experimental well. The LPS- and ATP-challenged cells were treated with SPA4 peptide as per (A) pre-ATP and (B) post-ATP treatment models. Z-VAD-FMK, a caspase inhibitor, was included as a positive control. Bars represent means ± sem of pooled results from 3 experiments performed in triplicate separately on different occasions.

Figure 9. SPA4 peptide suppresses IL-1β in cell-free supernatant of dendritic cells challenged with LPS and ATP and treated with SPA4 peptide as per the (A) pre-ATP and (B) post-ATP treatment models.

Cell-free medium supernatants were harvested at 5 h. IL-1β levels were measured by ELISA. Z-VAD-FMK was used as a positive control. Bars represent means ± sem of pooled results from 3 experiments performed in triplicate at different times.

SPA4 peptide suppresses cellular pools of NLRP3 protein, pro-IL-1β, caspase activity, and secretion of IL-1β in primary mouse alveolar macrophages

We also determined that the activity of SPA4 peptide was not limited to dendritic cells. We evaluated the effect of SPA4 peptide on NLRP3 expression, NLRP3 inflammasome formation, caspase-1 activity, and secreted levels of IL-1β in freshly harvested mouse alveolar macrophages. Adherent alveolar macrophages were challenged with LPS and ATP and were treated with SPA4 peptide, as described above. We found increased expression of pro-IL-1β (Fig. 10B and C) and NLRP3 protein (Fig. 10D) in alveolar macrophages challenged with LPS alone or in combination with ATP. Treatment with SPA4 peptide suppressed the expression of pro-IL-1β and NLRP3 protein in lysates of cells challenged with LPS alone or in combination with ATP. To detect the active unit of caspase-1, we precipitated the protein from the medium supernatant using the methanol–chloroform method and then immunoblotted for the p10 unit of caspase-1. We observed that the challenges with LPS and ATP induced the release of the p10 unit of active caspase-1 in the medium supernatant. SPA4 peptide treatment, however, suppressed the release of the active caspase-1 (Fig. 10E). These results were consistent with a significant decrease in the levels of secreted IL-1β (Fig. 10F).

Figure 10. SPA4 peptide reduces cellular NLRP3 protein, caspase-1 activation, and IL-1β secretion in primary mouse alveolar macrophages.

(A) Mouse alveolar macrophages were harvested from bronchoalveolar lavage fluids of normal mice. The cell population was stained with Wright-Giemsa stain and photomicrographed using a ×40 objective. The figure is representative of 1 of the 3 experiments. (B and D) Twenty micrograms of cell lysate protein was separated on SDS-PAGE gel, transferred on the nitrocellulose membrane, and immunoblotted with IL-1β or NLRP3-specific antibodies. The membrane was then stripped and immunoblotted with actin-specific antibody. The densitometry was performed on the immune-complexes. Arbitrary densitometric units for pro-IL-1β or NLRP3 proteins were normalized with those of actin and plotted as a bar chart. (E) Total protein in cell-free medium supernatants was precipitated by the methanol–chloroform method and immunoblotted against the p10 subunit of caspase-1. Immunoblots are from 1 representative experiment of 3 experiments. (C and F) Cellular expression of pro-IL-1β (picogram per microgram of total cellular protein) and secreted levels of IL-1β (picogram per milliliter) in cell-free medium supernatants of alveolar macrophages. Bars represent means ± sem of pooled results from 3 experiments performed in triplicate separately on different occasions.

SPA4 peptide suppresses IL-1β through TLR4

We determined whether reduction of IL-1β secretion occurs through the TLR4-immunomodulatory activity of SPA4 peptide in dendritic cells transfected with WTTLR4 or TLR4DN plasmid DNA constructs. We found that dendritic cells transfected with WTTLR4 produced 3 or more times the IL-1β as did TLR4DN-transfected or untransfected dendritic cells, respectively (Fig. 11). Treatment with the SPA4 peptide significantly reduced the expression of IL-1β in the supernatants of untransfected and WTTLR4-transfected dendritic cells but had no effect on TLR4DN-transfected dendritic cells (Fig. 11).

Figure 11. SPA4 peptide suppresses IL-1β through its interaction with TLR4.

Untransfected, WTTLR4-transfected, or TLR4DN-transfected dendritic cells were challenged with LPS or LPS and ATP and treated with SPA4 peptide. The cell-free supernatants were collected and subjected to measurement of secreted levels of IL-1β. The secreted levels of IL-1β were normalized with cellular protein content. Bars represent means ± sem of results are from 1 representative experiment of 5 experiments performed in triplicate separately.

DISCUSSION

During an active infection or noninfectious tissue injury, several PAMPs and DAMPs, which are recognized by different PRRs, are released. Therefore, crosstalk among different PRRs results in induction of the innate immune response as part of our natural host-defense mechanism. The inflammatory response is an integral part of innate immunity. However, an exaggerated inflammatory response can lead to severe tissue injury, multiple organ failure, or chronic inflammatory conditions [2, 3]. Thus, it is important to understand the complex network of molecules and signaling that is responsible for an inflammatory response. It is now widely accepted that crosstalk between TLR4 and the NLRP3 inflammasome in response to LPS and ATP stimuli is associated with an inflammatory response [4, 35, 36]. In our previously published reports [14, 15], we identified the SPA4 peptide that binds to TLR4 and down-regulates LPS-TLR4-MYD88-NF-κB and the inflammatory cytokine response. In light of the recognition of the crosstalk between TLR4 and NLRP3 inflammasome, here, we report that SPA4 peptide treatment affects the NLRP3 inflammasome in conjunction with suppression of TLR4 signaling for its anti-inflammatory activity.

We used LPS as a TLR4-ligand and ATP as an NLRP3-inflammasome inducer [4, 37]. The combination of LPS and ATP as inflammatory stimuli is biologically relevant in the context of noninfectious endotoxic shock-induced lung injury. The inflammatory response through crosstalk between TLR4 and NLRP3 inflammasome requires 2 steps. The first step is the priming of cells by TLR4; the second step is the activation of NLRP3 inflammasome assembly, which leads to caspase-1 induction and production of IL-1β and IL-18 (Fig. 3) [4, 6, 31]. We speculated that the suppression of LPS-TLR4-NF-κB by the SPA4 peptide may further affect the NLRP3 inflammasome, and exert anti-inflammatory effects.

The LPS-induced TLR4–NF-κB primes the cells and induces the mRNA expression of IL-1β and NLRP3. MYD88 and NF-κB are required for NLRP3 induction [23, 24, 38, 39]. NF-κB is associated with the transcription of inflammatory-pathway-related genes, including IL-1β and NLRP3 [23, 24]. Our results demonstrate that the SPA4 peptide does not affect the binding of its ligand LPS to TLR4 (Fig. 1). Rather, the binding of SPA4 peptide to TLR4 suppresses MYD88-dependent NF-κB activity and the inflammatory cytokine response [14–16]. Consistent with these findings, the mRNA expression of IL-1β and NLRP3 is reduced and eventually limits the protein expression of cellular pro-IL-1β and NLRP3 (Figs. 2–5 and 10).

Upon activation, NLRP3 undergoes oligomerization and forms a large complex structure that leads to recruitment of the adapter-protein ASC. Interaction of NLRP3 and ASC through the pyrin–pyrin domains leads to the formation of speck-like structures [7, 8]. The specks recruit procaspase-1 and induce the conversion of procaspase-1 into active caspase-1. Active caspase-1 then acts on pro-IL-1β and causes secretion of a biologically active form of IL-1β. Here, we demonstrate that the SPA4 peptide inhibits inflammasome formation, caspase activity, and IL-1β production. From the Western immunoblot analysis, we also confirmed that SPA4 peptide treatment inhibits the production of active caspase-1 (Fig. 10). These results suggest that down-regulation of TLR4 signaling by the SPA4 peptide at the priming step most likely brings down the cellular pools of pro-IL-1β and NLRP3, which are required for the formation of inflammasome.

Although our investigations support the SPA4 peptide interference at the priming step of inflammasome formation, it is difficult to rule out the possibility of the SPA4 peptide affecting other associated parameters or pathways. We have included a limited set of challenges with LPS and ATP. Other DAMPs, such as hyaluronan or high-mobility group B protein, may also contribute to TLR4 signaling, inflammasome formation, and the overall pathology of endotoxic shock-induced lung injury [40–42]. Because the SPA4 peptide is effective in reducing inflammation and tissue injury when administered therapeutically to LPS-challenged cells and in a mouse model of LPS-induced inflammation [16], we believe that the challenges with LPS and ATP and the timing of SPA4 peptide treatment closely mimic the biologic scenario. Future studies in genetic mouse models and other pathologic conditions will help to delineate the anti-inflammatory mechanisms of the SPA4 peptide.

During the past decade, the role of the NLRP3 inflammasome has been widely studied for its critical role in the pathogenesis of several diseases and is considered a potential target for therapeutic intervention. Currently available therapies target IL-1β: anakinra (Kineret; Amgen, Thousand Oaks, CA, USA)—a recombinant, nonglycosylated form of the human interleukin-1 receptor antagonist; rilonacept (Arcalyst; Regeneron Pharmaceuticals, Tarrytown, NY, USA)—a dimeric fusion protein consisting of the ligand-binding domains of the extracellular portions of the human interleukin-1 receptor component and IL-1 receptor accessory protein linked in-line to the Fc region of human IgG1; and canakinumab (Ilaris; Novartis Pharmaceuticals, Whippany, NJ, USA)—a neutralizing monoclonal IL-1β antibody [3]. Therapeutic intervention at different steps of signaling, from priming to inflammasome activation, could also suppress IL-1β levels and an overall biologic response against infectious and inflammatory stimuli. Pharmacological agents Bay 11-7082, parthenolide, glyburide, cytokine release inhibitory drug 3, and pralnacasan (VX-740 and VX-765) have recently been reported to suppress inflammasome [22, 43–47]. The results presented here suggest that the TLR4-interacting SPA4 peptide reduces the inflammasome at the priming step in a noninfectious, LPS- and ATP-challenge model. More-detailed studies are warranted to further investigate the activity of SPA4 peptide in models of severe challenges with a combination of PAMPs, DAMPs, and infectious organisms.

AUTHORSHIP

V.R. performed binding assay, challenges, and treatments in dendritic cells and macrophages and respective assays in harvested cells and medium supernatants; compiled the results; and wrote the first draft of the manuscript. S.A. conceptualized and designed the experiments, trained V.R. in harvesting the BALF and macrophages and in performing the experiments, and supervised V.R. in analyzing the results and in writing the manuscript.

Glossary

ASC

apoptosis-associated speck-like protein

BALF

bronchoalveolar lavage fluid

BODIPY

boron-dipyrromethene

DAMP

damage-associated molecular pattern

MFI

mean fluorescence intensity

PAMP

pathogen-associated molecular pattern

PRR

pathogen recognition receptors

TLR4

Toll-like receptor 4

TLR4DN

dominant-negative mutant of TLR4

WTTLR4

wild-type mouse TLR4

Z-VAD-FMK

carbobenzoxy-valyl-alanyl-aspartyl-(O-methyl)-fluoromethylketone

DISCLOSURES

The authors declare no competing financial interests.