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

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.

Keywords: anti-inflammatory activity; immunomodulation; surfactant protein A.

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.

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.

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.

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.

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.

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.

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.

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.