Investigating the Role of the NLRP3 Inflammasome Pathway in Acute Intestinal Inflammation: Use of THP-1 Knockout Cell Lines in an Advanced Triple Culture Model

Abstract

The NLRP3 inflammasome plays an important role in intestinal homeostasis as well as inflammation. However, in vivo studies investigating the role of the NLRP3 inflammasome in inflammatory bowel disease (IBD) report contrasting results, leaving it unclear if the NLRP3 inflammasome augments or attenuates intestinal inflammation. To investigate the role of the NLRP3/caspase-1 pathway in a model of acute intestinal inflammation, we modified a previously established in vitro triple culture model of the healthy and inflamed intestine (Caco-2/HT29-MTX-E12/THP-1). Using THP-1 knockout cell lines, we analyzed how the NLRP3 inflammasome and its downstream enzyme caspase-1 (CASP1) affect inflammatory parameters including barrier integrity and cytotoxicity, as well as gene expression and secretion of pro-inflammatory cytokines and mucus. Furthermore, we investigated differences in inflammation-mediated cytotoxicity towards enterocyte-like (Caco-2) or goblet-like (HT29-MTX-E12) epithelial cells. As a complementary approach, inflammation-related cytotoxicity and gene expression of cytokines was analyzed in intestinal tissue explants from wildtype (WT) and Nlrp3^-/- mice. Induction of intestinal inflammation impaired the barrier, caused cytotoxicity, and altered gene expression of pro-inflammatory cytokines and mucins in vitro, while the knockout of NLRP3 and CASP1 in THP 1 cells led to attenuation of these inflammatory parameters. The knockout of CASP1 tended to show a slightly stronger attenuating effect compared to the NLRP3 knockout model. We also found that the inflammation-mediated death of goblet-like cells is NLRP3/caspase-1 dependent. Furthermore, inflammation-related cytotoxicity and upregulation of pro-inflammatory cytokines was present in ileal tissue explants from WT, but not Nlrp3^-/- mice. The here presented observations indicate a pro-inflammatory and adverse role of the NLRP3 inflammasome in macrophages during acute intestinal inflammation.

Keywords: HT29-MTX-E12; IBD; barrier; caco-2; gastrointestinal tract; inflammatory disease; macrophages; mucus.

Figure 1

Secretion of (A) IL-1β, (B) IL-8 and (C) TNF-α into the supernatant by differentiated THP-1 cells (wild type, CASP1^-/- or NLRP3^-/- ) after 24 h incubation with 10 µg/cm² amine-modified polystyrene nanobeads (PS-NH₂), 200 µg/cm² crystalline silica (DQ12), LPS (10 ng/ml) or LSP/IFN-γ (both 10 ng/ml). Mean ± SD of N = 3; *p < 0.05, compared to the untreated control; ^#p < 0.05, compared to the WT of the same treatment group.

Figure 2

TEER at (A) t=24 h and (B) t=48 h of stable (full bars) and inflamed (open bars) triple-cultures with WT, CASP1^-/- or NLRP3^-/- THP-1 cells. Mean ± SD of N = 3; *p < 0.05, compared to the respective WT control; ^#p < 0.05 compared to the respective stable culture.

Figure 3

LDH activity in the apical compartment after 48 h of triple culture with WT, CASP1^-/- or NLRP3^-/- THP-1 cells. Mean + SD of N = 3. *p < 0.05 compared to the respective WT triple culture, ^#p < 0.05 compared to the respective stable culture.

Figure 4

Cytokine release into the basolateral compartment after 48 h of stable (full bars) and inflamed (open bars) triple culture with WT, CASP1^-/- or NLRP3^-/- THP-1 cells, as assessed by ELISA. Mean ± SD of N = 3. *p < 0.05, compared to the respective WT triple culture. “b.d.” = below detection limit.

Figure 5

Gene expression in the epithelium or THP-1 cells after 48 h of triple culture. (A) Gene expression in the inflamed WT culture compared to the stable WT culture. (B) Gene expression in the stable knockout cultures compared to the stable WT culture. (C) Gene expression in the inflamed knockout cultures compared to the inflamed WT culture. Mean ± SD of N = 3. ^#p < 0.05, compared to the stable control; *p < 0.05, compared to the WT control.

Figure 6

(A) Representative images of MUC5AC-stained epithelial layers of stable (top row) and inflamed (bottom row) triple cultures with WT, CASP1^-/- or NLRP3^-/- THP-1 cells. Blue: nuclei (Hoechst 33342), green: MUC5AC (Alexa Fluor 488). Images were acquired at 100x magnification. Scale bar (100 µm) applies to all images. (B) Quantitative analysis of MUC5AC-stained area in stable (full bars) or inflamed (open bars) triple cultures. Mean ± SD of N = 3, ^#p < 0.05 compared to the respective stable culture.

Figure 7

Cytotoxicity in Caco-2 (A) or HT29-MTX-E12 (B) monocultures after treatment with basolateral supernatants ( = 48h; relative concentration 0.25, 0.5 or 1) from stable (full bars) or inflamed (open bars) triple cultures (WT, CASP1^-/- or NLRP3^-/- THP-1 cells) for 48 (h) Cytotoxicity was assessed via LDH assay. Mean ± SD of N = 5, *p < 0.05 compared to the untreated control.

Figure 8

(A) LDH activity in the supernatants of LPS/IFN-γ-treated ileal tissue explants from WT or Nlrp3^-/- mice after 1, 3, or 6 (h) (B) Gene expression in ileal tissue explants after 6 h of treatment. Mean ± SEM of N = 4, ^#p < 0.05 compared to the untreated control of the corresponding time point.