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. 2024 Feb 5;81(1):77.
doi: 10.1007/s00018-024-05124-3.

Decreased expression of the NLRP6 inflammasome is associated with increased intestinal permeability and inflammation in obesity with type 2 diabetes

Gema Frühbeck  1   2   3   4 , Javier Gómez-Ambrosi  5   6   7 , Beatriz Ramírez  5   6   7 , Sara Becerril  5   6   7 , Amaia Rodríguez  5   6   7 , Amaia Mentxaka  5 , Víctor Valentí  6   7   8 , Rafael Moncada  6   7   9 , Gabriel Reina  10 , Jorge Baixauli  8 , Marcos Casado  5 , Camilo Silva  6   11 , Javier Escalada  6   11 , Victoria Catalán  12   13   14
Affiliations

Decreased expression of the NLRP6 inflammasome is associated with increased intestinal permeability and inflammation in obesity with type 2 diabetes

Gema Frühbeck et al. Cell Mol Life Sci. .

Abstract

Background: Obesity-associated dysfunctional intestinal permeability contributes to systemic chronic inflammation leading to the development of metabolic diseases. The inflammasomes constitute essential components in the regulation of intestinal homeostasis. We aimed to determine the impact of the inflammasomes in the regulation of gut barrier dysfunction and metabolic inflammation in the context of obesity and type 2 diabetes (T2D).

Methods: Blood samples obtained from 80 volunteers (n = 20 normal weight, n = 21 OB without T2D, n = 39 OB with T2D) and a subgroup of jejunum samples were used in a case-control study. Circulating levels of intestinal damage markers and expression levels of inflammasomes as well as their main effectors (IL-1β and IL-18) and key inflammation-related genes were analyzed. The impact of inflammation-related factors, different metabolites and Akkermansia muciniphila in the regulation of inflammasomes and intestinal integrity genes was evaluated. The effect of blocking NLRP6 by using siRNA in inflammation was also studied.

Results: Increased circulating levels (P < 0.01) of the intestinal damage markers endotoxin, LBP, and zonulin in patients with obesity decreased (P < 0.05) after weight loss. Patients with obesity and T2D exhibited decreased (P < 0.05) jejunum gene expression levels of NLRP6 and its main effector IL18 together with increased (P < 0.05) mRNA levels of inflammatory markers. We further showed that while NLRP6 was primarily localized in goblet cells, NLRP3 was localized in the intestinal epithelial cells. Additionally, decreased (P < 0.05) mRNA levels of Nlrp1, Nlrp3 and Nlrp6 in the small intestinal tract obtained from rats with diet-induced obesity were found. NLRP6 expression was regulated by taurine, parthenolide and A. muciniphila in the human enterocyte cell line CCL-241. Finally, a significant decrease (P < 0.01) in the expression and release of MUC2 after the knockdown of NLRP6 was observed.

Conclusions: The increased levels of intestinal damage markers together with the downregulation of NLRP6 and IL18 in the jejunum in obesity-associated T2D suggest a defective inflammasome sensing, driving to an impaired epithelial intestinal barrier that may regulate the progression of multiple obesity-associated comorbidities.

Keywords: Inflammasome; Inflammation; Intestinal integrity; Jejunum; NLRP; Obesity; Type 2 diabetes.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Effect of obesity and obesity-associated T2D on circulating levels of main markers related to intestinal dysfunction. Fasting plasma concentrations of A endotoxin, B lipopolysaccharide binding protein (LBP), C flagellin, D zonulin, E lactoferrin, F S100 calcium-binding protein A8/calprotectin A (S100A8), G interleukin-18 (IL-18), H interleukin-18 binding protein (IL-18BP), I ratio IL-18/IL-18BP, J interleukin-1β (IL-1β), K interleukin-6 (IL-6) and L C–C motif chemokine ligand 5 (CCL5/RANTES) in normal-weight (NW) volunteers (n = 17–20), patients with obesity with normoglycemia (OB-NG) (n = 21) and with obesity-associated type 2 diabetes (OB-T2D) (n = 39). M Heatmap of the associations between markers of intestinal dysfunction, gene expression levels of NLRP6 and IL18 in jejunum and anthropometric parameters, glucose profile as well as HDL-cholesterol and AST/ALT ratio. Bars represent the mean ± SEM. Differences between groups were analyzed by one-way ANOVA followed by Tukey’s tests. *P < 0.05 and **P < 0.01. ALT alanine aminotransferase, AST aspartate aminotransferase, BMI body mass index, HOMA homeostatic model assessment, NLRP nucleotide-binding oligomerization domain, leucine rich repeat and pyrin, QUICKI quantitative insulin sensitivity check index, WHtR waist-to-height ratio
Fig. 2
Fig. 2
Impact of obesity-associated T2D on jejunum expression levels of the main inflammasome components. A Bar graphs show the gene expression levels of key inflammasome components (NLRP1, NLRP3 and NLRP6) and its effectors (IL1B and IL18) in jejunum samples from patients with obesity with normoglycemia (OB-NG) (n = 5) and with obesity-associated type 2 diabetes (OB-T2D) (n = 10). B Protein expression levels of NLRP3 and NLRP6 in jejunum samples from patients with OB-NG (n = 5) and with OB-T2D (n = 10). C Representative immunostaining (n = 3) and quantification (n = 5 per group) for NLRP3 and NLRP6 in jejunum samples from patients with OB-NG and with OB-T2D [scale bar (100 × : 50 µm; 200 × : 25 µm)]. Values are the mean ± SEM. Differences between groups were analyzed by unpaired two-tailed Student’s t test. *P < 0.05. NLRP, nucleotide-binding oligomerization domain, leucine rich repeat and pyrin; IL, interleukin
Fig. 3
Fig. 3
Bar graphs show the mRNA levels of A key intestinal inflammation-related genes and B markers associated with the integrity of the epithelial intestinal barrier in jejunum samples from patients with obesity with normoglycemia (OB-NG) (n = 5) and with obesity-associated type 2 diabetes (OB-T2D) (n = 10). C Representative immunostaining (n = 3) and quantification (n = 5 per group) for CD68 in jejunum samples from patients with OB-NG and with OB-T2D [scale bar (100 × : 50 µm; 200 × : 25 µm)]. Values are the mean ± SEM. Differences between groups were analyzed by unpaired two-tailed Student’s t test. *P < 0.05. ADIPOQ adiponectin, CCL2 monocyte chemoattractant protein-1, CLDN1 claudin 1, IL interleukin, KLF4 Kruppel like factor 4, MUC2 mucin 2, NGAL lipocalin 2, NOD2 nucleotide binding oligomerization domain containing 2, OCLN occluding, S100A9 S100 calcium-binding A9, SPP1 osteopontin, STEAP4 STEAP4 metalloreductase, TLR4 toll-like receptor-4, TJP1 tight junction protein-1
Fig. 4
Fig. 4
Gene expression levels of the inflammasome components (NLRP3 and NLRP6), their main effectors (IL1B and IL18) and the intestinal integrity genes (MUC2 and TJP1) in human enterocytes CCL-241 cells treated with different concentrations of A tumor necrosis factor (TNF)-α, B interleukin (IL)-1β, C adipocyte conditioned media (ACM) and D taurine and histamine during 24 h (n = 6 per group). E Effect of lipopolysaccharide (LPS), parthenolide (PTL) and LPS for 3 h followed by PTL for another 4 h in the regulation of inflammasome components and inflammation and intestinal integrity genes in CCL-241 cells. Values are the mean ± SEM (n = 6 per group). Differences between groups were analyzed by one-way ANOVA followed by Dunnett’s tests. *P < 0.05, **P < 0.01 and ***P < 0.001 vs unstimulated cells. NLRP nucleotide-binding oligomerization domain, leucine rich repeat and pyrin, IL interleukin, MUC2 mucin 2, TJP1 tight junction protein-1
Fig. 5
Fig. 5
Effect of blocking NLRP6 expression in A the gene expression levels of interleukin-18 (IL18), mucin 2 (MUC2), tight junction protein 1 (TJP1), adiponectin (ADIPOQ) and interleulin-1β (IL1B) and B the release of mucin-2 into the culture media. CCL-241 cells were transfected with or without 200 pmol/L NLRP6 siRNA/2 × 105 cells/well for 24 h. Values are the mean ± SEM (n = 6 per group). Differences between groups were analyzed by unpaired Student’s t-test. *P < 0.05 and **P < 0.01
Fig. 6
Fig. 6
Gene expression levels of NLRP3, NLRP6 and MUC2 in A CCL-241 cells and C visceral adipocytes after the incubation with heat-inactivated Akkermansia muciniphila and with the bacteria-conditioned medium (BCM) (40%) for 24 h. Mucin-2 concentrations in the culture media of B CCL-241 cells and D visceral adipocytes incubated in the presence of heat-inactivated A. muciniphila and with the bacteria-conditioned medium (BCM) (40%) for 24 h. E Gene expression levels of CLDN1 and OCLN in CCL-241 cells after the incubation with heat-inactivated Akkermansia muciniphila and with the bacteria-conditioned medium (BCM) (40%) for 24 h. Values are the mean ± SEM (n = 6 per group). Differences between groups were analyzed by one-way ANOVA followed by Tukey’s tests. *P < 0.05, **P < 0.01 and ***P < 0.001. BHI brain heart infusion, CLDN1 claudin 1, MUC2 mucin 2, NLRP nucleotide-binding oligomerization domain, leucine rich repeat and pyrin, OCLN occludin
Fig. 7
Fig. 7
The downregulated expression of NLRP6 and IL18 and increased levels of pro-inflammatory factors (TLR4, CCL2, SPP, and CD68) in the jejunum from patients with obesity-associated T2D suggest a defective inflammasome sensing, driving to an impaired epithelial intestinal barrier, also evidenced by the increased circulating levels of intestinal damage markers (endotoxin, LBP, zonulin, lactoferrin, S100A8) and inflammatory factors (IL-6, CCL5) favoring the development of multiple obesity-associated comorbidities. In addition the secretome of adipocytes from patients with obesity-associated T2D decreased the expression of MUC2 and TJP1 in intestinal cells, strengthening the role of adipocytes in the maintenance of the intestinal barrier homeostasis and the crosstalk between these types of cells. CCL2, monocyte chemoattractant protein-1; CCL5, C–C motif chemokine ligand 5; NLRP nucleotide-binding oligomerization domain, leucine rich repeat and pyrin, IL interleukin, LBP lipopolysaccharide binding protein, MUC2 mucin 2, S100A8 S100 calcium-binding A8, SPP1 osteopontin, TJP1 tight junction protein-1, TLR4 toll-like receptor-4
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