Mitochondrial impairment drives intestinal stem cell transition into dysfunctional Paneth cells predicting Crohn's disease recurrence

Abstract

Objective: Reduced Paneth cell (PC) numbers are observed in inflammatory bowel diseases and impaired PC function contributes to the ileal pathogenesis of Crohn's disease (CD). PCs reside in proximity to Lgr5⁺ intestinal stem cells (ISC) and mitochondria are critical for ISC-renewal and differentiation. Here, we characterise ISC and PC appearance under inflammatory conditions and describe the role of mitochondrial function for ISC niche-maintenance.

Design: Ileal tissue samples from patients with CD, mouse models for mitochondrial dysfunction (Hsp60^Δ/ΔISC) and CD-like ileitis (TNF^ΔARE), and intestinal organoids were used to characterise PCs and ISCs in relation to mitochondrial function.

Results: In patients with CD and TNF^ΔARE mice, inflammation correlated with reduced numbers of Lysozyme-positive granules in PCs and decreased Lgr5 expression in crypt regions. Disease-associated changes in PC and ISC appearance persisted in non-inflamed tissue regions of patients with CD and predicted the risk of disease recurrence after surgical resection. ISC-specific deletion of Hsp60 and inhibition of mitochondrial respiration linked mitochondrial function to the aberrant PC phenotype. Consistent with reduced stemness in vivo, crypts from inflamed TNF^ΔARE mice fail to grow into organoids ex vivo. Dichloroacetate-mediated inhibition of glycolysis, forcing cells to shift to mitochondrial respiration, improved ISC niche function and rescued the ability of TNF^ΔARE mice-derived crypts to form organoids.

Conclusion: We provide evidence that inflammation-associated mitochondrial dysfunction in the intestinal epithelium triggers a metabolic imbalance, causing reduced stemness and acquisition of a dysfunctional PC phenotype. Blocking glycolysis might be a novel drug target to antagonise PC dysfunction in the pathogenesis of CD.

Keywords: Crohn's disease; energy metabolism; inflammatory bowel disease; intestinal epithelium; intestinal stem cell.

Figure 1

Paneth cell dysfunction and reduced Lgr5-expression correlate with CD-like inflammation in TNF^ΔARE mice Ileal tissue sections from TNF^ΔARE mice with different levels of inflammation and IEC isolates derived from TNF^ΔARE mice and WT littermates were analysed. (A) Immunofluorescence (IF) costaining of Lysozyme (red) and E-cadherin (IEC borders, grey) counterstained with Dapi (nuclei, blue), lower panel: higher magnification of the indicated sections. Numbers above the pictures indicate the HS of the respective tissue section. (B) Representative H&E images for HS 0 (non-inflamed), 2 (moderate inflammation), 4 (severe inflammation). (C) Correlation analysis (Pearson) of the number of Lysozyme positive (Lyz⁺) cells and HS and (D) proportions of highly granular Lyz⁺ cells (≥2 granules) and HS. Right: representative Lyz staining depicting Lyz⁺ cells with low and high granularity, respectively. (E) qRT-PCR analysis of IECs for genes involved in PC function (n=5) (F) Representative pictures of Lgr5 in situ hybridisation, including magnifications; numbers indicate the respective HS. (G) Illustration of Lgr5 transcript quantification; each dot, indicated by a black arrow represents one Lgr5 transcript. (H) Correlation analysis (Pearson) of the proportion of crypts with high Lgr5 expression (≥10 transcripts) and HS. (I) qRT-PCR analysis of IECs for Lgr5 (n=5). Statistics were performed by unpaired t-test. Bars represent mean+SEM. Asterisks indicate significant differences *p<0.05, **p<0.01, ***p<0.001. ARE, AU-rich (adenosin-uracil) elements; CD, Crohn’s disease; HS, histopathological score; IEC, intestinal epithelial cell; PC, Paneth cell; TNF, tumour necrosis factor; WT, wild type.

Figure 2

Aberrant Paneth cell phenotype and LGR5-expression correlate with disease activity in ileal tissue margins of patients with CD Ileal tissue sections of patients with CD undergoing resection surgery were analysed and tissue margins classified as non-inflamed and inflamed, respectively, at time of surgery were compared. (A) Overview of CD patient numbers for baseline and endoscopic follow-up disease classification. (B) Quantification of the total number of LYZ⁺ cells, highly granular LYZ⁺ cells (≥2 granules) and number of LYZ⁺ cells in upper crypt based on LYZ IF staining. (C) Representative pictures of IF costaining of Lysozyme (red) and E-cadherin (IEC borders, grey) counterstained with Dapi (nuclei, blue), right panel: higher magnification of the indicated sections. (D) Representative pictures of LGR5 in situ hybridisation, including magnifications. (E) Quantification of LGR5 in situ hybridisation giving the proportion of crypts with high LGR5 expression (≥15 LGR5 transcripts) and of crypts with LGR5 expression in upper crypt. Statistics were performed by unpaired t-test. Bars represent mean+SEM. Asterisks indicate significant differences *p<0.05, **p<0.01, ***p<0.001. CD, Crohn’s disease; IEC, intestinal epithelial cell; LYZ⁺, Lysozyme positive.

Figure 3

PC phenotype and LGR5-expression predict disease recurrence in patients with non-inflamed CD Ileal tissue sections classified as non-inflamed at time of surgery of patients with CD undergoing resection surgery were analysed. Tissue sections were stained for LYZ by IF and for LGR5 by in situ hybridisation, respectively, and expression patterns were quantified. Numbers of highly granular LYZ⁺ cells (≥2 granules), numbers of LYZ⁺ cells in upper crypt, proportion of crypts with high LGR5 expression (≥15 LGR5 transcripts) and proportion of crypts with LGR5 expression in upper crypt were determined. Patients with CD with endoscopic recurrence (Rutgeerts score ≥i2) 6–12 months after surgery were compared with patients with CD not experiencing recurrence. (A, C, E, G) Overall comparison of recurrent versus non-recurrent patients for the respective marker; (B, D, F, H) From left to right: distribution of the respective marker among patients with recurrent (red circles) and non-recurrent (black circles) CD with median indicated; probability of patients with CD to experience recurrence if above or below the median for the respective marker; representative pictures showing sections from patients with CD above or below median. (A, B) Number of highly granular LYZ⁺ cells (≥2 granules), (C, D) Number of LYZ⁺ cells in upper crypt, (B, D) IF costainings of Lysozyme (red) and E-cadherin (IEC borders, grey) counterstained with Dapi (nuclei, blue). (E, F) Proportion of crypts with high LGR5 expression (≥15 LGR5 transcripts), (G, H) proportion of crypts with LGR5 expression in upper crypt, (F, H) LGR5 in situ hybridisation. (A, C, E, G) Statistics were performed by unpaired t-test. Bars represent mean+SEM. Asterisks indicate significant differences *p<0.05, **p<0.01, ***p<0.001. (B, D, F, H) Statistical analysis was performed via χ² test. CD, Crohn’s disease; IEC, intestinal epithelial cell; LYZ⁺, Lysozyme positive; PC, Paneth cell.

Figure 4

Inflammation in TNF^ΔARE mice is associated with mitochondrial dysfunction in ileal crypts Isolated ileal crypts and tissue sections from TNF^ΔARE mice and WT littermates were analysed. (A) ATP content of primary isolated crypts relative to life cell protease activity measured by a fluorescence assay (n=10/7). (B) Overview of crypt structure and illustration of the area used for protein quantification. (C) IF images of Hsp60 (green) counterstained with Dapi (nuclei, blue), including magnifications. Numbers above the pictures indicate the HS of the respective tissue section. Right: corresponding quantification. (D) Costaining of Pkr (green) and E-cadherin (IEC borders, grey) counterstained with Dapi (nuclei, blue), including magnifications. Numbers above the pictures indicate the HS of the respective tissue section. Right: corresponding quantification. (E) qRT-PCR analysis of primary ileal crypts for genes involved in mitochondrial MT-UPR, mitochondrial signalling and (F) ER stress (n=6). Statistics were performed by unpaired t-test. Bars represent mean+SEM. Asterisks indicate significant differences *p<0.05, **p<0.01, ***p<0.001. (G) Transmission electron microscopy of ileal crypt bases. Panel A: WT; A1-2: PCs display abundant, apical, electron-dense SGs with narrow halos (arrow). Asterisks mark secretory granules with small electron dense cores and wide rims of flocculent material of low electron-density. A-3. Unaltered appearance of rER and mitochondria (M) in a WT-PLC. B: non-inflamed TNF^ΔARE mice; B-1–6: PC ultrastructure essentially resembles WT mice. N: nucleus. C: inflamed TNF^ΔARE mice. C-1–2: Few remaining cells with PLC-typical location and morphology often show vacuolation (V) and broadened halos of secretory granules. IC: infiltrating inflammatory cell C-4–6: Mitochondrial lesions in PLC include intramitochondrial inclusions (C-4, C-5), mitochondrial swelling with dissolution and disruption of cristae, and loss of matrix density and also distension of the rER (C-6). ARE, AU-rich (adenosin-uracil) elements; CD, Crohn’s disease; HS, histopathological score; IEC, intestinal epithelial cell; PC, Paneth cell; rER, rough endoplasmic reticulum; SG, secretory granule; TNF, tumour necrosis factor; WT, wild type.

Figure 5

Mitochondrial impairment in ISC causes transition towards dysfunctional Paneth cells Ileal tissue sections and IEC isolates of Hsp60^flox/flox mice and Hsp60^Δ/ΔISC mice were analysed at different time points after end of tamoxifen treatment. (A) qRT-PCR analysis of IECs for Hsp60 and genes involved in mitochondrial MT-UPR and mitochondrial signalling at day 0. (B) Lgr5 in situ hybridisation (brown) and lysozyme (Lyz, turquoise) immunehistochemistry (IHC) costaining, lower panel: magnifications, dotted lines indicate crypt and cell borders, arrow-heads indicate Lyz⁺ cells, arrows indicate Lgr5—Lyz double-positive cells. (C) Quantification of the proportion of crypts with high Lgr5 expression (≥10 Lgr5 transcripts), the proportion of Lgr5—Lyz double-positive cells, and the proportion of Lgr5 negative, Lyz single-positive cells over time. (D) Representative pictures of Lgr5—Lyz double-positive cells, and Lgr5 negative, Lyz single-positive cells; dotted lines indicate crypt and cell borders, arrows indicate Lgr5—Lyz double-positive cells, arrow-heads indicate Lgr5 negative, Lyz single-positive cells. (E) IF costaining of Lyz (red) and E-cadherin (IEC borders, grey) counterstained with Dapi (nuclei, blue) showing granular and non-granular staining pattern. (F) Quantification of the proportion of highly granular Lyz⁺ cells (≥2 granules). (G) qRT-PCR analysis of IECs from day 0 for PC-derived AMP and Dll4. (H) Quantification of the proportion of Hsp60 negative, Lyz single positive cells. (I) Representative pictures of IHC costaining for Hsp60 (brown) and Lyz (turquoise) used for quantification; dotted lines indicate crypt and cell borders, arrow indicates Hsp60—Lyz double positive cells, arrow-heads indicate Hsp60 negative, Lyz single-positive cells. (J) IF costaining of Hsp60 (green), Lyz (red), and E-cadherin (IEC borders, grey) counterstained with Dapi (nuclei, blue), including magnifications. Asterisks indicate Hsp60—Lyz double positive, highly granular Paneth cells, crosses indicate Hsp60 negative, Lyz single-positive cells, depicting a diffuse Lyz staining. (K) Schematic representation of the main findings in this figure. Statistical analyses were performed by one-way ANOVA followed by Tukey test or unpaired t-test. Bars represent mean+SEM. Asterisks indicate significant differences *p<0.05, **p<0.01, ***p<0.001. AMP, antimicrobial peptides; ANOVA, analysis of variance; IEC, intestinal epithelial cell; MT-UPR, mitochondrial unfolded protein response;

Figure 6

Mitochondrial respiration determines stemness and PC functionality under inflammatory conditions (A–E) Ileal tissues and crypts derived from non-inflamed and inflamed TNF^ΔARE mice were analysed. (A) Representative pictures of ileal sections; top: H&E staining with histopathological score indicated; bottom: IF costaining of Lyz (red) and E-cadherin (grey) counterstained with Dapi (nuclei, blue) showing absence of PCs in inflamed tissue. (B) qRT-PCR analysis of primary ileal crypts for Lgr5 and Lyz (n=6). (C, D) Characterisation of intestinal organoids derived from ileal (C) and jejunal (D) crypts at day 5 of ex vivo culture. Left: Proportion of living organoids; middle: quantification of de novo crypt formation (budding); right: representative bright field pictures. (E) qRT-PCR analysis of primary ileal crypts for metabolism-determining genes (n=6). (F) Schematic representation of oligomycin and DCA targets. (G, H) Ileal organoids derived from WT mice were treated with oligomycin for 24 hours. (G) Left: bright field and IF costaining of Lyz (red) and E-cadherin (IEC borders, grey) counterstained with Dapi (nuclei, blue). Dotted lines indicate cell borders of Lyz⁺ cells; asterisks indicate Lyz⁺ cells magnified in the pictures on the right side. Right: quantification of Lyz⁺ cell numbers per crypt (upper graph) and the proportion of highly granular (≥2 granules) Lyz⁺ cells (lower graph). (H) qRT-PCR analysis of intestinal organoids for Lgr5 and Paneth cell function-associated genes (upper panel) and for genes associated with mitochondrial signalling (lower panel, n=6). (I) Same analysis as in (H) for human organoids derived from the small intestine and treated with oligomycin for 24 hours (n=6). (J–L) Ileal organoids derived from inflamed TNF^ΔARE mice were analysed (n=6). (J) From left to right: experimental scheme (D0=isolation of primary crypts and start of culture), non-treated and DCA-treated organoids were compared; proportion of living organoids at day 3 and day 8 of culture; quantification of de novo crypt formation at day 3 and day 8. (K) From left to right: experimental scheme, DCA-treated organoid cultures were passaged at day 8, and subsequently cultured in control (-DCA) and DCA-containing medium, respectively, until day 14; proportion of living organoids at day 10 and day 14 of culture; quantification of de novo crypt formation at day 14. (L) Representative bright field pictures of organoids at day 8 (before passaging) and at day 14 of culture. (M) WT and inflamed TNF^ΔARE mice-derived ileal organoids were treated with DCA for 14 days. qRT-PCR analysis for Lgr5 and Paneth cell function-associated genes. Statistics were performed by unpaired t-test when comparing two groups and by one-way ANOVA followed by Tukey test for three-group comparisons, respectively. Statistics for budding analyses were performed by Kruskal–Wallis test on ranks. Bars represent mean+SEM. Asterisks indicate significant differences *p<0.05, **p<0.01, ***p<0.001. ANOVA, analysis of variance; ARE, AU-rich (adenosin-uracil) elements; DCA, dichloroacetate; Lyz⁺, Lyz positive; OXPHOS, oxidative phosphorylation; WT, wild type.

Figure 7

Mitochondrial impairment drives ISC transition towards dysfunctional PCs predicting Crohn’s disease recurrence Schematic representation of the main findings of this work. Targeted disruption of mitochondrial function in ISCs leads to transition of ISCs into dysfunctional PCs. Under inflammatory conditions, mitochondrial impairment in the ISC niche results in ISC exhaustion and generation of dysfunctional PCs characterised by loss of Lyz positive granules, concomitant to aberrant Lgr5 and Lyz expression in upper crypts. These alterations precede tissue pathology and serve as predictive markers for early endoscopic recurrence in CD. Ex vivo, the inhibition of mitochondrial respiration (OXPHOS) in intestinal organoids reflects the impact of an inflammatory environment on the ISC niche, whereas reinforcement of OXPHOS by inhibition of glycolysis is able to override inflammation-imprinted changes of the ISC niche. ARE, AU-rich (adenosin-uracil) elements; CD, Crohn’s disease; ISC, intestinal stem cell; Lyz, Lysozyme; OXPHOS, oxidative phosphorylation; PC, Paneth cell; TNF, tumour necrosis factor.