Reduced Paneth cell function and Lgr5-expression correlate with CD-like inflammation

In TNF^ΔARE mice, a deletion in the tumour necrosis factor (TNF) AU-rich (adenosin-uracil) elements (ARE) leads to loss of translational control of TNF, resulting in a microbiota-driven transmural inflammation of the distal ileum.20 CD-like tissue inflammation gradually develops in TNF^ΔARE mice and has been linked to PC dysfunction.20 PCs contain distinct cytoplasmic granules for exocytosis of AMPs,27 and lysozyme-positive (Lyz⁺) secretory granule morphology is used as a functional marker for PC phenotype classification.13 14 We took advantage of the heterogeneity in the grade of CD-like inflammation in TNF^ΔARE mice to characterise PCs across a broad range of histopathological scores (HS). Ileal tissue sections derived from TNF^ΔARE mice with no (HS 0), moderate (HS <4) and severe (HS>4) inflammation were stained for Lyz and PCs were subdivided into a highly granular (≥2 Lyz⁺ cytoplasmic granules) and a lowly granular (<2 Lyz⁺ cytoplasmic granules and/or diffuse cytoplasmic Lyz staining) phenotype (figure 1A). Representative H&E stainings of tissue sections at different HS are given in figure 1B. Total numbers of Lyz⁺ PCs per crypt (figure 1C) and the percentage of highly granular PCs (figure 1D) inversely correlated with the grade of inflammation and signs of PC abnormalities were already visible in moderately inflamed mice (figure 1C,D). Comparing tissues from non-inflamed TNF^ΔARE mice and wild type (WT) littermates, we observed no differences in the total numbers of PCs. However, non-inflamed TNF^ΔARE mice showed a mild reduction in PC granularity (online supplementary figure 1A,B). Confirming reduced PC function in TNF^ΔARE mice, transcriptional levels of Lyz1 and PC-derived AMPs alpha-defensin 5 (Defa5) and Ang4 and ISC-niche supporting Dll4 were reduced (figure 1E). To depict the impact of ileitis-associated changes in PC function on CBC ISC marked by Lgr5 we performed in situ hybridisation for Lgr5 (figure 1F). The number of dots, representing Lgr5 transcripts, were counted (figure 1G) and crypts were classified as crypts with high Lgr5 expression (≥10 dots) and low Lgr5 expression (<10 dots), respectively. Consistent with reduced PC function, Lgr5 ⁺ ISCs were diminished under inflammatory conditions (online supplementary figure 1C) and numbers of crypts with high Lgr5 expression were inversely correlated with the degree of histopathology (figure 1H). Reduced stemness in inflamed TNF^ΔARE mice was confirmed by qPCR analysis of Lgr5 (figure 1I). No differences were observed in crypt morphology and Lgr5 expression between WT and non-inflamed TNF^ΔARE mice (online supplementary figure 1C,D). In situ hybridisation for Olfactomedin (Olfm) 4, a broader marker of active ISCs, confirmed reduced stemness under inflammatory conditions (online supplementary figure 1E-G). The parallel decrease of PC function and stemness indicates the tight functional interrelation of the ISC niche under inflammatory conditions.

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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.

Aberrant PC phenotype and LGR5-expression correlate with active ileal CD

To verify the relevance of TNF^ΔARE mice, we obtained samples from 70 patients with CD undergoing surgical resection from a previously published prospective study.28 Early postoperative lesions observed by ileocolonoscopy best predict the course of disease and ileocolonoscopy is currently the gold standard to assess the risk of clinical recurrence.29 Ileal tissue margins were collected during resection surgery and a postoperative endoscopy was performed 6–12 months later to assess the endoscopic recurrence according to the Rutgeerts score. Endoscopic recurrence was defined as a Rutgeerts score ≥i2. Classification of patients with CD according to the presence or absence of inflammation in the resected tissue samples at baseline, and classification of postoperative endoscopic recurrence, is schematically shown in figure 2A, and patients’ characteristics at the time of surgery are compiled in online supplementary table 1. Consistent with TNF^ΔARE mice, numbers of LYZ⁺ PCs and highly granular PCs (≥2 LYZ⁺ cytoplasmic granules) were significantly reduced in tissue margins classified as inflamed at time of surgery compared with those classified as non-inflamed (figure 2B,C). Interestingly, the number of LYZ⁺ cells in ‘upper crypt’ (above +6 position) significantly increased in inflamed compared with non-inflamed tissue margins, suggesting an aberrant ISC niche architecture under inflammatory conditions (figure 2B, right). Reflecting the CD-like mouse model, inflamed tissue margins displayed a reduction in crypts with high LGR5 expression (≥15 dots), and concomitant to LYZ⁺ cells above the +6 position, LGR5 expression in upper crypts was induced (figure 2D,E).

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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.

Paneth cell phenotype and LGR5-expression pattern predict recurrence in patients with CD

As previously shown,28 inflammation in resected ileal tissue at baseline was predictive for endoscopic recurrence, yet 44% of the analysed patients with CD with non-inflamed tissue margins still developed recurrent disease (figure 2A). Analysing regions of the intestine free of severe active or chronic inflammation has been proposed to more accurately reflect disease development due to the potential presence of early molecular and pathologic changes.13 Therefore, we tested in ileal margins classified as non-inflamed at the time of surgery if abnormal PC phenotypes and aberrant LGR5⁺ expression predict endoscopic recurrence after 6–12 months. Indeed, a low proportion of highly granular PCs (figure 3A,B) and high numbers of LYZ⁺ cells in upper crypts (figure 3C,D) correlated with the risk of recurrence, while the total number of LYZ⁺ PCs in crypts were not different between recurrent and non-recurrent groups (online supplementary figure 2A,B). In line, a low proportion of crypts with high LGR5 expression (figure 3E,F) and enhanced LGR5 expression in upper crypts (figure 3G,H) were further predictive for endoscopic recurrence. Assigning numbers of highly granular PCs, PCs in upper crypt, highly Lgr5 expressing crypts and Lgr5 expression in upper crypts as risk factors (0–4), we demonstrated a strong cumulative effect of the number of risk factors on the probability of disease recurrence (online supplementary figure 2C). Smoking, that had been identified previously as a risk factor for recurrence in this cohort,28 was associated with reduced PC granularity, a low proportion of crypts with high LGR5 expression and enhanced LGR5 expression in upper crypts (online supplementary figure 3A–C). Yet, within patients with CD experiencing recurrence, smoking had no additional impact on the risk factors analysed (online supplementary figure 3D,E). CD-associated risk alleles of NOD2 and ATG16L1 have been reported to impact PC phenotype;13 however, we observed no impact of the single nucleotide polymorphisms ATG16L1 rs6752107 and NOD2 rs2066845 or rs2066844 on the ISC niche appearance, probably due to a high proportion of risk allele carriers (online supplementary figure 3F-H). Contrarily, in tissue margins classified as inflamed at the time of surgery, aberrant PC phenotypes and stemness failed to better stratify disease recurrence (online supplementary figure 4). In summary, these data show a strong predictive value for alterations of the ISC niche for early endoscopic recurrence, indicating these phenotypic changes as first molecular signs of inflammatory changes, preceding macroscopic lesions.

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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.

Inflammation in TNF^ΔARE mice is associated with impaired mitochondrial function

Phenotypic changes of PCs have been described concomitantly to structurally impaired mitochondria,14 and mitochondrial stress signalling (MT-UPR) is apparent in IEC from patients with IBD and mouse models of intestinal inflammation.25 Accordingly, adenosine triphosphate (ATP) levels were reduced in isolated ileal crypts from TNF^ΔARE mice, along with increased levels of the MT-UPR marker proteins Hsp60 and (dsRNA-activated protein kinase) Pkr25 under inflammatory conditions (figure 4A–D). Concomitantly, transcriptional levels of genes involved in signalling pathways related to disturbed mitochondrial function, including MT-UPR (Trb3, Atf5, Chop), antioxidative response (Hif1a) and low ATP levels (Prkaa2, AMP-kinase) were upregulated in ileal crypts (figure 4E), while expression of Grp78, a surrogate marker of ER stress remained unaltered (figure 4F). In line, transmission electron microscopy showed markedly reduced numbers of PCs with typical morphological features at the ileal crypt bases of inflamed TNF^ΔARE mice. The few remaining Paneth-like cells often exhibited secretory granules with broadened halos, intracytoplasmic vacuolations, dilation of the rough ER, as well as degenerative mitochondrial alterations, including mitochondrial swelling with dissolution and disruption of cristae, loss of matrix density and occasionally formation of intramitochondrial electron-dense inclusions (figure 4G). Autophagy contributes to the mobilisation of cellular energy stores and alterations in autophagy-related proteins have been linked to the disruption of the PC granule exocytosis pathway and degenerating mitochondria.14 Thus staining for the autophagy marker LC3, an increase in LC3 expression in crypt bases of inflamed TNF^ΔARE mice was observed accompanying metabolic and morphologic alterations (online supplementary figure 5A,B).

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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.

Mitochondrial impairment in ISC causes transition towards dysfunctional Paneth cells

PC and Lgr5 ⁺ ISC alterations concurrent to mitochondrial impairment were present under inflammatory conditions in TNF^ΔARE mice. Hence, we characterised the role of mitochondrial function on ISC niche appearance using a mouse model in which Hsp60 can be specifically deleted in Lgr5 ⁺ ISC (Hsp60 ^flox/flox × Lgr5-eGFP-IRES-CreER^T2-Tg) via administration of tamoxifen (Hsp60^Δ/ΔISC).26 Hsp60 represents a target gene of MT-UPR signalling and constitutes the main chaperone of the mitochondrial matrix.30 Thus, loss of Hsp60 leads to disturbed proteostasis in mitochondria and subsequent activation of MT-UPR signalling.26 Consequently, Hsp60 deficiency results in mitochondrial dysfunction, including reduced mitochondrial respiration and a drop in cellular ATP content.26 Confirming MT-UPR activation on Hsp60 loss, transcription of the cochaperone and surrogate marker of MT-UPR, Hsp10 increased early after induction of Hsp60 deletion in parallel with induction of Trb3, Chop, Hif1a and Prkaa2 (figure 5A). Following Lgr5 and Lyz expression in the ileum of Hsp60^Δ/ΔISC mice for up to 6 days after the end of tamoxifen treatment, transient changes of the ISC niche were characterised. At day 2, the proportion of highly Lgr5 expressing crypts dropped (figure 5B,C). Parallel efforts to characterise PC phenotypes demonstrated that Lgr5 ^- Lyz⁺ cell numbers decreased, while numbers of Lgr5 ⁺ Lyz⁺ double positive cells increased, starting at day 0 after end of tamoxifen treatment (figure 5B–D). Along with diminished expression of markers for ISCs and PCs, PC granularity and expression of PC-derived AMPs were reduced in response to Hsp60 loss-induced mitochondrial dysfunction in ISCs (figure 5E–G). Reflecting inflamed TNF^ΔARE mice, LC3 staining in crypt bases of Hsp60^Δ/ΔISC mice depict induction of autophagy (online supplementary figure 5C) along with metabolic and morphological alterations of the ISC niche. To exclude the possibility of mitochondrial dysfunction-mediated cell death of Lgr5 ⁺ ISC and Lyz⁺ cells, tissue sections from day 2, when loss of Lgr5 expression was most pronounced, were costained for TUNEL and eGFP or Lyz, respectively. Furthermore, tissue sections were stained for the apoptosis marker cleaved caspase (CC) 3, confirming absence of enhanced apoptosis at day 2 after the end of tamoxifen treatment, but indicating increased apoptosis in crypt basis at days 4 and 6 (online supplementary figure 6). Hsp60 deficiency in Lgr5 ⁺ ISCs was associated with a complete loss of proliferation as indicated by Ki67 staining (online supplementary figure 7A). However, expression of Olfm4 and Hopx, a marker of +4 reserve stem cells potentially contributing to Lgr5⁺ ISC replenishment,31 was retained and transiently enhanced in crypts (online supplementary figure 7B,C). In line, cells above the crypt basis remained Hsp60 and Ki67 positive throughout the observed time points (online supplementary figure 7A). Crypt-based cells regained Hsp60 and Ki67 expression from day 4 on, indicating apoptotic cell death and extrusion of Hsp60 deficient cells. Lgr5 ⁺ ISC can differentiate into PCs, and vice versa, dedifferentiation of PCs and acquisition of stem-like features has been described following loss of CBC ISCs.9 10 To determine if dysfunctional PCs arise from mitochondrial function-impaired ISCs, we stained for Lyz and Hsp60. Hsp60 negative cells directly originated from Lgr5 ⁺ ISCs that had suffered from mitochondrial dysfunction. The ratio of Lyz⁺ cells in which Hsp60 could not be detected was increased at day 0 and day 2 (figure 5H,I), indicating Lgr5 ⁺, Hsp60^- ISCs to acquire Lyz expression via mechanisms initiated by mitochondrial dysfunction. Furthermore, reduced PC granularity and diffuse Lyz staining was associated with lack of Hsp60 in PCs (figure 5J). In summary, these data suggest that mitochondrial impairment, including reduced mitochondrial respiration, initiates a transition of Lgr5 ⁺ ISCs towards a PC-like phenotype. However, the inability of these Lgr5 ⁺ Lyz⁺ double positive cells to adjust their mitochondrial function to the new cell-phenotypic demand (due to lack of Hsp60), seems to hamper differentiation into functionally mature PCs (figure 5K). Interestingly, costaining of Hsp60 with Mucin 2 (Muc2), Chromogranin A (ChgA) or doublecortin-like kinase 1 (dclk1) as markers for the secretory IEC types (goblet cells, enteroendocrine cells and tuft cells, respectively) indicated that ISC suffering from mitochondrial dysfunction only acquired Lyz expression, since Muc2, ChgA and dclk1 expressing cells remained Hsp60 positive (online supplementary figure 8A–C). At day 6 after tamoxifen treatment, ISCs and PCs regained normal phenotypes comparable to Hsp60 ^flox/flox mice (figure 5B,C,E,F,H). At this time point, Hsp60 mRNA expression in IEC returned to control levels (figure 5A). The complete regain of Hsp60⁺, Lgr5 ⁺ ISCs and a normal ISC niche phenotype indicates replenishment of Lgr5 ⁺ ISCs from reserve ISC populations as described before.9 32

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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;

Crypts from inflamed TNF^ΔARE mice fail to grow into organoids

Further investigating the role of mitochondrial function on ISC niche-regulation under inflammatory conditions, primary crypts of non-inflamed and inflamed TNF^ΔARE mice were isolated and seeded in medium without Wnt factors.33 Addition of Wnt factors to the medium is not essential for small intestinal (SI) organoid culture and has been linked to the presence of PCs providing these signals naturally.34 Ileal crypts from inflamed mice displayed reduced Lgr5 and Lyz expression, confirming figure 1 (figure 6A,B). Consistently, ileal crypts from inflamed, but not non-inflamed TNF^ΔARE mice almost completely failed to grow into organoids. Remaining organoids showed a severe defect in de-novo crypt formation (budding) (figure 6C). In non-inflamed TNF^ΔARE mice and WT littermates, seeding efficiency and budding were comparable (online supplementary figure 9A), confirming inflammation and not genotype as responsible for impaired growth. Of note, crypts derived from the jejunum, directly adjacent to the inflamed ileum but showing no tissue pathology in TNF^ΔARE mice, also displayed a reduced ability to form organoids (figure 6D). Applying an intestinal organoid culture medium supplemented with Wnt factors directly after crypt isolation did not rescue growth (online supplementary figure 9B). Hence, it is likely that disturbances of both Lgr5 ⁺ ISCs and PCs contribute to the inflammation-induced growth defect.

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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.

Mitochondrial respiration is required to maintain stemness and PC functionality

The metabolism of cells constituting the ISC niche is coordinated to support each other’s demand and particularly mitochondrial oxidative phosphorylation (OXPHOS) is fine tuned to meet the requirements of different IEC subtypes.8 24 Since crypts derived from an inflammatory environment and Hsp60^Δ/ΔISC organoids both display mitochondrial dysfunction, ISC niche alterations and a growth defect (figures 4 and 6A–D, online supplementary figure 9C,D), we determined mRNA expression levels of key factors involved in OXPHOS and glycolysis in primary crypt derived from non-inflamed and inflamed TNF^ΔARE mice.

Pdha, belonging to the pyruvate dehydrogenase complex (PDC), Yy1, a transcriptions factor regulating mitochondrial complex I genes and cytochrome c oxidase (Cox) IV were consistently reduced (figure 6E). PDC acts as gatekeeper of metabolism by linking cytoplasmic glycolysis to the TCA cycle and OXPHOS, Hence, oligomycin, blocking OXPHOS and dichloroacetate (DCA), targeting PDC to shift ATP generation from glycolysis to OXPHOS35 (figure 6F), were used to characterise the role of metabolism on ISC niche function. Murine ileal WT organoids and human SI organoids were treated with sublethal doses of oligomycin (online supplementary figure 9E) or DCA. Organoids treated with oligomycin depict lower numbers of Lyz⁺ cells per crypt and reduced PC granularity (figure 6G). Concomitantly, transcriptional levels of Lgr5, Lyz and Ang4 were reduced, while Chop, Hif1a and Prkaa were upregulated (figure 6G). Noteworthy, these results were mirrored in human intestinal organoids (figure 6H). In contrast, DCA had only minimal effects on these readouts (online supplementary figure 10).

Reinforcing mitochondrial respiration restores inflammation-imprinted dysfunction of the ISC niche

Finally, we tested if the inflammation-associated growth defect in TNF^ΔARE mice-derived crypts could be reversed by DCA. Indeed, addition of DCA to the organoid culture medium directly after seeding rescued the ability of inflamed TNF^ΔARE mice-derived ileal crypts to grow into organoids and form de-novo crypts (figure 6J). Strikingly, DCA withdrawal subsequent to passaging at day 8 of culture conferred no negative effects to organoid growth compared with continued DCA treatment (figure 6K,L). Comparing DCA-exposed organoids derived from inflamed TNF^ΔARE mice and WT mice, transcriptional level of ISC and PC-associated genes largely converged (figure 6M). These data demonstrate that activation of OXPOHOS via inhibition of glycolysis is sufficient to restore inflammation-imprinted metabolic dysfunction of the ISC niche.

The persisting regain of stemness after withdrawal of DCA underlines the reversible nature of ISC alterations under inflammatory conditions, making ISC niche metabolism an attractive target for therapeutic interventions (figure 7).

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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.