Advanced oxidation protein products induce Paneth cells defects by endoplasmic reticulum stress in Crohn's disease

Summary Paneth cells (PC) play a key role in the innate immune response of intestine epithelium, and PC defects contribute to the pathogenesis of Crohn’s disease (CD). In this study, we utilized active CD tissues and advanced oxidation protein products (AOPP)-challenged C57BL/6 mouse model to investigate the effect of AOPP on PC defects in CD. We found that AOPP accumulated in active CD tissues and was negatively associated with lysozyme expression, while positively correlated with the presence of ER stress markers. Furthermore, AOPP treatment induced PC defects mainly through excessive ER stress in vivo, and AOPP also caused mitochondria-associated ER membranes formation and mitochondrial dysfunction. In addition, the effects of AOPP could be attenuated by the administration of ER stress inhibitor, TUDCA. These findings suggest a pathogenic role of AOPP contributing to PC defects and may provide the basis for developing new strategies to managing CD.

In active CD tissue, AOPP accumulation associated with PC defects and ER stress AOPP challenge resulted in PC defects and ER stress in mice TUDCA, an ER stress inhibitor, effectively mitigated AOPPmediated PC defects TUDCA alleviated AOPPinduced MAM formation and mitochondrial dysfunction INTRODUCTION Crohn's disease (CD) is a multifactorial chronic relapsing inflammatory disease of the gastrointestinal tract, exhibiting loss of intestinal epithelial cell (IEC) barrier integrity and dysregulated immune cell responses. 1 Paneth cells (PC), a kind of special epithelial cells located at the base of crypts, play an important role in regulating intestinal homeostasis by interceding host-microbial relations. PC routinely secrets antimicrobial peptides (lysozyme, cryptdins, and phospholipase A) to protect the host from intestinal pathogenic bacteria and shape the composition of the microflora, which colonizes the small intestine. 2 PC also functions in maintaining the regeneration of the small intestinal epithelium. 3 PC defects, represented by the reduced number of PC and impaired production of antimicrobial peptides, contribute to microbiota changes and poor clinical outcomes. [4][5][6] It is regarded that PC defects is a probable hallmark of intestinal inflammation, thus PC-targeted therapeutics may provide a novel strategy for CD. 7 However, the pathogenic factors and underlying mechanisms driving PC defects remain poorly understood.
It is indicated that endoplasmic reticulum (ER) stress and its associated pathological alternation could play a role in PC defects. 8 ER stress arises from the insufficiency of ER to deal with protein folding. 9 To secrete antimicrobial peptides routinely, ER of PC must deal with a very high amount of protein folding, providing more susceptibility to excessive ER stress. 10,11 In cases where ER stress cannot be reversed, as intimate organelles of ER, mitochondria-associated ER membranes (MAM) and mitochondria would cooperate to lead to cell death. 10 In specimens and plasma with active CD, our previous studies confirmed that there was a marked increase of advanced oxidation protein products (AOPP) and further manifested that AOPP could function in the pathogenesis and progression of CD. [12][13][14][15] Furthermore, Luceri et al. also demonstrated that AOPP may represent a pathogenic factor and a potential therapeutic target of CD. 16 While AOPP can induce IEC and crypt cell death, it remains unclear whether these protein products can cause PC defects and if the underlying mechanism is ER stress-dependent. 14 Here, we showed that AOPP-induced PC defects in vivo via ER stress could be ameliorated by tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor. These results together indicated that AOPP was involved in triggering PC defects and disrupting intestinal homeostasis during CD.

Analysis of AOPP expression associated with PC defects in active CD tissue
To investigate the potential role of AOPP in PC defects, AOPP levels were evaluated in biopsy CD tissues. A total of seven CD patients and five healthy subjects were enrolled in this study (Tables 1 and 2).Lysozyme (Lyz) expression was used to assess PC function and identify PC areas in intestinal tissues. 6,8,17 Immunohistochemistry (IHC) staining indicated that Lyz was predominantly located in the intestinal crypts in which PC were located. IHC analysis found that Lyz expression was 2.63-fold lower in CD lesions than in normal tissues (p < 0.01; Figure 1A). Immunofluorescence (IF) revealed that the expression of Lyz was weaker and abnormally distributed in CD tissues, including lower expression in normal secretory granules and diffuse staining in the PC cytoplasm ( Figure 2A).
In accordance with our previous study, 14 AOPP was mainly detected in inflammatory cells in the lamina propria layer and in the cytoplasm of intestinal crypt cells, where Lyz is also distributed. AOPP immunostaining was nearly 3-fold higher in CD versus healthy intestinal tissue (p < 0.01; Figure 1B). Furthermore, Pearson correlation and linear regression analyses revealed that AOPP accumulation was negatively correlated with Lyz expression in CD tissues (R = À0.869, p = 0.011; Figure 1E). In contrast, AOPP accumulation was not significantly correlated with Lyz expression in healthy controls ( Figure S1A). These results suggest that AOPP deposition in intestinal tissues may be involved in the development of PC defects.

AOPP accumulation correlated with the expression of ER stress markers in active CD tissue
Excessive ER stress contributes to the deterioration of cell function and eventual cell death. This study assessed the presence of ER stress-associated protein markers in the PCs of active CD tissues. ER stress-associated proteins including glucose-regulated protein 78 (GRP78), CAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP), and inositol-requiring enzyme 1a (IRE1a) were detected by IHC or immunofluorescence. Positive immunostaining for GRP78 and CHOP was observed in PC, and the staining intensity was higher in CD lesions than in normal tissues (p < 0.01) ( Figures 1C and 1D). The intestinal crypts in active CD tissues also exhibited increased IRE1a staining ( Figure 2B).
In our previous study, AOPP deposition correlated with intestinal damage and the epithelial-mesenchymal transition (EMT) in CD, suggesting that AOPP may correlate with the presence of ER stress in active CD iScience Article crypt epithelial cells. 15 Statistical analysis was performed to confirm the relationship between AOPP and ER stress markers and the results suggested that AOPP accumulation was positively correlated with GRP78 and CHOP expression in CD intestinal crypts (R = 0.793, p = 0.033; Figure 1F and R = 0.828, p = 0.022; Figure 1G). Meanwhile, AOPP accumulation was not significantly correlated with GRP78 and CHOP expression in healthy controls (Figures S1B and S1C). These findings suggest that there is a correlation between AOPP and ER stress and that the accumulation of AOPP may have functional significance in ER stress-induced PC defects.

Effect of AOPP challenge on PC defects and ER stress in mice
To investigate whether AOPP affected PC defects by inducing ER stress in vivo, normal male C57BL/6 mice were treated with a daily intraperitoneal injection of AOPP for 28 days. The intestinal histopathology and expression of PC and ER stress markers were assessed.
The AOPP-treated mice showed signs of intestinal inflammation and injury. HE staining revealed that chronic AOPP treatment resulted in a reduction in PC (p < 0.01; Figure 3A). IHC analysis showed that Lyz was widely and strongly expressed in intestinal crypts in vehicle (PBS)-treated mice but had reduced expression in AOPP-treated mice at 28 days (p < 0.01; Figure 3B). AOPP treatment also significantly reduced Lyz protein and mRNA expression in the isolated intestinal crypts of mice (p < 0.01; Figures 3C and 3D) and decreased mRNA expression of crpytdin (also called defensin), an antimicrobial protein produced by PC, suggesting loss of PC function (p < 0.01; Figure 3E). 6,8,17 AOPP treatment significantly augmented expression of the ER stress markers, GRP78 and CHOP, in the intestinal crypts of mice (p < 0.01; Figure 4A and 4B

TUDCA effectively mitigated AOPP-mediated PC defects
To confirm the role of ER stress in AOPP-induced PC defects, mice were pretreated with TUDCA, an inhibitor of ER stress, prior to AOPP treatment. TUDCA treatment reversed AOPP-induced reduction in PC numbers in intestinal crypts (p < 0.01; Figure 5A) and increased Lyz expression by PC (p < 0.01; Figure 5B). TUDCA also restored the AOPP-induced reduction of lysozyme and cryptdins in the isolated intestinal crypts (p < 0.05; Figures 5C-5E). To determine the effect of TUDCA on ER stress, the expression of ER stress markers was assessed after TUDCA treatment, and the levels of GRP78, CHOP, and IREa were shown to be significantly reduced (p < 0.05; Figures 6A-6E) These findings suggested that AOPP-induced PC defects are associated with an increase in ER stress.

TUDCA alleviated AOPP-induced MAM formation and mitochondrial dysfunction
Evidence suggests that excessive ER stress can increase cell death by altering MAM and causing mitochondrial dysfunction. To assess this, the morphology of MAM and mitochondria were visualized in the intestinal crypts of control and AOPP-treated mice using transmission electron microscopy. AOPP induced obvious nuclear pyknosis, with increased MAM formation and swollen mitochondria in the intestinal crypts (p < 0.05; Figure 7A). The increased formation of MAM was also demonstrated by Western blotting (p < 0.05;  ATP production was lower in isolated mitochondria from the intestinal crypts of AOPP-treated mice (p < 0.01; Figure 8A). Mitochondrial Ca 2+ levels and the opening of MPTP in intestinal crypts were also investigated. By measuring expression of the mitochondrial Ca 2+ indicator, Rhod-2, quantitative fluorescence analysis revealed a higher mitochondrial Ca 2+ concentration in the isolated intestinal crypts of AOPP-treated mice than in control mice (p < 0.01; Figure 8B). Flow cytometry showed that the induction of aberrant MPTP opening was also significantly enhanced in AOPP-treated mice (p < 0.01; Figure 8C).

DISCUSSION
CD is characterized by excessive damage to the intestinal barrier and abnormal immune responses resulting from commensal bacteria in genetically predisposed individuals. 18 PC defects contribute to microbiome changes and poor clinical outcomes. In our previous study, aberrant deposition of AOPP in the iScience Article plasma and intestine was shown to contribute to the pathogenesis and development of CD. These findings suggested that AOPP may be a pathogenic factor and thus, a potential target for CD treatment. 16 However, the effect of AOPP on PC defects as well as the underlying mechanisms of this relationship has not yet been clarified. The current study found that AOPP and excessive ER stress were associated with PC defects in CD intestinal crypts. AOPP also triggered PC defects in vivo which could be ameliorated by TUDCA, an ER stress inhibitor. TUDCA was able to ameliorate AOPP-induced MAM formation and mitochondrial dysfunction. To our knowledge, this is the first study to identify the effects of AOPP on PC, revealing another role for AOPP in the pathogenesis of CD.
PC defects are considered the central element of CD pathogenesis, which causes dysfunction of the microbiome, intestinal epithelial barrier, and immune response. 19,20 Our prior studies illustrated that AOPP, a proinflammatory mediator and a novel biomarker of oxidative damage, could induce G1 phase arrest, EMT, and IEC death, thus contributing to CD development and pathogenesis. [13][14][15] The current study showed that AOPP is predominantly colocalized with PC in CD intestinal crypts, and its accumulation is negatively correlated with the expression of Lyz, a secretory granule produced by PC. Thus, it is probable that AOPP may participate in CD development by inducing PC defects. The remarkable secretory activity of PC requires a strong and stable ER to handle large amounts of protein folding. 21 ER stress is an adaptive reaction that reduces the unfolded protein load to maintain cell viability and function. When this process cannot be reversed, cellular functions deteriorate, often leading to cell death. The current study study found that the deposition of AOPP by PC caused excessive ER stress in active CD tissues. iScience Article Identifying the underlying mechanism for AOPP-induced PC defects in CD is required to inform the development of novel methods to treat CD. PC are vulnerable to excessive ER stress. Indeed, ischemia/reperfusion damage and alcohol are also shown to trigger PC defects through ER stress. 22 AOPP induces cell death through oxidative stress-related signaling and can mediate apoptosis and EMT transition through the reactive oxygen species (ROS)-induced ER stress pathway. [23][24][25][26][27] The current study found that AOPP was capable of inducing excessive ROS production, which in turn triggered apoptosis, EMT, and G1 phase arrest in IEC. In addition, the chronic administration of AOPP-induced PC defects in a mouse model by reducing the number of PC, impairing the production of antimicrobial mRNA, including Lyz and crpytidins, by PC, and augmenting the mRNA and protein expression of the ER stress markers, GRP78, and CHOP, in the intestinal crypts. These results together suggested that chronic AOPP challenges could trigger PC defects and ER stress in mice. As a iScience Article recognized ER stress inhibitor, TUDCA has been effectively used to treat cholestatic liver disease. 28,29 TUDCA alleviates intestinal inflammation by suppressing ER stress and was used in a translational phase I study to reduce the symptoms of active ulcerative colitis. 30 The current study found that TUDCA could effectively reverse AOPP-induced PC defects. TUDCA likely functions by directly inhibiting ER stress.
The maintenance of cellular homeostasis involves the participation of multiple organelles. Increasing evidence suggests that the ER, MAM, and mitochondria can cooperate to cause cellular defects in response to various pathologic stimuli. 22,31,32 ER stress is shown to induce MAM formation which leads to the release of a high level of Ca 2+ into mitochondria, the aberrant opening of MPTP, and reduced ATP production, eventually causing mitochondrial dysfunction and cellular defects. 33,34 The current study found that iScience Article AOPP-induced MAM formation, high Ca 2+ release, an MPTP decrease, and mitochondrial dysfunction.
These findings indicate that AOPP may induce PC defects by promoting an ER stress-MAM-mitochondrial dysfunction axis. TUDCA was shown to inhibit ER stress and prevent mitochondrial dysfunction to protect PC from the effects of AOPP exposure.
In summary, AOPP may cause PC defects by inducing an ER stress-MAM-mitochondrial dysfunction axis. Targeting AOPP-mediated ER stress-PC defects may thus be a novel approach for treating CD ileitis.

Limitations of the study
This study provides evidence that AOPP exposure induces PC defects, which may be mediated by ER stress activation. AOPP also caused mitochondria-associated ER membrane formation and mitochondrial dysfunction, all of which were attenuated by administration of the ER stress inhibitor, TUDCA. Considering the multifaceted effects of TUDCA, further study is necessary to elucidate the specific role of ER stress in AOPP-induced PC defects and to comprehensively understand the mechanisms underlying the interaction between TUDCA, ER stress, and PC function.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

Materials availability
This study did not generate new unique reagents.
Data and code availability d Data reported in this paper will be shared by the lead contact upon request.
d This paper does not report original code.
d Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Clinical samples
Intestinal tissues were collected from 7 patients clinically diagnosed with CD and 5 healthy volunteers, who underwent endoscopy at Nanfang Hospital of Southern Medical University (Guangzhou, China) in 2021.
The clinical characteristics of patients with CD are summarized in Table 1 35 Briefly, 1.0 3 10 9 /mL isolated crypts were washed twice with cold PBS in a 50 mL polypropylene tube and centrifuged at 600g for 5 min at 4 C. The supernatant was discarded. The pellet was resuspended in 20 mL of cold IBcells-1 (225 mM mannitol, 75 mM sucrose, 0.1 mM EGTA and 30 mM Tris-HCl pH 7.4). and then was disrupted by dounce homogenization. The homogenate was spun at 600g for 5 min at 4 C; the supernatant was collected and further centrifuged at 7,000g for 10 min at 4 C. The pellet containing mitochondria was gently resuspended in 20 mL of cold IBcells-2 (225 mM mannitol, 75 mM sucrose and 30 mM Tris-HCl pH 7.4) and purified further by centrifuging twice at 7,000g and 10,000g for 10 min at 4 C respectively. The obtained crude mitochondrial pellet was resuspended gently in 2 mL of cold MRB buffer (250 mM mannitol, 5 mM HEPES (pH 7.4) and 0.5 mM EGTA) and purified by centrifugation at 95,000g for 30 min on a Percoll medium (Sigma-Aldrich). MAM was identified as the diffused white band located above the mitochondrial fraction on the Percoll medium and obtained by differential centrifugation as described. Protein concentration was measured using the BCA Protein Quantitation Kit (Shenergy Biocolor) and 25 mg of protein was analyzed by Western blotting as indicated above.

Transmission electron microscopy
The intestinal tissues of mice were fixed with 2.5% glutaraldehyde for 1 h at room temperature and 3 h at 4 C, then replaced the glutaraldehyde with PBS. After dehydration in a graded series of alcohol (15 min each; 50%, 70%, 80%, 90%, 95%, 100% 3 2), the tissues were embedded in Resin mixture and generated 70 nm ultrathin sections using a glass knife on a Leica Ultracut R cutter. The sections were stained with uranyl acetate and lead citrate for 15 min each，and examined in a Hitachi HT-7700 transmission electron microscopy (TEM; Hitachi High-Technologies Europe GmbH). The ER mitochondrial contacts were quantified as described previously. 36 Briefly, the images were analyzed using ImageJ. The mitochondrial and ER membranes were delineated using the freehand tool. The selected areas were converted to masks and the total number, the perimeter of ER and mitochondria were calculated. Two independent investigators quantified the images blindly. For the acquisition of MAM quantification, we normalized the total ER connected to mitochondria to total mitochondrial perimeter.

Mitochondria fractionation from mice intestinal crypts
Mitochondrial fractions of intestinal crypts from mice were prepared using the Cell Mitochondria Isolation Kit (Beyotime Institute of Biotechnology) by the manufacturer's protocol. Briefly, intestinal crypts were collected and washed twice with cold PBS and resuspended with Lysis buffer. After vortex and incubation on ice for 15 min, the pellets were homogenized for 25 St and the homogenates were centrifuged at 650 g for 10 min at 4 C. The supernatants were centrifuged at 11,000g for 15 min at 4 C. The resulting pellets were mitochondria fractionations.

Detection of adenosine triphosphate (ATP) levels
ATP levels in mitochondria of intestinal crypts from mice were measured using an ATP Assay Kit (Beyotime, Shanghai, China) according to the manufacturer's protocols. The isolated mitochondria were lysed and centrifuged at 12,000g for 5 min at 4 C. After mixing the supernatants (100 mL) with ATP detection working iScience Article solution (100 mL), the luminescence produced was measured with a luminometer counter (Molecular Devices, Shanghai, China), and the concentration of ATP was calculated using an ATP standard curve.

Mitochondrial Ca 2+ measurement
For the functional study, isolated intestinal crypts were then resuspended in cold PBS. After that, mitochondrial Ca 2+ levels in intestinal crypts of mice were measured with Rhod-2 probe (AAT Bioquest, Inc.) according to the manufacturer's instructions. We added 100 mL of the dye-working solution into the wells already containing 100 mL of culture medium and incubated the dye-loading plate at room temperature for 30 min. Then replaced the dye working solution with the HHBS buffer with 1.0 mM Probenecid. Finally, put the plate into a fluorescence microplate reader (Molecular Devices, Shanghai, China) and ran the experiment as Ex/ Em = 549/578 nm to collect Rhod-2 signals.

Assays of mitochondrial permeability transition pore
For the functional study, single-cell suspension of intestinal crypts was obtained. Mitochondrial permeability transition pore (MPTP) in intestinal crypts of mice was measured using a BBcellProbeTM CA1 Assay Kit (BestBio, Shanghai, China) in accordance with the manufacturer's instructions. Prepared isolated intestinal crypts (1 3 10 6 /mL) and divided the crypts into three parts (500 mL per tube), and recorded them. Added 3 mL of BBcellProbeTM CA1 probe working solution into the tubes respectively. Next, added 5 mL of quenching agent into two of the tubes, and added 5 mL of permeable agent into one of the tubes. After incubating in the dark at 37 C for 15 min, added 3 mL of 1x MPTP dye buffer into the tubes respectively and centrifuged to collect crypts. The crypts were resuspended in cold PBS and collected by flow cytometry (BD FACSCalubur, USA). Quantitative analysis of flow cytometry data was determined using FlowJo (v10) software.

QUANTIFICATION AND STATISTICAL ANALYSIS
Values were expressed as the median and range and all experiments were repeated at least in triplicate. Statistical analyses were performed using SPSS 20.0 software (SPSS, Inc., Chicago, IL). The Student's t test was used to assess the differences between two groups. Pearson correlation and linear regression analyses were used to determine the correlation between AOPP and Lysozyme, GRP78, or CHOP. To compare treatment with vehicle, AOPP alone, and TUDCA + AOPP, a one-way analysis of variance (ANOVA) and pairwise comparisons were conducted and evaluated using the LSD or Dunnett's T3 method, as appropriate. Significance was set at p < 0.05.