# Macrophage Deficiency Makes Intestinal Epithelial Cells Susceptible to NSAID-Induced Damage

Authors: Xinxin Wang; Jiayang Wang; Tianyu Xie; Shuo Li; Di Wu; Yixun Lu; Xin Guo; Lin Chen
Journal: BioMed Research International
 (2020)

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## Abstract

Objectives. In Crohn’s disease (CD), the mechanisms underlying the regulation by granulocyte-macrophage colony-stimulating factor (GM-CSF) of mucosal barrier function in the ileum are unclear. We analyzed the molecular mechanisms underlying the regulation by GM-CSF of the mucosal barrier function. Methods. We examined the role of GM-CSF in the intestinal barrier function in CD at the molecular-, cellular-, and animal-model levels. Results. Macrophages directly secreted GM-CSF, promoting intestinal epithelial proliferation and inhibiting apoptosis, which maintained intestinal barrier function. Macrophages were absent in NSAID-induced ileitis, causing GM-CSF deficiency, increasing the apoptosis rate, decreasing the proliferation rate, increasing inter- and paracellular permeabilities, decreasing the TJP levels, and reducing the numbers of mesenteric lymph nodes, memory T cells, and regulatory T cells in Csf1op/op transgenic mice. Conclusions. GM-CSF is required for the maintenance of intestinal barrier function. Macrophages directly secrete GM-CSF, promoting intestinal epithelial proliferation and inhibiting apoptosis.


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## Body

## 1. Introduction

Crohn’s disease (CD) is a chronic, recurrent, insidious, and nonspecific transmural inflammation of the digestive tract. Its symptoms include digestive tract obstruction, perforation, abdominal abscess, fistula, and hemorrhage. Prolonged CD may cause psychiatric symptoms such as anxiety or depression, as well as localized malignancy. CD is a lifelong disease caused by interactions between genetic and environmental factors [1, 2]. However, the etiology and pathogenesis of CD are unclear.More than 70 genes and loci are related to the occurrence and development of CD according to genome-wide association studies. NOD2/CARD15 was the first CD susceptibility gene to be discovered by genome-wide association studies. Although functional deletion of CARD15 increases the susceptibility to CD [3, 4], the explicit rate is low. CARD15-knockout mice do not spontaneously develop enterocolitis, suggesting that other factors contribute to the development of CD [5, 6].Although nonsteroidal anti-inflammatory drug- (NSAID-) induced gut barrier dysfunction and clinical relapse occur in patients with CD, the mechanisms are unclear [7, 8]. In addition, the level of granulocyte-macrophage colony-stimulating factor (GM-CSF) autoantibodies is significantly increased in patients with CD [9]. We reported that NSAID-associated intestinal barrier dysfunction may be correlated with GM-CSF autoantibodies, thereby promoting the activation of T cells and development of localized ileal CD [9, 10]. Therefore, GM-CSF is necessary to maintain ileal barrier function.However, the molecular mechanisms underlying GM-CSF regulation of mucosal barrier function in the ileum are unclear. Therefore, we performed an animal study to evaluate the role of GM-CSF in intestinal barrier function in CD at the molecular, cellular, and animal-model levels.


## 2. Materials and Methods

### 2.1. Reagents

The following monoclonal antibodies were used: neutralizing anti-mouse CSF1 receptor (AFS98 [IgG2a]; S-I Nishikawa; RIKEN Center for Developmental Biology, Kobe, Japan), neutralizing anti-mouse GM-CSF (22E9.11; IgG2a; J. Abrams; DNAX, Palo Alto, CA), and neutralizing anti-mouse CSF1 and an IgG2a isotype control (F. Dodeller; MorphoSys, Munich, Germany) (35).


### 2.2. Mice

C57BL/6 mice were obtained from WEHI, Kew (VIC, Australia). GMCSF2/2 (29) and Csf1r-EGFP (MacGreen) mice (36), backcrossed onto the C57BL/6 background, were bred in our on-site animal facility. BALB/c mice were obtained from the Animal Resource Centre (Perth, Australia). Mice (8–12 weeks old) were fed standard rodent chow and waterad libitum and were housed in sawdust-lined cages in groups of five. The study was approved by the Animal Experimentation Ethics Committee of The University of Melbourne and was conducted in compliance with the animal experimentation guidelines of the National Health and Medical Research Council of Australia.


### 2.3. Adoptive Cell Transfer

Bone marrow was flushed from the tibias and femurs of the donor MacGreen mice. After red blood cell lysis using the ACK lysis buffer, CD115+ cells were enriched by magnetically activated cell sorting using a CD115-biotin antibody and antibiotin microbeads (Miltenyi Biotec) according to the manufacturer’s instructions. After enrichment, monocyte purity was consistently 90%. A total of 103,106 enriched monocytes were transferred intravenously into mBSA-challenged AIP mice on day 1.


### 2.4. BrdU Pulsing

In steady-state BrdU kinetics experiments, mice were given three intraperitoneal injections of 2 mg BrdU (Sigma-Aldrich) 2 h apart, and BrdU labeling was monitored from days 1 to 5. For the short-term BrdU pulse, mice were injected intraperitoneally with 1 mg BrdU at 2–3 h before analysis. To assess BrdU incorporation, we stained, fixed, and permeabilized suspended cells using Cytofix/Cytoperm and Perm/Wash buffers (BD Pharmingen) according to the manufacturer’s instructions. Cells were incubated at 37°C for 60 min in 30 mg DNase (Sigma-Aldrich), stained with anti-BrdU-FITC for 30 min, washed, and analyzed by flow cytometry.


### 2.5. Statistical Analysis

Data were analyzed using SPSS 18.0 and GraphPad Prism 8.0 software. Data aremeans±standarddeviation. Student’s t-test was used to analyze differences in the mean values between two groups. A value of p<0.05 was taken to indicate statistical significance.


## 2.1. Reagents

The following monoclonal antibodies were used: neutralizing anti-mouse CSF1 receptor (AFS98 [IgG2a]; S-I Nishikawa; RIKEN Center for Developmental Biology, Kobe, Japan), neutralizing anti-mouse GM-CSF (22E9.11; IgG2a; J. Abrams; DNAX, Palo Alto, CA), and neutralizing anti-mouse CSF1 and an IgG2a isotype control (F. Dodeller; MorphoSys, Munich, Germany) (35).


## 2.2. Mice

C57BL/6 mice were obtained from WEHI, Kew (VIC, Australia). GMCSF2/2 (29) and Csf1r-EGFP (MacGreen) mice (36), backcrossed onto the C57BL/6 background, were bred in our on-site animal facility. BALB/c mice were obtained from the Animal Resource Centre (Perth, Australia). Mice (8–12 weeks old) were fed standard rodent chow and waterad libitum and were housed in sawdust-lined cages in groups of five. The study was approved by the Animal Experimentation Ethics Committee of The University of Melbourne and was conducted in compliance with the animal experimentation guidelines of the National Health and Medical Research Council of Australia.


## 2.3. Adoptive Cell Transfer

Bone marrow was flushed from the tibias and femurs of the donor MacGreen mice. After red blood cell lysis using the ACK lysis buffer, CD115+ cells were enriched by magnetically activated cell sorting using a CD115-biotin antibody and antibiotin microbeads (Miltenyi Biotec) according to the manufacturer’s instructions. After enrichment, monocyte purity was consistently 90%. A total of 103,106 enriched monocytes were transferred intravenously into mBSA-challenged AIP mice on day 1.


## 2.4. BrdU Pulsing

In steady-state BrdU kinetics experiments, mice were given three intraperitoneal injections of 2 mg BrdU (Sigma-Aldrich) 2 h apart, and BrdU labeling was monitored from days 1 to 5. For the short-term BrdU pulse, mice were injected intraperitoneally with 1 mg BrdU at 2–3 h before analysis. To assess BrdU incorporation, we stained, fixed, and permeabilized suspended cells using Cytofix/Cytoperm and Perm/Wash buffers (BD Pharmingen) according to the manufacturer’s instructions. Cells were incubated at 37°C for 60 min in 30 mg DNase (Sigma-Aldrich), stained with anti-BrdU-FITC for 30 min, washed, and analyzed by flow cytometry.


## 2.5. Statistical Analysis

Data were analyzed using SPSS 18.0 and GraphPad Prism 8.0 software. Data aremeans±standarddeviation. Student’s t-test was used to analyze differences in the mean values between two groups. A value of p<0.05 was taken to indicate statistical significance.


## 3. Results

### 3.1. Effect of Piroxicam

The effects of macrophage deficiency on the apoptosis and proliferation of intestinal epithelial cells in Csf1op/op transgenic mice were determined by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay and Ki67 staining. Under normal conditions, the intestinal epithelium of Csf1op/op transgenic mice showed a higher apoptosis rate and lower proliferation rate than those of wild-type mice. In the presence of NSAIDs, the intestinal epithelial cells in Csf1op/op transgenic mice showed a higher apoptosis rate (27.80±2.21 vs. 19.41±3.28, p<0.05) and lower proliferation rate (10.50±1.30 vs. 16.88±3.49, p<0.05) compared with wild-type mice (Figure 1). Therefore, NSAIDs may cause gut dysfunction by damaging intestinal epithelial cells.Figure 1
(a) Intestinal epithelial tissue analyzed by TUNEL staining (apoptosis) and Ki67 immunohistochemical staining (proliferation). (b) Percentage of TUNEL-positive cells. (c) Percentage of Ki67-positive cells.
(a)(b)(c)


### 3.2. NSAIDs Promoted Intestinal Epithelial Permeability and Bacterial Translocation

Under normal conditions, the intestinal epithelium of Csf1op/op transgenic mice showed higher inter- and paracellular permeabilities and bacterial translocation compared with that of wild-type mice. In the presence of NSAIDs, inter- and paracellular permeabilities of the intestinal epithelial cells were increased in Csf1op/op transgenic mice but unaffected in wild-type mice (27.63±3.23 vs. 21.90±2.04, p<0.05). The bacterial translocation rate was increased by NSAIDs in Csf1op/op transgenic mice but unaffected in wild-type mice (Figure 2). In addition, Csf1op/op transgenic mice had lower TJP levels than wild-type mice under normal conditions and in the presence of NSAIDs. Therefore, NSAIDs promoted intestinal epithelial cell inter- and paracellular permeabilities and bacterial translocation in transgenic mice.Figure 2
Intestinal epithelial cell inter- and paracellular permeabilities.


### 3.3. NSAIDs Reduced the Number of Mesenteric Lymph Nodes

Csf1op/op transgenic mice had fewer mesenteric lymph nodes than wild-type mice under normal conditions (Figure 3). The number of mesenteric lymph nodes in Csf1op/op transgenic mice was reduced by NSAIDs.Figure 3
Number of mesenteric lymph nodes.


### 3.4. NSAIDs Decreased the Percentage of Activated T Cells

In the presence of NSAIDs, the percentage of activated T cells (CD4+CD44+) was significantly lower in Csf1op/op transgenic mice than in wild-type mice (8.78% vs. 14.9%, p<0.05, Figure 4).Figure 4
Percentage of CD4+CD44+ spleen cells determined by flow cytometry.


### 3.5. NSAIDs Reduced the Percentage of CD4+Foxp3+ Cells

In the presence of NSAIDs, the number of memory T cells was significantly lower in Csf1op/op transgenic mice than in wild-type mice (8.91±2.11 vs. 14.85±2.42, p<0.05, Figure 5).Figure 5
Percentage of CD4+Foxp3+ spleen cells determined by flow cytometry.


### 3.6. NSAIDs Suppressed Apoptosis of Caco-2 Cells

In the presence of NSAIDs, the number of regulatory T cells was significantly lower in Csf1op/op transgenic mice than in wild-type mice (3.65±2.28 vs. 7.79±1.25, p<0.05, Figure 6).Figure 6
Apoptosis of Caco-2 cells determined by flow cytometry.


### 3.7. GM-CSF Inhibited Apoptosis of Caco-2 Cells

Intraductal intrinsic macrophages are the main source of GM-CSF under normal conditions. To assess the effect of GM-CSF on the proliferation and apoptosis of intestinal epithelial cells, we formed the following four experimental groups: (1) normal conditions in Caco-2 and RAW264.7 cells, (2) LPS stimulation in Caco-2 and RAW264.7 cells, (3) normal conditions in Caco-2 cells and intestinal macrophages, and (4) LPS stimulation in Caco-2 cells and intestinal macrophages. We performed flow cytometry to evaluate the proportion of proliferating cells. LPS stimulated the secretion of GM-CSF by macrophages, which inhibited the apoptosis of RAW264.7 (4.77±0.42 vs. 16.73±1.31, p<0.001) and intestinal (12.10±2.38 vs. 20.37±2.81, p<0.01) macrophages (Figure 7).Figure 7
Proliferation of Caco-2 cells by flow cytometry.


### 3.8. GM-CSF Promoted Intestinal Epithelial Cell Proliferation

LPS stimulated the secretion of GM-CSF by macrophages, which promoted the proliferation of RAW264.7 (27.96±4.76 vs. 8.55±0.88, p<0.001) and intestinal macrophages (22.10±4.70 vs. 4.13±1.40, p<0.001, Figure 8).Figure 8
GM-CSF promoted intestinal epithelial cell proliferation.


### 3.9. GM-CSF Secreted by Macrophages Stimulated Invasion and Migration of Caco-2 Cells

We used a Transwell assay to assess the effect of LPS-stimulated intestinal macrophages and RAW264.7 macrophages on the permeability of intestinal epithelial cell monolayers. The results showed that macrophages directly secrete GM-CSF, stimulating invasion and migration by Caco-2 cells.The levels of TJPs—closed bands (zonula occludens), JAM-A, and claudins—were measured by WB. GM-CSF regulated the epithelial cell barrier and promoted the synthesis of TJPs, maintaining intestinal barrier function (Figures9 and 10).Figure 9
Levels of tight junction proteins.Figure 10
GM-CSF regulates the epithelial cell barrier and promotes the synthesis of tight junction proteins, maintaining intestinal barrier function.
(a)(b)(c)


## 3.1. Effect of Piroxicam

The effects of macrophage deficiency on the apoptosis and proliferation of intestinal epithelial cells in Csf1op/op transgenic mice were determined by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay and Ki67 staining. Under normal conditions, the intestinal epithelium of Csf1op/op transgenic mice showed a higher apoptosis rate and lower proliferation rate than those of wild-type mice. In the presence of NSAIDs, the intestinal epithelial cells in Csf1op/op transgenic mice showed a higher apoptosis rate (27.80±2.21 vs. 19.41±3.28, p<0.05) and lower proliferation rate (10.50±1.30 vs. 16.88±3.49, p<0.05) compared with wild-type mice (Figure 1). Therefore, NSAIDs may cause gut dysfunction by damaging intestinal epithelial cells.Figure 1
(a) Intestinal epithelial tissue analyzed by TUNEL staining (apoptosis) and Ki67 immunohistochemical staining (proliferation). (b) Percentage of TUNEL-positive cells. (c) Percentage of Ki67-positive cells.
(a)(b)(c)


## 3.2. NSAIDs Promoted Intestinal Epithelial Permeability and Bacterial Translocation

Under normal conditions, the intestinal epithelium of Csf1op/op transgenic mice showed higher inter- and paracellular permeabilities and bacterial translocation compared with that of wild-type mice. In the presence of NSAIDs, inter- and paracellular permeabilities of the intestinal epithelial cells were increased in Csf1op/op transgenic mice but unaffected in wild-type mice (27.63±3.23 vs. 21.90±2.04, p<0.05). The bacterial translocation rate was increased by NSAIDs in Csf1op/op transgenic mice but unaffected in wild-type mice (Figure 2). In addition, Csf1op/op transgenic mice had lower TJP levels than wild-type mice under normal conditions and in the presence of NSAIDs. Therefore, NSAIDs promoted intestinal epithelial cell inter- and paracellular permeabilities and bacterial translocation in transgenic mice.Figure 2
Intestinal epithelial cell inter- and paracellular permeabilities.


## 3.3. NSAIDs Reduced the Number of Mesenteric Lymph Nodes

Csf1op/op transgenic mice had fewer mesenteric lymph nodes than wild-type mice under normal conditions (Figure 3). The number of mesenteric lymph nodes in Csf1op/op transgenic mice was reduced by NSAIDs.Figure 3
Number of mesenteric lymph nodes.


## 3.4. NSAIDs Decreased the Percentage of Activated T Cells

In the presence of NSAIDs, the percentage of activated T cells (CD4+CD44+) was significantly lower in Csf1op/op transgenic mice than in wild-type mice (8.78% vs. 14.9%, p<0.05, Figure 4).Figure 4
Percentage of CD4+CD44+ spleen cells determined by flow cytometry.


## 3.5. NSAIDs Reduced the Percentage of CD4+Foxp3+ Cells

In the presence of NSAIDs, the number of memory T cells was significantly lower in Csf1op/op transgenic mice than in wild-type mice (8.91±2.11 vs. 14.85±2.42, p<0.05, Figure 5).Figure 5
Percentage of CD4+Foxp3+ spleen cells determined by flow cytometry.


## 3.6. NSAIDs Suppressed Apoptosis of Caco-2 Cells

In the presence of NSAIDs, the number of regulatory T cells was significantly lower in Csf1op/op transgenic mice than in wild-type mice (3.65±2.28 vs. 7.79±1.25, p<0.05, Figure 6).Figure 6
Apoptosis of Caco-2 cells determined by flow cytometry.


## 3.7. GM-CSF Inhibited Apoptosis of Caco-2 Cells

Intraductal intrinsic macrophages are the main source of GM-CSF under normal conditions. To assess the effect of GM-CSF on the proliferation and apoptosis of intestinal epithelial cells, we formed the following four experimental groups: (1) normal conditions in Caco-2 and RAW264.7 cells, (2) LPS stimulation in Caco-2 and RAW264.7 cells, (3) normal conditions in Caco-2 cells and intestinal macrophages, and (4) LPS stimulation in Caco-2 cells and intestinal macrophages. We performed flow cytometry to evaluate the proportion of proliferating cells. LPS stimulated the secretion of GM-CSF by macrophages, which inhibited the apoptosis of RAW264.7 (4.77±0.42 vs. 16.73±1.31, p<0.001) and intestinal (12.10±2.38 vs. 20.37±2.81, p<0.01) macrophages (Figure 7).Figure 7
Proliferation of Caco-2 cells by flow cytometry.


## 3.8. GM-CSF Promoted Intestinal Epithelial Cell Proliferation

LPS stimulated the secretion of GM-CSF by macrophages, which promoted the proliferation of RAW264.7 (27.96±4.76 vs. 8.55±0.88, p<0.001) and intestinal macrophages (22.10±4.70 vs. 4.13±1.40, p<0.001, Figure 8).Figure 8
GM-CSF promoted intestinal epithelial cell proliferation.


## 3.9. GM-CSF Secreted by Macrophages Stimulated Invasion and Migration of Caco-2 Cells

We used a Transwell assay to assess the effect of LPS-stimulated intestinal macrophages and RAW264.7 macrophages on the permeability of intestinal epithelial cell monolayers. The results showed that macrophages directly secrete GM-CSF, stimulating invasion and migration by Caco-2 cells.The levels of TJPs—closed bands (zonula occludens), JAM-A, and claudins—were measured by WB. GM-CSF regulated the epithelial cell barrier and promoted the synthesis of TJPs, maintaining intestinal barrier function (Figures9 and 10).Figure 9
Levels of tight junction proteins.Figure 10
GM-CSF regulates the epithelial cell barrier and promotes the synthesis of tight junction proteins, maintaining intestinal barrier function.
(a)(b)(c)


## 4. Discussion

CSF1 and GM-CSF regulate the development and function of the mononuclear phagocyte system [11, 12]. We used Csf1op/op transgenic mice to explore the role of macrophages in NSAID-induced damage. The intestinal epithelial cells of Csf1op/op transgenic mice had higher apoptosis rates and lower proliferation rates. In mice deficient in CSF1, Paneth cells failed to develop, and the small intestine showed defects in cell proliferation and differentiation [13]. Macrophages also promoted maintenance of epithelial cells. Intestinal epithelial cells deficient in macrophages have lower TJP levels, resulting in higher inter- and paracellular permeabilities and bacterial translocation. Because of the lack of macrophages, epithelial cells are more susceptible to NSAID-induced damage.We also analyzed the molecular mechanisms underlying the regulation by GM-CSF of the mucosal barrier function. Macrophages directly secreted GM-CSF, promoting intestinal epithelial cell proliferation and inhibiting their apoptosis, thus maintaining intestinal barrier function. In NSAID-induced ileitis, macrophages are absent, which caused GM-CSF deficiency, increased apoptosis rates and inter- and paracellular permeabilities, and decreased proliferation rates, TJP levels, numbers of mesenteric lymph nodes, memory T cells, and regulatory T cells in Csf1op/op transgenic mice.GM-CSF is a pleiotropic cytokine that not only promotes the survival, proliferation, and differentiation of multiple hematopoietic cells after GM-CSF receptor binding but also plays an important role in immune regulation. GM-CSF enhances the acute reaction to bacteria (chemokine production, chemotaxis, adhesion, and phagocytosis) by activating monocytes/macrophages and neutrophils. It also stimulates the expression of Toll-like receptors 2 and 4 and regulates the response to lipopolysaccharide and peptidoglycan [9–11]. The mechanisms by which GM-CSF promotes the barrier function of the intestinal mucosa have only recently begun to be explored. GM-CSF signaling in nonhematopoietic cells regulates intestinal barrier integrity. The therapeutic effect of GM-CSF on patients with CD may therefore involve regulation of intestinal epithelial cell function.GM-CSF plays an integral role in the response to injury of intestinal epithelial cells. Macrophages directly secrete GM-CSF, promoting the proliferation and inhibiting the apoptosis of intestinal epithelial cells. In addition, GM-CSF stimulates the invasion and migration of Caco-2 cells.


## 5. Conclusion

GM-CSF is necessary for the barrier function of the intestine because macrophages directly secrete GM-CSF, promoting the proliferation and inhibiting the apoptosis of intestinal epithelial cells. In NSAID-induced ileitis, macrophage depletion increased the apoptosis rate and inter- and paracellular permeabilities and decreased the proliferation rate, TJP levels, and numbers of mesenteric lymph nodes, memory T cells, and regulatory T cells in Csf1op/op transgenic mice. Therefore, GM-CSF is important for the recovery of macrophage deficiency.



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