Analysis of α-Defensin 5 Secretion in Differentiated Caco-2 Cells: Comparison of Cell Bank Origin

Abstract

α-Defensin 5 has a particularly broad antibacterial spectrum; it eliminates pathogenic microorganisms and regulates intestinal flora. Although Caco-2 cells are similar to small intestinal cells, it is unclear whether they secrete α-defensin 5. Therefore, we investigated whether Caco-2 cells secrete α-defensin 5 and determined the secretion mechanism using cells from three cell banks (ATCC, DSMZ, and RIKEN). The Caco-2 cell proliferation rate increased with the number of culture days, irrespective of cell bank origin. On the other hand, the alkaline phosphatase activity, which affects cell differentiation and the mRNA levels of several cytokines, such as interleukin 8 (IL-8), IL-6, IL-1β, tumor necrosis factor-α (TNF-α), and IL-2, in the Caco-2 cells fluctuated with the number of culture days, and differed for each cell bank. α-Defensin 5 secretion was detected in all three cell bank Caco-2 cells; particularly, the ATCC Caco-2 cells grew linearly depending on the cell culture day as well as the levels of IL-8 and TNF-α mRNA. This suggested that α-defensin 5 secretion in the ATCC Caco-2 cells was associated with fluctuations in the mRNA levels of various cytokines, such as IL-8 and TNF-α. In conclusion, Caco-2 cells may be a simple model for screening health food components and drugs that affect α-defensin 5 secretion.

INTRODUCTION

α-Defensin is an antimicrobial peptide secreted by Paneth cells in the crypts of the small intestine, and has two isoforms in humans: α-defensin 5 and 6. α-Defensin 5 has a particularly broad antibacterial spectrum; it eliminates pathogenic microorganisms and regulates intestinal flora.¹⁾ α-Defensin 5 expression is known to decrease in the intestines of patients with Crohn’s disease, which is an inflammatory bowel disease (IBD), causing necrotizing enteritis; this suggests that α-defensin 5 may regulate intestinal function.^2,3) Therefore, it is expected that an increase in α-defensin 5 secretion in the intestinal tract of IBD patients will be a therapeutic target. Models that can be used to evaluate α-defensin 5 mRNA expression levels in vivo have been reported⁴⁾; however, few studies have measured the α-defensin 5 secretory ability in vitro. A simple model for the evaluation of α-defensin 5 secretion would enable easier screening for components that affect this peptide. Recent reports indicate that the exposure of α-defensin 5 peptides to Caco-2 cells increases the secretion of interleukin 8 (IL-8); however, the association between α-defensin 5 and cytokines has not been fully examined.^5,6) Although α-defensin 5 is not present in normal large intestines,⁷⁾ Caco-2 cells, which are gastrointestinal model cells, express α-defensin 5 and alkaline phosphatase (ALP), a differentiation marker for cells similar to small intestinal cells; the ALP activity fluctuates with the number of culture days.^8–10) Therefore, we focused on whether Caco-2 cells could be used as a model for evaluating α-defensin 5 secretion. To this end, we examined the effect of cell differentiation in Caco-2 cells on α-defensin 5 secretory ability and the mRNA levels of various cytokines in an attempt to determine the appropriate conditions for using Caco-2 cells as a model for measuring α-defensin 5 secretion. Caco-2 cells obtained from the American Type Culture Collection (ATC C) and Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) cell banks have been reported to exhibit different activities of glucose transport, which is a major energy source.¹¹⁾ Hence, we speculated that Caco-2 cells from three cell banks, namely, ATC C, DSMZ, and RIKEN, would exhibit differences in the expression of α-defensin 5 and various genes.

In this study, we examined the cell proliferation capacity, cytokine mRNA levels, and α-defensin 5 secretion ability of Caco-2 cells obtained from three cell banks to establish a simple model for measuring α-defensin 5 secretion.

MATERIALS AND METHODS

Cell Culture

Cells of the human gastrointestinal model cell line (Caco-2) were obtained from three cell banks, namely, ATC C (passage: 30–40), DSMZ (passage: 26–36), and RIKEN (passage: 54–64), and were maintained in plastic culture flasks. The medium used was Dulbecco’s modified Eagle’s medium (Sigma, U.S.A.) supplemented with 10% fetal bovine serum (ICN Biomedicals, U.S.A.), 100 IU/mL penicillin–100 µg/mL streptomycin (Sigma), and 1% nonessential amino acids (Gibco, U.S.A.). Monolayer cultures were grown in an atmosphere of 5% CO₂–95% air at 37 °C. The cells were provided fresh growth medium every 1–2 d. When the Caco-2 cells reached confluence, they were harvested with 0.25 mM trypsin and 0.2% ethylenediaminetetraacetic acid (EDTA) (0.5–1 min at 37 °C), re-suspended, and seeded into a new flask.

Real-Time Quantitative PCR

Total RNA was prepared from Caco-2 cells using ISOGEN (Nippon Gene, Japan). cDNA was prepared from 1 µg total RNA via reverse transcription using ReverTra Ace (Toyobo, Japan). PCR was performed using 40 cycles of 95 °C for 30 s, 95 °C for 10 s, 59 °C for 30 s, and 72 °C for 10 s. Supplementary Table 1 shows the primer sequences used in this study. The levels of PCR products were normalized to those amplified from the internal reference genes (18S).

ALP Activity and α-Defensin 5 Secretion Assay

The ALP activity in the culture supernatant of each type of Caco-2 cell was measured using a QuantiFluo™ Alkaline Phosphatase Assay kit (BioAssay Systems, U.S.A.) according to the manufacturer’s protocol. The secretion levels of α-defensin 5 were measured using an α-defensin 5 enzyme-linked immunosorbent assay (ELISA) kit (LS-F37993, Lifespan Biosciences, U.S.A.) according to the manufacturer’s instructions. ATC C and RIKEN Caco-2 cells were seeded at 1.8 × 10⁴ cells/well, and Caco-2 cells obtained from DSMZ were seeded at 0.6 × 10⁴ cells/well in 96-well plates to achieve confluence in 7 d. ALP activity and α-defensin 5 secretion in the culture supernatant was measured.

Cell Viability

The growth of Caco-2 cells was assessed by the 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Caco-2 cells were seeded in a 96-well plate as described in section 2.3, and MTT stock solution (5 mg/mL) in phosphate buffered saline was added to each well at a final concentration of 0.25 mg/mL in medium and incubated for 1 h at 37 °C. After removing the medium, 200 µL of dimethyl sulfoxide was added to each well to dissolve the formazan crystals, and the absorbance was measured at 562 nm using a microplate reader (TECAN 200 PRO M).

Data Analysis

Student’s t-test was used to determine the significance of differences between two group means. Statistical significance among the means of more than two groups was evaluated using Dunnett’s test. Statistical significance was defined as p < 0.05.

RESULTS AND DISCUSSION

Influence of Number of Culture Days on Cell Viability and ALP Secretory Capacity

Prior to determining the α-defensin 5 secretion ability of Caco-2 cells, the effect of the number of culture days on cell viability was investigated by comparing Caco-2 cells of each cell bank. As shown in Fig. 1A, the cell proliferation rate increased with the number of culture days for all types of Caco-2 cells. To ascertain the relationship between the number of culture days and cell differentiation, we detected the ALP activity in the culture supernatant of each cell type, since it has been reported that an increase in the ALP activity in Caco-2 cells is strongly associated with cell differentiation.¹⁰⁾ The ALP activity in ATC C Caco-2 cells increased with the number of culture days, and peaked 14 d after cell seeding. On the other hand, those of the DSMZ and RIKEN Caco-2 cells continued to increase with the number of culture days (Fig. 1B). Since the increase in ALP activity in Caco-2 cells is known to resemble that of a brush border marker,¹²⁾ we inferred that the ATC C Caco-2 cells had fully differentiated after 14 d. Then, we focused on various cytokines in each cell, such as IL-8, a well-known inflammatory cytokine associated with cell proliferation in colon cancer cell line models.¹³⁾

Fig. 1. Effect of the Number of Culture Days on Cell Viability, Which Was Normalized to the Viability at 7 d, in ATCC Caco-2 Cells (A) and Alkaline Phosphatase (ALP) Activity Levels in Caco-2 Cells from Different Cell Banks (B)

Data are presented as means with standard deviation (S.D.) (error bars) of nine determinations.

Influence of Number of Culture Days on mRNA Levels of Various Cytokines

Before evaluating the mRNA levels of several markers via quantitative (q)PCR, we assessed the mRNA levels of housekeeping genes, as the levels might change with the number of culture days. Since the 18S expression level in each cell did not significantly change, we selected 18S as the reference gene in subsequent evaluations (Supplementary Fig. 1). We examined the effects of the number of culture days of Caco-2 cells from ATC C, DSMZ, and RIKEN on the IL-8 mRNA levels. Figure 2 shows the IL-8 mRNA levels in each Caco-2 cell type, particularly in cells from ATC C and RIKEN; they increased with the number of cell culture days (Fig. 2). Then, we examined the mRNA levels of other cytokines such as IL-6, IL-1β, tumor necrosis factor-α (TNF-α), and IL-2. As shown in Fig. 3, the IL-6 mRNA levels significantly varied in each Caco-2 cell culture after 28 d, unlike those after 7 d. The IL-1β and TNF-α mRNA levels were significantly elevated in each cell after 21 and 28 d compared with those after 7 d. The association between the IL-2 mRNA level and number of cell culture days differed for cells from different cell banks (Fig. 3). The results suggested that the number of cell culture days affected various cytokine mRNA levels which regulate the immunity of the human intestine in Caco-2 cells. Recent reports indicate that the exposure of α-defensin 5 peptides to Caco-2 cells increases the secretion of IL-8.^5,6) Therefore, we investigated the effect of the number of culture days on α-defensin 5 secretion.

Fig. 2. Effect of Number of Culture Days on IL-8 mRNA Levels in Caco-2 Cells from Different Cell Banks

Transcript levels were normalized to those of the 18S housekeeping gene. Data are presented as means with standard error (S.E.) (error bars) of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. cell culture for 7 d.

Fig. 3. Effect of Number of Culture Days on IL-6, IL-1β, TNF-α and IL-2 mRNA Levels in Caco-2 Cells from Different Cell Banks

Transcript levels were normalized to those of the 18S housekeeping gene. Data are presented as means with S.E. (error bars) of three independent experiments. * p < 0.05, ** p < 0.01 vs. cell culture for 7 d.

Influence of Number of Culture Days on α-Defensin 5 Secretion

Figure 4 shows the α-defensin 5 secretion in Caco-2 cells from DSMZ and RIKEN peaked 14 d after cell seeding. On the other hand, the secretion in the cells from ATC C increased linearly from Days 7 to 28. Therefore, we suggest that ATC C Caco-2 cells may be a simple model for evaluating α-defensin 5 secretion. Since the IL-8 or TNF-α mRNA levels in ATC C Caco-2 cells correlated with the number of cell culture days, α-defensin 5 secretion from these cells may be associated with fluctuations in the mRNA levels of IL-8 or TNF-α. To clarify the effect of α-defensin 5 on cytokine mRNA levels, we examined the mRNA levels of IL-8, IL-6, IL-1β, TNF-α, and IL-2 in Caco-2 cells treated with α-defensin 5 peptides. The concentration of α-defensin 5 peptides in the human ileum is 6–30 µg/mL.³⁾ We selected an α-defensin 5 peptide concentration of 18 µg/mL. The mRNA levels of all the selected cytokines increased significantly in ATC C Caco-2 cells (Supplementary Fig. 2). Exposure of ATC C Caco-2 cells to α-defensin 5 peptides induced IL-8 secretion in via nuclear factor-kappa B (NF-κB),⁵⁾ and TNF-α-induced IL-8 secretion levels are dependent on NF-κB signaling.¹⁴⁾ However, exposure to a low concentration of α-defensin 5 peptides (0.36 µg/mL) for 24 h had no effect on IL-8, IL-6, IL-1β, TNF-α, and IL-2 mRNA levels (data not shown). Although we speculated that long-term exposure to α-defensin 5 peptides may affect cytokine mRNA levels, leading to altered α-defensin 5 secretion and cytokine mRNA expression, this was not verified in the current study. Miles et al. reported that microRNAs (miRNAs) such as miR-124 and miR-924 regulated α-defensin 5 expression and might be associated with cytokines.¹⁵⁾ Moreover, Zeng et al. reported that engineering probiotic-producing human defensin 5 downregulated the expression of several inflammation-related molecules and preserved the epithelial barrier integrity.¹⁶⁾ Further investigation is in progress to elucidate the details of a direct association between α-defensin 5 secretion and the expression levels of cytokines. In conclusion, this small intestine cell model that secretes α-defensin 5 makes it possible to easily screen health food components and drugs that affect the secretion of this peptide in vitro.

Fig. 4. Effect of Number of Culture Days on α-Defensin 5 Secretion Levels in Caco-2 Cells from Different Cell Banks

Data are presented as means with S.D. (error bars) of nine determinations.

Acknowledgments

This work was supported by the OTC Self-Medication Promotion Foundation (Grant provided to M.K.) and the Japan Society for the Promotion of Science (JSPS) KAKENHI (Grant No. 17K00871, provided to N.S.).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

REFERENCES

• 1) Salzman NH, Hung K, Haribhai D, Chu H, Karlsson-Sjöberg J, Amir E, Teggatz P, Barman M, Hayward M, Eastwood D, Stoel M, Zhou Y, Sodergren E, Weinstock GM, Bevins CL, Williams CB, Bos NA. Enteric defensins are essential regulators of intestinal microbial ecology. Nat. Immunol., 11, 76–83 (2010).
• 2) Wehkamp J, Salzman NH, Porter E, Nuding S, Weichenthal M, Petras RE, Shen B, Schaeffeler E, Schwab M, Linzmeier R, Feathers RW, Chu H, Lima H Jr, Fellermann K, Ganz T, Stange EF, Bevins CL. Reduced Paneth cell α-defensins in ileal Crohn’s disease. Proc. Natl. Acad. Sci. U.S.A., 102, 18129–18134 (2005).
• 3) Elphick D, Liddell S, Mahida YR. Impaired luminal processing of human defensin-5 in Crohn’s disease: Persistence in a complex with chymotrypsinogen and trypsin. Am. J. Pathol., 172, 702–713 (2008).
• 4) Kubota A, Kobayashi M, Sarashina S, Takeno R, Okamoto K, Narumi K, Furugen A, Suzuki Y, Takahashi N, Iseki K. Reishi mushroom Ganoderma lucidum modulates IgA production and alpha-defensin expression in the rat small intestine. J. Ethnopharmacol., 214, 240–243 (2018).
• 5) Lu W, de Leeuw E. Pro-inflammatory and pro-apoptotic properties of Human Defensin 5. Biochem. Biophys. Res. Commun., 436, 557–562 (2013).
• 6) Zhao A, Lu W, de Leeuw E. Functional synergism of Human Defensin 5 and Human Defensin 6. Biochem. Biophys. Res. Commun., 467, 967–972 (2015).
• 7) Wehkamp J, Schwind B, Herrlinger KR, Baxmann S, Schmidt K, Duchrow M, Wohlschläger C, Feller AC, Stange EF, Fellermann K. Innate immunity and colonic inflammation: Enhanced expression of epithelial α-defensins. Dig. Dis. Sci., 47, 1349–1355 (2002).
• 8) Takahashi N, Kobayashi M, Ogura J, Yamaguchi H, Satoh T, Watanabe K, Iseki K. Immunoprotective effect of epigallocatechin-3-gallate on oral anticancer drug-induced α-defensin reduction in Caco-2 cells. Biol. Pharm. Bull., 37, 490–492 (2014).
• 9) Santamaria MH, Perez Caballero E, Corral RS. Unmethylated CpG motifs in Toxoplasma gondii DNA induce TLR9-and IFN-β-dependent expression of α-defensin-5 in intestinal epithelial cells. Parasitology, 143, 60–68 (2016).
• 10) Wang X, Li L, Zhang G. Impact of deoxynivalenol and kaempferol on expression of tight junction proteins at different stages of Caco-2 cell proliferation and differentiation. RSC Adv., 9, 34607–34616 (2019).
• 11) Steffansen B, Pedersen MDL, Laghmoch AM, Nielsen CU. SGLT1-mediated transport in Caco-2 cells is highly dependent on cell bank origin. J. Pharm. Sci., 106, 2664–2670 (2017).
• 12) Matsumoto H, Erickson RH, Gum JR, Yoshioka M, Gum E, Kim YS. Biosynthesis of alkaline phosphatase during differentiation of the human colon cancer cell line Caco-2. Gastroenterology, 98, 1199–1207 (1990).
• 13) Ning Y, Manegold PC, Hong YK, Zhang W, Pohl A, Lurje G, Winder T, Yang D, LaBonte MJ, Wilson PM, Ladner RD, Lenz HJ. Interleukin-8 is associated with proliferation, migration, angiogenesis and chemosensitivity in vitro and in vivo in colon cancer cell line models. Int. J. Cancer, 128, 2038–2049 (2011).
• 14) Son DO, Satsu H, Shimizu M. Histidine inhibits oxidative stress- and TNF-alpha-induced interleukin-8 secretion in intestinal epithelial cells. FEBS Lett., 579, 4671–4677 (2005).
• 15) Miles DR, Shen J, Chuang AY, Dong F, Wu F, Kwon J. Alpha-defensin 5 expression is regulated by microRNAs in the Caco-2 intestinal epithelial cell line. J. Inflamm. Bowel Dis. Disord., 1, 105 (2016).
• 16) Zeng L, Tan J, Xue M, Liu L, Wang M, Liang L, Deng J, Chen W, Chen Y. An engineering probiotic producing defensin-5 ameliorating dextran sodium sulfate-induced mice colitis via inhibiting NF-kB pathway. J. Transl. Med., 18, 107 (2020).