Colonoids

Colonoid cultures using the well-established and successful protocols in M’Koma Lab was used. Colonoids were purchased from Cellesce Limited (Medicenter, Cardiff, United Kingdom [UK]). The original colonoids were licensed from the Director of Organoid Biology, Professor Trevor Dale’s Laboratory at Cardiff University, UK. Cellesce (Cardiff, UK) (https://pubmed.ncbi.nlm.nih.gov/31033964) is a Biotrech company that has a patented bioprocessing technology for the growing and expansion of commercialized organoid models enabling drug discovery and genetics research [7].

Human colonoids functionally recapitulate normal intestinal physiology and pathophysiology and are three-dimensional (3D) in-vitro-grown cell culture with near-native microanatomy that arise from self-organizing mammalian pluripotent or adult stem cells. Recent advances in the 3D culture of isolated intestinal crypts have enabled the generation of human gastrointestinal epithelial organoids. Gastrointestinal organoids recapitulate the human in vivo physiology because of all the intestinal epithelial cell types that differentiated and proliferated from tissue resident stem cells. Thus far, gastrointestinal organoids have been extensively used for generating gastrointestinal disease.

Immortalized colonic epithelial cells

The immortalized colonic epithelial cells, (NCM460) is a human adult, normal-colon epithelium-derived cell lines, derived from the normal colon of a 68-year-old Hispanic male [8]. The NCM460 was received by a Material Transfer Agreement with INCELL Corporation (San Antonio, Texas, USA). The cells express cytokeratins, villin, and other colonic epithelial cell antigens associated with other cell types. Some of the cells are positive for mucin synthesis. They do not grow in soft agar and are non-tumorigenic. They have fastidious growth requirements and must be maintained in INCELL’s enriched M3:10™ medium for long-term in vitro culture with maintenance of an appropriate phenotype.). Human colon epithelial cells, NCM460 (https://www.incell.com/product/ncm460d-cell-line) were derived from the normal colon mucosa from a 68-year-old Hispanic male individual selected for in vitro growth [8]. This cell-line was not infected or transfected with any exogenous genetic information. We were interested to use this cell-line to find out whether the release of UC or CC associated cytokines is triggered by the same mechanisms that stimulate DEFA5 secretion by PCs. It is also not clear whether the effects of DEFA5 and CC-associated cytokines are synergistic in protecting the colon epithelium. To study the mechanisms underlying the secretion of DEFA5, as an alternative to the colonoids, we maintained the normal epithelial immortalized colonic epithelial cells, NCM460 in 2D or 3D cultures and assessed the effects of CC and UC-associated signatures/cytokines on the secretion of DEFA5, and downstream signaling effectors. The cells express cytokeratins, villin, and other colonic epithelial cell antigens associated with other cell types. Some of the cells are positive for mucin synthesis. They do not grow in soft agar and are non-tumorigenic. They have fastidious growth requirements and must be maintained in INCELL’s enriched M3:10™ medium for long-term in vitro culture with maintenance of an appropriate phenotype.

Reagents, antibodies, and kits.

The supplies (reagents, antibodies, and kits) used in experiments are herewith summarized in Table 1.

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Table 1. List of items used in experiments.

Reagents, antibodies, kits, vendors and catalog numbers.

https://doi.org/10.1371/journal.pone.0246393.t001

Immunohistochemistry and western blotting

Five colon tissue protein extracts and staining of DEFA5 per disease by IHC was done as previously described [9,10]. Quantification of HD5 staining was analyzed manually by microscopy and automatically quantified using Nikon’s Eclipse Ti microscope with built-in NEARAS [10].

Western blot was used to assess any differences in DEFA5 protein levels. Protein was extracted from a minimum of 10 colon biopsy samples each from UC, CC, and IC. Whole cell lysates were extracted from full-thickness colon samples using T-PER (Thermo Fisher Scientific) per manufacturer’s protocol. Bradford Assays (Bio-Rad) were run to determine protein concentration, and protein was loaded onto a 4–20% SDS-PAGE tris/glycine gel (Bio-Rad). Proteins were transferred to PVDF (Bio-Rad), and Western blots for DEFA5 and β-actin loading control were performed with primary and secondary antibodies (Santa Cruz, Dallas, TX) per manufacturer’s protocol. Blots were visualized with Opti-4CN colorimetric detection kit (Bio-Rad) and imaged with ChemiDoc XRS+ imaging system (Bio-Rad). Band intensities were measured, and data analysis performed with Image Lab Software (Bio-Rad).

ELISA, cytokines assays, lysozyme ELISA kit and enterotoxins (LPS, LTA, SAE) was purchased from R&D Systems, BD Biosciences (www.bdbiosciences.com), Cell Technology LTD (www.immunospot.com), Abcam (http://www.abcam.com), ThermoFisher Scientific and Sigma-Aldrich (www.sigmaaldrich.com). Specificity of antibodies was tested using positive and negative controls provided by companies and available in the laboratories of PIs in the respective assays.

Colectomy tissue samples from IBD patients

In order to carryout proteomic and gene tissue profiling of differentially expressed proteins/genes in IBD, we first sought ethical approval from the Meharry Medical College (IRB file #: 100916AM206) and Vanderbilt University Medical Center (IRB file #s: 080898 and 100581) Institutional Review Boards. Informed consent was provided, and patient participation in the study was voluntary. Samples were obtained from the NIH-funded Digestive Disease Research Center (PI: David A. Schwartz), the Vanderbilt Gastrointestinal Biospecimen Repository, and the Cooperative Human Tissue Network at Vanderbilt University Medical Center (PI: Mary K. Washington), in Collaboration with the Meharry Medical College Human Tissue Acquisition Shared Resource Core (PI: Billy R. Ballard) [11,12]. How discovery of DEFA5 as a potential diagnostic biomarker for determining whether a patient suffering from IBD has UC or CC is previously described [5]. Twenty-one (#21) de-identified adult patient samples were identified. These samples comprised of endoscopy pathology biopsy tissues with inconclusive IC diagnosis at Vanderbilt University Medical Center (VUMC) between 2000 and 2007, and were reevaluated for disease course in 2014 (after a mean surveillance follow-up, 8.7±3.7 (range, 4–14) years, to identify the rates of diagnosis resolution to clinically authentic CC or UC. Diagnosis of each patient was determined based on standard clinical and pathologic features as previously described [13,14] by three gastrointestinal pathologists blinded to clinical diagnosis reconciled and confirmed colitis diagnosis for each patient and represented a consensus among treating physicians. Patients who clinically did or did not changed and maintained the IC diagnosis were tested simultaneously via IHC and NEARAS for DEFA5 quantitative cell staining to determine if DEFA5 could be used to identify CC versus UC and IC cohort into UC or CC. For each selected sample, medical records data on patient demographics, medical and surveillance of endoscopy and clinical findings history were retrieved and reviewed retrospectively. Samples included in all experiments were taken from various parts of the colon; all inflamed tissue unless otherwise indicated. The availability of a detailed IBD patient database registry at VUMC made chart review and follow-up surveillance possible.

Immortalized colonic epithelial cells culture and treatment with DEFA5

To establish the dose for our experiments we treated Immortalized colonic epithelial cells (NCM 460) with DEFA5 at 3 different concentrations (5, 10 and 15 μM), collected the supernatant at 6 hr. and 24 hr. and ran ELISA in triplicates for IL 6 and TNF-α (Bioligand). After looking at the results, 10 μM DEFA5 was used in all future experiments.

NCM 460 were received by a Material Transfer Agreement from INCELL Corporation (San Antonio, Texas, USA) [8]. NCM 460 cells (1x10⁶ cells) were plated in a 100 mm culture dish prior to starting the experiment. At the end of the treatment period, the cell culture supernatant was collected and frozen at –80°C until supernatants from all the treatment time-points were collected. Abcam Cytokine Antibody Array (catalog no. ab133998) was used for the simultaneous detection of multiple cytokines following the manufacturer’s protocol. Briefly, the cytokine array membranes were blocked by incubating with 2 ml 1X blocking buffer at room temperature (RT) for 30 min. Blocking buffer was aspirated, and undiluted cell culture supernatants (1 ml) were added to each well and incubated overnight at 4°C. After sample incubation, membranes were thoroughly washed and incubated with 1 ml of 1X Biotin-Conjugated Anti-Cytokines overnight at 4°C. Next, the membranes were washed again and incubated with 2 ml of 1X HRP-Conjugated Streptavidin for 2 hr. at RT. The membranes were then washed, and the cytokines expressed were detected by chemiluminescence.

Human colonoids culture and treatment with DEFA5

Colonoids have been used previously to study the interaction between intestinal pathogens and intestinal epithelium [15–19]. In this study, human colonoids were purchased from Cellesce Limited (Medicentre, Cardiff, United Kingdom) [7]. Colonoid culture was performed as per instructions provided by the manufacturer (Cellesce, Cardiff, UK). Two thousand (#2000) colonoids per well were plated in a 12-well plate. After 2 days of growth in complete medium, the colonoids were treated with purified Human DEFA5 (5 ug/ml) for 6 hr. and 24 hr. The control colonoids were not treated. At the end of the treatment period, the colonoids culture supernatant was collected and frozen at –80°C until supernatants from all the treatment time-points were collected. Abcam Cytokine Antibody Array (catalog no. ab133998) was used for the simultaneous detection of multiple cytokines following the manufacturer’s protocol. Briefly, the cytokine array membranes were blocked by incubating with 2 mL 1X blocking buffer at room temperature (RT) for 30 min. Blocking buffer was aspirated and undiluted cell culture supernatants (1ml) were added to each well and incubated overnight at 4°C. After sample incubation, membranes were thoroughly washed and incubated with 1ml of 1X Biotin-Conjugated Anti-Cytokines overnight at 4°C. Then the membranes were washed again and incubated with 2 ml of 1X HRP-Conjugated Streptavidin for 2 hours at room temperature. The membranes were then washed, and the cytokines expressed were detected by chemiluminescence.

Human colonoids culture and treatment with bacterial enterotoxins

Colonoid culture was performed as per instructions provided by the manufacturer (Cellesce, UK). Two thousand colonoids per well were plated in a 12-well plate. After 2 days of growth in complete medium, the colonoids was treated with enterotoxin following a time course for up to 2 wks. At each time point, we collected both the colonoids and the culture supernatants. Then, we determined the expression and/or secretion of DEFA5 by ELISA. Untreated colonoids served as control. We also seeded the colonoids at low density in triplicates and allow them to attach overnight. We then treated one set with enterotoxin and leave the other set untreated. We allow the colonoids to form colonies and then, we stained the colonies with anti-DEFA5 antibody to determine if the enterotoxin treatment leads to differentiation into CCLCs. We then determined the expression of DEFA5 and the secretion of DEFA5-induced signatures (cytokines/chemokines/growth factors) by ELISA. At the end of the treatment period, the colonoids culture supernatant was collected and frozen at –80°C until supernatants from all the treatment time-points were collected. Equal numbers of colonoids were treated with/without enterotoxins (LPS, catalog no. L7770, concentrations—1μg/ml); LTA, catalog no L4015, concentrations– 20 μg/ml; and SAE, CAS Number 11100-45-1, concentrations—1 μg/ml) following a time course for up to 14 days. The experiments were repeated twice. At each time point, we collected both the colonoids and the spent medium and determined the expression and/or secretion of DEFA5 by ELISA and IHC staining for lysosomes. We also seeded the colonoids at low density in triplicate, allowed to attach overnight, and then treated one set with enterotoxins and the other set without and allowed the colonoids to form colonies for up to 14 days. Colonies were stained with anti-DEFA5 antibodies to determine whether: (1) the treatment lead to differentiation into CCLCs; and (2) the expression and/or secretion of DEFA5 by ELISA, as well as other signatures, occurred. For colonoid experiments with DEF5A duplicates were used, we only had enough colonoids to do duplicates. The catalog numbers: IL 6—Biolegend Catalog no 430508, TNF-a—Biolegend Catalog no 430201, and DEFA5—Origene Catalog no TP310219. We have indicated these changes in the methods section of the manuscript.

Lysozyme assay procedure

The assay was performed using the lysozyme ELISA kit from Abcam (catalog no. ab108880) per manufacturer’s instructions. Briefly, 50 μl of the standards and samples were pipetted in appropriate wells of the 96-well plate and incubated at RT for 2 hr. Next, the plate was washed five times with 200 μl of the 1X wash buffer, and 50 μl of the biotinylated lysozyme antibody was added to each well and incubated for 1 hr. at RT. The plate was again washed five times with wash buffer, and 50 μl of 1X SP conjugate was added to each well. The plate was again incubated for 30 min at RT. After washing the SP conjugate, 50 μl of chromogen substrate was added to each well, and the plate was incubated for another 20 min. The reaction was stopped by adding 50 μl of stop solution to each well, and the plate was read at 450 nm. The standard curve was generated from the obtained results, and the concentration of lysozyme in unknown samples was calculated using the standard curve.

DEFA5 association with ulcer-associated cell lineage (UACL)?

We examined the association between injury to the mucosae of the IBD colon and emergence of UACL and/or CCLCs as an active reparative and/or defensive response to ulceration and/or enterotoxins. We used mild disease activity of CC formalin-fixed paraffin-embedded (FFPE) tissue sections for MUC6 vs. DEFA5 IHC staining. Tissue IHC for DEFA5 detection antibody was accomplished as previously described [5] and for Mucin 6 (CLH5: sC-33668) IHC was done according to the instructions provided by the manufacturer (Santa Cruz Biotech, Inc).

Bacterial toxins information

Circumventing IC into authentic CC or UC

To determine if DEFA5 could be used to assess whether IC patients could be delineated into a diagnosis of either UC or CC instantly, we assessed levels of DEFA5 in surgical pathology colon samples using IHC. in all these 21 IC patients, in each instance of a final diagnosis of CC, DEFA5 high NEARAS counts agreed with that diagnosis, Fig 3A & 3B. We also found that when the 6 patients that remained clinically and histologically inconclusive (by an attending gastroenterologist and by 3 blinded pathologists) and retained unchanged IC diagnosis were analyzed using DEFA5 IHC NEARAS profile counts, Fig 3A. Three patients (n = 3) showed high DEFA5 count and agreed with the final diagnosis of CC (red front circle) and another 3 patients had low DEFA5 count and were in agreement with the final diagnosis of UC (green front circle). Further, bioinformatically, a fit logistic model depicts group CC vs. UC as the outcome and DEFA5 as independent variable discriminator biomarker, Fig 3C. The R2 of the model fit is 1 and area under the ROC curve is 1. The area under the ROC curve was not plotted since it will look similar as the orange curve in ROC plot.

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Fig 3. Multivariable logistic regression model for probability of being CC.

A, twenty-one (#21) IC patients were followed for 8.7±3.7 (range, 4–14) years. Although most IC resolved to UC or CC, but the mean diagnostic delay was 7 (range, 4–14) years. 28.5% of the patients could still not be diagnosed into authentic UC or CC. The mean area fraction of DEFA5 count (%) by NEARAS IHC staining agrees with final diagnostic outcome of every IC patient samples tested. B, Clinical data of these 21 IC patients who correlate with 3A data could have been diagnosed using DEFA5 immunoreactivity during the first endoscopy biopsy to avoid diagnosis delay. C, Bioinformatic data depicting a fit logistic model with group CC vs. UC (in 3A) as the outcome and DEFA5 as independent variable discriminator. The R2 of the model fit is 1 and area under the ROC curve is 1. The area under the ROC curve was not plotted since it will look similar as the orange curve in ROC plot. Three statistical methods were used: (1) Univariate analysis: Mann Whitney U test between two groups for each peak, adjusting p-values by FDR; (2) LASSO; and (3) Elastic net. As the scale of all measures were small, we multiplied all values by 10,000.

https://doi.org/10.1371/journal.pone.0246393.g003

Bacterial enterotoxins treatment on immortalized colonic epithelial cells (NMC460) and colonoids

We observed that DEFA5 was significantly higher in lysate during the end of the first week of treatment with LPS and LTA compared with controls (Fig 4A) and correlated to enterotoxins and duration of treatment, P< 0.0001. The reverse was true in the supernatant (Fig 4B). During the second week of SAE treatment, DEFA5 in supernatant was increased 3-fold, P< 0.0001 and was not detected (nd) in the lysate (Fig 4B). This suggests that immune stimuli are a stem cell niche precursor for DEFA5-secreting CCLCs expansion. Lysozyme analysis of the supernatant tested positive, an indication that CCLCs have differentiated (Fig 4C). In patients with IBD, this hypothesizes that bacteria are enriched at the inflamed colonic mucosal surface. The complex relationship between microbial distribution and its influence on the host has yet to be elucidated. In that light, we recently developed colonoid culture to reveal the mechanistic basis for genetic susceptibility to CC, which will also provide a diagnostic platform for therapeutics, including individualized therapeutics, and understanding the effects of immune and microbial stress on epithelial barrier function.

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Fig 4. Treatment of colonoids with bacterial enterotoxins.

A–B, Equal numbers of colonoids were treated with/without enterotoxins (LPS, LTA, SAE) following a time course of up to 14 days. At each time point, we collected both the colonoids and the spent medium and determined the expression and/or secretion of DEFA5 by ELISA and IHC staining for lysozymes. We also seeded the colonoids at low density in triplicate, allowed to attach overnight, and then treated one set with enterotoxins and the other set without. We allowed the colonoids to form colonies for 14 days. Colonies were stained with anti-DEFA5 to determine whether the treatment led to differentiation into apparent CCLCs. The expression and/or secretion of DEFA5 and proinflammatory cytokines, etc. was determined by ELISA. C, Lysozyme assay: The assay was performed using lysozyme ELISA kit firm abcam (catalog no. ab108880 as per manufacturer’s instructions.

https://doi.org/10.1371/journal.pone.0246393.g004

DEFA5 treatment on immortalized colonic epithelial cells and colonoids

Immortalized colonic epithelial cell is an epithelial cell monolayer lining of the colon while a colonoid cell is a miniaturized and simplified version of a colon organ produced in vitro in three dimensions that shows realistic micro-anatomy. Treatment of DEFA5 on immortalized colonic epithelial cells and colonoids induced expression of various subtype signatures (Fig 5A–5C). We graphically present the cytokines, chemokines, and growth factors (signatures) measured on immortalized colonic epithelial cells and colonoids at 24 hr. We observed that at 24 hr., certain signatures were disparately secreted between the immortalized colonic epithelial cells and colonoids, specifically MIP-3α, TNF-β, MIF, IFN-γ, and IL-1β; MIP-1D, GRO, IL-8, Eotaxin, TARC; EGF, TIMP-2, TIMP-1, and Angiogenin (Fig 5A).

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Fig 5. Treatment of immortalized colonic epithelial cells and colonoids with DEFA5.

We graphically present the data on the signatures for immortalized colonic epithelial cells and colonoids separately. We did not perform statistical tests on these data. Immortalized colonic epithelial cells (NCM 460) were received by a Material Transfer Agreement from INCELL Corporation (San Antonio, Texas, USA) [8]. NCM 460 cells (1x10⁶ cells) were plated in a 100 mm culture dish prior to starting the experiment. At the end of the treatment period, the cell culture supernatant was collected and frozen at –80°C until supernatants from all the treatment time-points were collected. Abcam Cytokine Antibody Array (catalog no. ab133998) was used for the simultaneous detection of multiple cytokines following the manufacturer’s protocol. Colonoids were purchased from Cellesce Limited (Medicentre, Cardiff, UK). Organoid culture was performed as per instructions provided by the manufacturer (Cellesce). Two thousand colonoids per well were plated in a 12-well plate. After 2 days of growth in complete medium, the colonoids were treated with purified Human DEFA5 (5 μg/ml) for 6hr. and 24hr. The control colonoids were not treated. At the end of the treatment period, the colonoids culture supernatant was collected and frozen at –80°C until supernatants from all the treatment time-points were collected. The experiments were repeated twice. At each time point, we collected both the colonoids and the spent medium and determined the expression and/or secretion of DEFA5 by ELISA and IHC staining for lysozymes. We also seeded the colonoids at low density in triplicate, allowed to attach overnight, and then treated one set with enterotoxins and the other set without and allowed the colonoids to form colonies for up to 14 days. Cytokines, chemokines, and growth factor measurements (signatures) are depicted: A, 24hr measurement; B, (24hr measurement) minus (6hr measurement) and C, (24hr measurement) minus (Control 24hr measurement) respectively.

https://doi.org/10.1371/journal.pone.0246393.g005

At 24 hr. vs. 6 hr. measurement analyses, we further observed a wider range of intensity secretion disparity especially for MIP-3α, MIF, IFN-γ, IL-1β, TNF-α, Oncostatin M, IL-15, GM-CSF, TNF-β, and IL-16; MIP-1D, GRO,Eotaxin-2, IL-8, RANTES, GRO-α, MCP-1, TARC, ENA-78, MIP-1β, MIG, SDF-1; EGF, TIMP-2, TIMP-1, Angiogenin, PDGF-BB, BDNF, VEGF, IL-3, Osteopontin, Osteoprotegerin, IGF-I, and FGF-9 (Fig 5B).

Finally, we calculated the cytokines differential expression, chemokines, and growth factors measured on immortalized colonic epithelial cells or colonoids and control at 24 hr. (Fig 5C). Fig 5C depicts disparity at 24 hr. vs. control 24 hr. measurements, MIP-3α, TNF-β, MIF, IL-1β, IL-15; MIP-1D, Exotaxin-2, GRO, TARC, IL-8; EGF, TIMP-2, TIMP-1, and Angiogenin. Among these, GM-CSF (a stem-cell stimulator that works to produce granulocytes and monocytes. It is interesting that this cytokine is increased in an area where we are seeing differentiation of stem cells in IBD. Eotoxin-3, and IL-10 were the most highly secreted in colonoids. This disparate release of inflammatory proteins is intriguing, given the important role of defensins in regulating the immune system.

Our study finds the high expression levels of DEFA5 in colonoid cells treated with bacterial enterotoxins, which can be found in patients with CC with presence of enriched bacterial enterotoxins. Cytokines differential expression observed in colonoids and their disparate are depicted Fig 5A–5C.

Presented conferences

In parts, The Annual Congress of The American Society of Colon and Rectal Surgeons, Hollywood, Florida May 2–6, 2009, Receiving Award for best Basic Science Poster Presentation, The New Jersey Society of Colon and Rectal Surgeons; at Annual Congress of The Digestive Disease Week, New Orleans, LA, May 2–5, 2010; at The Annual Congress of The American Society of Colon and Rectal Surgeons, Minneapolis, MN, May 15–19, 2010; at The 102^nd Annual Congress, the American Association for Cancer Research, Orlando Florida 2–6 April, 2011; at the Center to Reduce Cancer Health Disparities Conference, Bethesda North, Rockville, MD, July 12–15, 2011; at NCI Translational Science Conference “From Molecular Information to Cancer Medicine”, Washington, DC, July 28–29, 2011; at The 103^nd Annual Congress, the American Association for Cancer Research, Chicago, IL 31 March– 4 April, 2012; at AACR Special Conference on Molecular Targeted Therapies: Mechanisms of Resistance, San Diego, CA May 9–12, 2012; at the Fifth AACR Conference on The Science of Cancer Health Disparities San Diego, CA October 27–30, 2012; at The Annual congress of The American Society of Colon and Rectal Surgeons, 30 April–4 May, 2016, Receiving Award for Best Clinical Podium Presentation, The New England Society of Colon and Rectal Surgeons, Los Angeles, CA; and at Keystone Symposia on Molecular and Cellular Biology, Host and the Environment in IBD: Scientific Advances Leading to New Therapeutics (A2), Taos, New Mexico, Jan 13–17, 2019.