Investigation of Inflammation and Tissue Patterning in the Gut Using a Spatially Explicit General-Purpose Model of Enteric Tissue (SEGMEnT)

The mucosa of the intestinal tract represents a finely tuned system where tissue structure strongly influences, and is turn influenced by, its function as both an absorptive surface and a defensive barrier. Mucosal architecture and histology plays a key role in the diagnosis, characterization and pathophysiology of a host of gastrointestinal diseases. Inflammation is a significant factor in the pathogenesis in many gastrointestinal diseases, and is perhaps the most clinically significant control factor governing the maintenance of the mucosal architecture by morphogenic pathways. We propose that appropriate characterization of the role of inflammation as a controller of enteric mucosal tissue patterning requires understanding the underlying cellular and molecular dynamics that determine the epithelial crypt-villus architecture across a range of conditions from health to disease. Towards this end we have developed the Spatially Explicit General-purpose Model of Enteric Tissue (SEGMEnT) to dynamically represent existing knowledge of the behavior of enteric epithelial tissue as influenced by inflammation with the ability to generate a variety of pathophysiological processes within a common platform and from a common knowledge base. In addition to reproducing healthy ileal mucosal dynamics as well as a series of morphogen knock-out/inhibition experiments, SEGMEnT provides insight into a range of clinically relevant cellular-molecular mechanisms, such as a putative role for Phosphotase and tensin homolog/phosphoinositide 3-kinase (PTEN/PI3K) as a key point of crosstalk between inflammation and morphogenesis, the protective role of enterocyte sloughing in enteric ischemia-reperfusion and chronic low level inflammation as a driver for colonic metaplasia. These results suggest that SEGMEnT can serve as an integrating platform for the study of inflammation in gastrointestinal disease.

fate is quickly chosen, and as the cell exits the proliferative zone of high Wingless-related integration site (Wnt) activity, it differentiates. The cell then continues to climb the villus (directed by the EphB receptor-Ephrin B ligand gradient) until it de-attaches from the base membrane and undergoes anoikis, a special type of apoptosis [89,90]. Intestinal Stem Cells (ISC): Each crypt contains 4 ISCs in an azimuthally symmetric distribution located 4 cell lengths above the base of the crypt [32]. In order to maintain homeostasis, ISC's divide asymmetrically approximately every 2 hours. If an ISC undergoes necrosis, it typically leads to the total death of the crypt as SEGMEnT lacks a mechanism for ISC renewal.
Transit Amplifying (TA) Cells: Undifferentiated cells follow a decreasing EphB receptor gradient as they migrate up the crypt with a speed of approximately 15 µm/hr. TA cells divide probabilistically with P (divide) = 0.15/hr, thus, a TA cell created by an ISC will divide an average of approximately four times as it traverses 25 cell lengths (1 cell length = 10 µm). This corresponds to a crypt that has a depth of 300 µm, typical in the human ileum. TA cells are twice as susceptible to necrosis as differentiated enterocytes.
Enterocytes: Differentiated enterocytes follow an increasing Ephrin-B ligand gradient as they migrate up the villus. Enterocytes' sole function is to interact with the signaling network. Enterocytes respond to inflammatory signals with 0.25 the strength of an inflammatory cell.
Neutrophils, Macrophages: Upon exposure to damage-associated molecular pattern molecules (DAMPs), blood vessels start secreting neutrophils and macrophages. Every 15 minutes, a neutrophil or macrophage has the opportunity to adhere to the tissue outside of the blood vessel with a probability based on the amount of DAMP present:

Implementation of Signaling Networks
Absolute measurements of signaling molecules are not required in SEGMEnT; relative chemokine concentrations dictate the system's behavior, therefore, all signaling molecules are measured in arbitrary units. Figure S1) This mechanism prevents infinite crypt growth with a self-stimulating Wnt source.

Morphogenesis Pathways (see Blue Components in
A cell's immediate response to Wnt signaling is the inactivation of its β-catenin  Hh signaling also provides a redundant apoptotic regulatory mechanism to assist the PTEN pathway. Hh activity induces transforming growth factor beta (TGF-β) activity [58], which leads to the production of SMAD3, a known apoptotic agent [84][85][86].
Hh receptors are considered to exist in abundance, and thus a cell can receive an arbitrary amount of Hh signal. When 1 u of Hh molecule complexes with its associated receptors, the cell begins the transcription for 1 u of SFRP1, and causes the immediate release of 1 u of TGF-β, which leads to the transcription of 1 u of SMAD3. Figure S1)  [89,90]. By regulating apoptosis, the PTEN/PI3K pathway also controls villus height. We posit that PTEN is up-regulated by inflammation (for which we use STAT3 as a proxy), and it is this up-regulation that is responsible for the known down-regulation of Hh [101]. RIP Kinase: RIP Kinase is another molecule that plays contradictory roles in the model. RIP1 induces cell death through necrosis, but at the same time up-regulates NF-κB expression, increasing a cell's survivability [94,95]. RIP is secreted in response to TNF exposure or TLR4 signaling [94]. RIP binds to TNF receptors and in doing so change the form of cell death from apoptosis to necrosis [96]. Biologically, the polyubiquitination of RIP is induced upon RIP binding to the TNF receptor, TNFR1. This causes the activation of NF-κB. When this ubiquitination is reversed, the functionality of RIP is changed to promote cell death through necrosis [95,97]. 1 u of RIP prevents the transcription of 1 u of NF-κB. 1 u of RIP has a necrotic contribution of 1 (when combined with an equal amount of TNF-α. 1 u of ROS has a necrotic contribution of 10.

Inflammatory Pathways (see Orange Components in
When an enterocyte has a necrotic value of 100, it undergoes necrosis. Interleukins: IL-6, IL-10, IL-13, and IL-15 are represented in the model. This class of molecules is secreted primarily by inflammatory cells in response to TLR4 signaling. IL-10 acts as an anti-inflammatory agent by inhibiting the activity of TNF-α. IL-10 signaling is regulated by TNF as well as TLR4 signaling [62]. IL-6 disposes a cell towards apoptosis by up-regulating the expression of STAT3 [98,102]. IL-13 is also stimulated by TNF signaling and encourages apoptosis [103]. IL-15 prevents apoptosis through an up-regulation of IFN-γ [104]. IL-6, IL-10, IL-13, and IL-15 are secreted at a constant rate (unless modified by external signaling) of 2u/hr by inflammatory signals in response to TLR4 signaling.

Signaling Time Delays:
The delay from signaling event to its effect is a result of transcriptional, translational, and transportation delays [105,106]. Experiment is combined with accepted measurements regarding these delays to estimate signaling time delays. mRNA is transcribed at a rate of 20 nucleotides/sec; there is an approximately 5 minute delay as the RNA is transferred from the nucleus to the cytoplasm; the translation rate when synthesizing the target protein is 6 nucleotides/sec; each intron requires 5-10 minutes to remove. As these well-known delays are not sufficient to reproduce experiment, an experimental delay from the Wnt signaling event to β-catenin related transcription of 5-8 hours is added. Delays used in SEGMEnT are shown in Table 3.

Diffusion of Secreted Mediators
SEGMEnT implements a 1 st order discrete approximation to the diffusion equation: !"(!,!)
As we are on a 2d grid, this becomes: where is the diffusion constant. We have adopted the notation !!!,! = + ∆ , , . This leads to the final approximation: We approximate the diffusion constant for all molecules to be 100 µm 2 s -1 , equal to that of BMP-2 molecule [107].

Simulation Details
A 0.64 mm 2 section of ileal tissue comprising 8 crypts and 8 villi was simulated for all simulation experiments.

Wnt Inhibitor
SEGMEnT simulates a Wnt inhibitor by decreasing the amount of Wnt produced by the Wnt source at the base of the crypt by 25% per hour, decreasing the amount of Noggin produced at the Wnt source by 25% per hour, and eliminating the ability of intestinal cells to have self-stimulating Wnt activity.

Hh Inhibitor
SEGMEnT simulates an Hh inhibitor by removing the ability to synthesize Hh from the differentiated enterocytes. This allows the crypt to grow by eliminating the Wnt inhibitor, SFRP1. Villus height is maintained by the redundant PTEN apoptosis pathway.

PTEN Inhibitor
SEGMEnT simulates a PTEN inhibitor by removing the ability to synthesize PTEN from the differentiated enterocytes. This forces the Hh pathway to become the sole regulator of villus height.

Local Mucosal Wound
To simulate tissue injury/trauma SEGMEnT applies 10 u of DAMP and 10 u of ROS to the villi in the simulation. This causes the immediate necrosis of all affected cells. Blood vessels register the inflammation, causing macrophages and neutrophils to activate and begin to clear the necrotic tissue.

Intestinal Ischemia/Reperfusion
Ischemia is represented by deactivating all blood vessels in the affected tissue; this results in a 70% decrease in ISC proliferation, a 90% decrease in TA cell proliferation, a 10% increase in inflammatory potential per hour in GEC's. During the ischemic period, TA cells and differentiated enterocytes increase their inflammatory response [108] such that their response has 0.8 the strength of an inflammatory cell. Crypts rapidly shrink after the onset of ischemia, due primarily to the retarded proliferation [108][109][110]. Ischemic GECs produce and secrete fatty acid binding protein (FABP). This compound accumulates during the ischemic period, and upon reperfusion acts to initiate sloughing of associated GECs [55]. Upon reperfusion ischemic cells produce a series of pro-inflammatory factors (platelet activating factor (PAF), interleukin-8 (IL-8) and various endothelial adhesion factors) currently aggregated as "neutrophil aggregation factor," that attracts and activates neutrophils and initiates the inflammation seen with reperfusion. In the absence of sloughing the ischemic/necrotic GECs remain on the villus for a longer length of time, continuing to secrete damage associated molecular pattern molecules (DAMPs) and neutrophil aggregation factors in response to the reperfusion inflammation until they have been removed via phagocytosis. Inflammatory cytokines thus generated build up in the crypt until the undifferentiated cells begin to necrose. After a critical mass of cells has necrosed, the inflammation becomes self-sustaining (i.e., "cytokine storm"). Upon reperfusion, proliferation rates return to normal and the system enters a period of crypt hyperplasia as there are fewer differentiated cells to antagonize the Wnt differentiation pathway through Hh. As the villus regrows, the crypt returns to a normal size and the system returns to homeostasis.

Colonic Metaplasia of the Ileum
Simulation of the pathogenesis of colonic metaplasia in SEGMEnT is based on the hypothesis that there is a chronic low-level inflammatory stimulus to the enteric mucosa [27], and implemented as the presence of DAMP as a stimulus to GEC TLR4 receptors.
10 u of DAMP are applied to each cell on the villus, and this is maintained indefinitely.