REGULATION OF INTESTINAL EPITHELIAL
BARRIER AND IMMUNE FUNCTION BY
ACTIVATED T CELLS
By NGA LE
Submitted in partial fulfillment of the requirements for the degree of
Doctor of Philosophy
Dissertation Advisor: Alan Levine, Ph.D.
Department of Molecular Biology and Microbiology
CASE WESTERN RESERVE UNIVERSITY
January 2021
CASE WESTERN RESERVE UNIVERSITY
SCHOOL OF GRADUATE STUDIES
We, the thesis committee, hereby approve the thesis/dissertation of
NGA LE
Candidate for the PhD degree*.
Committee Chair John Tilton
Committee Member Henry Boom
Committee Member Donald Anthony
Committee Member Jerrold Turner
Thesis Advisor Alan D. Levine
Date of Defense 08/10/2020
*We also certify that written approval has been obtained for any proprietary material contained therein.
2 Table of Contents
Table of Contents ...... 3
List of Figures ...... 5
Abstract ...... 7
I. INTRODUCTION ...... 9
1.1. Gastrointestinal (GI) tract and epithelial cells ...... 9
1.2. Tight Junctions (TJ) ...... 14
1.3. Cytokines and Intestinal Epithelial Function...... 17
1.4. Mucosal T cells ...... 18
II. MATERIALS AND METHODS ...... 24
III. RESULTS ...... 31
3.1. Polarized IECs attract the migration of activated T cells ...... 31
3.2. Persistently-activated T cells disrupt the permeability of an IEC monolayer ...... 32
3.3. Defining the activation status of pulse- and persistently-activated T cells 38
3.4. Exposure of an epithelial monolayer to activated T cells regulates the expression of TJ protein and mRNA ...... 44
3 3.5. Structural changes to the TJ complex due to activated T cell engagement ...... 51
3.6. Contribution of epithelial cell morphology to paracellular permeability ...... 58
3.7. Reciprocity in the mucosa: IECs after contact with activated T cells may modulate innate and adaptive inflammatory responses ...... 61
IV. DISCUSSION ...... 64
4.1. Initial contact with activated T cells strengthens barrier integrity (Phase 1)
67
4.2. Long-term communication with activated T cells yields dichotomous
changes in barrier permeability depending on the character of the immune
stimulation (Phase 2) ...... 69
4.3. Communication with activated T cells modulates the immune properties of
the epithelium and vice versa ...... 71
V. CONCLUSION AND FUTURE DIRECTION ...... 75
5.1. Primary IEC monolayer ...... 77
5.2. Mucosal T cells and commensal pathogens ...... 78
5.3. Communication via exosomes/microvesicles (extracellular vesicles; EVs) 79
BIBLIOGRAPHY ...... 82
4 List of Figures
Figure 1: The anatomy of the GI tract including related organs...... 10
Figure 2: The anatomy of the gastrointestinal mucosa in small intestine and colon ...... 12
Figure 3: Classification of IELs ...... 22
Figure 4: Three-dimensional co-culture model to study the interaction between T cells and IECs ...... 34
Figure 5: Activated T cells modulate the pore and leak permeability pathways of an intestinal epithelial cell monolayer in two-dimensional co-culture model ...... 35
Figure 6: Activated T cells modulate the pore, leak and unrestricted paracellular permeability pathways of an intestinal epithelial cell monolayer...... 37
Figure 7: Expression of surface proteins and cytokines by pulse- and persistently-activated T cells after co-culture with an intestinal epithelial monolayer...... 39
Figure 8: Co-culture of T cells with an epithelial monolayer modulates the T cell subsets, the expression of gut homing markers and cytokines differently based on the activation status of the T cells ...... 42
Figure 9: Exposure of an epithelial monolayer to activated T cells modestly regulates the expression of TJ mRNA and proteins ...... 46
Figure 10: Activated T cells reduce the height of an epithelial monolayer in Phase 1 and modulate the arrangement of TJ strands and the location TJ proteins ...... 48
Figure 11: Activated T cells modulate the arrangement of TJ strands and the location CLDN1 ...... 49
Figure 12: Activated T cells reduce the height of an epithelial monolayer in Phase 1 and modulate the arrangement of TJ strands and the location of CLDN4 ...... 50
Figure 13: Activated T cells modulate the arrangement of TJ strands and the location CLDN2 ...... 52 Figure 14: Activated T cells reduce the height of an epithelial monolayer in Phase 1, as revealed by staining for OCLN ...... 54
Figure 15: Activated T cells increase the size of the cells within an epithelial monolayer in both Phases 1 and 2, as revealed by staining for ZO-1 ...... 55
5 Figure 16: Co-culture of an epithelial monolayer with persistently-activated T cells results in a loss of actin from the microvilli and stimulates a diffuse distribution of CLDN1 from the apical to the basolateral surface in Phase 2 ...... 57
Figure 17: Co-culture with pulse- and persistently-activated T cells increase the size of IECs within a monolayer and persistently-activated T cells induce epithelial cell death ...... 60
Figure 18: IECs in contact with activated T cells may modulate innate and adaptive inflammatory responses...... 63
Figure 19: The summary of the regulation of T cells on epithelium ...... 66
Figure 20: Changes in IEC barrier permeability is dependent on T cell concentration and independent of CD4+ versus CD8+ T cell subsets ...... 76
Figure 21: Effect on EVs on the IEC barrier...... 81
6 Regulation of Intestinal Epithelial Barrier and Immune
Function by Activated T Cells
Abstract
By NGA LE
Background and Aims: Communication between T cells and the intestinal epithelium is altered in many diseases, causing T cell activation, depletion, or recruitment and disruption of the epithelium. We hypothesize that activation of T cells regulates epithelial barrier function by targeting the assembly of the tight junction complex.
Methods: In a 3-dimensional and 2-dimensional co-culture model of activated T cells subjacent to the basolateral surface of an epithelial monolayer, the pore, leak, and unrestricted pathways were evaluated using transepithelial resistance and flux of fluorescently-labelled tracers. T cells were acutely and chronically activated by cross-linking the TCR. Tight junction assembly and expression were measured using qPCR, immunoblot, and immunofluorescence confocal microscopy.
Results: Co-culture with acutely and chronically activated T cells decreased the magnitude of ion flux through the pore pathway, which was maintained in the presence of acutely-activated T cells. Chronically activated T cells after 30 h
7 induced a precipitous increase in the magnitude of both ion and molecular flux, resulting in an increase in the unrestricted pathway, destruction of microvilli, expansion in cell surface area, and cell death. These fluctuations in permeability were due to changes in the assembly and expression of tight junction proteins, cell morphology, and viability. Co-culture modulated the expression of immune mediators in the epithelium and T cells.
Conclusion: Bi-directional communication between T cells and epithelium mediates a bi-phasic response in barrier integrity that is facilitated by the balance between structural proteins partitioning in the mobile lateral phase versus the tight junction complex and cell morphology.
8 I. INTRODUCTION
1.1. Gastrointestinal (GI) tract and epithelial cells
The lining of the GI tract is the largest barrier separating the mammalian host from the external environment. The GI tract, whose functions include digestion, absorption, excretion, and protection, consists of the mouth, esophagus, stomach, small intestine, large intestine, and anus (Figure 1). The small intestine consists of the duodenum, the jejunum, and the ileum, while the large intestine is subdivided into the cecum, the colon, and the rectum. In the small intestine, the intestinal epithelium provides a layer of absorptive enterocytes and secretory cells, forming villi (decorated with goblet cells, enteroendocrine cells, and tuft cells) and crypts
(decorated with Paneth cells). In the large intestine, mostly crypts are formed. In addition, Paneth cells are absent, and the enterocytes are replaced with absorptive colonocytes. Enterocytes and colonocytes help to absorb water, solutes and nutrients including vitamins. Enteroendocrine cells produce and release peptide hormones. Goblet cells secrete a variety of mucins, and Paneth cells function as the factory of digestive enzymes, growth factors, and antimicrobial peptides. Tuft cells, in the other hand, are linked to infectious diseases and the host immune responses [1, 2].
9
Figure 1: The anatomy of the GI tract including related organs
The human gastrointestinal (GI) tract, begins with the mouth and ends with the anus. Accessory digestive organs include salivary gland, liver, gallbladder, spleen and pancreas [3].
10 There are many physicochemical mechanisms to protect the surface of the intestine against the possible damage by the pathogens. One of them is the selectively secretion of immunoglobulins, mostly IgA, to the lumen, which originate from two main sources: (i) by serum immunoglobulins and (ii) by intestinal plasma cells. This secretion, which called transcytosis of through intestinal epithelial cells
(IECs), generates by the polymeric immunoglobulin receptor (pIgR) expressed on the intestinal cells. The secretion IgA can modulate microbial communities and pathogenic microbes through specific and non-specific pathogen interactions [4-
6].
The epithelium, a monolayer of polarized intestinal epithelial cells, serves not only as an impermeable barrier to intestinal microbiota, but also as a selectively permeable filter for nutrient absorption and waste secretion [7]. The IECs are held together by tight junctions (TJs), adherens junctions, desmosomes, and gap junctions. While most junctions merely connect cells to each other, the TJs form a tight seal between cells and separate the intestinal lumen from the lamina propria
(LP) where immune cells are located. Two functionally distinct zones of intestinal epithelium are recognized: (i) the proliferative zone, in which an expandable stem cells population resides, and (ii) the functional zone, in which the stem cells, called crypt base columnar cells, differentiate into terminal functional lineages. The IECs proliferate and turn over quickly. The life span of IECs is around 3-5 days.
Absorptive IECs are shed into the lumen, where they undergo apoptosis. The continuous production of new IECs in the crypt is following by the elimination of older cells, resulting in a rapid turnover of IECs [8, 9] (Figure 2). Imbalance of
11
Figure 2: The anatomy of the gastrointestinal mucosa in small intestine and colon
IECs move upwards to the crypt and when reaching the tip of the villus (small intestine) or the crypt (colon), they undergo apoptosis and are then shed to the lumen. On the contrary, Paneth cells migrate downwards to the base of the crypt to provide stem cells with growth factors. Beneath the IECs, stromal cells (myofibroblasts), and other immune cells, such as B cells, IgA-producing plasma cells, macrophages, dendritic cells and T cells reside in the lamina propria, maintaining a hyporesponsive state [10].
12 epithelial proliferation and turnover is observed in diseases such as inflammatory bowel disease (IBD).
Transportation through the epithelial barrier may occur by two routes: (i) the transcellular pathway across the plasma membranes is directional, energy dependent, and regulated by transporters and channels on cell membranes of
IECs, and (ii) the paracellular pathway across the space between cells, which shows passive diffusion, size and charge selectivity, and is different in various epithelia. These routes both contribute to the permeability of the epithelium, but the paracellular pathway is the main determinant of ion and solute leakiness, and thus is a more important contributor to overall tissue-specific transportation [11].
Permeability of an epithelium is regulated by paracellular flux through two distinct mechanisms: the pore and leak pathways [12-14]. The pore pathway is size and charge selective and can transport molecules with a maximal diameter of
∼5-10 Å [13, 15-18]. In contrast, the leak pathway is not charge selective, but is limited in capacity, allowing molecules with diameters up to 125 Å to pass [13, 14,
19]. When the epithelium is damaged, both large and small molecules, as well as microbes, can easily cross the barrier via a route termed the unrestricted pathway
[20, 21].
There are many methods to evaluate the permeability of an IEC barrier. One of the most widely-used methods is using tracer molecules of different sizes to measure the pore, leak, and unrestricted pathway in vitro and in vivo. A
13 volt/ohmmeter is used for measurements of transepithelial electrical resistance
(TER) of a cultured barrier in vitro. The equivalent resistance (Rtotal) is given by the combination of the resistance through the transcellular pathway (Rtc) and the paracellular pathway (Rpc) as: 1/Rtotal = 1/Rtc + 1/Rpc. Because the resistance of cell membranes is very high, Rtotal approximately equals Rtc.
1.2. Tight Junctions (TJ)
Intercellular tight junctions (TJ), which seal the epithelial monolayer, are the key structures that regulate paracellular intestinal permeability. Extracellular strands of TJs form a series of appositions extending in a belt-like network that surrounds each cell and attaches it to neighboring cells [22]. In this way, TJs serve as a barrier, or gate, that selectively regulates the diffusion of water, ions, and other molecules through the paracellular space. At the IEC cell membrane, the presence of the dense TJ structure also subdivides the plasma membrane into two distinct compartments (an apical and a basolateral domain) and functions as a molecular fence to maintain the polarity of the epithelial cells. In addition to their barrier function, the ability of the TJs to interact with both intracellular and extracellular components allows them to act as a platform to transfer bidirectional signals between the interior of the cells and their external environment. These signals in turn, regulate TJ assembly, function, and, consequently, affect epithelial permeability [23-26].
14 TJ assembly is Ca2+ concentration dependent and linked to cell polarity, which leads to cell surface polarization to establish of apical and basolateral domains.
Many TJ proteins interact with the actin cytoskeleton, and the regulation of its dynamics is essential for the formation and function of the cell junction. For example, myosin light chain kinase (MLCK), an actomyosin regulator, stimulates the increase of intestinal paracellular permeability by junctional remodeling and occludin internalization during inflammation [27, 28].
At the molecular level, TJs are assembled from multiple transmembrane proteins that include members of the very large claudin protein family and occludin.
Occludin and several claudin proteins help seal the intercellular space, while other claudin proteins help form the paracellular channels [29, 30]. Other integral membrane proteins, such as the zonula occludens (ZO) proteins and the tight- junction-associated MARVEL protein (TAMP), are crucial factors in the regulation and maintenance of the epithelial permeability [31-33]. However, the roles of each
TJ or TJ-related proteins are still underrepresented by literature.
Claudins are major structural components of the tight junction.
Overexpression, knockdown, knockout, or mutation of claudins, all cause changes in paracellular permeability. Similar to occludin, claudins have four transmembrane domains with a molecular weight of ∼24 kDa. The PDZ define this domain in the
C terminal of claudin interacts with the PDZ domain of ZO. Nearly 30 different human claudin isoforms have been identified and different tissues express different
15 sets of claudin isoforms [34]. In addition to their structural role, claudins also play a large role in the selectivity of the epithelium. While claudins appear to form similar structures, they can exert highly divergent effects on transepithelial resistance. For example, claudin-1, -3, -4, -5, and -6 are barrier-forming and decrease the permeability to cations, while claudin-7, -19 decrease the permeability to anions.
Claudin-2, -10b, and -15 create cation pores, while claudin-10a and -17 favor anion pores. Some structures require the interaction of different claudins. For instance, claudin-16 and -19 form cation pores, while claudin-4 and -8 form anion pores [22].
The expression of TJ proteins is tissue-specific and dependent with the cell physiology or status. During the inflammation or cancer stage, TJ proteins are deregulated and delocalized as a consequence of epigenetic factors, growth factors and cytokines [35]. The expression of TJ proteins may be regulated at the transcriptional level by a number of different factors. For example, occludin can be regulated by Ras pathway or inflammatory cytokines; and claudins can be affected by factors β-catenin/Tcf complex and Cdx homeodomain protein/hepatocyte nuclear factor-1α [25, 36-39].
The signaling through TJs to the cell interior functions to regulate gene expression, cell proliferation, differentiation and morphogenesis. In particular, ZO proteins, which can shuttle between the nucleus and plasma membrane and recruit many cytosolic molecules, play a vital role to regulating the localization of transcription factors, such as ZONAB/DbpA and AP-1, and many other proteins to control gene expression [40]. Additionally, when the density of cells reaches the
16 certain threshold, the proliferation of the IECs is inhibited through ZO protein linking to ZONAB's interactions with the cell division kinase CDK4 [41]. Therefore, mutations or aberrant signaling mechanism related to these proteins can disturb the proper cell functioning, thus are widely correlated with carcinomas and any changes like gut inflammation.
1.3. Cytokines and Intestinal Epithelial Function
Cytokines, chemokines, and immune mediators are important factors supporting homeostasis of epithelium, and therefore play a critical role in intestinal inflammation and inflammation-associated damage. Cytokines have a variety of effects on the epithelium, including (i) promoting (Tumor Necrosis Factor or TNF,
Interleukin-6 or IL-6, and IL-17) or inhibiting (Transforming Growth Factor-b or
TGF-b, Interferon type I) proliferation, (ii) supporting differentiation (IL-13, IL-4, and IL-33), (iii) regulating apoptosis (IFN, TNF, TGF-b1), (iv) regulating the integrity of the barrier (IL-6, IL-10, IL-17), and (V) modulating epithelial immune properties (TNF-α-induced IL-8 expression) [42, 43].
In some diseases, such as in Crohn’s disease, ulcerative colitis, and irritable bowel syndrome, the leaky barrier allows an increase antigen penetration into the lamina propria (LP – the connective tissue under the basal of lining epithelium).
This prompts an inflammatory response and leads to an increase in the secretion of pro-inflammatory cytokines and mediators and recruitment of circulating
17 inflammatory cells. The pro-inflammatory cytokines produced during an inflammatory response – TNF-α, IFN-γ, IL-1β – disrupt intestinal TJs and increase epithelial permeability; while anti-inflammatory cytokines, such as IL -10 and TGF-
β, may attenuate intestinal inflammation partly by preserving the TJ barrier function
[44]. Studies in vitro also show that cytokines can remodel TJs by two main mechanisms: (i) alteration of TJ composition when TJ proteins that make “tight” barriers are replaced with those with “leaky” properties, and vice versa; and (ii) contraction of actin cytoskeleton via phosphorylated myosin light chain (MLC) induced by activation of protein kinase C (PKC), which possibly alters paracellular permeability by creating small gaps between cells, and endocytosis of TJ proteins, which happen simultaneously [45-48]. Moreover, cell culture systems also reveal that some cytokines have contradictory effects on epithelial permeability when experiments are performed with different cell lines. While studies in vitro only focus on individual cytokines, it should be recognized that in some bowel diseases, the natural complexity of the cytokine environment makes it difficult to isolate the effects of inflammation and cytokines on the TJs [49].
1.4. Mucosal T cells
In addition to its function as a selectively permeable barrier, the epithelial monolayer plays a pivotal role in innate and adaptive immunity. IECs are in continuous communication with subjacent immune cells ensuring the maintenance of a healthy and immunologically protective physical barrier [50, 51]. IECs express
18 a variety of receptors for cytokines and chemokines [52]. When stimulated by pathogenic luminal microbes or inflammatory signals produced by immune cells in the underlying mucosa, IECs secrete cytokines and chemokines, demonstrating that IEC can respond to and regulate mucosal T and other immune cells.
IECs also may present antigenic peptides to both CD4+ and CD8+ T lymphocytes, thus priming the immune system by triggering and modifying the activation and differentiation of T cells, dendritic cells, and B cells [53].
Complementing the chemical and physical epithelial barrier, T lymphocytes in the underlying LP orchestrate the second line of defense against invading pathogens and maintain gut homeostasis [54]. The number of T cells present in the human intestine is greater than that in the remainder of the body. Mucosal T cells have a tolerant activated phenotype which can easily toggle from high and low thresholds of activation [55], enabling them to maintain the necessary equilibrium between effective immunity and physiological immune tolerance to preserve tissue homeostasis in the presence of commensal microflora [56, 57]. Thus, mucosal T cells support immunological protection without compromising organ integrity.
Mucosal T cell arise through the unique activation of circulating naïve T cells as they transit into intestinal lymphoid tissues, such as Peyer’s patches and mesenteric lymph nodes. These educated T cells then acquire gut homing properties through the upregulation of several adhesion molecules including integrin α4β7 and CCR9. They migrate to LP and adapt to the gut mucosa environment [58, 59].
19 Mucosal T cells form two main subsets: (i) conventional (or type a) mucosal T cells, which express TCRαβ alongside CD4 or CD8αβ, and which are mainly found in LP, and (ii) non-conventional (or type b) mucosal T cells, which express CD8αα homodimers along with either TCRαβ+ or TCRγδ, and which are more prevalent in the mucosal epithelium. Mucosal T cells could be seen as long-lived effector memory T cells. In the lamina propria lymphocyte (LPL) population, CD4+ T cells are dominant and, in addition to the helper subsets, are also enriched for both
Foxp3-expressing regulatory T cells (Tregs) and Th17 cells [57, 60].
The CD8αβ LPL and intraepithelial lymphocytes (IELs) have an activated phenotype but display reduced proliferation and inflammatory cytokine production.
Specifically, IELs are described as exhibiting an ‘activated yet resting’ phenotype.
They express high levels of expression of both effector and regulatory molecules, such as CD69, NK-inhibitory and activating receptors such as Ly49 and KIR families, cytotoxic genes such as Granzyme B, and inhibitory receptors like LAG-
3; thus, they are consequently highly cytolytic but without strong stimulators, they possess quiescence phenotype rather than productive immunity. Therefore, they are ready for an immediate response but, at the same time, their activation is highly regulated to avoid uncontrolled activity and tissue destruction [57, 60, 61].
IELs include a variety of cell types with distinct effector functions such as cytotoxic, helper, and regulatory properties. They are one of the largest populations of lymphocytes which have been intensively investigated (Figure 3)
20 but their functions remain incompletely understood. They located interspersed between epithelial cells with the ratio 1 per 10 IECs, and potentially contribute as a first line of immune defense against enteric pathogens. IELs express either
TCRαβ or TCRγδ and mostly CD8αα. The majority of TCRγδ+ IELs is predominantly associated with CD8αα+ homodimer and is very different from
TCRγδ+ T cells located in lymphoid tissues, which are primarily lack of CD8 expression. The CD8αα+ does not function as a coreceptor but instead influences their functional characteristics, influences the activation of T cells and possibly extinguishes the response to high-affinity antigens to maintain the partially activated state of these cells [62-65].
Disruption of communication between T cells and IECs is associated with many chronic inflammatory diseases, including IBD, whose pathogenesis is linked to excessive activity of mucosal T cells and a defect in tolerance mechanisms mediated by resident mucosal and infiltrating blood T cells. For instance, in Crohn's disease, T helper 1 lymphocytes, which are characterized by increased production of IL-2, IL-12 and IFN-γ, are predominant while the abolishing the production of regulatory cells is abolished [66]. Furthermore, mucosal inflammation in IBD is also accompanied by the activation and proliferation of peripheral T cells, possibly as a
21
Figure 3: Classification of IELs
TCR+ IELs can be further divided into by conventional (induced) and nonconventional (natural) subsets [62].
22 result of increased intestinal permeability, epithelial cell apoptosis, and consequent translocation of microbial products into the LP and the circulation [67]. Though it is well known that cytokines secreted by T cells can regulate the permeability of IEC barrier through TJs, and IECs can express MHC-I like /MHC-II to present antigen to T cells as well as secrete a wide range of cytokines and chemokines to target many immune cell types; the mechanism by which T cells communicate with the epithelium and maintain barrier integrity is poorly understood.
To investigate the mutual and bidirectional communication between activated T cells and IECs and identify T cell-derived inflammatory pathways that regulate paracellular permeability and modulate barrier integrity, we developed two in vitro two-dimensional and three-dimensional co-culture models, using intestinal epithelial cell monolayers and spheroid to represent the colonic barrier. Our results reveal that activated T lymphocytes initiate a biphasic response in the epithelium that initially increases barrier integrity. This observed enhanced barrier function is either maintained or rapidly reversed, depending on the activation state of the T cells. Persistently strong T cell activation leads to increased paracellular permeability by altering the balance of claudin family proteins in the TJ, disassembling the TJ, reducing IEC proliferation, and inducing cell death.
Our study reveals that the intensity and duration of interaction of the T cells and
IECs would decide the fate of the barrier. However, in the limits of the of study, the genetic pathways would lead to this consequence remains unclear.
23 II. MATERIALS AND METHODS
Caco-2 monolayer and Transepithelial Resistance (TER)
Caco-2 BBe cells, a human colorectal adenocarcinoma cell line [68], a generous gift from Dr. Turner (Harvard Medical School, Boston, MA), were grown in complete medium [RPMI-1640 (Corning, Corning, NY), supplemented with 10%
FBS (Corning), 20 mM HEPES (Genesee, San Diego, CA)] at 37°C and 5%
2 humidified CO2. Cells were seeded at 20,000 cells/transwell on 0.33 cm polyester transwell filters with 0.4 μm pores (Corning) on the underside of the transwell filter and allowed to attach for one day before the transwell filter was flipped and cultured in the normal configuration. Permeability was determined measuring transepithelial resistance (TER) with a voltohmmeter (World Precision
Instruments, Sarasota, FL). TER values were corrected by subtracting a reading obtained from an empty transwell and multiplied by the area of the filter (Ω.cm2).
Medium was replenished every 2-3 days for 2 weeks and then daily thereafter.
After reaching maturity the TER of the monolayer remained stable for 14 days during which time the experiment was completed.
Peripheral blood T cell isolation and stimulation
Human T cells were isolated from the peripheral blood of healthy donors drawn into heparin-coated tubes using the EasySep™ Direct Human T Cell
Isolation Kit (StemCell, Vancouver, Canada). T cells were rested for 24 h in complete medium before activation with anti-CD3/CD28 dynabeads
24 (ThermoFisher, Waltham, MA) for 2 d. After activation, T cells were washed once.
T cells were persistently-activated if they were used directly without removing the dynabeads, or pulse-activated if the dynabeads were removed using magnetic separation before co-culture.
Caco-2 cell – T cell co-culture
Activated T cells were suspended in complete medium and added to the upper chamber at 0.5 million/transwell. The upper and lower chambers were filled with 200 µl and 1000 µl medium, respectively. TER was measured 3-4 times during the first 24 h, then once per day. Medium was replenished daily by replacing half of the medium in the upper chamber and all of the medium in the lower chamber.
Flux through the monolayer
Monolayers were washed several times with complete medium to remove
T cells and incubated with a mixture of 3 dyes in the lower chamber (apical surface): 31.2 µM fluorescein (Sigma-Aldrich, St. Louis, MO), 78 µM CF-350- labeled 3.5 kDa dextran (Biotium, Fremont, CA), and 156 µM TRITC-labeled 70 kDa dextran (ThermoFisher). Every 2 h, 50 μl from the upper chamber (basolateral surface) was transferred to a black 96-well plate, and fluorescence intensity was measured using the Victor 3V 1420 fluorescence plate reader (Perkin Elmer,
Waltham, MA). The 50 μl of sampled medium was returned to the culture plate to maintain equilibrium. Both the ratio of fluorescein or 3.5 kDa dextran flux relative to 70 kDa dextran and the net permeability flux are presented.
25 Calculation of Apparent Permeability (Papp)
The Papp (cm/s) of each probe is calculated using the equation � = , where dQdt is permeability rate (mmol/s), A is the surface area of the filter (cm2) and C0 is the initial concentration in the donor chamber (mM)[69].
Caco-2 microspheres and dextran flux
Fully dispersed 4x103 Caco-2 BBe cells were mixed with 100 �l of cold 80%
(v/v) Matrigel and grown in a No 1.5 glass bottom 24-well plate (Mattek Corp.,
Ashland, MA) for 2-3 weeks to allow microspheres to form. Medium was refreshed every 2 days. The outside of the spheres (the basolateral surface) was exposed to
150 mM FITC-labeled 4 kDa dextran (FD4, Sigma-Aldrich) in complete medium.
Similarly sized well-developed cysts were chosen randomly for analysis without bias for their position in the Matrigel layer. Live imaging was performed on a Leica
SP8 confocal microscope in a humidified incubator at 37°C. Images were taken every 10 min. Fluorescence intensity on the luminal side (interior of the sphere) and basal medium was determined and expressed as a ratio. The change in fluorescence ratio over time was used to calculate the slope, representing the rate of dextran influx[70].
Immunoblotting
Caco-2 monolayers were washed once in situ with HBSS, and the cells were lysed and removed from the transwell filter with 50 µl RIPA buffer
(ThermoFisher), supplemented with Halt Protease and Phosphatase Inhibitor
26 Cocktail, EDTA-free (ThermoFisher). After homogenization by vortexing, the lysates were mixed with reducing sample buffer (ThermoFisher) and boiled for 5 min. Proteins were fractionated on a 12 or 14% polyacrylamide gel by electrophoresis and electrotransferred onto a PVDF membrane (Immobilon-PSQ
PVDF, Millipore Sigma, Burlington, MA). The immunoblot was developed using the same antibodies as immunofluorescence staining and IRDye 800CW donkey anti- rabbit or anti-mouse IgG (H + L) (LI-COR, Lincoln, NE). The membrane was scanned with an Odyssey Imaging system (LI-COR), and protein concentration in each band was quantified with Image Studio Lite software (LI-COR). The densitometric intensity of each target protein was normalized to the intensity of the
GAPDH loading control (mouse anti-human GAPDH clone 6C5, Millipore Sigma).
RNA expression
Caco-2 BBe monolayers were washed in situ with HBSS. Cells attached to the plastic walls of the transwell were removed before lysis buffer was applied directly to the filter. Resting, pulse-, and persistently-activated T cells were harvested from the transwells 8 h before the Phase 1 peak or 24 h before the end of phase 2 and washed with PBS, prior to RNA isolation. Total RNA was isolated using the Purelink RNA micro scale kit (ThermoFisher) and quantified by Nanodrop
(ThermoFisher). 20-40 ng of Caco-2 RNA or 100 ng of T cell RNA was copied to cDNA by reverse transcription using a High-Capacity RNA-to-cDNA Kit
(ThermoFisher). mRNA levels were quantified using the TaqMan master mix and assays (ThermoFisher) for qPCR. Relative expression was calculated using the -
27 2DDCt method based on RPLP0 as the endogenous control for Caco-2 and 18S for
T cells.
Immunofluorescence staining
Caco-2 BBe monolayers were washed twice in situ with HBSS with Ca2+
(Lonza, Walkersville, MD) and fixed with paraformaldehyde (0.5% for 30 min for
TJ proteins or 4% for 20 min for actin) at room temperature (RT) in 10 mM MES pH 6.1, 138 mM KCl, 3 mM MgCl2, 2 mM EGTA, 0.32 M sucrose buffer. The low concentration of paraformaldehyde was used to avoid antigen masking to detect
TJ proteins. After washing with HBSS, the transwell filter was cut from the transwell insert and placed into a 48 well plate for staining. Monolayers were permeabilized with 0.1% saponin (Sigma-Aldrich) in PHEM buffer (60mM PIPES, 25mM HEPES,
10mM EGTA, and 4mM MgSO4, pH 6.9) for 30 min at RT, blocked with 10% goat serum (ThermoFisher) for 1 h at RT, then incubated with either rabbit anti-claudin-
1 (SAB4200534, Sigma-Aldrich), rabbit anti-claudin-4 (ab53156, Abcam,
Cambridge, MA), mouse anti-claudin-2 (32-5600, ThermoFisher), mouse anti- occludin (clone OC-3F10, 33-1500, ThermoFisher), or anti-ZO1 (33-9100,
ThermoFisher) antibody in the presence of 0.05% saponin overnight at 4oC. The signal was revealed with either Alexa Fluor 488-conjugated goat anti-rabbit or goat anti-mouse (Jackson ImmunoResearch Laboratories, West Grove, PA) or Alexa
Fluor 647-conjugated phallodin (ThermoFisher) in 0.05% saponin in PHEM buffer for 1 h at RT. The transwell filter was washed several times with HBSS and mounted in ProLong Diamond Antifade Mountant with DAPI (ThermoFisher).
28 Imaging was performed with a Leica SP8 confocal microscope, using an oil- immersion 40x magnification objective. Z-stack images (0.05 μm thickness) were analyzed with Leica LAS X software or Fiji. Aspect ratio for the Z view was 5.7:1, automatically generated by the Leica LAS X software based on the conversion of one x-y slice to one pixel.
Flow cytometry
T cells were left untreated, harvested after 48 h of activation with anti-
CD3/CD28 dynabeads, or collected at the end of Phase 1 and Phase 2 of co- culture, washed once with PBS, and stained with the Zombie Aqua™ Fixable
Viability Kit, followed by antibody stain: PE/Cy7 anti-human CD69, Brilliant Violet
711™ anti-human CD25, FITC anti-human CD4, Alexa Fluor® 700 anti-human
CD8a, PerCP/Cyanine5.5 anti-human CD3, APC anti-human/mouse Integrin β7, all from Biolegend (San Diego, CA). Cells were fixed in 1% paraformaldehyde and analyzed using an LSRFortessa (BD) in FACS buffer (0.1% BSA, 0.05% sodium azide in PBS). Flow cytometry samples were gated on singlets via SSC, and then lymphocytes using FSC and SSC. Gates were drawn to exclude CD14+ CD19+ and dead cells, and expression levels and percent positive cells quantified using
FlowJo v10.4 software (BD).
29 Expression of active Transforming Growth Factor-b and interleukin-1b
Medium from basolateral and apical chambers was collected at the end of
Phase 1 and 2 to measure the active forms of TGFb (Biolegend) and IL-1b (R&D
Systems, Minneapolis, MN) by ELISA, following the manufacturer’s instructions.
Cell Viability
Dead cells were detected by the LIVE/DEAD™ Viability/Cytotoxicity Kit for mammalian cells after optimization (ThermoFisher).
Statistical Analysis
Statistical analysis was performed using the Brown-Forsythe and Welch
ANOVA for multiple comparisons or the Student’s t-test using Prism 8.0 (GraphPad
Software, San Diego, CA), as indicated in the legends. All values are provided as mean ± sd. P-values less than 0.05 were considered significant (ns, p > 0.05; *, p≤
0.05; **, p≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001).
30 III. RESULTS
3.1. Polarized IECs attract the migration of activated T cells
To investigate the capability of an IEC monolayer to modulate the migration of blood-derived T cells into sites of mucosal inflammation, we developed a 3- dimensional colonoid-like co-culture model. The human intestinal epithelial Caco-
2 cell subclone BBe was cultured in Matrigel to support the formation of organoid- like cysts surrounded by a single layer of polarized IECs (Figure 4A and 4B).
Human peripheral blood CD3+ T cells were activated by crosslinking the TCR with anti-CD3/CD28 beads. The epithelial culture was overlaid with a thin layer of
Matrigel containing activated T cells (Figure 4A). After two days, pulse-activated
T cells migrate into the spheroid-containing layer and establish a close and continuous interaction with the basolateral surface of the epithelial monolayer
(Figure 4C). In contrast, persistently-activated T cells migrate more slowly and after two days, only a small number were detected in the lower Matrigel layer, and few were associated with the cysts (Supplemental Movies).
• S1 https://ars.els-cdn.com/content/image/1-s2.0-S2352345X20301107-mmc1.mp4
• S2 https://ars.els-cdn.com/content/image/1-s2.0-S2352345X20301107-mmc2.mp4
• S3 https://ars.els-cdn.com/content/image/1-s2.0-S2352345X20301107-mmc3.mp4
31 3.2. Persistently-activated T cells disrupt the permeability of an IEC
monolayer
We investigated the ability of these T cells to modulate the permeability of the epithelial barrier, recording the flux of FITC-labeled 4 kDa dextran (FD4) from the basolateral surface to the cyst apical lumen by quantitative confocal microscopy.
Pulse-activated T cells induced a minimal increase in FD4 flux to the apical lumen over 3 h (Figure 4D), with no evidence of harm to the epithelium after three days
(Figure 4E). In contrast, persistently-activated T cells dramatically increased FD4 flux (Figure 4D), explained by partial breakdown in cyst structure, decreased translucence, and epithelial cell death (Figure 4E).
To define the mechanism by which activated T cells modulate barrier integrity, we cultured Caco-2 BBe cells in an inverse configuration on a semi-permeable filter in a transwell culture dish (Figure 5A), creating a polarized monolayer in which the upper chamber is adjacent to the basolateral surface. Both pulse- and persistently-activated human T cells added to the upper chamber stimulated a steady increase in TER between 15 to 24 h of co-culture, signifying a reduction in ion flux across the barrier (Figure 5B). Once maximal resistance was reached, monolayers co-cultured with pulse-activated T cells sustained this elevated level of resistance, while monolayers co-cultured with persistently-activated T cells initiated a second phase of the response, in which there was a continuous decrease in resistance over the following 4 d, often dropping to below the control
32 level (Figure 5B). This biphasic response was observed with all donors (two representative donors shown), so it is not dependent on an MHC-match between
IECs and T cells. The additional of anti-CD3/CD28 Dynabeads alone to the monolayer had no effect on TER (Figure 6A). We will refer to Phase 1 as when the TER increases within the first 30 h in response to all activated T cells, and
Phase 2 is defined by the steady state resistance maintained by pulse-activated T cells or the decline in resistance induced by persistently-activated T cells.
To evaluate the leak pathway, T cells were removed at the end of Phase 1 or
Phase 2 and a mixture of fluorescein and fluorescently-labeled dextrans of different molecular weight, and hence diameter, were added to the basolateral chamber.
When the leak pathway was analyzed as Apparent permeability (Papp), an untreated monolayer was slightly permeable to fluorescein (11 Å) and 3.5 kDa dextran (28 Å) and impermeable to 70 kDa dextran (116 Å) (Figure 6B). There was no noticeable difference in the rate of passage of any probe between untreated and activated T cell-exposed IECs in Phase 1 (Figure 6B). Because the untreated monolayer allows limited small molecule permeability at baseline, co- culture with activated T cells could not further increase its barrier strength, as was observed in Phase 1 for the pore pathway. At the end of Phase 2, pulse-activated
T cells, which sustain elevated TER (i.e., reduced ion flux via the pore pathway), induced a minimal increase in leak pathway permeability for smaller probes (11 and 28 Å) and had no effect when evaluated with the larger probe (116 Å, Figure
6C). In contrast, persistently-activated T cells in Phase 2 induced extremely
33
Figure 4: Three-dimensional co-culture model to study the interaction between T cells and IECs
Co-culture model was constructed with three layers of Matrigel in a glass bottom 24 well plate. From top to bottom, imbedded T cells, a thin blank layer of Matrigel, and imbedded epithelial cysts (A). Structure of Matrigel-imbedded Caco-2 cysts grown for 2 weeks (B). Evidence of migration of pulse-activated T cells (white) from the top layer to the bottom layer to interact with cysts (dark spheres) on their basolateral surface (C). Co-culture with activated T cells increases the permeability of the epithelial cysts to FITC- labeled 4 kDa dextran (FD4). Each dot represents a random cyst. (D). Structure of the cysts after a 3-d co-culture with pulse- or persistently-activated T cells (E).
34
Figure 5: Activated T cells modulate the pore and leak permeability pathways of an intestinal epithelial cell monolayer in two-dimensional co-culture model
Two-dimensional, anatomically correct co-culture model of a transwell filter coated with a polarized monolayer (A). Pulse-activated (blue) and persistently-activated (red) T cells induce a biphasic response on the TER versus a control (green) monolayer, independent of T cell donor
(Phase 1 - P1; Phase 2 - P2) (B). The ratio of normalized fluorescent intensity for the passage of fluorescein (11 Å) and dextran 3.5 kDa (28 Å) relative to the flux intensity of 116 Å dextran 70 kDa was unchanged by co-culture with pulse- or persistently-activated T cells in Phase 1 (C).
Persistently-activated T cells induce a significantly higher rate of fluorescein and dextran 3.5 kDa flux relative to dextran 70 kDa flux in Phase 2 (D).
35 high apparent permeability through the epithelium for all size ranges, noting, in particular, permeability for a 70 kDa probe, which measures the unrestricted pathway. To evaluate if the increase in apparent permeability of the smaller probes in the persistently-activated T cell co-culture is due to increased damage to the barrier and thus passage through the unrestricted pathway, we determined the ratio of the levels of fluorescein or 3.5 kDa dextran flux relative to 70 kDa dextran flux (Figure 5C and 5D). Consistent with measurements of apparent permeability, the net flux of all probes is not affected by T cell co-culture in Phase 1 (Figure 6B and 6D), while the increased permeability of both smaller probes in Phase 2, when the monolayer is co-cultured with persistently-activated T cells (Figure 6C and 6E), is indeed mediated by passage through the leak and unrestricted pathway (Figure
6B). Thus, sustained contact with persistently-activated T cells, modeling chronically inflamed T cells, damages the barrier, dysregulating the flux of ions and macromolecules.
36
Figure 6: Activated T cells modulate the pore, leak and unrestricted paracellular permeability pathways of an intestinal epithelial cell monolayer
While pulse- (blue) and persistently-activated (red) T cells induce a biphasic response in an epithelial monolayer, adding anti-CD3/CD28 dynabeads alone (gold) to the transwell culture has no effect on TER (A). The permeability of the epithelial barrier was evaluated by following the paracellular flux of fluorescent probes (B to E). Pulse-activated (blue) and persistently-activated (red) T cells do not modulate the passage of small (11 Å), medium (28 Å), and large (116 Å) probes versus a control (green) monolayer in Phase 1 (B), while persistently-activated T cells significantly increase the apparent permeability (Papp) of all three probes in Phase 2 (C).The net flux of the fluorescently-labelled tracers, normalized to t=0, is shown for Phase 1 (D) and Phase 2 (E).
37 3.3. Defining the activation status of pulse- and persistently-
activated T cells
To characterize the immunological properties that distinguish the pulse- from the persistently-activated T cell populations, we evaluated their proliferative capacity, predisposition toward apoptosis, cytokine synthesis profile, and homing capabilities after co-culture with IECs during both Phase 1 and Phase 2. Setting input T cells immediately after activation as control, both pulse- and persistently- activated T cells during Phase 1 express similar levels of Ki67 mRNA (124 ± 8.08%
116 ± 8.79% of control, respectively). However, in Phase 2, pulse-activated T cells show significant 7-fold reduced Ki67 mRNA levels (13.7 ± 1.93%), while Ki67 expression in persistently-activated T cells remain relatively unchanged at 86.7 ±
7.68%. These findings suggest that pulse-activated T cells become quiescent in
Phase 2, while persistently-activated T cells remain stimulated. To assess if these
T cells are undergoing apoptosis, their live/dead ratio was determined by flow cytometry. The percentage of dead cells remain consistently at less than 4% through the co-culture in all T cell subtypes (data not shown), indicating that cell death is unlikely to contribute to the decline of barrier integrity in Phase 2 for persistently-activated T cells.
38
Figure 7: Expression of surface proteins and cytokines by pulse- and persistently- activated T cells after co-culture with an intestinal epithelial monolayer.
Expression of the activation markers CD69 and CD25 and gut homing receptor integrin b 7 in pulse- and persistently-activated T cells in Phase 1 (P1) and Phase 2 (P2) was assessed by flow cytometry. Untreated T cells and T cells after 48 hours of activation with anti-CD3/CD28 dynabeads (Input) were stained as controls (A). RNA expression of cytokines from Input and pulse- activated T cells harvested 8 h before the Phase 1 peak and persistently-activated T cells harvested 24 h before the end of Phase 2 was measured by RT-qPCR, normalized to unstimulated T cells (B).
39 As expected, activating T cells by cross-linking CD3/CD28 induced expression of the cell surface markers CD69 and CD25, and their surface expression levels remained elevated in Phase 1 for both T cell subtypes (Figure
7A). Consistent with the loss of proliferative potential of pulse-activated T cells in
Phase 2, CD69 and CD25 levels begin to return to baseline, while these activation markers remain high in persistently-activated T cells (Figure 7A). Furthermore, mRNA expression of the activation marker PD-1 followed the same kinetic response (data not shown). In Phase 1 the ratio of CD4+ to CD8+ T cells remains unchanged, while in Phase 2 the ratio of CD4+ to CD8+ decreases from 1.6 to 1.2 for pulse activation co-cultures and increases to 2.0 in persistent activation co- cultures. Pulse and persistent activation co-culture with the monolayer slightly favored the expansion of CD8+ T cells and the decline of CD8+ T cells, respectively, in Phase 2 (Figure 8A). These results, together with those to be discussed in Fig. 18, suggest there is reciprocal communication between T cells and IECs.
40
41
Figure 8: Co-culture of T cells with an epithelial monolayer modulates the T cell subsets, the expression of gut homing markers and cytokines differently based on the activation status of the T cells
Percent of CD4+, CD8+, double negative (DN) and double positive (DP) with and without co- culture by flow cytometry (n = 3) (A). RNA expression of integrin b7, integrin aL, and chemokine receptor CCR9 measured by RT-qPCR in activated T cells that had been co-cultured an epithelial monolayer at the end of Phase 1 or Phase 2 (n = 3-4). The data was normalized to 18S RNA and
T cells immediately after anti-CD3/CD8 activation without co-culture (Input) were used as the baseline to calculate the fold change in expression (n=3) (B). A heatmap showing the ratio of T cell cytokine mRNA expression between pulse- and persistently-activated T cells co-cultured with a
Caco-2 monolayer and those harvested at the time equivalents of 8 h before the Phase 1 peak and
24 h before the end of Phase 2 without co-culture, where downregulation was denoted in yellow and upregulation was quantified in blue (C).
42 We investigated the expression of homing markers and chemokine receptors on activated T cells to potentially explain their migration pattern. The mean fluorescent intensity of the gut homing integrin b7 immediately after activation was modestly increased, and this increase persisted throughout co-culture. The percentage of pulse-activated T cells expressing integrin b7 also increased in both
Phase 1 and Phase 2, while this increase was not observed for persistently- activated T cells, suggesting elevated homing potential for pulse-activated cells
(Figure 7A). We detected a similar increase in migration potential for pulse- activated T cells when measuring mRNA levels for b7, CD11a (integrin aL), and
CCR9, whose levels decreased in persistently-activated T cells (Figure 8B). This result is consistent with the migration pattern seen in the 3-D co-culture (Fig. 4 and
Supplemental movie 2) in which pulse-activated T cells demonstrated a higher capacity to migrate to the colonic organoids.
The functional consequence of T cell activation was evaluated by measuring a wide range of cytokine mRNA synthesis after IEC T cell co-culture, normalized to an unstimulated T cell. As expected, all cytokine levels (except TGF-b) were elevated 10-fold or more immediately after T cell activation (input cells). Pulse- activated T cells harvested 8 h before the Phase 1 peak had dramatically down- regulated all cytokine mRNA expression and were entirely quiescent 24 h before the end of Phase 2. In contrast, persistently-activated T cells maintained elevated cytokine mRNA production throughout Phase 1 and only slightly decreased expression 24 h before the end of Phase 2 (Figure 7B). These results are
43 consistent with the proliferative capacity and activation markers expression on the cells, indicating that the loss of barrier integrity in Phase 2 by persistently-activated
T cells is associated with persistent T cell activation. In keeping with the theme of bi-directional communication we evaluated the impact of IECs on T cell cytokine synthesis (Figure 8C). In Phase 1, Caco-2 co-culture was able to either up- or down-regulate cytokine synthesis by pulse-activated T cells, while co-culture only upregulated persistently-activated T cell cytokine expression (TNF-a, in particular).
Yet, in phase 2, the Caco-2 monolayer downregulated cytokine mRNA during persistent activation.
3.4. Exposure of an epithelial monolayer to activated T cells
regulates the expression of TJ protein and mRNA
To define the molecular mechanisms that mediate changes in the pore, leak, and unrestricted pathways due to T cell co-culture, we evaluated the expression of key TJ proteins and mRNA in the monolayer. Claudin-1 mRNA levels modestly increase in co-culture at the peak of Phase 1 after exposure to both pulse- and persistently-activated T cells and return to baseline at the end of Phase 2 (Figure
9A). In contrast, claudin-2 (Figure 9B) and occludin (Figure 9D) mRNA levels do not change during Phase 1, yet are significantly higher in Phase 2. Claudin-4 follows a third pattern with increased mRNA in both phases (Figure 9C). A fourth temporal pattern was revealed by ZO-1, which decreased slightly in Phase 1 and returned to baseline in Phase 2 (Figure 9E). Gene expression changes for each of these TJ mRNAs were independent of the activation scheme for the T cells.
44 These results demonstrate that regulation of TJ gene expression is mediated by the presence of T cells, yet due to the intricacy of the TJ complex, following a limited number of components does not capture the observed biological effect on barrier integrity.
To confirm that activated T cell communication with an epithelial monolayer also modulates steady state intracellular protein levels, we examined regulation by pulse- and persistently-activated T cells of protein concentrations for the same claudin family members and occludin (Figure 9F). Co-culture with pulse-activated
T cells induced higher claudin-2 and claudin-4 protein expression in Phase 2, while co-culture with persistently-activated T cells increased claudin-2 and claudin-4 in both phases. There were no significant differences in the levels of claudin-1 between the phases or treatment regimens, yet there were more claudin-1 degradation products in Phase 2 after persistent T cell activation (Figure 9F). The expression of occludin did not change in Phase 1, but increased in Phase 2 on co- culture with pulse-activated T cells (Figure 9F). The lack of concordance between
TJ mRNA synthesis and steady state protein levels, in response to activated T cells, underscores the complexity of TJ regulation, assembly, degradation, and function.
45
Figure 9: Exposure of an epithelial monolayer to activated T cells modestly regulates the expression of TJ mRNA and proteins
RNA expression of claudin-1, -2, -4, occludin, and ZO-1 in a monolayer co-cultured with activated T cells at the end of Phase 1 or Phase 2 (n = 3-4). (A to E). Protein expression by immunoblot of claudin-1, -2, -4, and occludin in a Caco-2 monolayer co-cultured with activated T cells at the end of Phase 1 or Phase 2. GAPDH expression was used as an internal standard. Results from different transwells are shown (F).
46
47 Figure 10: Activated T cells reduce the height of an epithelial monolayer in Phase 1 and modulate the arrangement of TJ strands and the location TJ proteins
Both pulse- and persistently-activated T cells reduce the cell height of a Caco-2 monolayer in Phase 1, as revealed by CLDN1 staining (green) (aspect ratio 5.7:1). The red line is a reflection of phalloidin staining from the transwell filter. The scale bar represents 10 �m (A). Cell height was quantified using ImageJ. (B). Immunofluorescence staining for CLDN1 and CLDN4 in TJ strands in co-culture with activated T cells at the end of Phase 1 or Phase 2, shown by an image of the top ∼20% of the x-z slices close to the apical surface. The intensity profile represents a scan of the signal strength shown by the yellow bar (C). Immunofluorescence staining for OCLN in TJ strands and the mobile lateral subpool, in co-culture with activated T cells at the end of Phase 1 or Phase 2, shown by a total Z stack representation. The sharp, solid green strand is the TJ; the fuzzy, diffuse staining is the mobile lateral subpool. The intensity profile represents a scan of the signal strength shown by the yellow bar in the squares marked in the upper right of each image (D).
48
Figure 11: Activated T cells modulate the arrangement of TJ strands and the location CLDN1
Immunofluorescence staining for CLDN1 in TJ strands and the mobile lateral subpool, in co-culture with activated T cells at the end of Phase 1 or Phase 2, shown by a total Z stack representation. The sharp, solid green strand is the TJs; the fuzzy, diffuse staining is the mobile lateral subpool. The scale bar represents 10 �m.
49
Figure 12: Activated T cells reduce the height of an epithelial monolayer in Phase 1 and modulate the arrangement of TJ strands and the location of CLDN4
Both pulse- and persistently-activated T cells reduce the cell height of a Caco-2 monolayer in
Phase 1, as revealed by CLDN4 staining (green) (aspect ratio 5.7:1). The red line is a reflection of phalloidin staining from the transwell filter. The scale bar represents 10 �m (A).
Immunofluorescence staining for CLDN4 in TJ strands and the mobile lateral subpool, as described in the legend for Figure 10 (B).
50 3.5. Structural changes to the TJ complex due to activated T cell
engagement
Regulation of TJ protein expression does not directly address the assembled, functional TJ, since TJ proteins associate with the TJ complex and are found in a mobile lateral subpool that does not contribute to barrier integrity [22]. Claudin-1 and -4 proteins are highly expressed in the mobile lateral subpool in a resting monolayer and were assembled into TJs in Phase 1 (~3-fold increase in intensity), but not Phase 2, after co-culture with either pulse- or persistently-activated T cells
(Figure 10C). By immunofluorescent staining their total expression increased in both phases, consistent with the RNA and immunoblot data shown in Fig. 9.
Additionally, upon T cell co-culture claudin-1 and -4 displayed a punctate, intracellular cytosolic staining pattern (Figure 11 and 12). In Phase 2, we also observed an elevation in claudin-1 and -4 staining at the apical surface (Figure
10A and Figure 12A). Overall, these findings indicate that not only do activated T cells stimulate the expression of claudin-1 and -4 in an epithelial monolayer, they induce their re-localization.
Similar to claudin-1 and -4, claudin-2 contributes to TJ assembly; upon T cell co-culture, claudin-2 preferentially localizes to TJ strands. However, in Phase 2, the claudin-2 staining pattern appears to be scattered and disorganized, most notably in the presence of persistently-activated T cells (Figure 13). Co-culture with pulse- or persistently-activated T cells dramatically decreases the occludin
51
Figure 13: Activated T cells modulate the arrangement of TJ strands and the location CLDN2
Immunofluorescence staining for CLDN2 in TJ strands, as described in the legend to Figure
10.
52 mobile fraction and lateral/cellular subpool and favors accumulation into the TJ complex in Phase 1 (Figure 10D). In phase 2 the pulse-activated culture showed an increased mobile fraction and lateral/cellular subpool of occludin while, with persistent activation, occludin levels in the TJ complex were decreased and the subpool depleted (Figure 10D and Figure 14). We detected higher intracellular punctate occludin staining, especially in Phase 2 of the pulse-activated culture and both phases of the persistently-activated model (Figure 10D), possibly reflecting a mechanism for the re-localization or degradation of the mobile lateral subpool.
There is no difference in ZO-1 staining under any co-culture conditions, reflecting its role as an intracellular protein binding to the C-terminus of transmembrane TJ proteins (Figure 15). There was one additional and intriguing feature revealed in this analysis. In the persistently-activated co-cultures in Phase
2, there was a notable loss of actin signal (Figure 16A and 16B). This finding indicates that the interaction of actin with TJ proteins and the apical brush border microvilli, which are actin-based protrusions, is disrupted or abnormal in the setting of an inflammatory T cell signal, consistent with reports from IBD intestinal epithelium [71].
53
Figure 14: Activated T cells reduce the height of an epithelial monolayer in Phase 1, as revealed by staining for OCLN
Both pulse- and persistently-activated T cells reduce the cell height of a Caco-2 monolayer in Phase 1, as revealed by OCLN staining (green), as described in the legend for Figure 7.
54
Figure 15: Activated T cells increase the size of the cells within an epithelial monolayer in both Phases 1 and 2, as revealed by staining for ZO-1
Both pulse- and persistently-activated T cells increase the cell size in a Caco-2 monolayer at the end of Phase 1 and even greater at the end of Phase 2, as revealed by ZO-1 staining (green), shown by a total Z stack representation. The scale bar represents 10 �m.
55
56 Figure 16: Co-culture of an epithelial monolayer with persistently-activated T cells results in a loss of actin from the microvilli and stimulates a diffuse distribution of CLDN1 from the apical to the basolateral surface in Phase 2
An x-z image of an untreated and treated monolayer (aspect ratio 5.7:1), was stained for actin (phalloidin in red) and CLDN1 (green) in Phase 2. Top panels are actin only, middle panels are CLDN1, and the bottom panels are for co-localization (A). An image in the x-y plane of a composite Z stack shows colocalization (merged) of actin (red) with CLDN1 (green) in TJ strands in a control monolayer, a slight loss of colocalization in a pulse-activated T cell monolayer co-culture, and a complete loss of the actin signal induced by persistently-activated T cells in Phase 2 (B). The scale bar represents 10 �m.
57 3.6. Contribution of epithelial cell morphology to paracellular
permeability
The complexities revealed by our analysis of TJ mRNA expression, protein levels, and immunofluorescent patterns in a monolayer exposed to activated T cells suggest that studying the overall junctional complex in isolation may not provide an exact depiction of the forces that regulate barrier integrity. Classically, an increase in claudin-1 and claudin-4 levels decreases the permeability of the barrier through their sealing function, while excess claudin-2 typically increases permeability through the pore pathway. Thus, the changes in TJ proteins may not explain why we observe a reduction in ion flux via the pore pathway (Fig. 5B) in
Phase 1 and a decline in quality of the leak and unrestricted pathway in Phase 2 with persistently-activated T cell exposure (Fig. 5D). In addition to the contribution of TJs, permeability of the barrier is also affected by cell morphology [12].
Immunofluorescence staining for claudin-1 revealed a noticeable decrease in the height of the epithelial monolayer in Phase 1 after co-culture with both pulse- and persistently-activated T cells (Figure 10A and 10B). This shortening of the cells was reversed in Phase 2. Thus, when considering the mechanisms of barrier function, one must also consider possible changes in cell morphology.
Co-culturing the monolayer with activated T cells decreased the number of cells per microscopic field in both phases (Figure 17A and 17B), coincident with the down-regulation of Ki67 expression, a marker for cell proliferation (Figure
58 17C), indicating an inhibition of epithelial cell growth or recovery. Notably this decrease in cell number and corresponding flattening of the cells (Fig. 10A) occur in Phase 1, consistent with the observed increase in TER. We conclude that these
“shortened, fatter” cells with decreased cell membrane-to-cell membrane contact per unit area account in part for the decline in paracellular permeability in Phase
1. In contrast, in Phase 2, co-culture with persistently-activated T cells induced cell death, as revealed by increased DNA staining by the cell-impermeant viability indicator, ethidium homodimer-1 (EthD-1) (Figure 17D). In addition, in both phases of co-culture with persistently-activated T cells calcium flux may be disrupted due to the upregulation of TRPV-6, a calcium influx protein channel, and downregulation of the PMCA1b channel, encoded by the ATP2B1 gene, which extrudes calcium (Figure 17E). These results indicate that in a persistently- activated T cell co-culture (i.e., an inflammatory setting), prolonged incubation disrupts TJ protein gene expression, alters TJ assembly, induces TJ protein degradation and entry into the cytosol, distorts cell morphology, and promotes cell death, thus dramatically increasing the unrestricted pathway and leading to high barrier permeability.
59
Figure 17: Co-culture with pulse- and persistently-activated T cells increase the size of IECs within a monolayer and persistently-activated T cells induce epithelial cell death
Pulse- and persistently-activated T cells increase the size of Caco-2 cells within a monolayer in Phase 1 and Phase 2, as revealed by ZO-1 staining (A). Reduced cell number per field (n = 9-
10 fields), induced by pulse- and persistently-activated T cells, was quantified by counting the number of nuclei detected by DAPI stain (B). Reduced IEC proliferation, due to pulse- and persistently-activated T cell co-culture, was confirmed by quantifying mRNA expression of the proliferation marker, Ki67 (MKi67) (C). Co-culture with persistently-activated T cells in Phase 2 leads to cell death, as revealed by increased EthD-1 staining (D). Increased RNA expression of the membrane calcium channel TRPV6, involved in the first step in calcium absorption, and decreased
RNA expression of ATP2B1, coding for the PMCA1b channel that functions to remove calcium from the cell, in a Caco-2 monolayer in co-culture with activated T cells at the end of Phase 1 or Phase
2 using TaqMan qPCR, normalized to RPLP0 as the endogenous control (n = 3-4) (E).
60 3.7. Reciprocity in the mucosa: IECs after contact with activated T
cells may modulate innate and adaptive inflammatory responses
A key hallmark of the intestinal mucosa is bidirectional host defense regulation between the surface epithelium and the underlying lamina propria immune cells.
We investigated if co-culture with either pulse- or persistently-activated T cells induces a functional immune phenotype within the monolayer. As an example,
Th17 cells play a critical role in the mucosa against extracellular pathogens by regulating the integrity of epithelial barrier [72]. Since TGF-β, IL-6, and IL-1β contribute to the polarization of Th17 cells, we studied their expression in these co-culture models (Figure 18A). Expression of TGF-β and IL-1β mRNA by the monolayer is induced by pulse- and persistently-activated T cells in both phases, suggesting the presence of an immunological feedback mechanism, by which activated T cells engage the epithelium to increase its immunoregulatory function.
Since both TGF-β and IL-1β must be proteolytically processed into an active form, we also measured active protein in the conditioned media from these co- culture assays using commercially available paired antibodies. Secretion of active
IL-1β was below the limit of detection. TGF-β is a well-known key regulator of epithelial regeneration and a product of both T cells and IECs. The active form of
TGF-β accumulated predominantly at the apical surface (Figure 18B). As shown with mRNA measurements, its synthesis is induced by both pulse- and persistently-activated T cells, apart from persistently-activated T cells at the end of
61 Phase 2, which may reflect IEC cell death at this timepoint (Figure 18B). Activated
T cells in isolation secrete minimal amounts of active TGF-β (data not shown).
This proposed reciprocity was confirmed when the expression of molecules related to the capacity for antigen presentation was profiled (Figure 18A).
Expression of the inhibitory costimulatory molecule programmed death ligand 1
(PD-L1), the principal ligand of programmed death 1 (PD-1), is induced in both
Phase 1 and 2 by persistently-activated T cells, while b2-microglobulin (B2M), a component of the MHC Class I system, was upregulated on the monolayer after co-culture with both types of activated T cells (Figure 18A). Consistent with the stimulation of B2M in the monolayer, CD1d and HLA-C are also upregulated, with slightly different kinetics. Thus, the ability of IECs to present antigen to CD8+ T cells is enhanced in response to T cell engagement. Ironically, HLA-DMA is down- regulated in the same monolayer, indicating reduced antigen presentation to CD4+
T cells. While clarification of this dichotomy requires further investigation, overall these results highlight the bidirectional communication between T cells and the epithelium as they work together toward host defense.
62
Figure 18: IECs in contact with activated T cells may modulate innate and adaptive inflammatory responses
RNA expression of cytokines, PD-L1, or MHC components in a monolayer co-cultured with activated T cells at the end of Phase 1 or Phase 2 (n = 3-4) (A). Accumulation of active TGF-b in basolateral and apical conditioned media at the end of Phase 1 and Phase 2 was measured by
ELISA (B).
63 IV. DISCUSSION
Chronic inflammation of the GI tract is often associated with the loss of epithelial barrier integrity and resultant microbial translocation, as demonstrated in many diseases including IBD, Irritable Bowel Syndrome, and HIV infection[67, 73,
74]. Events that contribute to the pathology associated with intestinal permeability include (i) direct damage to the epithelium, (ii) dysbiosis of the luminal microbiome, and (iii) local mucosal inflammation, with an important role for T cell activation in disease progression and tissue damage [75, 76]. Earlier reports demonstrated that a single cytokine, such as TNF-a or IFN-g, can modulate the integrity of the intestinal barrier through alteration of TJ composition due to increased expression of incompatible claudin isoforms that exchange with the mobile fraction and lateral/cellular subpool [70, 77, 78]. However, the dynamics of interaction between the epithelium and the underlying immune cells are far more complex than secretion of a single cytokine, and the cooperative, mutually beneficial communication between the two compartments must be evaluated. Therefore, we established two anatomically correct co-culture models, one in two dimensions and another in three dimensions, that enable us to characterize the dual effects of distinctly activated T cells on the function of the epithelial barrier as well as the corresponding potential effect of the epithelium on the subjacent immune cells.
Results from our in vitro models reveal that the capacity of activated T lymphocytes to modulate the integrity of an intestinal barrier is complex and
64 depends on (i) the activation state of the T cell and (ii) the kinetics of the cell-cell communication (Figure 19). These perturbations in paracellular permeability induced by activated T cells control both the leak and pore pathways, and may, under specific conditions, also change the morphology and health of the monolayer and thereby enhance the unrestricted pathway. One may perceive a drawback of this report is the use of blood-derived T cells, but that choice was intentional due to the proven migration of these cells to the gut during acute and chronic inflammatory disease. When we evaluated the ability of activated blood-derived T cells to express gut chemokine and homing receptors, we found that CCR9 and the b7 chain that associates with integrins a4b7, aEb7, and aLb7 are induced and differentially regulated in pulse- versus persistently-activated T cells, possibly explaining their divergent ability to migrate toward colonic spheroids, as shown in
Fig. 4 and the Supplemental movies. A highly effective treatment for IBD is administration of a blocking antibody to the a4b7 integrin that directs T cell homing to the gut [79].
65
Phase 1 Phase 2
OR Pulse Persistent Pulse Persistent
Permeability: Pore↓ Leak ↔ Permeability: Pore↓ Leak↑ Permeability ↑↑: Unrestricted ↑
TJ assembly and expression: ↑↓ TJ assembly and expression: ↑ TJ: Dysregulated Actin: ↔ Actin: ↔ Actin: Dysregulated Cell Height: ↓ Cell Height: ↔ Cell Height: ↔ Cell density: ↓ Cell density: ↓ Cell density: ↓ Cell death: ↔ Cell death: ↔ Cell death: ↑ ↓ increase, ↑ decrease, ↔ no change
Figure 19: The summary of the regulation of T cells on epithelium
66
4.1. Initial contact with activated T cells strengthens barrier
integrity (Phase 1)
Increased TER of the monolayer upon co-culture with either pulse- or persistently-activated T cells reflects a reduction in ion flux through the pore pathway regulated by the TJ complex. Since conductance through the cell is sufficiently low due to the high resistance of the plasma membrane, the paracellular pathway normally dictates the overall TER. However, when the cell membrane is perturbed by an external stimulus, cell morphology may cause a consequential contribution to the TER [80]. A monolayer with larger cells has a lower percentage of its cell surface shielded by the TJ complex through which ions can pass. Thus, a monolayer with larger cells, that has maintained a TJ complex with similar electrical properties, will exhibit a higher TER due to less ion flux.
Because the untreated monolayer established a sturdy leak pathway with limited molecular flux, our model was unable to reveal if the leak pathway also tightened upon exposure to activated T cells. Thus, we are unable to state unequivocally that
TJ structure or composition alone contributed to the increased TER. As seen in
Phase 1, expression of TJ mRNAs and proteins, as well as the subcellular localization of these proteins, after either pulse- or persistently-activated T cell co- culture, demonstrated no predictable pattern of claudin or occludin usage or location to fully explain the increased resistance. Immunofluorescence staining of the TJ proteins, CLDN1, CLDN2, CLDN4, OCLN, and ZO-1, revealed that TJ
67 strand intensity and its ratio to the highly mobile/low stability protein subpool varied dramatically for each condition and each targeted protein. However, there is a clear accumulation of occludin at the tricellular junction, which may contribute to a strengthened barrier. Furthermore, the slight difference in ZO-1 staining in Phase
1 documented in Fig. 15 resembles that seen with silencing of HIF1B [81], suggesting that one mechanism by which activated T cells may regulate the barrier is by inducing hypoxia or metabolic changes in the IEC. One additional consistent parameter was the decreased number of cells per unit area in the monolayer in
Phase 1. We propose that lower cell density, in combination with changes in the pore and leak pathway, yielded the higher resistance.
This “loss” of cells, coupled to their increase in breadth and reduction in height, indicates a major change in cell morphology. The alteration in morphology likely required a re-arrangement of new TJs for the expanded cell boundary. It is possible that some of the observed changes in mRNA and protein expression for claudin proteins and occludin may have been in response to these morphological demands. Combining the changes in morphology with the decreased expression of the proliferation marker, Ki67, in Phase 1 for both modes of T cell activation, suggests that activated T cells induced the differentiation and morphogenesis of the epithelium and remodeling of the TJ, which stimulated a strengthening of the intestinal barrier function. These results underscore that an acute proinflammatory microenvironment may lead to enhanced host defense and reduced epithelial permeability.
68
4.2. Long-term communication with activated T cells yields
dichotomous changes in barrier permeability depending on the
character of the immune stimulation (Phase 2)
In the 3D model and in Phase 2 of the 2D model, barrier permeability evaluated by the leak and unrestricted pathways significantly increased in co-culture with persistently-activated T cells. Persistent activation led to a collapse of the 3D cyst structures and considerable cell death in the 2D model. The inability of persistently- activated T cells to maintain a strong epithelial barrier is reflected by the observation that pulse-activated T cells rapidly lose their ability for cytokine synthesis in Phase 1 and return to a quiescent state, as revealed by their decrease in proliferative potential and activation markers. In contrast, persistently-activated
T cells, upon co-culture, maintain and – for some cytokines – increase their rate of synthesis, continue to proliferate, and remain activated. These findings are consistent with the study of cytokines, such as TNF-a or IFN-g, on 3D epithelium, which confirms their roles in increasing paracellular permeability via apoptosis or cell shedding [70]. In contrast, quiescent, pulse-activated T cells in the 3D culture modestly affected the leak pathway, and the increased quality of the pore pathway
(i.e., increased TER) was maintained in Phase 2 of the 2D co-culture. Thus, the pattern of mediators expressed by activated T cells leads to distinct epithelial responses.
69 A number of factors explain the capacity of persistently-activated T cells to increase the flux of ions and molecules through the pore and leak pathways.
Immunofluorescence staining highlights a reduction in the intensity of TJ strands after co-culture and higher proportion of TJ proteins in the mobile fraction and lateral/cellular subpool, each of which contribute to a leakier epithelium [77]. Our results add a new twist to these findings. The apical brush border microvilli are actin-based protrusions, yet the relocation of claudins to the apical surface appears to be accompanied by a dramatic loss in actin staining, indicating that the microvilli structure is disrupted or abnormal in a chronic inflammatory setting. Although it is unclear how the microvilli are altered in patients with IBD, there is clear evidence that the microvilli decrease in length is accompanied by ultrastructural defects [71].
The inappropriate accumulation of claudin proteins at the apical surface leads us to hypothesize that epithelial polarization is compromised, affecting the architecture of the epithelial barrier.
Persistently-activated co-culture conditions also lead to an increase in cell size.
Compared to Phase 1, the number of cells per unit area dropped from 80% of control to 35%. In addition, persistent activation led to barrier damage due to cell death and, as a consequence, permeability via the unrestricted pathway allowed free passage of ions, macromolecules, and pathogens. Epithelial cell death and an enhanced unrestricted pathway have been reported previously, induced by cytokines or chronic inflammation, as seen in IBD, diverticulitis, and HIV infection
[82-84]. Importantly, microbial translocation, due to the unrestricted pathway,
70 subsequently initiates chronic systemic immune activation and a sequela of complications in the cardiovascular, liver, coagulation, lipid metabolism, and nervous systems [85, 86]. The mechanism of cell death is unknown, yet it is well established that the accumulation of intracellular calcium is required for the activation of many pathways, including cell death [87]. One possible mechanism, among many others, for cell death in Phase 2 might be a result of accumulation of intracellular calcium due to the coordinated upregulation of TRPV-6 calcium influx channels, and downregulation of the PMCA1b channel (ATP2B1 gene), which extrudes calcium. Supporting this intriguing possibility, we found that a monolayer in the presence of pulse-activated T cells demonstrates the same potential calcium imbalance in Phase 1, but this imbalance is ablated in Phase 2, when the monolayer maintains its elevated TER and shows no sign of cell death. While our data do not point to a specific mechanism that would lead the epithelial monolayer to resist lethal consequences of pulse-activated T cells, our results do indicate that under an appropriate mode and time of interaction, the barrier may be able to suppress the damage caused by mediators from activated T cells.
4.3. Communication with activated T cells modulates the immune
properties of the epithelium and vice versa
When co-culturing with IECs, CD4+ vs CD8+ T cell compartment was altered during Phase 2 and this result proved IECs played a role in the regulation of T cells differentiation and polarization, which was dependent with the activation status of T cells. The communication between IECs and T cells modulated the
71 cytokine expression of epithelium shown in Fig. 18 and this data provided a possible mechanism in which IECs regulate the T cell subsets via cytokine secretion. Furthermore, in IBD, the CD4+ T cell population increased and were thought to be the main driver in perpetuating chronic intestinal inflammation, and perturbing the balance in the intestinal mucosa [88, 89]. In consistent with this observation from literature, our result showed there was an increasing in CD4+ population with persistent versus pulse activation in Phase 2, indicating the chronic activation of T cells disrupts the homeostasis of the gut and is the underlying factor of many bowel diseases like IBD.
Upon T cell co-culture, we also observed an upregulation of IL-1β and TGF-β expressed by the epithelial monolayer. TGF-β is best known as an immunosuppressive cytokine for its regulatory activity and induction of peripheral tolerance as well as epithelial regeneration, while IL-1b functions as a proinflammatory factor inducing the expansion of differentiated CD4+ T cells and other immune cells. The predominant secretion of TGF-β from the apical surface of epithelial cells reflects its critical role in facilitating the recovery of the epithelium from injury. In a healthy polarized epithelium, TGF-β is secreted apically, whereas the type I and type II TGFβ receptors are localized specifically at the basolateral surface [90]. Disruption of the epithelial barrier by injury enables apically-derived
TGF-β to activate its receptors via the Smad2–Smad4 complex and initiate the repair process[91]. Although there is some evidence that IL-1β and TGF-β are
72 functionally antagonistic [92, 93], the production of both cytokines indicates an important, yet unknown, immunoregulatory activity for an activated epithelium.
T cell cytokine expression was altered in distinct ways when co-cultured with
IECs: (i) TGF-β, TNF-a, and IL-17A were upregulated, (ii) IL-2, IL-22, and IL-6 were either up or down-regulated, depending on the activation status and Phase, and (iii) IFN-g and IL-1β were dramatically down-regulated in Phase 2 during persistent activation, as the monolayer was dying. This complexity of intestinal epithelial-mediated regulation of T cell cytokine synthesis is consistent with the literature, in which mucosal T cells are distinguished with a unique phenotype that maintains the delicate balance between immune activation and tolerance [60].
The expression of PD-L1 increased several hundred-fold with co-culture. PD-
L1 is upregulated in the presence of cytokines like IFN-g and TNF-a, during sepsis, and infection. PD-L1 binds to programmed death 1 (PD-1), which is highly expressed on persistent activated T cells, inhibits T cell activation, inflammatory cytokine production, and T cell proliferation and survival, induces the generation of T regulatory cells, and controls inflammation and tolerance in the GI tract [94].
Recent studies show elevated PD-L1 expression suppresses Th1 cells in ulcerative colitis, while loss of PD-L1 in Crohn’s Disease contributes to the persistence of Th1 inflammation [95]. Nevertheless, elevation in PD-L1 in a cytokine-treated Caco-2 monolayer is associated with an increase in epithelial
73 permeability, which can be reversed with a PD-L1 blocking antibody; however, the mechanism remains unclear.
Activated T cells also modulate the expression of MHC-I/II and MHC-I like molecules on the epithelium. These molecules, when bound to antigen, are important ligands for T cell survival, development, homeostasis, and activation. In our 2D model, there is no direct interaction between the epithelium and activated
T cells due to the 10 µm transwell filter; therefore, modulation of epithelial expression of immune molecules is mediated by soluble factors. Upregulation of
B2M, CD1d, and HLA-C, and downregulation of MHC-II after T cell co-culture, suggests a role of the activated epithelium in immune tolerance, mediated by the activity of CD8+, CD4+, or NKT cells. A recent report showed that expression of
MHC-II is higher in Lgr5+ epithelial stem cells than in differentiated IECs [96]. Thus, we propose that mediators from T cells induce IEC differentiation, accompanied by a reduction in Ki67. Nevertheless, the role for expression of these immunoregulatory proteins to the integrity of the epithelium, host defense, immune tolerance, and homeostasis awaits further study.
74 V. CONCLUSION AND FUTURE DIRECTION
We provide mechanistic evidence that IEC-T cell communication modulates epithelial paracellular permeability by regulating TJ assembly and cell morphology and alters the homeostatic immune properties of the epithelium. Overall, our results highlight that, in the setting of acute intestinal inflammation, the epithelium initially responds by strengthening its barrier function, yet chronic immune exposure disrupts the epithelium, enhancing microbial translocation.
To begin to evaluate the details of the bi-directional interaction of T cells with
IECS, we varied the quantity of activated T cells co-cultured with the barrier monolayer and showed that the effect was T cell dependent (Figure 20A). In addition, we contrasted the effectiveness of purified activated CD4+ versus CD8+ peripheral T subsets on barrier integrity. These results indicated that the alteration of IEC permeability was responsive to treatment with either CD4 or CD8 T cells
(Figure 20B). However, we have not yet analyzed other molecular alterations to further contrast each T cell subset. It is important to note that the experiments shown in Fig. 20 were performed with the parental Caco-2 cell line and must be repeated with the same Caco-2 subclone used throughout this dissertation to confirm the findings and correlate them with the other conclusions we have drawn.
75 1500 A 0.72 million T cells 0.36 million T cells 1000 0.18 million T cells 0.09 million T cells 0.045 million T cells Ohm.cm2 500
Corrected TER Media
0 0 100 200 300 Hours of coculture CD3/4/8 B 1500 CD3+ CD4+ 1000 CD8+ Media
Ohm.cm2 500 Corrected TER
0 0 100 200 300 Hours of coculture
Figure 20: Changes in IEC barrier permeability is dependent on T cell concentration and independent of CD4+ versus CD8+ T cell subsets
Persistently-activated T cells and the parental Caco-2 cell line were used for the experiments on T cell titration (A) and T cell subsets (B).
76 Some of the data which were not shown are the removal and transfer of T cells.
Removal of T cells in phase 1 and 2 abolished the effect of T cells on IECs barrier.
This confirmed T cells were the culprit for the observation and the barrier when T cells were removed could recover though phase 2 took more time. With the transferring experiments, persistently activated T cells at the end of phase 2 were transferred to a new fresh barrier and they also induced the biphasic curve on the
IECs. This proved the alteration of ICEs possibly underwent two steps, and the collapse of IECs on phase 2 was possible the consequence when the intensity of the T cell effect exceeded the threshold of IEC endurance.
Finally, the study will be advanced in the future by evaluating this bi-directional communication using primary epithelial cells and/or mucosal T cells. Moreover, we will investigate the method of communication through exosomes and microvesicles
(EVs).
5.1. Primary IEC monolayer
A method for the culture of organoids has been recently optimized. Enteroid or colonoid cultures are derived from G-protein coupled receptor 5 (Lgr5)+ stem cells located in the crypts of donor biopsies and are maintained in a Matrigel matrix. The
3D organoids are dissociated and applied to a transwell filter, then differentiated to generate the intestinal barrier. The human monolayer cultures, which recapitulate the diversity of intestinal cell types and possess properties similar to
77 primary intestinal epithelium, provide an advantage in studying the cross-talk between IECs and other immune cells [97, 98]. This model will overcome the weaknesses of using a cancer cell line that depends very much on culture technique. We will exploit this technique to generate the barrier from colonoids generate from healthy biopsies. The experiments will be done similarly with ones from Caco-2 and be repeated with at least three donors to see if we have the same effects with Caco-2. Barrier and immune function will be analyzed to address the same question. Moreover, we will apply single cell sequencing which is an advanced method to study the effect on each epithelial cell subset. This can reveal the possible pathways and mechanisms by which T cells alter the IECs.
5.2. Mucosal T cells and commensal pathogens
The properties of blood T cells are quite different from those of mucosal T cells which are almost activated and adopt new phenotype to adapt to the mucosa environment. While the isolation of mucosal T cells is currently technically challenging, the use of peripheral blood T cells allowed us to reveal the effect of multiple cytokines on the properties of the epithelial barrier. In the future, we aim to develop a method to purify mucosal T cells from healthy colon biopsies in order to investigate the crosstalk between mucosal T cells and IEC barriers. We then will perform similar experiment with mucosal T cells instead of using peripheral blood
T cells to coculture with the IECs in the present or absent of commensal antigens.
These communications would better approximate interactions found in the gut.
78 This system would also allow us to investigate how the presence of commensal antigens influence these communications.
5.3. Communication via exosomes/microvesicles (extracellular
vesicles; EVs)
Signaling via EVs, cell-derived membrane structures found in bodily fluids, is a method for communication, regulation, and defense among cells. EVs are enriched in cytokines, chemokines, RNAs, and proteins, suggesting that EVs play a role as a vehicle to concentrate and transport these signaling molecules [99] [100]. In addition to cytokines, exosomal RNAs (in particular, miRNAs) are the most well- studied. These molecules are of major interest and play important roles in post- transcriptional regulation of expression. They can be taken up by target cells and subsequently modulate recipient cells. There is evidence showing the essential roles of EVs in cell-cell crosstalk and the contribution of EVs to inflammation of the epithelium [101]. Thus, we aim to investigate the effect of T cell EVs on the epithelium and analyze miRNAs from the EV in the co-culture media in order to find molecules that regulate the functions of the barrier.
Our preliminary data show that concentrated EVs, harvested from activated T cells, can induce an increase in the TER of the Caco-2 monolayer (Figure 21A), similar to that seen with conditioned media alone (Fig. 5). Interestingly though, EV- depleted media reduced TER (Figure 21B). It is important to note that the
79 experiments presented in Fig. 21 were performed with the parental Caco-2 cell line and must be repeated with the same Caco-2 subclone used throughout this dissertation. In addition, new more refined methods for the isolation and quantitation of EVs now exist and future analyses require that more sophisticated techniques be applied to the question of the contribution of EVs to barrier integrity before a final conclusion is drawn. However, these exciting, though untested, preliminary findings suggest that EVs are potentially the vehicles for the cross-talk between T cells and IECs.
We have collected the media from the coculture in three different conditions
(control, pulse and persistent), then the EVs were purified and sequenced for miRNA by a commercial service. We expected the data would reveal the pathways which T cells and IECs communicates via the EVs. From the preliminary data, the content of the miRNA form EVs included miRNAs, transcripts (fragments), antisense transcripts (fragments), lincRNAs, ncRNAs, tRNAs and rRNAs. Majority of miRNAs in EVs were originated from T cells and small percent was from Caco-
2 BBe. However, the main population of coding RNA in EVs were from Caco-2.
Moreover, co-culture with Caco-2 BBe suppressed 80% of T cell-derived miRNAs.
Some of these miRNAs were associated with cell growth and some were related to inflammatory pathways. Nevertheless, biological repeats will be performed to further confirm these observations and to analyze the differential expression of pulse versus persistent activation.
80 A 300 Media EVs from complete RPMI 200 EVs from T cell conditoned media
Ohm.cm2 100 Corrected TER
Treatment 0 0 50 100 150 Hours of treatment
B Pellet from 3-day or 4- day T cell activation
Ultracentrifugation Vesicle-depleted media from 3-day 3 day or 4 day activation or 4-day T cell activation
80 P=0.0007 Media (n=6) 60 Pellet from 3-day activated T cells (n=2), p= 0.0043 40 P=0.0043 Pellet from 4-day activated T cells (n=2), p= 0.0007 20 P<0.0003 P<0.0001 EV-depleted media from 2-day activated T cell (n=2), p<0.0003 0 Media EV-depleted media from 4-day activated T cell (n=2), p<0.0001 TER change (%) -20 P: value compared with control -40
Figure 21: Effect on EVs on the IEC barrier.
EVs harvested from the conditioned media of T cells activated with Dynabeads were concentrated by ultracentrifugation. EVs were added to the co-culture three times as indicated by the Treatment arrows. EVs from activated T cell conditioned media, but not the EVs from RPMI complete media, stimulated an increase in the TER of the monolayer (A). While EVs increase TER, as shown in Fig 21A and 21B (blue), EV-depleted media reduces the TER of the barrier (pink) (B). Parental Caco-2 clone was used for both experiments.
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