DISCUSSION
As the incidence and awareness of FA increases, there is a demand for appropriate models of FA for mechanistic studies and the development of therapeutics. This study aims to generate a human gut tissue-based model of acute, IgE-mediated FA. Using the PCIS system, allergic responses that occur within GI tissues may be characterized.
Cellular composition is key for accurate inferences on the nature of an investigated response. To ensure the representation of distinct cell subsets, scNuqSeq was employed to define the cellular composition of the PCIS. The resulting map of cellular distribution confirmed that all relevant cell types were present in the samples, including stromal and epithelial cells as well as key immune cell populations that are commonly described in human colon tissue.36,37 Not only is this dataset useful in this context, but forms the framework for future functional assessments and adds to single cell libraries for healthy infant colon tissue, a relatively understudied group.
In addition to confirmation of cellular diversity, establishing tissue viability and metabolic activity in the tissue is essential to ensure accurate readouts. Using routine LDH and WST-1 assays, PCIS maintained consistent viability for at least 24h in culture.38This model was specifically designed for acute allergic responses, which did not warrant investigations beyond 24h. Previous reports have shown that the viability and morphology of human PCIS can be maintained up to 48h20 and even 72h.19,39
Smooth muscle contraction was chosen as the readout for allergic responses in the PCIS model, as allergic reactions stemming from food exposure often result in symptoms centralized to the GI tract.40 Moreover, food allergen-induced muscle contraction in sensitized individuals has been reproducibly demonstrated in both animal and human models.41–43 The inflammatory mediators stored in mast cell granules, which includes histamine, serotonin and tryptase, have all been shown to directly cause smooth muscle contraction in the gut.5,44,45 Similar studies using precision cut lung slices have also utilized bronchoconstriction to model allergic responses to aeroallergens.46–48 To measure the contraction response in PCIS, a novel analysis program was developed internally, as other commercial motion tracking software were unable to reflect the patterns observed in the videos. This program, designed to quantify the muscle contraction responses in PCIS, also has the potential to be applied beyond this system. Any model in which movement is a central aspect of response could utilize this program to quantify results. The configurations can also be set based on controls and adjusted to accommodate changes in lighting during filming.
PCIS generated from non-allergic donor tissue underwent passive sensitization to induce sensitivity to the allergen of the allergic plasma donor. Passive sensitization is a common experimental procedure used to induce sensitivity in non-allergic cells or tissue.47,49–51 Use of this process not only reduces the need for relatively rare allergic tissue donors, but also extricates the humoral component of the allergic response. PCIS passively sensitized with clinically confirmed peanut allergic plasma stimulated with PE or positive controls displayed a strong contraction response that was significantly greater than the baseline movement due to enteric reflexes.52 This response was allergen-specific, as demonstrated by the lack of response to a clinically irrelevant allergen (OVA). In contrast, PCIS passively sensitized with plasma from a peanut-sensitized non-allergic donor did not display a strong response to PE stimulation, indicating that the PCIS model reflects the clinical phenotype of the donor. Although the overall patterns of response were clear between stimuli, the kinetics and magnitude of the muscle contraction responses may vary between individual PCIS. This was expected as the gut slices differ in terms of smooth muscle composition and mast cell distribution due to variability between tissue donors and along an individual GI tract.
Tryptase release from intestinal mast cells occurs immediately upon activation.53 Measurements of tryptase in the PCIS culture supernatant confirmed inflammatory mast cell-derived mediators were released following stimulation with a relevant allergen (peanut) or FcɛRI-crosslinker but not an irrelevant allergen (OVA), further highlighting the specificity of responses.
As observed in the PCIS model, histamine is a key mediator of the allergic response that affects smooth muscle contraction. Thus, antihistamines were the drug of choice to demonstrate the utility of this ex vivo model for testing anti-allergic therapeutics in development, as illustrated by the suppression of contraction responses. While anti-allergic drugs are commonly tested in animal models,41,54,55 the use of a human tissue-based model either in place of or in addition to animal testing would allow for more translatable and clinically relevant results. Of note, the PCIS model has often been used to study pharmacokinetics in the context of human gut tissue.14–16,20,21
Passive sensitization of PCIS with plasma from a peanut allergic donor pre-OIT resulted in a robust contraction response following stimulation with PE, while a comparatively diminished response was observed post-peanut OIT as well as in combination. OIT has become a standard treatment option for FA and has been broadly effective in treating children with established FA. During OIT, levels of allergen-specific IgG, IgG4 and IgA increase, and are considered to act as protective antibodies, whereas levels of allergen-specific IgE decline over time.56,57 These results demonstrate the use of PCIS as a model of FA in potentially assessing responses to therapy and immunomodulation. Moreover, at the start and end of OIT, an OFC is completed to determine the threshold of response and the development of desensitization respectively; however, severe reactions may occur. As PCIS reflects allergic status, this model may also be used as a proxy for OFCs to prevent higher risk challenges.
In summary, a novel human gut tissue-based FA model has been developed. PCIS generated from non-allergic tissue, passively sensitized with plasma from allergic donors, displayed visible and quantifiable allergen-specific smooth muscle contractions upon allergen stimulation. This model has great potential as a valuable experimental tool in FA research as it can be used to differentiate sensitized allergic versus sensitized non-allergic individuals, test anti-allergic drugs within a relevant environment and observe the progression of allergen-specific immunotherapy. As mast cells are difficult to isolate in peripheral blood, and the complex interactions between immune cells and structural cells in the gut are not easily observed, the utility of this FA model addresses a relevant research need.
As with all models, there are several constraints. A significant limitation is the lack of tissue availability from appropriate sources. Additionally, gut tissue is sensitive to culture conditions and has a limited viability. While this is appropriate for acute, short-term outcomes, this model cannot be used without modifications for long-term studies or for the study of delayed allergic responses. Additional considerations include the isolated nature of the PCIS model, which cannot replicate circulation or migration of cells from other tissue, as well as equal exposure to stimulants on both surfaces (apical/basal) which does not reflect the human system. Importantly, as the tissue is from non-atopic donors, it cannot reflect the pre-existing inflammation observed within the GI tract of allergic patients.
Despite these limitations, this human tissue-based model is directly translatable in contrast to individual cell culture models or animal models of disease which cannot replicate the human system. It contains all resident cell types interacting in a physiologically accurate environment. This allows for observation of cellular mechanisms in tissues that are not represented by peripheral blood samples and may also be adapted to investigate other GI-based diseases. The promising potential of PCIS as a model of FA will also expand to future studies including early changes in gene expression following allergen exposure.