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.