Abstract

   Slow progress in the fight against neurodegenerative diseases (NDs)
   motivates an urgent need for highly controlled in vitro systems to
   investigate organ-organ– and organ-immune–specific interactions
   relevant for disease pathophysiology. Of particular interest is the
   gut/microbiome-liver-brain axis for parsing out how genetic and
   environmental factors contribute to NDs. We have developed a
   mesofluidic platform technology to study gut-liver-cerebral
   interactions in the context of Parkinson’s disease (PD). It connects
   microphysiological systems (MPSs) of the primary human gut and liver
   with a human induced pluripotent stem cell–derived cerebral MPS in a
   systemically circulated common culture medium containing CD4^+
   regulatory T and T helper 17 cells. We demonstrate this approach using
   a patient-derived cerebral MPS carrying the PD-causing A53T mutation,
   gaining two important findings: (i) that systemic interaction enhances
   features of in vivo–like behavior of cerebral MPSs, and (ii) that
   microbiome-associated short-chain fatty acids increase expression of
   pathology-associated pathways in PD.

INTRODUCTION

   The gut-brain axis operates as a bidirectional communication system
   integrating the central nervous system (CNS) with endocrine, metabolic,
   and immune signaling pathways ([60]1). As a vital participant in this
   system, the microbiome and its metabolic products, including
   short-chain fatty acids (SCFA), directly and indirectly affect the
   broader gut-immune-liver-brain axis. Accumulating data implicate
   dysregulation of the gut-brain axis in a variety of pathologies from
   inflammatory bowel disease to neurodegenerative diseases (NDs) ([61]2,
   [62]3).

   The causality of multifactorial diseases involving the gut-brain axis
   are difficult to parse in animal models, as the highly convoluted
   nature of the systemic interactions are combined with interspecies
   differences in metabolism and immunology. For example, SCFA produced by
   fermentation in the human proximal colon (up to 600 mmol/day) can
   influence gut-brain communication and function directly or indirectly
   through immune, endocrine, vagal, and other humoral pathways ([63]3).
   Microbial bioactives can affect the axis via local interactions with
   enteric nerves transduced to the CNS (vagal pathway) or, separately,
   via systemic circulation to organs and tissues (humoral pathway) and
   immune cells (immune pathway). The liver is prominently involved in the
   humoral and immune pathways as the first draining point of the large
   intestine. In animal models, those individual routes of action and
   connections to pathology are difficult to deconvolute. Furthermore, the
   influence of individual microbiome-derived metabolites cannot be
   readily separated from the context of the entire gut milieu. This
   drives a need for causality-focused, human-based, preclinical models
   that incorporate engineering conceptualization of diseases such as new
   platform technologies designed to capture the crucial yet complex
   physiological phenomena in vitro in a systematic and scalable manner.

   Parkinson’s disease (PD) is prototypical of NDs, with links to the gut
   microbiome and systemic immune function, for which etiologies and
   effective therapies remain poorly defined. PD is a late age-onset,
   chronic, neurodegenerative disorder characterized by inflammation,
   accumulation of Lewy bodies in neurons, and cell death. Approximately
   90% of PD cases are sporadic ([64]4). However, familial PD has been
   linked to dominant mutations, such as the A53T mutation, causing
   misfolding and aggregation of α-synuclein (αSyn) with formation of Lewy
   bodies ([65]5). Neurons throughout the nervous system are affected,
   causing especially pronounced damage to dopaminergic neurons in the
   brain and associated symptomatic loss of motor control. Environmental
   and genetic factors have been associated with development of PD and
   other NDs.

   A potentially important signaling link between the gut microbiome and
   the brain in the context of PD involves SCFA. A previous study with
   gnotobiotic mice implicated the presence of SCFA to faster progression
   toward PD in a mouse model of the disease ([66]1). Intriguingly, recent
   data in mice also implicate the microbiome in increased inflammation
   related to amyotrophic lateral sclerosis—a phenomenon that is reduced
   in mice treated with broad-spectrum antibiotics ([67]6). SCFA exert
   pleiotropic effects that may contribute to a brain phenotype: They are
   linked to the development of microglia, provide an important energy
   source for the brain, and influence neuronal function ([68]2).
   Moreover, SCFA modulate several major metabolic pathways in the liver
   that alter blood lipids and sugars and influence the inflammatory
   phenotypes of immune cells in the intestine, liver, and circulation
   ([69]7, [70]8), thus indirectly influencing the microenvironment of the
   nervous system in ways that might potentiate or protect from
   development of PD or other NDs in humans.

   Experiments performed in gnotobiotic mice cannot be translated directly
   into a protocol for experimentation on human patients. We therefore
   designed an in vitro all-human physiomimetic model that captures
   salient features observed in those studies as a demonstration for how
   continuously interlinked microphysiological systems (MPSs) can bring
   insights into human disease pathophysiology. MPSs are in vitro models
   that, under perfusion, mimic facets of physiological organ behavior
   ([71]9). The goal of physiomimetic models is to define the essential
   elements of complex disease states involving multiple organ systems and
   capture these in the simplest possible MPS experimental configuration
   that will reveal useful insights. Here, we first define the
   physiomimetic model for parsing causality in the interconnection
   between the microbiome and early stages of NDs according to the
   following phenomena: (i) humoral and immune pathways connecting
   microbial metabolites (but not microbes) to the gut-liver-brain axis
   independently of endocrine and vagal pathways; (ii) how interaction
   between MPSs of the gut, liver, and circulating CD4^+ T cells affects
   maturation of neurons, astrocytes, and microglia; (iii) the effects of
   bioaccessible SCFA on cerebral MPSs that represent certain features of
   familial PD and those of healthy controls; and (iv) how increase or
   reduction of inflammatory mediators via SCFA and inclusion of CD4^+ T
   cells affects the PD phenotype.

   On the basis of this conceptualization of the disease, we designed an
   experimental platform linking three complex MPSs—gut/immune,
   liver/immune, cerebral/immune—via a common culture media containing
   circulating immune cells in continuous coculture. The platform design
   simultaneously addresses several challenges required to accomplish the
   desired physiological integration, including (i) open system
   accommodating standard culture models for each MPS (e.g., Transwell
   insert for mucosal barrier), thus facilitating access to individual
   compartments for fluid sampling and comparison with literature for
   behaviors of individual MPSs; (ii) continuous recirculation of medium
   within each MPS to facilitate molecular and cellular transport between
   tissues and circulating medium with flow rates tailored to the needs of
   each individual MPS; (iii) continuous recirculation of a relatively
   small volume of common culture medium (5 ml) between MPSs at flow rates
   sufficient to exchange the entire culture volume over five times per
   day, with flow patterns that provide physiologically scaled ratios of
   systemic (25%) and portal (75%) circulation to the liver MPS; (iv) in
   addition to continuous flow between MPSs, independent fluid mixing
   within each MPS to enhance local molecular and cellular transport; (v)
   continuous recirculation of functionally viable immune cells; (vi)
   minimal loss of lipophilic medium components to platform adsorption;
   and (vii) moderate throughput on each platform, with three complete
   independent circuits per platform. While individual constraints have
   been addressed in other platforms, this is the first demonstration of
   integration of all components. The crucial elements of the approach
   include (i) unique on-board microfluidic pumps that are safe for immune
   cells and can be multiplexed on the platform, with individually
   addressable flow rates, to eliminate external tubing and minimize
   circulating medium volumes; (ii) elimination of polydimethylsiloxane
   (PDMS) by machining the platform from polycarbonate and using a
   non-PDMS elastomer on the pumps; and (iii) inclusion of gravity flow
   connections as part of the fluidic circuit, to offset any slight
   differences in pump flow rates arising from minor fabrication variances
   and thus control fluid volumes in individual compartments while
   minimizing the overall platform footprint (see details in Materials and
   Methods and Results). We take advantage of this on-board,
   platform-integrated pumping technology to conduct a proof-of-principle
   experiment demonstrating its potential utility for coculture with
   circulating immune cells, thus illuminating facets of complex systemic
   immune system–mediated processes.

   Our findings using this physiomimetic model of early-onset PD indicate
   that the interaction of healthy cerebral MPS controls with MPSs of the
   gut-liver axis in the presence of circulating CD4 regulatory T cells
   (T[regs]) and T helper 17 (T[H]17) cells beneficially affects the
   cerebral MPS’s phenotype. This includes increased expression of genes
   associated with maturity of neurons, astrocytes, and especially
   microglia. We observed pathology-related effects of systemic SCFA
   unique to MPSs of PD, but not in healthy controls. Hence, engineered
   human physiomimetic models can aid in our understanding of
   multifactorial NDs and complement in vivo animal models as tools to
   investigate disease causality.

RESULTS

Conceptualization of key biological phenomena informing experimental platform
design

   At a coarse-grain view, we considered the following biological
   phenomena in the conceptualization of a physiomimetic model of the
   gut-liver-brain axis ([72]Fig. 1A and fig. S1A): (i) SCFA adsorption
   through a colon mucosal barrier incorporating innate immune cells,
   where the SCFA may be partially metabolized to influence production of
   soluble signaling molecules; (ii) transport through the portal
   circulation to the liver, where additional metabolism occurs by
   hepatocytes, and where the SCFA exert influence on innate immune
   Kupffer cells in the liver; and (iii) transport of soluble metabolites
   and inflammatory mediators through the systemic circulation to the
   brain along with (iv) migration of adaptive immune CD4^+ T cells via
   systemic circulation between the gut, liver, and the brain.

Fig. 1. Human 3XGLB physiomimetic system of the gut-liver-brain axis.

   Fig. 1
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   (A) Schematic representation of the design rationale for the
   experimental approach and description of individual MPSs included in
   this study. (B) Top left: pneumatic plates machined in acrylic; top
   right: mesofluidic plate machined from monolithic polysulfone; bottom:
   3X Gut-Liver-Brain (3XGLB) platform composed of pneumatic and fluidic
   plates with elastomeric polyurethane membrane in between them to form a
   pumping manifold with integrated fluid channels. The platform allows
   three-way interaction in three replicates where the center
   liver-specific MPS can be fluidically linked to two additional
   Transwell-based MPSs. Photo credit: Martin Trapecar, MIT. (C) Top view
   of the 3XGLB with identified fluidic and pumping properties as well as
   operational parameters (for more details, see fig. S1A).

   As MPSs represent relatively reductionist models of complex organ
   systems, we structured individual MPSs to reflect the above-described
   physiological features and scaled the relative cell numbers according
   to their physiological functions ([74]Fig. 1A). The gut serves many
   functions, of which nutrient absorption and regulation of immune
   tolerance toward the commensal microbiome are among the most important.
   Cell lines that are often used to model some features of the colon
   barrier, such as the cancer-derived Caco-2 line, are not appropriate
   for modeling SCFA effects because of the stark differences in their
   metabolism and signaling compared to human primary tissues ([75]10). We
   thus designed the gut MPS based on primary colon epithelial cells that
   were propagated as organoids and seeded as single cells on 12-well
   Transwells. Myeloid cells, like macrophages and dendritic cells, are
   both the first line of defense against pathogens in the gut and crucial
   modulators of epithelial homeostasis and tissue repair; hence, their
   integration into MPSs of the gut is an essential biological feature
   ([76]11). We seeded peripheral blood mononuclear cell (PBMC)–derived
   macrophages and dendritic cells on the basolateral side of the
   Transwells harboring a differentiated primary epithelial monolayer
   (fig. S1B).

   The liver receives most of its blood from the gut and plays a pivotal
   role in the metabolism of and immunity against gut-derived products.
   The liver MPS was designed to capture the metabolic transformation of
   SCFA as well as features of the immunological environment. The
   microtissue comprised a coculture of human primary cryopreserved
   hepatocytes and Kupffer cells at physiological 10:1 ratio maintained in
   a culture medium permissive for retention of inflammation responses
   ([77]12, [78]13). Although it is technically feasible to include
   additional primary nonparenchymal cells ([79]14), or cell lines
   representing them ([80]15), immune-metabolic cross-talk can be
   adequately represented in long-term culture by primary hepatocytes and
   Kupffer cells ([81]12). Cells were seeded on a microperfused liver
   scaffold that allows for optimal oxygenation and nutrient flow (fig.
   S1C) ([82]12).

   Key players in perpetuating inflammation-related cerebral PD pathology
   are neurons, astrocytes, and microglia. We therefore adapted a
   well-established, robust model comprising human PD patient-derived
   induced pluripotent stem cells (iPSCs) differentiated into neurons,
   astrocytes, and microglia cultured in a Transwell format amenable to
   incorporation on a mesofluidic platform ([83]16). We used a neural
   differentiation method that gives rise to various types of CNS cells,
   as PD-associated pathology is not limited to the substantia nigra and
   dopaminergic neurons, but rather affects a wide variety of CNS cell
   types ([84]17). The cerebral cell types used in this study were derived
   from human iPSCs (hiPSCs) that carry either the A53T mutation in αSyn
   (PD) or hiPSC corrected (PD-C) to wild-type healthy status ([85]5), to
   enable isogenic comparison of disease and healthy cerebral tissue.

   Microbiome-derived metabolites and their derivatives have been shown to
   affect the differentiation and function of T lymphocytes, most notably
   the balance between CD4^+ T[regs] and T[H]17 cells ([86]8), both of
   which are implicated in PD. These two immune cell types play an
   important role in maintaining the balance between autoimmunity and
   immune tolerance where T[regs] dampen inflammation by producing
   transforming growth factor–β (TGF-β) on one side and T[H]17 cells
   (T[H]17s) support inflammation by releasing the cytokines
   interleukin-17 (IL-17) and IL-22 ([87]18) on the other. An increased
   number of T[H]17 cells versus the frequency of T[regs] in circulation
   has been observed in patients with PD, yet the exact contribution of
   this phenomenon is yet to be defined ([88]19, [89]20). Moreover, in
   advanced stages of PD, T[H]17 cells have been shown to be the first
   effector T cells to cross the disrupted blood-brain barrier (BBB). In
   this current study, we therefore integrated circulating PBMC-derived
   T[reg] and T[H]17 cells as a feature of the adaptive immune system that
   has been implicated in the progression of PD.

   Capturing the biological phenomena described above in an in vitro
   experimental setting requires a platform technology that enables (i)
   continuous fluidic communication between MPSs as well as controllable
   circulation within each compartment, (ii) undisrupted continuous
   circulation of CD4^+ immune cells, and (iii) the incorporation of
   well-defined and validated cell culturing systems such as Transwell
   inserts that are routinely used by many medical researchers and (iv)
   that are engineered on a mesofluidic scale that allows for the
   interrogation of bigger media volumes and tissue mass as those offered
   by microfluidic setups.

   The resulting 3XGLB (3X Gut-Liver-Brain) human physiomimetic system of
   the gut-liver-brain axis allows for the selective integration of key
   physiological features and MPSs ([90]Fig. 1B and movie S1). The
   physiomimetic 3XGLB system was engineered to house three sets of three
   fluidically interconnected MPSs with adjustable, pneumatic intra- and
   intercompartmental circulation ([91]Fig. 1C). The system features
   low-volume pumps that can circulate culture medium containing adaptive
   immune cells between the individual compartments, preserving immune
   cell viability (fig. S1D). Cell ratios and numbers of all MPSs were
   kept constant across all interaction studies (scaled as described in
   [92]Fig. 1A) and are described in detail in Materials and Methods.

Physiomimetic interaction improves markers of cerebral MPS phenotype and
maturity of neurons, astrocytes, and microglia

   First, we aimed to understand how the physiomimetic interaction of
   cerebral MPSs with the gut and liver MPSs, as well as T[reg]/T[H]17
   cells, affects expression of genes specific to the maturation and
   function of neurons, astrocytes, and microglia. We evaluated
   differential gene expression (DGE), enriched pathways in PD-C MPS
   tissue, and cytokine/chemokine concentrations in the shared common
   medium (CM) during different modes of a 4-day interaction ([93]Fig.
   2A). Canonical functional tissue phenotypic markers, gut barrier
   integrity, and liver albumin production were preserved throughout
   tissue interaction (fig. S1, B and C). These functions were also
   maintained during interactions involving the PD MPSs and SCFA described
   below.

Fig. 2. Physiomimetic interaction increases in vivo markers of cerebral MPSs.

   Fig. 2
   [94]Open in a new tab

   (A) Schematic presentation of conditions compared in [95]Fig. 1 (B to
   F) and tables S1 and S2. Top: Control PD-C cerebral MPS in isolation;
   middle: Control PD-C cerebral MPS in interaction with the gut and liver
   MPSs; bottom: Control PD-C MPS in interaction with the gut and liver
   MPSs and T[reg]/T[H]17 cells. (B to D) We jointly harvested neurons,
   astrocytes, and microglia of three separate replicates of control PD-C
   cerebral MPSs after a 4-day interaction with the gut and liver MPSs in
   the absence or presence of T[reg]/T[H]17 cells. (B) We compared DGE of
   neuron-, astrocyte-, and microglia-related genes between cerebral MPSs
   in isolation versus those in interaction. Significance is expressed as
   *P < 0.05, **P < 0.001, ****P < 0.00001. (C and D) PANTHER pathway
   enrichments in cerebral PD-C MPSs in interaction over those in
   isolation. Data represent averages of three replicates. Pathways are
   ranked on the basis of a combined z and P value score. (E)
   Concentrations of cytokines measured in the CM shared between MPSs
   after 96 hours of gut-liver-cerebral (PD-C) interaction studies with or
   without circulating T[reg]/T[H]17 cells and indicated reported values
   of the same proteins in human plasma (for exact values and references,