3.3. LPS-induced changes in gut barrier and BBB
To simulate the inflammatory response of gut-brain axis, our GBA chip
was treated with LPS. The TEER values of the gut and the BBB were
decreased in response to LPS treatment, with increased permeability of
both barriers (Fig. 6A~D). LPS can be found on the cell
walls of Gram-negative bacteria (Schumann,
1992), which is known to disrupt gut integrity and can penetrate
systemic circulation across the gut barrier
(Ghosh, Wang, Yannie, & Ghosh, 2020;
Hirotani et al., 2008). This endotoxin
can also impair BBB integrity and increased circulating LPS is
associated with brain-related issues (Banks
& Erickson, 2010; Qin et al., 2007;
Zhu et al., 2017). Although the gut
epithelium and BBB showed a similar trend in overall, the extent of
response to LPS was different depending on the culture condition. For
example, both cells seemed less sensitive to LPS in transwell condition
than chip condition. In chip condition, physiological properties of
Caco-2 such as barrier function, absorptive property and enzyme activity
can be altered (Choe et al., 2017), which
suggests that the response of Caco-2 cells to LPS may be affected by the
flow.
It is also notable that the changes in absorption permeability was more
significant than the changes in TEER in response to LPS. In the gut chip
developed by Chi et al., Caco-2 cells featured 0.9 fold change in TEER
and 2 fold change in permeability when exposed to flow
(Chi et al., 2015). In the study
conducted by Hirotani et al., a similar trend was observed, where the
absorption permeability changed more dramatically than the values of
TEER (Hirotani et al., 2008). The TEER
value reflects the ionic conductance of the paracellular pathway in the
cell monolayer while the flux of non-electrolyte tracers represents the
paracellular water flow and the pore size of the tight junctions
(Srinivasan et al., 2015). As TEER and
flux of non-electrolyte tracers indicate different entities, the change
in TEER value does not always correspond to the change in permeability
of paracellular pathway (Zucco et al.,
2005).
A proinflammatory cytokine, IL-8 is thought to engage in several
inflammatory processes (Ehrlich et al.,
1998), and BBB could be a crucial source of them, which could influence
brain microenvironment (Banks & Erickson,
2010; Chen et al., 2001;
Verma, Nakaoke, Dohgu, & Banks, 2006).
The bEnd.3 cell line used in our study is a murine cell line, which
shows inflammatory response different from human cells
(Asfaha et al., 2013). Therefore, we used
the primary human brain microvascular endothelial cell, hBMECs, to study
inflammatory response in our GBA chip (Fig 6E~G). The
IL-8 assay was conducted to confirm if proper alteration in the cytokine
secretion can be observed after the endotoxin stimulation in the gut.
Caco-2 cells in the well did not show significant changes. In contrast,
hBMECs in the well showed significant increase of the cytokine after the
stimulation. This result implies that the hBMECs were more sensitive to
LPS treatment and the induced inflammatory response observed in our GBA
chip originate mostly from brain endothelial cells.