The HMI™ module: a new tool to study the Host-Microbiota Interaction in the human gastrointestinal tract in vitro

Abstract

Background: Recent scientific developments have shed more light on the importance of the host-microbe interaction, particularly in the gut. However, the mechanistic study of the host-microbe interplay is complicated by the intrinsic limitations in reaching the different areas of the gastrointestinal tract (GIT) in vivo. In this paper, we present the technical validation of a new device--the Host-Microbiota Interaction (HMI) module--and the evidence that it can be used in combination with a gut dynamic simulator to evaluate the effect of a specific treatment at the level of the luminal microbial community and of the host surface colonization and signaling.

Results: The HMI module recreates conditions that are physiologically relevant for the GIT: i) a mucosal area to which bacteria can adhere under relevant shear stress (3 dynes cm(-2)); ii) the bilateral transport of low molecular weight metabolites (4 to 150 kDa) with permeation coefficients ranging from 2.4 × 10(-6) to 7.1 × 10(-9) cm sec(-1); and iii) microaerophilic conditions at the bottom of the growing biofilm (PmO2 = 2.5 × 10(-4) cm sec(-1)). In a long-term study, the host's cells in the HMI module were still viable after a 48-hour exposure to a complex microbial community. The dominant mucus-associated microbiota differed from the luminal one and its composition was influenced by the treatment with a dried product derived from yeast fermentation. The latter--with known anti-inflammatory properties--induced a decrease of pro-inflammatory IL-8 production between 24 and 48 h.

Conclusions: The study of the in vivo functionality of adhering bacterial communities in the human GIT and of the localized effect on the host is frequently hindered by the complexity of reaching particular areas of the GIT. The HMI module offers the possibility of co-culturing a gut representative microbial community with enterocyte-like cells up to 48 h and may therefore contribute to the mechanistic understanding of host-microbiome interactions.

Figure 1

Scheme of the HMI module for long-term studies of the host-microbiota interaction in the GIT. A polyamide semipermeable membrane and a mucus layer form a double functional layer that separates the luminal compartment (upper one) from the lower compartment containing enterocyte cell lines. The HMI module allows to study the bacterial adhesion under relevant shear forces and microaerophilic conditions. It allows the reciprocal exchange of signals and metabolites between compartments and it allows the exposure of cell lines to a complex microbial community, representative for the human colon, for up to 48 h.

Figure 2

MTT values (expressed as Optical Density – OD) of Caco-2 cells directly exposed for 2 h to the complex microbial community of the ascending colon of a SHIME reactor (direct contact), exposed to the same microbial community within a HMI module (HMI 1 and 2) or to sterile SHIME medium (control) for 48 h. Values are averages ± standard deviation (n = 2). * = statistically different from the control condition according to a Student’s two-tailed t-test (p < 0.05).

Figure 3

Scheme of the adapted SHIME system (consisting of stomach, small intestine and ascending colon - AC - compartments) used for the long-term study. Two HMI modules have been connected in parallel to the vessel simulating the AC compartment in order to obtain information on bacterial adhesion and host response after 24 and 48 h. The SHIME system was fed three times per day with SHIME feed; the medium in the lower compartment of the HMI modules (containing Caco-2 cells) was fully replaced every 6 hours by means of an automatic pump. The exhausted medium was collected in order to analyze the concentration of IL-8.

Figure 4

Scheme of the long-term experiment and of the relative sampling points for the different analyses. The experiment consisted of a 2-week startup period, 1-week control and 1-week treatment. The HMI modules were connected to the ascending colon compartment of a SHIME system during the last 3 days of the control and treatment periods. Samples from the lumen of the SHIME were collected for SCFA and DNA analyses. Samples from the surface of the double functional layer of the HMI modules were collected for DNA analyses. Samples from the lower compartment of the HMI module were collected for IL-8 measurements. DNA = qPCR and DGGE. DNA* = qPCR, DGGE and FISH (the latter only at 48 h).

Figure 5

DGGE fingerprinting analysis for bifidobacteria (a) lactobacilli (b) and composite data set of the gels for bifidobacteria. lactobacilli and total bacteria, including bootstrap analysis with 1000 samplings (c). A = control period (Cluster II); B = treatment period (Cluster I). L = luminal samples collected from the SHIME reactor; M = mucus sample collected from a fraction of the membrane inside the HMI module. 0, 24 and 48 indicate the hours that the HMI modules have been connected to the SHIME system during the control and treatment periods (as illustrated in Figure 3). Clustering analysis was based on the Pearson product–moment correlation coefficient and dendrograms were created by using UPGMA linkage.

Figure 6

FISH analyses a) positioning of F. prausnitzii (left panel - fluorescent microscopy) and bifidobacteria (right panel - Confocal Laser Scanning Microscopy) in the microbial biofilm with respect to the membrane and mucus layer (M), as indicated by the white arrows. Oxygen concentration (O₂) is assumed to decrease from the bottom to the top of the biofilm. The green background is auto-fluorescence of the matrix: EPS, and non-responding bacteria in the left panel, while in the right panel it corresponds to bacteria stained with the EUB338 probe FITC labeled, and also some auto-fluorescent EPS. b) Concentration of F. prausnitzii (F.p.) and Bifidobacterium spp. (Bif.) in the lumen of the SHIME (L) and mucus layer (M) of the HMI module during the treatment period determined by specific qPCR (n = 3).

Figure 7

Cytokine production and enterocytes (a) data related to the IL-8 production along the experiment (n = 2). Data are expressed as (pg mL⁻¹)/h; the standard deviation was calculated on the readings of the two parallel setups. (b) Microscopy scans of the cell lines collected from the HMI modules of the SHIME treated with the test product to evaluate the morphology at time 0, 24 and 48 h.