Lipopolysaccharide from Crypt-Specific Core Microbiota Modulates the Colonic Epithelial Proliferation-to-Differentiation Balance

Abstract

We identified a crypt-specific core microbiota (CSCM) dominated by strictly aerobic, nonfermentative bacteria in murine cecal and proximal colonic (PC) crypts and hypothesized that, among its possible functions, it may affect epithelial regeneration. In the present work, we isolated representative CSCM strains using selective media based upon our initial 16S rRNA-based molecular identification (i.e., Acinetobacter, Delftia, and Stenotrophomonas). Their tropism for the crypt was confirmed, and their influence on epithelial regeneration was demonstrated in vivo by monocolonization of germfree mice. We also showed that lipopolysaccharide (LPS), through its endotoxin activity, was the dominant bacterial agonist controlling proliferation. The relevant molecular mechanisms were analyzed using colonic crypt-derived organoids exposed to bacterial sonicates or highly purified LPS as agonists. We identified a Toll-like receptor 4 (TLR4)-dependent program affecting crypts at different stages of epithelial differentiation. LPS played a dual role: it repressed cell proliferation through RIPK3-mediated necroptosis of stem cells and cells of the transit-amplifying compartment and concurrently enhanced cell differentiation, particularly the goblet cell lineage.IMPORTANCE The LPS from crypt-specific core microbiota controls intestinal epithelium proliferation through necroptosis of stem cells and enhances cell differentiation, mainly the goblet cell lineage.

Keywords: LPS; homeostasis; intestinal stem cells; necroptosis.

FIG 1

In vivo analysis of mice monoassociated with CSCM members. (A) Percentage of EdU⁺ cells in proximal colonic (PC) tissue from mice monocolonized with CSCM members or with B. fragilis on day 15 with a representative EdU staining of proximal colonic tissue from germfree mice as a control (Ctrl). (B) Percentage of apoptotic/dead cells in PC tissue from mice monocolonized with CSCM members on days 15 and 30 or monoassociated with B. fragilis on day 15. nd, not determined. (C to H) Representative pictures of apoptotic/dead cells of proximal colonic tissue from germfree mice (C) or from mice colonized for 15 days with A. modestus (D), A. radioresistens (E), D. tsuruhatensis (F), S. maltophilia (G), and B. fragilis (H). Magnification, 400×. (I) Proximal colonic crypt length of mice monocolonized with CSCM members A. modestus (Am), A. radioresistens (Ar), D. tsuruhatensis (Dt), and S. maltophilia (Sm). Data are expressed as means plus standard deviations (error bars). There were five mice in each group. Values that were significantly different from the value for the nonstimulated control group are indicated by asterisks as follows: *, P < 0.05; **, P < 0.01. Data from at least 50 crypts per section were examined for all histological parameters.

FIG 2

Effects of sonicated CSCM members on PC organoids. (A and B) Ratio of WT murine PC colonospheres (A) and colonoids (B) stimulated with sonicated CSCM isolates on day 7. The CSCM members are A. modestus (Am), A. radioresistens (Ar), D. tsuruhatensis (Dt), and S. maltophilia (Sm). There were four mice in each group. The ratio was calculated by normalizing the values (numbers) for stimulated organoids by the number of organoids without stimulation as a control (Ctrl). (C) Real-time PCR (RT-PCR) showing the gene expression in crypt cultures stimulated with sonicated CSCM isolates. (D) Ratio of total organoids from TLR4^−/− crypts stimulated with sonicated CSCM isolates. (E) Ratio of total organoids from WT crypts during stimulation with a sonicate from Lactobacillus casei ATCC 334. Data are expressed as means plus standard deviations (error bars). There were three mice in each group. Values that were significantly different (P < 0.05) from the value for the nonstimulated control group are indicated by an asterisk.

FIG 3

Structures of lipid A from Acinetobacter, Delftia, and Stenotrophomonas LPSs. The A. modestus and A. radioresistens lipid A differ by the presence of a C₁₀ or C₁₂ secondary fatty acid, respectively. The S. maltophilia lipid A is a blend of several species in which all the fatty acids can differ by one carbon atom and range from C₁₀ to C₁₂. The respective MALDI MS spectra are shown in the supplemental material (Fig. S4).

FIG 4

Effects of the LPSs purified from CSCM members on PC organoids. (A and B) Ratio of viable PC colonospheres (A) and colonoids (B) derived from WT crypts were calculated on days 5 and 7. PC organoids were stimulated with different concentrations (0.01 to 10 μg/ml) of LPS. (C and D) Ratio of colonospheres (C) and colonoids (D) during stimulation of organoids derived from WT mice (black bars) and TLR4^−/− mice (white bars) with LPS purified from CSCM members (1.0 μg/ml). (E and F) Ratio of colonospheres (E) and colonoids (F) during stimulation of organoids derived from WT mice with LPS (black bars) or with KOH-treated LPS (white bars) (1.0 μg/ml). The CSCM members are A. modestus (Am), A. radioresistens (Ar), D. tsuruhatensis (Dt), and S. maltophilia (Sm). There were six mice in each group. Values that were significantly different from the value for the nonstimulated control (Ctrl) group are indicated by asterisks as follows: *, P < 0.05; **, P < 0.01.

FIG 5

Effects of the LPS purified from CSCM members on size and transit-amplifying (TA) cells of PC organoids. (A) Representative bright-field micrographs of colonoids grown in matrigel for 5 days with LPS from CSCM members (1.0 μg/ml). The CSCM members are A. modestus (Am), A. radioresistens (Ar), D. tsuruhatensis (Dt), and S. maltophilia (Sm). (B) Individual area of day 5 PC organoid stimulated with LPS (1.0 μg/ml) in randomly selected fields. Red bars indicate averaged area of PC organoid. Values are means ± SE. n = 32 in non stimuli group, n = 29 in A. modestus group, n = 50 in A. radioresistens group, n = 54 in D. tsuruhatensis group, and n = 33 in S. maltophilia group as indicated. (C) Percentage of Ki-67⁺ TA cells in day 5 PC organoid stimulated with LPS. There were eight mice in each group. Values that were significantly different (P < 0.05) from the value for the nonstimulated control group are indicated by an asterisk. (D) RT-PCR showing Axin2 and Ascl2 mRNA abundances in WT PC organoids stimulated with LPS (1.0 μg/ml). Data are expressed as means plus standard deviations. There were three mice in each group. Values that were significantly different (P < 0.05) from the value for the nonstimulated control group are indicated by an asterisk.

FIG 6

LPSs purified from CSCM members induce necroptosis and damage-associated molecular patterns (DAMPs) and IL-33. The CSCM members are A. modestus (Am), A. radioresistens (Ar), D. tsuruhatensis (Dt), and S. maltophilia (Sm). (A) Gene expression of Ripk1 and Ripk3 in LPS-stimulated organoids. (B) Ratio of colonospheres and colonoids during stimulation of organoids derived from WT mice (black bars) and RIPK3^−/− mice (white bars) with LPS purified from CSCM members (1.0 μg/ml). There were eight mice in each group. *, P < 0.05. (C) RT-PCR showing the expression of TNF-α, IL-33, S100A8, S100A9, and S100A14. There were six mice in each group indicated on the x axis. *, P < 0.05 versus nonstimulated control (Ctrl) group.

FIG 7

LPSs purified from CSCM members induce the differentiation/maturation of PC epithelial cells and act on Lgr5⁺ cells. (A) LPSs purified from CSCM strains induce mRNA abundance associated with maturated colonocyte transcripts. The CSCM members are A. modestus (Am), A. radioresistens (Ar), D. tsuruhatensis (Dt), and S. maltophilia (Sm). RT-PCR showing the expression of Alpi, Klf4, Tff, and keratin 20 (krt20) as mature epithelial cell markers and Car1, Slc9a2, and Slc9a3 as markers of colonoids cultured from WT crypts. There were three mice in each group. *, P < 0.05 versus the nonstimulated control (Ctrl) group. (B) Representative micrographs of alkaline phosphatase (Alpi) immunostaining (red) of PC colonoids stimulated with LPSs from CSCM members. Nuclei were stained with 4′,6′-diamidino-2-phenylindole (DAPI) (blue). Quantitative analysis of the ratio of Alpi-stained area to DAPI-stained area were measured using the ImageJ software from day 5 PC colonoid stimulated with LPS (1.0 μg/ml). In the graph in the middle of panel B, the red bars indicate the average ratios of Alpi-positive area/DAPI-positive area of the PC organoid for the group. In the graph to the right in panel B, values are means plus standard errors (SE) (error bars). (C) Effects of LPSs from CSCM members on terminal differentiation of goblet cells (GC) in PC organoids. Fluorescence-activated cell sorting (FACS) analysis showing the ratios of WGA⁺ UEA-1⁺ cells, which are located in the middle of the PC crypt, WGA-1^low UEA-1⁺ cells, which are located at the top of the PC crypt, and c-kit⁺ WGA⁺ goblet-like cells (GLC) in doublet and dead cells depleted of EpCam⁺ CD45.2⁻ cells from day 5 PC organoids. The organoids were from mice given A. modestus LPS (1.0 μg/ml), A. radioresistens LPS (1.0 μg/ml), D. tsuruhatensis LPS (1.0 μg/ml), or S. maltophilia LPS (1.0 μg/ml). There were six mice in each group. *, P < 0.05 versus nonstimulated control group. (D) Gene expression level of colonoids treated with CHIR99021 and valproic acid showing the upregulation of Lgr5 expression and downregulation of genes involved in differentiation. (E) Ratio of PC colonoids during stimulation of organoids derived from C57BL/6 mice cultured with CHIR99021 and vaproic acid with LPSs from CSCM members (1.0 μg/ml).