1. Physics of Living Systems

High-molecular-weight polymers from dietary fiber drive aggregation of particulates in the murine small intestine

  1. Asher Preska Steinberg
  2. Sujit S Datta
  3. Thomas Naragon
  4. Justin C Rolando
  5. Said R Bogatyrev
  6. Rustem F Ismagilov  Is a corresponding author
  1. California Institute of Technology, United States
  2. Princeton University, United States
https://doi.org/10.7554/eLife.40387
Share this article
Cite this article
  1. Asher Preska Steinberg
  2. Sujit S Datta
  3. Thomas Naragon
  4. Justin C Rolando
  5. Said R Bogatyrev
  6. Rustem F Ismagilov
(2019)
High-molecular-weight polymers from dietary fiber drive aggregation of particulates in the murine small intestine
eLife 8:e40387.
https://doi.org/10.7554/eLife.40387
  2. Download BibTeX
  3. Download .RIS
7 figures, 9 tables and 1 additional file

Figures

Figure 1 with 1 supplement
Download asset Open asset
PEG-coated particles aggregate in the murine small intestine (SI).

(A and B) Fluorescent scanner image of gastrointestinal tract (GIT) from a mouse orally administered a suspension of 1 µm diameter PEG-coated particles (green). Scale bar is 0.5 cm. (see Figure …

https://doi.org/10.7554/eLife.40387.002
Figure 1—figure supplement 1
Download asset Open asset
Overview of image processing for fluorescent scanner images appearing in Figure 1.

(A) Unmodified fluorescent scanner images of the gastrointestinal tract of a mouse gavaged with 1 µm-diameter PEG-coated particles (prior to the contrast and color-adjustments shown in Figure 1A-B). …

https://doi.org/10.7554/eLife.40387.003
Figure 2
Download asset Open asset
PEG-coated particles aggregate in fluid from the murine small intestine (SI) ex vivo.

The 1-µm-diameter PEG-coated particles form aggregates in fluid collected from the upper (A-C) and lower (D) SI in ~10 min. (A and D) Maximum z-projections of 10 optical slices taken on a confocal …

https://doi.org/10.7554/eLife.40387.005
Figure 3
Download asset Open asset
Gel permeation chromatography (GPC) of fluid from the small intestine (SI) and aggregation of PEG-coated particles in fractionated fluid from SI.

(A and D) Chromatograms of samples from the upper (A) and lower (D) SI from a group of three chow-fed mice. Dashed lines indicate the three retention volumes the chromatograms were divided into for …

https://doi.org/10.7554/eLife.40387.006
Figure 4 with 3 supplements
Download asset Open asset
Aggregation of PEG-coated particles in model polymer solutions shows complex dependence on molecular weight (MW) and concentration of PEG.

(A) Aggregates of 1 µm diameter PEG-coated particles in a 1 MDa PEG solution with a polymer concentration (c) of 1.6 mg/mL. Image is a maximum z-projection of 10 optical slices taken on a confocal …

https://doi.org/10.7554/eLife.40387.007
Figure 4—figure supplement 1
Download asset Open asset
pH measurements of luminal fluid from different sections of the gastrointestinal tract.

Measurements were conducted on pooled samples of luminal fluid collected from two groups of mice. Each measurement was repeated three times, and the error bars are the standard deviation across the …

https://doi.org/10.7554/eLife.40387.008
Figure 4—figure supplement 2
Download asset Open asset
Aggregation of PEG-coated particles in model polymer solutions with different pH.

(A) Volume-weighted average sizes for serial dilutions of 1 MDa PEG solutions in a phosphate buffered saline solution with pH=6.0±0.1 (labeled pH = 6.0) and in Hanks' balanced salt solution (HBSS) with pH=7.6±0.1 (s…

https://doi.org/10.7554/eLife.40387.009
Figure 4—figure supplement 3
Download asset Open asset
Aggregation of PEG-coated particles in model polymer solutions from Figure 4D normalized by polymer overlap concentration.

Volume-weighted average sizes for serial dilutions of 1 MDa PEG solutions in Hanks' balanced salt solution (HBSS). Volume-weighted average sizes are plotted on the vertical axis in units of number …

https://doi.org/10.7554/eLife.40387.010
Figure 5 with 1 supplement
Download asset Open asset
Quantification of the aggregation of particles in the small intestine (SI) in MUC2 knockout (MUC2KO) and wild-type (WT) mice.

(A and B) Volume-weighted empirical cumulative distribution functions (ECDFs) comparing aggregation of the particles in undiluted, 30 µm filtered samples from the upper (A) and lower (B) SI of two …

https://doi.org/10.7554/eLife.40387.011
Figure 5—figure supplement 1
Download asset Open asset
Ex vivo aggregation in 0.45 µm-filtered luminal fluid from the small intestines (SI) of wild-type (WT) and MUC2 knockout (MUC2KO) mice.

(A and B) Volume-weighted empirical cumulative distribution functions (ECDFs) comparing aggregation of the particles in undiluted, 0.45-µm-filtered samples from the upper (A) and lower (B) SI of two …

https://doi.org/10.7554/eLife.40387.012
Figure 6 with 3 supplements
Download asset Open asset
Quantification of the aggregation of particles in the small intestine (SI) in immunoglobulin-deficient (Rag1KO) and wild-type (WT) mice.

(A and B) Volume-weighted empirical cumulative distribution functions (ECDFs) comparing aggregation of the particles in undiluted, 30 µm filtered samples from the upper (A) and lower (B) SI of two …

https://doi.org/10.7554/eLife.40387.015
Figure 6—figure supplement 1
Download asset Open asset
Western blots of 30 µm-filtered samples from the small intestine (SI) of wild-type (WT) and Rag1 knockout (Rag1KO) mice.

WT USI = WT upper SI; KO USI = KO lower SI; WT LSI = WT lower SI; KO USI = KO upper SI. For the detection of IgG, 1:10,000 dilutions of Li-Cor IRDye 800 CW Goat Anti-Mouse IgG was used. Because the …

https://doi.org/10.7554/eLife.40387.016
Figure 6—figure supplement 2
Download asset Open asset
Western blots of 30 µm-filtered samples from the small intestine (SI) of wild-type (WT) and Rag1 knockout (Rag1KO) mice.

WT USI = WT upper SI; KO USI = KO lower SI; WT LSI = WT lower SI; KO USI = KO upper SI. For detection of IgM, 1:10,000 dilution of Li-Cor IRDye 800CW Goat Anti-Mouse IgM was used. We do not detect …

https://doi.org/10.7554/eLife.40387.017
Figure 6—figure supplement 3
Download asset Open asset
Ex vivo aggregation in 0.45-µm-filtered luminal fluid from the small intestines (SI) of wild-type (WT) and Rag1 knockout (Rag1KO) mice.

(A and B) Volume-weighted empirical cumulative distribution functions (ECDFs) comparing aggregation of the particles in undiluted, 0.45-µm-filtered samples from the upper (A) and lower (B) SI of two …

https://doi.org/10.7554/eLife.40387.018
Figure 7
Download asset Open asset
Quantification of aggregation of PEG-coated particles in the small intestine (SI) of mice fed different polymers from dietary fiber.

(A) Volume-weighted average aggregate sizes (Vol Wt Avg Size) for serial dilutions of apple pectin and Fibersol-2. Volume-weighted average sizes are plotted on the vertical axis in terms of number …

https://doi.org/10.7554/eLife.40387.021

Tables

Table 1
Zeta potential and NMR measurements of PEG-coated particles.

For the zeta potential measurements, each particle solution was 0.1 mg/ml of particles in 1 mM KCl. Measurements were done on a Brookhaven NanoBrook ZetaPALS Potential Analyzer. Three trials were …

https://doi.org/10.7554/eLife.40387.004
Surface modification of PS particlesZeta potential (mV)Nanomoles PEG/mg particles
PEG 5 kDa−18.87 ± 1.785.5
PEG 5 kDa w/ mPEG 1 kDa backfill−7.66 ± 2.124.6
PEG 5 kDa w/ mPEG 350 Da backfill−9.99 ± 1.654.3
PEG 5 kDa w/ mPEG 5 kDa backfill−14.56 ± 1.784.0
PEG 2 kDa−39.59 ± 2.419.4
Carboxylate-coated (no PEG)−61.36 ± 12.400.0
Table 2
Estimates of physical parameters of polymers from gel permeation chromatography for liquid fractions from the upper small intestine of MUC2 knockout (MUC2KO) and wild-type (WT) mice.
https://doi.org/10.7554/eLife.40387.013
Retention volume (mL)11 to 1616 to 20>20
Mouse typeWTMUC2KOWTMUC2KOWTMUC2KO
Mw (kDa)3,560 ± 4105,420 ± 620162 ± 20147 ± 174.05 ± 0.462.96 ± 0.34
Mw/Mn1.361.592.162.433.5910.9
Rh (nm)49.145.56.315.951.181.02
Fract. Conc. (mg/mL)2.52 ± 0.291.18 ± 0.1324.6 ± 2.821.9 ± 2.588.7 ± 10.186.0 ± 9.8

  1. We calculated values with both dn/dc = 0.185 (for proteins) and dn/dc = 0.147 (pullulan). When the value varied with dn/dc, it is reported in the table as the mid-range values ± the absolute deviation between the two calculated values. Mw = the weight-average molecular weight; Mw/Mn = the dispersity; Rh = hydrodynamic radius; Fract. Conc. = Concentration of a given molecular weight fraction.

Table 3
Estimates of physical parameters of polymers from gel permeation chromatography for liquid fractions from the lower small intestine of MUC2 knockout (MUC2KO) and wild-type (WT) mice
https://doi.org/10.7554/eLife.40387.014
Retention volume (mL)11 to 1616 to 20>20
Mouse typeWTMUC2KOWTMUC2KOWTMUC2KO
Mw (kDa)4,730 ± 5405,180 ± 590219 ± 25155 ± 1813.7 ± 1.65.93 ± 0.68
Mw/Mn1.241.801.911.841.882.03
Rh (nm)57.049.28.457.581.891.35
Fract. Conc. (mg/mL)3.42 ± 0.392.36 ± 0.2723.0 ± 2.622.8 ± 2.654.8 ± 6.363.3 ± 7.2

  1. We calculated values with both dn/dc = 0.185 (for proteins) and dn/dc = 0.147 (pullulan). When the value varied with dn/dc, it is reported in the table as the mid-range values ± the absolute deviation between the two calculated values. Mw = the weight-average molecular weight; Mw/Mn = the dispersity; Rh = hydrodynamic radius; Fract. Conc. = Concentration of a given molecular weight fraction.

Table 4
Estimates of physical parameters of polymers from gel permeation chromatography for liquid fractions from the upper small intestine of immunoglobulin-deficient (Rag1KO) and wild-type (WT) mice.
https://doi.org/10.7554/eLife.40387.019
Retention volume (mL)11 to 1616 to 20>20
Mouse typeWTRag1KOWTRag1KOWTRag1KO
Mw (kDa)1,480 ± 1702,140 ± 250108 ± 1274.2 ± 8.52.84 ± 0.321.91 ± 0.22
Mw/Mn1.091.142.622.421.591.54
Rh (nm)31.839.84.772.511.0780.936
Fract. Conc. (mg/mL)1.07 ± 0.121.13 ± 0.1314.3 ± 1.613.9 ± 1.666.1 ± 7.670.5 ± 8.1

  1. We calculated values with both dn/dc = 0.185 (for proteins) and dn/dc = 0.147 (pullulan). When the value varied with dn/dc, it is reported in the table as the mid-range value ± the absolute deviation between the two calculated values. Mw = the weight-average molecular weight; Mw/Mn = the dispersity; Rh = hydrodynamic radius; Fract. Conc. = Concentration of a given molecular weight fraction.

Table 5
Estimates of physical parameters of polymers from gel permeation chromatography for liquid fractions from the lower small intestine of immunoglobulin-deficient (Rag1KO) and wild-type (WT) mice.
https://doi.org/10.7554/eLife.40387.020
Retention volume (mL)11 to 1616 to 20>20
Mouse typeWTRag1KOWTRag1KOWTRag1KO
Mw (kDa)1,080 ± 1202,490 ± 29066.9 ± 7.791.6 ± 10.53.64 ± 0.423.72 ± 0.43
Mw/Mn1.181.051.711.982.091.98
Rh (nm)34.647.14.674.851.1161.09
Fract. Conc. (mg/mL)1.52 ± 0.171.89 ± 0.2215.8 ± 1.814.1 ± 1.649.5 ± 5.755.1 ± 6.3

  1. We calculated values with both dn/dc = 0.185 (for proteins) and dn/dc = 0.147 (pullulan). When the value varied with dn/dc, it is reported in the table as the mid-range values ± the absolute deviation between the two calculated values. Mw = the weight-average molecular weight; Mw/Mn = the dispersity; Rh = hydrodynamic radius; Fract. Conc. = Concentration of a given molecular weight fraction.

Table 6
Gel permeation chromatography of Fibersol-2 and pectin in phosphate-buffered saline
https://doi.org/10.7554/eLife.40387.022
SampleFibersol-2Pectin
Mw (kDa)3.48232
Mw/Mn10.51.97
Rh (nm)1.2425.4

  1. Both fiber types were analyzed with dn/dc = 0.147 for polysaccharides. Mw = weight-average molecular weight; Mw/Mn = the dispersity; Rh = hydrodynamic radius

Table 7
Estimates of physical parameters of polymers from gel permeation chromatography for liquid fractions from upper small intestine of pectin and Fibersol-2 fed mice.
https://doi.org/10.7554/eLife.40387.023
Retention volume (mL)11 to 1616 to 20>20
Mouse typePectinFibersol-2PectinFibersol-2PectinFibersol-2
Mw (kDa)267 ± 31686 ± 7940.0 ± 4.535.3 ± 4.01.39 ± 0.161.67 ± 0.19
Mw/Mn1.501.082.152.642.451.48
Rh (nm)31.8N/C**5.522.880.819N/C**
Fract. Conc. (mg/mL)1.62 ± 0.190.516 ± 0.0599.00 ± 1.0323.3 ± 2.753.7 ± 6.177.0 ± 8.8

  1. We calculated values with both dn/dc = 0.185 (for proteins) and dn/dc = 0.147 (pullulan). When the value varied with dn/dc, it is reported in the table as the mid-range values ± the absolute deviation between the two calculated values. Mw = the weight-average molecular weight; Mw/Mn = the dispersity; Rh= hydrodynamic radius; Fract. Conc. = Concentration of a given molecular weight fraction. N/C** denotes values for which the concentration was too low to calculate.

Table 8
Estimates of physical parameters of polymers from gel permeation chromatography for liquid fractions from lower small intestine of pectin and Fibersol-2-fed mice.
https://doi.org/10.7554/eLife.40387.024
Retention volume (mL)11 to 1616 to 20>20
Mouse typePectinFibersol-2PectinFibersol-2PectinFibersol-2
Mw (kDa)282 ± 321680 ± 19030.2 ± 3.518.8 ± 2.21.12 ± 0.132.32 ± 0.27
Mw/Mn7.371.641.702.782.891.14
Rh (nm)29.026.45.282.160.7241.06
Fract. Conc. (mg/mL)2.48 ± 0.280.839 ± 0.0969.43 ± 1.153.6 ± 6.142.7 ± 4.988.3 ± 10.1

  1. We calculated values with both dn/dc = 0.185 (for proteins) and dn/dc = 0.147 (pullulan). When the value varied with dn/dc, it is reported in the table as the mid-range values ± the absolute deviation between the two calculated values. Mw = the weight-average molecular weight; Mw/Mn = the dispersity; Rh = hydrodynamic radius; Fract. Conc. = concentration of a given molecular weight fraction.

Key resources table
 Reagent type
(species)
or resource		DesignationSource or
reference		IdentifiersAdditional
information
MUC2KO,
C57BL/6 mice
(female)		MUC2KOEugene Chang Lab
(University of Chicago)
provided initial
breeding pairs which
were provided to
them
from Leonard
H. Augenlicht at the
Department of
Oncology
of Albert Einstein
Cancer Center		Genotyping was
performed by
Transnetyx Inc.;
Western blot was
done to confirm
lack of MUC2
(See Figure 5E)
Rag1KO,
C57BL/6
mice (male)		Rag1KOProvided by
Mazmanian
Lab at Caltech		RRID:IMSR_JAX:002216Western blot was
done to confirm
lack of IgA as
explained
in the text
(See Figure 6E)
C57BL/6 mice
(all male except for
WT controls in MUC2KO
experiments in Figure 5)		WTThe Jackson
Laboratory		RRID:IMSR_JAX:000664
AntibodyMUC2 polyclonal
antibody
(rabbit host)		BiomatikCat No:
CAU27315
AntibodyLi-Cor IRDye
800 CW Goat Anti
-Rabbit IgG		Li-CorP/N 925–32211;
RRID:AB_2651127
AntibodyLi-Cor IRDye
800 CW Goat Anti
-Mouse IgG		Li-CorP/N 925–32210;
RRID:AB_2687825
AntibodyLi-Cor IRDye
800 CW Goat Anti
-Mouse IgM		Li-CorP/N 925–32280
AntibodyGoat Anti-Mouse
IgA-unlabeled		Southern
Biotech		Cat No: 1040–01
AntibodyLi-Cor IRDye 800
CW Donkey
Anti-Goat IgG		Li-CorP/N 925–32214;
RRID:AB_2687553
Chemical
compound, drug		apple pectinSolgar Inc.‘Apple pectin
powder’;
SOLGB70120 00B
Chemical
compound, drug		Fibersol-2Archer Daniels
Midland/
Matsutani
LLC		Product code:
013100, Lot #:
CY4P28540
Chemical
compound, drug		USP grade
sucrose		Sigma-Aldrich
Chemical
compound, drug		Protease
inhibitor
cocktail		Roche cOmplete,
Mini, EDTA-free
Protease-Inhibitor
cocktail, Roche
Chemical
compound, drug		PEG 100 kDaDowPOLYOX
WSR N-10
Chemical
compound, drug		PEG 1 MDaDowPOLYOX
WSR N-12K
Chemical
compound, drug		PEG 3350BayerMiraLAX
Chemical
compound, drug		Hanks' Balanced
Salt Solution
(without calcium,
magnesium,
phenol red)		GE Healthcare
Life Sciences		Product code:
SH30588.02
Software,
algorithm		3D aggregate
analysis pipeline		This paper;
source code
available
through Dryad		Description in
Materials and
methods;
source
code provided
on Dryad
Othermesh-bottom
(or wire-bottom)
floors		Lab Products,
Inc.		P/N: 75016
Other1 μm diameter
PEG 5 kDa-coated
polysytrene beads		This paperDescription of
synthesis in
Materials
and methods
Other1 μm diameter
PEG 5 kDa-coated
polysytrene beads
with PEG 1 kDa
‘back-filling’		This paperDescription of
synthesis in
Materials
and methods
Otherstandard
chow diet		PicoLabPicoLab Rodent
Diet 20;
Product #5053
Otherautoclaved
chow diet		PicoLabLaboratory
Autoclavable
Rodent
Diet 5010

Additional files

Transparent reporting form
https://doi.org/10.7554/eLife.40387.025

Download links