Carrageenan oligosaccharides and their degrading bacteria induce intestinal inflammation in germ-free mouse

Background: Carrageenans (CGNs) are widely used in food and pharmaceutical industries. However, the safety of CGNs is still under debate, because degraded CGNs have been reported to promote an intestinal inflammatory response in animal models. Here, we studied the relationship among CGNs, human gut microbiota, and the host inflammatory response. Methods: TLC was selected for detecting the degradation of KCPs by human gut microbiota in vitro batch fermentation system. PCR-DGGE and real time PCR were used for studying bacterial community. ESI-MS was used for KCPs structure analysis. Hematoxylin-eosin staining (HE), immunohistochemistry (IHC) and RNA-seq were used to evaluated the KCPs on host inflammation response in germ-free mice. Results: Thin-layer chromatography (TLC) data showed that CGNs with a molecular weight (Mw) higher than 100 kDa are not degraded by human fecal microbiota, but low Mw CGNs with an Mw around ~4.5 kDa (KCOs) could be degraded by seven of eight human fecal microbiota samples. KCO degrading B. xylanisolvens was isolated from fecal samples, and PCR-DGGE profiling with band sequencing suggested that B. xylanisolvens was the key KCO degrader in the human gut . Two putative κ-carrageenase genes were identified in the genome sequence of B. xylanisolvens . However, their function on KCO degrading was not verified in vitro . And the sulfate group from KCO is not removed after in vitro degradation by human fecal microbiota, as shown by ESI-MS analysis. The effects of KCO and KCO degrading bacteria on the inflammatory response were investigated in germ-free mice. Increased numbers of P-P38-, CD3a-, and CD79a-positive cells were found in the colon and rectum in mice fed with KCO plus KCO degrading bacteria than in mice fed with only KCO or only B. xylanisolvens and E. coli , as shown by RNA-Seq analysis, HE staining, cutoff to The final obtained by rotary evaporation and lyophilization. The MW of KCP, SKCO, and KCO was measured using high-performance liquid chromatography (HPLC) (Agilent 1260, U.S.) with a Shodex OH pak SB-804 detected using a refractive index detector and multiangle laser light scattering. The

Accumulating evidence indicates that κ-CGNs, especially their low-MW degradation products, induce inflammation in the colon, resulting in a range of diseases in cell and animal models, including  38], gastroenterocolitis [39,40], arthritis [41,42], and inflammatory bowel disease. However, CGNs also show positive health effects, such as antitumor [43], antimicrobial [44], and antiviral [45,46]. The inconsistent effects of κ-CGN have been attributed to the varying MWs of compounds present in commercial κ-CGN products [1], but we propose that the variation in composition and function of human colonic microbiota may also contribute to the dilemma Therefore, in the present study, we investigated the interaction of κ-CGNs of different MWs with human colonic microbiota. The results clearly demonstrate that the κ-CGN degradation rate by human gut microbiota is correlated with the MW of the κ-CGNs. κ-CGNs with an MW over 100 kDa, which include both KCP and SKCO, cannot be utilized by human fecal microbiota in vitro (Supplementary Removal of sulfate esters from sulfated polysaccharides is considered one of the essential steps for microorganisms in the degradation of sulfurized polysaccharides [47]. The most diversified sulfatase genes have been discovered in marine bacteria [48], due to the higher contents of sulfate ester groups in marine polysaccharides. The human intestinal tract contains high amounts of sulfurized glycans. For example, mucins, heparan sulfate, and chondroitin sulfate, which are the main components of the extracellular matrix of mammalian cells, can be degraded by human colonic microbiota; bacterial sulfatases have also been identified from mucin degrading bacterial strains [49,50]. Bacteroides thetaiotaomicron, which has 28 putative sulfatase genes, is able to liberate free sulfate from chondroitin to provide it as the electron acceptor for the sulfate reducing bacterium Desulfovibrio piger in the mouse gut, releasing H 2 S as the end product [51]. Electrospray ionization mass spectrometry (ESI-MS) for sugar structure analysis The derivatives generated from digestion of CGN oligosaccharides were determined by gel filtration chromatography and analyzed using negative-ion electrospray ionization mass spectrometry (ESI-MS).
In brief, after removing the bacteria by centrifugation, the supernatants were separated on a Superdex Peptide 10/300 column. The sequence of each fraction was determined on a Thermo Large food residues were removed by passing the mixture through a 0.4 mm sieve. The human fecal slurry (7 ml) was inoculated into a bottle containing 63 ml of growth medium, and the bottle was incubated at 37 °C for 72 h in an anaerobic chamber (anaerobic workstation AW 500, Electrotek Ltd., U.K.). Fermentation products were collected at different time points for further analysis. The pH value after 48 h fermentation was measured by a pH probe (Eutech, Singapore).

Thin-layer Chromatography (tlc) And Total Carbohydrate Analysis
The degradation of KCP, SKCO, and KCO was detected by TLC analysis. Briefly, 0.2 µL of sample was loaded on pre-coated silica gel-60 TLC aluminum plates (Merck, Germany). After development with a solvent system consisting of formic acid/n-butanol/water (6:4:1, v:v:v), the plate was soaked in orcinol reagent and carbohydrates were visualized by heating at 120 °C for 3 min.
The total carbohydrate concentration in the fermentation samples was determined using the phenolsulfuric acid method, as described previously, using D-galactose as standard. Results are expressed as the mean amount of remaining carbohydrate relative to the total amount detected at 0 h.

Short-chain Fatty Acid (scfa) Analysis
Production of short-chain fatty acids (SCFAs) was determined by HPLC with an Aminex HPX-87H Exclusion Column. In brief, fermentation products were centrifuged at 14, 000 rpm for 15 min, and the supernatant was used for measurement. The detection condition included 5 mM H 2 SO 4 used as mobile phase at a flow rate of 0.6 ml/min. The column temperature was 50 °C and a refractive index detector was used at a wavelength of 215 nm.

Animal Experiments
Twenty-four three-week-old germ-free Kunming mice were randomly divided into four groups. KCO degrading bacteria (5 × 10 8 in 0.5 ml) were inoculated intragastrically to the GN and GNK groups on

Rna-seq Analysis
To identify genes that were differentially expressed (DEGs) in response to KCOs and KCO-degrading bacteria in germ-free mice at the transcription level, total RNA was isolated from rectum samples for 100% xylene for 20 minutes twice, and then immersed in paraffin at 58-60 °C for 3 hours. After the tissue was paraffin-embedded, 4-µm coronal serial sections were cut using a Microm HM-340E microtome (Microm, Walldorf, Germany). The sections were subjected to HE staining and mounted with neutral balsam. Subsequently, they were examined by microscopy to observe the changes in ulcer size and infiltration of inflammatory cells. Injuries to colon tissue were scored as previously described.

Immunohistochemical (ihc) Analysis
The specimens were stained with the EnVision™ two-step strategy and high-temperature antigen retrieval (pressure cooker, Supor Co, China). In brief, 4-µm-thick paraffin-embedded sections were deparaffinized twice (10 minutes each in 100% xylene) and then hydrated with 100% ethanol for 5 minutes twice, with 95% ethanol for 3 minutes, and with 80% ethanol for 5 minutes. After two 5minute soakings in distilled water, the slides were put into the pressure cooker filled with 1000 ml of boiling sodium citrate buffer (pH 9.0) and heated under pressure. After steaming, the pressure cooker was removed from the heat source and cooled down to room temperature with tap water. The slides were then rinsed twice for 3 minutes with PBS. The slides were incubated with 3% hydrogen peroxide for 10 minutes and rinsed twice in PBS for 3 minutes. Primary antibody (anti-CD3, 1:100, clone SP7, ab16669, Abcam; anti-VCAM1, 1:200, clone EPR5047, ab134047, Abcam; anti-PECAM1, 1:1000, clone EPR17259, ab182981, Abcam; anti-phospho-p38 MAPK (Thr180 and Tyr182), 1:400, clones D3F9 and 4511, Cell Signaling Technology) were applied for 60 minutes in a moist chamber at 37 °C. After rinsing twice for 3 minutes with PBS, the slides were incubated with HRP polymer for 30 minutes at 37 °C. After adding diaminobenzidine (DAB) chromagen, the slides were observed and examined for color change under a light microscope. This was followed by counterstaining with hematoxylin for 1 minute and rinsing with tap water for 1 minute. Two slides were treated with PBS instead of primary antibody and served as the negative control. Sections were observed using the double-blind method by a pathological physician. In each section, cells were selected in five randomly selected fields to calculate the percentage of positive cells.     Structural changes in mouse intestinal sections after treatment with KCO and KCO degrading bacteria. Four groups of germ-free mice were included in the experiment, which lasted for 6 weeks. GFK and GNK groups were exposed to KCO (5% in drinking water) for the entire experiment. Mice in the GNK group were inoculated by oral gavage with B.
xylanisolvens and E. coli (5×108) on day 0. The GN group was only inoculated with B.
xylanisolvens and E. coli (5×108) on day 0. The GF group was the control group. A.
were treated with KCO (5% in drinking water, GFK group), KCO plus B. xylanisolvens and E.

Supplementary Files
This is a list of supplementary files associated with this preprint. Click to download. KCOSupplementary20200501.pdf