Mucosal healing progression after acute colitis in mice

Table 1 Composition of the experimental diet

Ingredient, g/kg	P14
Acid casein (Armor Protéines®, ref. 139860)	112
Whey protein (Armor Protéines®, Protarmor 80, ref. 139805)	28
Corn starch	622.4
Sucrose	100.3
Cellulose	50
Soybean oil	40
Mineral mixture (AIN 93-M)	35
Vitamin mixture (AIN 93-V)	10
Choline	2.3
Metabolizable energy, kJ/g	14.5

Table 2 Colon morphometric and inflammatory markers

Parameter	d0	d7	d7 vs d0	d10	d10 vs d7	d13	d13 vs d10	d28	d28 vs d0	Statistical effect (time)
Colon length (cm)	6.5 ± 0.2	4.9 ± 0.3a	↓	5.4 ± 0.2a	-	5.5 ± 0.2a	-	5.7 ± 0.2a	↓	< 0.001
Colon weight/length (mg/cm)	16 ± 0.3	24 ± 0.4a	↑	31 ± 3.0ab	↑	34 ± 3.0ab	-	38 ± 8.0ab	↑	< 0.05
Osmolarity of colonic content	180 ± 8	311 ± 15a	↑	278 ± 8.0a	-	288 ± 13a	-	281 ± 15a	↑	< 0.01
Colonic water content (%)	64.6 ± 2.5	87.5 ± 2.0a	↑	75.5 ± 3.0ab	↓	76.3 ± 1.5ab	-	74.0 ± 1.9ab	↑	< 0.001
IL-1β (ng/mg of total colon protein)	9.4 ± 1.8	14.3 ± 2.4a	↑	13.7 ± 1.8a	-	16.2 ± 2.5a	-	15.4 ± 2.2a	↑	< 0.01
IL-6 (ng/mg of total colon protein)	1.5 ± 0.5	3.6 ± 0.6a	↑	4.2 ± 0.8a	-	4.9 ± 1.2a	-	2.3 ± 0.3bcd	-	< 0.001
MPO activity (UA/mg total colon protein)	1.2 ± 0.2	5.8 ± 1.2a	↑	5.3 ± 0.8a	-	3.1 ± 0.8abc	↓	1.9 ± 0.6bcd	-	< 0.001
Plasmatic LBP (µg/mL)	2.0 ± 0.2	5.8 ± 0.4a	↑	4.5 ± 0.5ab	↓	4.9 ± 0.6ab	-	4.6 ± 0.3c	↑	< 0.001

C57BL/6 male mice were given 3.5% dextran sodium sulfate (DSS) for 5 d in their drinking water in order to induce an acute inflammatory episode. Colon morphometric and inflammatory markers were analyzed at peak colitis (day 7) and during colitis resolution (day 10, day 13 and day 28). Day 0 values correspond to non-DSS treated mice. Values are means ± SE (n = 8-12).

^aP < 0.05 vs day 0;

^bP < 0.05 vs day 7;

^cP < 0.05 vs day 10;

^dP < 0.05 vs day 13.

DSS: Dextran sodium sulfate; LBP: Lipopolysaccharide-binding protein; MPO: Myeloperoxidase activity.

Table 3 Histological analysis scoring of inflammatory and healing parameters

Parameter	d7	d10	d10 vs d7	d13	d13 vs d10	d28	d28 vs d7	Statistical effect (time)
Inflammatory score	12.8 ± 0.8	9.3 ± 1.4b	↓	8.4 ± 1.3b	-	4.4 ± 1.7bcd	↓	< 0.001
Goblet cell depletion	2.27 ± 0.24	1.83 ± 0.21	-	1.08 ± 0.26bc	↓	0.88 ± 0.30bc	↓	< 0.001
Ulceration and erosion	2.09 ± 0.29	1.34 ± 0.31	-	1.17 ± 0.27	-	0.63 ± 0.32bcd	↓	< 0.001
Crypt damage	2.72 ± 0.19	1.58 ± 0.34b	↓	1.25 ± 0.30b	-	1.25 ± 0.45b	↓	< 0.001
Edema	2.09 ± 0.09	1.25 ± 0.22b	↓	1.42 ± 0.23b	-	0.0 ± 0.0bcd	↓	< 0.001
Inflammatory infiltrate (mononuclear cells)	1.91 ± 0.27	1.83 ± 0.30	-	1.75 ± 0.18	-	1.40 ± 0.33bcd	-	< 0.001
Inflammatory infiltrate (neutrophils)	1.73 ± 0.13	1.50 ± 0.29	-	1.75 ± 0.19	-	0.63 ± 0.32bcd	↓	< 0.001
Epithelial repair score	0.82 ± 0.22	2.83 ± 0.42b	↑	3.25 ± 0.43b	-	3.25 ± 0.25b	↑	< 0.001
Gland hyperplasia and presence of mitotic cells	0.0 ± 0.0	0.83 ± 0.24b	↑	0.92 ± 0.26b	-	0.88 ± 0.35b	↑	< 0.001
Re-epithelialization	0.54 ± 0.16	0.58 ± 0.15	-	0.58 ± 0.15	-	0.63 ± 0.33	-	NS
Crypt repair	0.27 ± 0.19	1.42 ± 0.34b	↑	1.75 ± 0.30b	-	1.75 ± 0.45b	↑	< 0.001

Hematoxylin-and-eosin stained colonic sections were coded for blind microscopic assessment by an external histological platform (Histalim, Montpellier, France), and microscopic changes were qualitatively described and scored using a severity scale (0 to 3). Measurements were performed at peak colitis (day 7) and during colitis resolution (day 10, day 13 and day 28). Values are means ± SE (n = 8-11).

^bP < 0.05 vs day 7;

^cP < 0.05 vs day 10;

^dP < 0.05 vs day 13.

NS: Non-significant difference.

Table 4 Kinetics of colonic mRNA expression level normalized to day 7 group (2-ΔΔCt)

	d0	d7	d7 vs d0	d10	d10 vs d7	d13	d13 vsd10	d28	d28 vsd0	Statistical effect (time)
Tight-junction protein
Cldn2	2.41 ± 0.29	1.24 ± 0.21a	↓	2.90 ± 0.15b	↑	1.94 ± 0.17b	-	2.26 ± 0.31b	-	< 0.001
Tjp1	1.45 ± 0.09	1.03 ± 0.08a	↓	1.17 ± 0.10b	↑	1.30 ± 0.06b	-	1.29 ± 0.06b	-	< 0.01
Ocln	2.92 ± 0.37	1.18 ± 0.24a	↓	1.36 ± 0.11a	-	1.96 ± 0.18abc	↑	2.21 ± 0.30bc	-	< 0.001
ECM remodeling
Mmp7	0.58 ± 0.16	1.46 ± 0.40a	↑	5.31 ± 1.37ab	↑	5.21 ± 1.22ab	-	3.69 ± 0.85ab	-	< 0.001
Mmp9	0.13 ± 0.01	1.35 ± 0.24a	↑	1.17 ± 0.27a	-	0.89 ± 0.15a	-	0.55 ± 0.20bcd	-	< 0.001
Timp1	0.05 ± 0.00	1.75 ± 0.49a	↑	0.84 ± 0.25ab	↓	0.41 ± 0.05b	-	0.23 ± 0.06bc	-	< 0.001
Col3a1	0.77 ± 0.07	1.60 ± 0.33a	↑	1.15 ± 0.15a	-	1.20 ± 0.12a	-	0.83 ± 0.16bcd	-	< 0.01
Wisp	0.31 ± 0.02	1.40 ± 0.35a	↑	0.78 ± 0.17ab	↓	0.52 ± 0.04b	-	0.43 ± 0.06b	-	< 0.001
Acta2	1.33 ± 0.17	1.25 ± 0.22	-	0.95 ± 0.16	-	0.79 ± 0.05a	-	0.85 ± 0.11	-	< 0.01
Goblet cell markers and mucins
Tff3	1.80 ± 0.24	1.29 ± 0.32	-	1.05 ± 0.12ad	-	1.62 ± 0.19c	↑	1.87 ± 0.13c	-	< 0.05
Muc2	3.77 ± 0.64	1.10 ± 0.16a	↓	1.72 ± 0.22a	-	3.03 ± 0.18bc	↑	3.38 ± 0.21bc	-	< 0.001
Klf4	2.31 ± 0.13	1.12 ± 0.22a	↓	1.52 ± 0.15a	-	1.73 ± 0.10a	-	2.56 ± 0.24bcd	-	< 0.001
Epithelial repair modulating factors
Igf-1	0.27 ± 0.02	1.23 ± 0.19a	↑	1.14 ± 0.22ad	-	0.77 ± 0.06ac	-	0.55 ± 0.09abcd	-	< 0.001
Il-15	4.59 ± 0.89	1.32 ± 0.51a	↓	1.67 ± 0.18a	-	3.61 ± 0.91bc	↑	4.73 ± 0.86bc	-	< 0.01
Il-22	0.33 ± 0.20	1.59 ± 0.44a	↑	0.36 ± 0.08b	↓	0.47 ± 0.14b	-	0.18 ± 0.04b	-	< 0.001
Il-33	0.08 ± 0.02	1.69 ± 0.43a	↑	0.60 ± 0.11ab	↓	0.43 ± 0.05ab	-	0.26 ± 0.06ab	↑	< 0.001
Saa	1.51 ± 0.27	1.21 ± 0.23	-	1.05 ± 0.16	-	3.40 ± 0.40abc	↑	3.18 ± 0.31abc	↑	< 0.001
Tgf-ß1	0.50 ± 0.03	1.29 ± 0.28a	↑	0.72 ± 0.07b	↓	0.51 ± 0.07b	-	0.43 ± 0.04b	-	< 0.001
Tgf-ß3	1.08 ± 0.11	1.47 ± 0.37	-	0.72 ± 0.10b	↓	0.93 ± 0.12	-	1.16 ± 0.16	-	< 0.01
Inflammatory markers
Il-1ß	0.04 ± 0.01	1.71 ± 0.50a	↑	0.39 ± 0.06ab	↓	0.45 ± 0.07ab	-	0.39 ± 0.09ab	↑	< 0.001
Il-6	0.01 ± 0.00	1.73 ± 0.51a	↑	0.84 ± 0.28ab	↓	0.41 ± 0.08b	-	0.15 ± 0.07b	-	< 0.001
Il-10	0.23 ± 0.03	1.37 ± 0.27a	↑	0.37 ± 0.04b	↓	0.31 ± 0.06b	-	0.25 ± 0.03b	-	< 0.001
Il-13	3.81 ± 0.75	1.26 ± 0.23a	↓	0.82 ± 0.08a	-	0.78 ± 0.10a	-	2.53 ± 0.39bcd	-	< 0.001
Tnf-α	0.13 ± 0.01	1.29 ± 0.22a	↑	0.46 ± 0.05ab	↓	0.50 ± 0.06ab	-	0.47 ± 0.07ab	↑	< 0.001
Ptgs	0.14 ± 0.01a	1.69 ± 0.39b	↑	0.84 ± 0.30c	↓	0.60 ± 0.10 c	-	0.29 ± 0.05a	-	< 0.001

Values are means ± SE (n = 8-12). Means with different superscripts within a row are significantly different.

^aP < 0.05 vs day 0;

^bP < 0.05 vs day 7;

^cP < 0.05 vs day 10;

^dP < 0.05 vs day 13. ECM: Extracellular matrix.

Table 5 Mucosa-adherent microbiota composition and cecal bacterial metabolic activity

Parameter		d0	d7	d7 vs d0	d10	d10 vs d7	d13	d13 vs d10	d28	d28 vs d0	Statistical effect (time)
Total bacteria (log/g mucosal content)		9.36 ± 0.35	10.4 ± 0.14a	↑	9.92 ± 0.16b	↓	10.6 ± 0.22ac	↑	10.2 ± 0.24ac	↑	< 0.001
Shannon index		6.46 ± 0.07	4.75 ± 0.32a	↓	4.56 ± 0.28a	-	4.44 ± 0.26a	-	5.03 ± 0.29a	↓	< 0.001
Chao index		794 ± 55.2	633 ± 43.7a	↓	593 ± 31.1a	-	635 ± 34.4a	-	606 ± 51.6a	↓	< 0.01
Actinobacteria (%)
Bifidobacteriaceae		0.02 ± 0.01	0.01 ± 0.00	-	0.01 ± 0.01	-	0.00 ± 0.00	-	0.60 ± 0.28abcd	↑	< 0.001
Coriobacteriaceae		0.01 ± 0.02	0.04 ± 0.02	-	0.07 ± 0.04	-	0.06 ± 0.04	-	0.83 ± 0.31abcd	↑	< 0.001
Bacteroidetes (%)
Bacteroidaceae		4.35 ± 1.24	5.6 ± 1.34	-	11.55 ± 2.77b	↑	3.08 ± 1.08c	↓	20.1 ± 4.98cd	↑	< 0.05
Porphyromonadaceae		12.1 ± 2.55	7.40 ± 2.08	-	2.53 ± 1.29	-	1.03 ± 0.35ab	-	12.1 ± 5.18d	-	< 0.001
Prevotellaceae		1.06 ± 0.33	0.01 ± 0.00a	↓	0.01 ± 0.01a	-	0.02 ± 0.01a	-	0.01 ± 0.01a	↓	< 0.01
	Alloprevotella	1.04 ± 0.32	0.00 ± 0.00a	↓	0.01 ± 0.01a	-	0.01 ± 0.01a	-	0.00 ± 0.00a	↓	< 0.05
Rikenelleceae		9.64 ± 1.79	0.33 ± 0.24a	↓	0.33 ± 0.24a	-	0.31 ± 0.26a	-	2.04 ± 1.20a	↓	< 0.001
	Alistipes	9.48 ± 0.72	1.04 ± 0.51a	↓	0.24 ± 0.15a	-	0.13 ± 0.09a	-	1.98 ± 1.20a	↓	< 0.001
Deferribacterres (%)				↓
Deferribacteraceae	Mucispirillum	3.53 ± 1.86	9.98 ± 2.33a	↑	10.16 ± 3.04a	-	14.82 ± 2.80a	-	3.49 ± 0.76bcd	-	< 0.001
Firmicutes (%)
Clostridiaceae	Roseburia	0.80 ± 0.17	0.01 ± 0.00a	↓	0.01 ± 0.01a	-	0.01 ± 0.01a	-	0.03 ± 0.01a	↓	< 0.001
	Clostridium XIVa	6.07 ± 0.84	1.09 ± 0.35a	↓	5.56 ± 2.73b	↑	8.54 ± 2.80abc	↑	4.01 ± 1.64b	-	< 0.05
	Butyrivibrio	0.28 ± 0.21	0.00 ± 0.00a	↓	0.00 ± 0.00a	-	0.00 ± 0.00a	-	0.00 ± 0.00a	↓	< 0.01
Eubacteriaceae		0.17 ± 0.05	0.04 ± 0.02a	↓	0.00 ± 0.00a	-	0.00 ± 0.00a	-	0.00 ± 0.00a	↓	< 0.001
Lachnospiraceae		35.0 ± 4.85	13.5 ± 2.91a	↓	21.7 ± 4.25a	-	23.2 ± 4.49a	-	23.6 ± 4.69	-	< 0.01
	Acetitafactor	5.46 ± 1.47	1.28 ± 0.55a	↓	0.30 ± 0.13a	-	1.11 ± 0.63a	-	1.00 ± 0.63a	↓	< 0.001
Lactobacillaceae		0.42 ± 0.19	0.24 ± 0.12	-	3.41 ± 2.82	-	0.63 ± 0.26	-	1.44 ± 0.88	-	NS
Ruminococcaceae		24.7 ± 4.5	30.4 ± 6.4	-	18.2 ± 3.6b	↓	26.7 ± 4.0	-	21.1 ± 4.0	-	< 0.05
Proteobacteria (%)
Desulfovibrionaceae		1.94 ± 0.39	1.68 ± 0.32	-	3.06 ± 0.64	-	3.64 ± 0.95ab	-	1.30 ± 0.31d	-	< 0.01
Enterobacteriaceae		0.24 ± 0.04	14.38 ± 5.93a	↑	6.28 ± 2.59a	-	5.99 ± 3.80a	-	0.55 ± 0.16bcd	-	< 0.01
	Escherichia Shigella	0.05 ± 0.03	14.08 ± 5.83a	↑	6.02 ± 2.53a	-	5.47 ± 3.46bc	-	0.30 ± 0.11bc	-	< 0.01
Pseudomonadaceae		0.11 ± 0.04	0.06 ± 0.01	-	0.06 ± 0.02	-	0.18 ± 0.09	-	0.21 ± 0.12	-	NS
Sutterellaceae		0.05 ± 0.40	1.02 ± 0.42	-	2.87 ± 0.87	-	4.97 ± 1.93	-	7.39 ± 2.97	-	NS
Rhodospirillaceae		0.14 ± 0.05	0.51 ± 0.26a	↑	0.02 ± 0.02b	↓	0.00 ± 0.00b	-	0.04 ± 0.02b	-	< 0.001
Tenericutes (%)
Anaeroplasmataceae		0.23 ± 0.13	0.00 ± 0.00a	↓	0.00 ± 0.00a	-	0.01 ± 0.01a	-	0.00 ± 0.00a	↓	< 0.01
Total SCFA (µmol/g)		12.4 ± 1.63	1.13 ± 0.78a	↓	0.06 ± 0.04a	-	0.14 ± 0.07a	-	4.88 ± 1.08abcd	↓	< 0.001
	Acetate	8.95 ± 1.16	0.94 ± 0.67a	↓	0.04 ± 0.04ab	↓	0.14 ± 0.07ab	-	3.40 ± 0.71abcd	↓	< 0.001
	Propionate	1.93 ± 0.29	0.04 ± 0.04a	↓	0.00 ± 0.00a	-	0.00 ± 0.00a	-	0.90 ± 0.26abcd	↓	< 0.001
	Butyrate	1.51 ± 0.22	0.15 ± 0.11a	↓	0.01 ± 0.01a	-	0.00 ± 0.00a	-	0.59 ± 0.13abcd	↓	< 0.001

Quantification of total bacteria in the mucosa adherent microbiota was determined by real-time qPCR. Values are expressed as log of gene copy numbers of the different bacterial groups per gram of mucosal content. Evolution of the relative abundance of mucosa-adherent microbiota families was assessed by Illumina MiSeq sequencing. Cecal short chain fatty acids concentrations were obtained by gas chromatography after esterification. Values are means ± SE (n = 8-12).

^aP < 0.05 vs day 0;

^bP < 0.05 vs day 7;

^cP < 0.05 vs day 10;

^dP < 0.05 vs day 13. NS: Non-significant difference; SCFA: Short chain fatty acids.