1. Researchers tested the effects of two commercially available batches of the probiotic VSL#3 (batches A and B) on mouse models of colitis induced by DSS and TNBS. 2. Only batch A attenuated weight loss, intestinal inflammation, and permeability, while batch B either had no effect or worsened symptoms. 3. Histological analysis and gene expression levels supported the findings, with batch A showing anti-inflammatory effects and batch B either no effect or pro-inflammatory effects.

1. Variability in industrial production affects probiotics activity: identification of batches of
probiotic VSL#3 that increases intestinal permeability and worsens colitis in rodents
Michele Biagioli 1, Luca Laghi 2, Adriana Carino 1, Sabrina Cipriani 3, Eleonora Distrutti 4, Silvia Marchianò 1 , Carola Parolin 5, Paolo Scarpelli 6,Beatrice Vitali 5, Stefano Fiorucci1.
1 Dipartimento di Scienze Chirurgiche e Biomediche, Università di Perugia, Perugia, Italy
2 Dipartimento di Scienze e Tecnologie Agro-Alimentari Centro Interdipartimentale di Ricerca Industriale Agroalimentare, Università di Bologna, Cesena, Italy
3 Dipartimento di Medicina, Università di Napoli, Napoli, Italy
4 SC di Gastroenterologia ed Epatologia, Azienda Ospedaliera di Perugia, Perugia, Italy
5 Dipartimento di Farmacia e Biotecnologie, Università di Bologna, Bologna, Italy
6 Dipartimento di Scienze Sperimentali, Laboratorio di Biotecnologia, Università di Perugia, Perugia, Italy
Background VSL#3 is a blend of 8 different probiotics that have been extensively used in the treatment of intestinal inflammation and is currently recommended for the treatment of chronic pouchitis. Recently, however, several in vitro and in vivo studies have shown a
lack of efficacy of VSL#3 in reducing inflammation in models of colitis (Lorén V, et al. Probiotics Antimicrob Proteins. 2016 Nov 10). Additionally, it has been reported that VSL#3 preparations currently available for consumers differ from the original preparation and more
than one blend is currently available for use (Cifone MG., et al. PLoS One. 2016 Sep 22;11(9):e016321).
Aim of the study. To test the efficacy of two commercially available batches of VSL#3 on a mouse model of colitis and compare their efficacy in modulating intestinal permeability.
Methods. Colitis was induced in Balb/c mice by administering the animals with 5% of DSS in drinking water for 8 days or by intra-rectal injection of TNBS (1 mg/mouse in 100μl solution 50% water and 50% ethanol). In both model mice were fed two different preparation of VSL#3
commercially available in Europe. The first batch coded TM091 (Batch A) had an expiration date of 09/10/2017 while the second batch coded 512058 (Batch B) had an expiration date of 12/2017. Both Batch A and B were administered to mice at the dose of 50 billion/day, starting
on the same day of administration of DSS and TNBS. The progression of disease was monitored daily by measuring body weight and CDAI score. At the time of the sacrifice the colons were excised, weighed, measured (length), and evaluated for macroscopic damage. Colon
samples and mesenteric lymph nodes were also collected for histopathology analysis (H&E), immunochemistry analysis and for evaluation of genes expression (RT-PCR). Furthermore in DSS model the intestinal permeability was measured on day 8 using a fluorescein
isothiocyanate conjugated dextran (FITC-dextran) (Sigma-Aldrich, catalog number: FD4). FITC-dextran was dissolved in PBS (100 mg/ml) and administered to each mouse at the dose of 44 mg/100 g body weight by oral gavage. In addition the two VSL#3 batches were cultured
and cell viability measured after 2 days of culture measured.
Results. Treating mice with VSL#3 Batch A attenuated the weight loss caused by DSS and TNBS; while treating mice with and VSL#3 Batch B had no effect on the body weight. The severity of intestinal inflammation as indicated by the CDAI was reduced in mice treated with
VSL#3 Batch A but not by those treated with VSL#3 Batch B. Batch A, but not Batch B, ameliorated the macroscopic scores: colon length, the ratio between weight and length of the colon and ulcer in comparison to DSS and TNBS. In contrast, VSL#3 Batch B failed to improve
any of the macroscopic parameters. Additionally VSL#3 Batch A reduced intestinal permeability compared to DSS, while treating mice with VSL#3 Batch B increased intestinal permeability. The histopathology examination of colon sections stained with H&E demonstrated that,
while VSL#3-A reduced the severity of histology scores, this protective effects were lost in TNBS mice administered VSL#3-B, as confirmed by immunohistochemistry analysis of OCLN and ZO-1 in the colonic lamina propria. VSL#3 Batch A reduced the expression of pro-
inflammatory cytokines and increased the expression of anti-inflammatory genes, an opposite pattern was observed in mice treated with VSL#3 B. Investigation of the expression of cytokines and markers of macrophages and Treg cells in the mesenteric lymph nodes,confirmed
these findings. While upon colitis induction, TNBS increased the expression of TNF-α and markers of a M1 phenotype and reduced the expression of IL-10,markers of M2 phenotype and FoxP3 gene, a marker for Treg cells, this pattern was fully reversed by treatment with
VSL#3-A, while VSL#3-B had no effect.
Culturing the VSL#3 resulted in a profound difference in bacterial viability. After 2 days in culture cell viability was 63 ± 4% % for Batch A and 44 ± 6.2 % for Batch B ( n= 6; P<0.059).
Conclusions. We report that two commercially available preparations of probiotic VSL#3 does not have the same effects and only one of the two formulation alleviates colitis induced by DSS and TNBS, while the other, worsens the severity of colitis and increases the intestinal
permeability.
NT DSS
DSS + VSL#3-A DSS + VSL#3-B
0
20
40
60
80
100
Moderate
Severe
Mild
Control DSS
Alone VSL#3-A VSL#3-B
Histologicscore
(%ofanimals)
0 1 2 3 4 5 6 7 8
70
80
90
100
DSS
NT
Days
DSS + VSL#3-B
DSS + VSL#3-A
%ofBodyWeight
0
5
10
15
NT
DSS
DSS + VSL#3-A
DSS + VSL#3-B
Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8
**
CDAI
NT DSS DSS + VSL#3-
A
DSS + VSL#3-
B
Anti-occludin
NT DSS DSS + Batch A DSS + Batch B
0
2
4
6
TGF-relativemRNAexpression
(x10^-2)
0
2
4
6
8
IL-10relativemRNAexpression
(x10^-4)
0
2
4
6
FoxP3relativemRNAexpression
(x10^-4)
0
50
100
150
*
*
TNF-relativemRNAexpression
(x10^-3)
0
10
20
30
40
1000
2000
3000
*
*
*
Il-1relativemRNAexpression
(x10^-4)
0
2
4
6
8
10
100
200
300
400 *
Il-6relativemRNAexpression
(x10^-4)
0
2
4
6
INF-relativemRNAexpression
(x10^-5)
0
2
4
6
8
10
NT
TNBS
TNBS + VSL#3-A
TNBS + VSL#3-B
*
*
OclnrelativemRNAexpression
(x10^-3)
A.
-1 0 1 2 3 4
70
80
90
100
TNBS
NT
Days
TNBS + VSL#3-B
TNBS + VSL#3-A
%ofBodyWeight
0
2
4
6
8
10
Day 1 Day 2 Day 3 Day 4
*
*
*
CDAI
0
2
4
6
*
ColonLength(cm)
0.00
0.02
0.04
0.06
0.08
0.10
*
RatioW/L(g/cm)
0
20
40
60
80
100 NT
TNBS
TNBS + VSL#3-A
TNBS + VSL#3-B
Ulcers(mm2
)
A. C.B. D. E.
NT TNBS TNBS + VSL#3-A TNBS + VSL#3-B
Anti- occludin
NT TNBS TNBS +
VSL#3-A
TNBS +
VSL#3-B
Anti-zonulin 1
0
20
40
60
80
100
Moderate
Severe
Mild
Control TNBS
Alone VSL#3-A VSL#3-B
Histologicscore(%ofanimals)
NT TNBS TNBS + VSL#3-A TNBS + VSL#3-B
0
1000
2000
3000
4000
15000
20000
25000 * *
*
*
FITC-dextranng/ml
0
2
4
6
8
*
N.S.
* *
ColonLength(cm)
0.00
0.02
0.04
0.06
*
* N.S.
N.S.
RatioW/L(g/cm)
0
50
100 *N.S.
N.S.
Ulcers(mm2
)
0
2
4
6
8
Cd38relativemRNAexpressiona
(x10^-3)
0
5
10
15
20
25
* *
*
Fpr2relativemRNAexpressiona
(x10^-3)
0
1
2
3
4
5
*
*
Egr2relativemRNAexpressiona
(x10^-3)
0.0
0.5
1.0
1.5
TNF-relativemRNAexpressiona
(x10^-2)
0
5
10
15
* *
* *
IL-10relativemRNAexpressiona
(x10^-4)
B
Anti-zonulin 1
A. B.
C. D. F.E.
G.
H.
I.
Figure 1. Mice were treated with DSS in drinking water and then administered with vehicle or one of the two
formulations of VSL#3 by gavage from day 0 to day 8. VSL#3-A attenuated the development of wasting disease, i.e
change in body weight (A) and CDAI score (B). VSL#3-B worsens of intestinal permeability: FITC dextran (C). Only
VSL#3-A reduced intestinal inflammatory score: colon length (D), ratio between colon weight and colon length (E) and
ulcers area (F). H&E staining on colon sections (G) and analysis of histological score (H) of control, DSS treated and
DSS plus one of the two formulations of VSL#3 (magnification 10x). Representative immunohistochemistry of colon
with anti-occludin (I) and anti-zonulin1 (J) antibody for each experimental group. Results are the mean ± SEM of 4-6
mice per group. (*P <0.05).
A. B.
Figure 1. Effects of VSL#3 on DSS colitis. Mice were treated with DSS in drinking water and then administered with vehicle or one of the two formulations of VSL#3 by gavage from day 0 to day 8. VSL#3-A attenuated the development of wasting disease, i.e change in body weight (A) and CDAI score (B). VSL#3-B worsens of intestinal permeability: FITC dextran (C). (D-F) Only VSL#3-A reduced intestinal inflammatory score: colon length (D), ratio between colon weight and colon length (E) and ulcers area (F). H&E staining on colon sections (G) and analysis of histological score (H) of control, DSS treated and DSS plus one of the two formulations of VSL#3 (magnification 10x). Representative immunohistochemistry of colon with anti-occludin (I) and anti-zonulin1 (J) antibody for each experimental group. Results are the mean ± SE
J.
Figure 2. Mice were treated with TNBS and then administered with vehicle or one of the two formulations of VSL#3
by gavage from day 0 to day 4. VSL#3-A attenuated the development of wasting disease, i.e change in body weight
(A) and CDAI score (B). (C-E) Only VSL#3-A reduced intestinal inflammatory score: colon length (C), ratio between
colon weight and colon length (D) and ulcers area (E).
Figure 3. H&E staining on colon sections (A) and analysis of histological score (B) of control, TNBS treated and
TNBS plus one of the two formulations of VSL#3 (10x). Representative immunohistochemistry of colon with anti-
occludin (C) and anti-zonulin1 (D) antibody for each experimental group. Results are the mean ± SEM of 4-7 mice
per group. (*P <0.05).
Effects of VSL#3 on DSS colitis. Effects of VSL#3 on TNBS colitis.
C.
D.
Effects VSL#3 preparations on biomarkers of intestinal inflammation on TNBS colitis.
Figure 4. Quantitative rtPCR analysis of pro-inflammatory genes TNF-α, IL-1β, IL-6 and IFN-γ; anti-inflammatory
genes, TGF-β, IL-10 and FoxP3 and occludin (A). Results are the mean ± SEM of 4-7 mice per group. (*P <0.05).
Quantitative rtPCR analysis on mesenteric lymph nodes (B) : pro-inflammatory cytokine TNF-α, anti-inflammatory
cytokine IL10, M1 macrophage markers Cd38 and Fpr2, M2 macrophage marker Egr2 and Treg cells marker
FoxP3 . Results are the mean ± SEM of 3-5 mice per group (*P <0.05).
Quantitative rtPCR analysis of pro-inflammatory genes TNF-α, IL-1β, IL-6 and IFN-γ (A-D), anti-inflammatory genes, TGF-β, IL-10 and FoxP3(E-G) and occludin (H). Results are the mean ± SEM of 4-7 mice per group. (*P <0.05)
0
10
20
30
40
NT
TNBS
TNBS + VSL#3-A
TNBS + VSL#3-B
* *
*
FoxP3relativemRNAexpressiona
(x10^-3)