doi: 10.1093/femsre/fux035 Review Article
R E V I E W A R T I C L E
Using murine colitis models to analyze
probiotics–host interactions
Rebeca Mart´ın
1
, Florian Chain
1
, Sylvie Miquel
2
, Jean-Paul Motta
3
,
4
,
Nathalie Vergnolle
4
, Harry Sokol
1
,
5
,
6
,
7
and Philippe Langella
1
,
∗
1
INRA, Commensals and Probiotics-Host Interactions Laboratory, Micalis Institute, INRA, AgroParisTech,
Universit ´e Paris-Saclay, 78350 Jouy-en-Josas, France,
2Laboratoire Microorganismes: G ´enome et
Environnement (LMGE), UMR CNRS 6023, Universit ´e Clermont-Auvergne, 63000 Clermont-Ferrand, France,
3
Department of Biological Science, Inflammation Research Network, University of Calgary, AB T3E 4N1,
Canada,
4IRSD, Universit ´e de Toulouse, INSERM, INRA, ENVT, UPS, F-31300 Toulouse, France,
5Sorbonne
University – Universit ´e Pierre et Marie Curie (UPMC), 75252 Paris, France,
6Institut National de la Sant ´e et de la
Recherche M ´edicale (INSERM) Equipe de Recherche Lab ´elis ´ee (ERL) 1157, Avenir Team Gut Microbiota and
Immunity, 75012 Paris, France and
7Department of Gastroenterology, Saint Antoine Hospital, Assistance
Publique – Hopitaux de Paris, UPMC, 75012 Paris, France
∗Corresponding author: INRA, Commensals and Probiotics-Host Interactions Laboratory, Micalis Institute, INRA, AgroParisTech, Universit ´e Paris-Saclay, 78350 Jouy-en-Josas, France. Tel+33134652070; E-mail:Philippe.langella@inra.fr
One sentence summary: Our aim is to highlight both the importance of the adequate selection of the animal model to test the potential probiotic strains and of the value of the knowledge generated by these in vivo tests.
Editor: Michiel Kleerebezem
ABSTRACT
Probiotics are defined as ‘live microorganisms which when administered in adequate amounts confer a health benefit on the host’. So, to consider a microorganism as a probiotic, a demonstrable beneficial effect on the health host should be shown as well as an adequate defined safety status and the capacity to survive transit through the gastrointestinal tract and to storage conditions. In this review, we present an overview of the murine colitis models currently employed to test the beneficial effect of the probiotic strains as well as an overview of the probiotics already tested. Our aim is to highlight both the importance of the adequate selection of the animal model to test the potential probiotic strains and of the value of the knowledge generated by these in vivo tests.
Keywords: TNBS; DSS; infectious colitis; spontaneous colitis; adoptive cell transfer colitis; inflammatory bowel disease
INTRODUCTION
Probiotics are ‘live microorganisms that, when administered in adequate amounts, confer a health benefit on the host’ (Hill et al.2014). Elie Metchnikoff was the first to suggest that, in hu-mans, improved health and longevity could result from the con-sumption of lactic acid bacteria (LAB) in fermented products (i.e.
yogurt). At present, most probiotic strains are lactobacilli and other LAB or bifidobacteria, although other species and genera are also used, including Escherichia coli Nissle 1917, Saccharomyces boulardii (de Vrese and Schrezenmeir2008) and Bacillus among others (Fijan2014). These genera are not dominant members of the human intestinal microbiota and can be considered as transient members of the intestinal microbiome. The human Received: 28 February 2017; Accepted: 8 June 2017
C
FEMS 2017. All rights reserved. For permissions, please e-mail:journals.permissions@oup.com
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gastrointestinal tract (GIT) contains one of the most densely populated microbial ecosystems known. The GIT microbiota is predominated by bacteria, with more than 1011 bacteria per gram of colonic content, many of which are extremely oxygen sensitive (Hooper and Gordon2001; Ley et al.2006). Members of the phyla Firmicutes (mainly Ruminococcus, Clostridium and Pep-tostreptococcus) and Bacteroidetes (mainly Bacteroides) dominate the human GIT microbiota, representing∼90% of the overall mi-crobiome (Martin et al.2014c). Next to these dominant phyla, several other phyla are present (e.g. Actinobacteria, Verrumicro-bia and several others), collectively constituting a highly diverse and complex microbial ecosystem, of which the composition is relatively stable over time, but substantially different between individuals (Eckburg et al.2005). Several researchers have charac-terized the human intestinal microbiota by cataloguing the ge-netic repertoire of the microbiome, i.e. its metagenome. In par-ticular, large scale publicly funded research programs such as the European MetaHIT project and the American Human Micro-biome Project have been instrumental in the unraveling of the human GIT metagenome (Qin et al.2010; Human Microbiome Project2012; Li et al.2014). These studies have revealed a new challenge for the research in probiotics as the most common probiotic strains used up to day do not appear to be dominant in the gut ecosystem. This observation opens the door to a new level where other scientific criteria based on microbiota com-position should be added to choose novel probiotic candidates rationally selected.
Regarding the mouse gut microbiota, the overall phylum-level composition was similar to that of the human gut micro-biome with Firmicutes, Bacteroidetes and Proteobacteria com-prising more than 70% of the gut microbiota (Xiao et al.2015). However, in this case, Lactobacilli are common inhabitants of proximal regions of the gut where they associate with non-secretory epithelial surfaces such as the forestomach (Tannock et al.2012).
The bacteria in the intestinal tract are interacting constantly with their human hosts in numerous ways (Sekirov et al.2010). The intestinal microbiota is known to modulate epithelium functions, protect against colonization by pathogenic bacteria and influence immune responses. We have recently reviewed these interactions (Martin et al.2013,2014c), and they are there-fore not the focus of this review. The importance of the hu-man intestinal microbiota is illustrated by the dysbiotic state (a microbial imbalance or maladaptation either on or inside the body that leads to perturbations to the structure of complex commensal communities) that is associated with a wide range of health problems. For example, GIT dysbiosis has been ob-served in patients suffering obesity, inflammatory bowel disease (IBD), irritable bowel syndrome (IBS) and antibiotic-associated diarrhea and specific pathogen-related colitis (Bakhtiar et al.
2013). In particular, various studies have indicated that different forms of IBD (BOX 1), which are characterized by chronic and reoccurring inflammation of the GIT, can provide clear exam-ples of how the microbiota could impact on human health as a large number of studies reported an altered bacterial compo-sition in IBD patients compared to healthy individuals (Hunter
2012; Bakhtiar et al.2013). For instance, at a lower taxonomic level the loss of some indicator species in IBD, like Faecalibac-terium prausnitzii (Sokol et al.2008), is well characterized. Fur-thermore, in IBD patients, inflammation occurs in regions with higher bacterial density (distal ileum and colon) with a direct correlation between the severity of the colitis and the bacte-rial density of the intestinal mucosa, antibiotics have shown some therapeutic efficacy in IBD patients and, notably, germ
Box 1. Inflammatory bowel disease Box
Inflammatory bowel disease (IBD): Group of gastrointestinal diseases characterized by chronic and relapsing inflamma-tion of the gastro-intestinal tract.
Crohn’s disease: Type of IBD that may affect any part of the gastrointestinal tract from mouth to anus. Majority of patients have an ileo-coecal involvement. Inflammation is patchy and can be transmural.
Ulcerative colitis: Type of IBD that may affect only the rec-tum and the colon. Inflammation is continuous and ho-mogenous and most of the time superficial.
IBD clinical symptoms often include abdominal pain, diar-rhea, fever and weight loss.
IBD histology can include ulceration, crypt abscesses, ab-normal crypt architecture, infiltration of the mucosa with immune cells and notably neutrophils and lymphocytes. Granuloma can be seen in Crohn’s disease only.
free animals do not develop ileitis (Sellon et al.1998; Sokol2014; Schaubeck et al.2016).
Thanks to the ever-greater body of knowledge on the human microbiome, research has identified several new potential can-didate probiotic species among the dominant members of the adult GIT microbiota. For example, species such as F. prausnitzii (Sokol et al.2008; Martin et al.2014b) and Akkermansia muciniphila (Everard et al.2013; Schneeberger et al.2015; Dao et al.2016; Gomez-Gallego et al.2016), which are depleted in IBD and obese patients respectively, are potential next-generation probiotics (NGPs).
To label a microorganism as a probiotic, it must be safe, survive production and storage conditions, as well as transit through the intestinal tract, where it should elicit a host re-sponse with a demonstrable health-promoting effect (Martin et al.2014c). Although some probiotic effects may be elicited by different strains and/or species (Hill et al.2014), most probiotic properties are considered strain specific. Therefore, one cannot assume that properties of a specific strain will be shared by other strains of the same species and, consequently, individual tests must be carried out for each strain, especially for NGPs (Pineiro and Stanton2007; Miquel et al.2015a). In functional analyses of the possible beneficial effects of bacteria, the classical pre-clinical approach encompasses two stages. The first stage takes place in vitro and commonly includes both microbiology exper-iments as well as interaction screening models employing cell lines and/or tissues. The second stage generally employs pre-clinical in vivo models, frequently employing rodent models of health and disease (Daniel et al.2006). Among the most com-monly used in vivo rodent models (mice or rats) are a variety of models that replicate different forms of colitis and allow the study of the impact of the microbiota and in particular of probi-otics, on colon inflammation, serving as a model for their poten-tial health-promoting effects in different forms of IBD or other diseases that are associated with intestinal mucosa inflamma-tion. Several studies have tried to link these preclinical in vitro and in vivo results to identify in vitro indicators that can predict in vivo benefits. For example, Foligne et al. (2007b) proposed the ratio of anti-inflammatory and proinflammatory cytokines (in-terleukin [IL]-10 and IL-12 respectively) produced by peripheral blood mononuclear cells (PBMCs) upon their in vitro exposure to probiotic strains could predict their protective effect in vivo in a chemically induced murine colitis model. However, it remains challenging to identify such in vitro tests that can reliably predict
the impact of microbes in rodent colitis models. In the context of that same translational pipeline, another important challenge in this field relates to the predictive quality of anti-inflammatory effects observed in the rodent colitis models for similar inflam-mation suppressing effects in human intestinal mucosa, which is essential to reliably identify and screen candidate probiotic strains for applications aimed to combat intestinal mucosa in-flammation problems like those observed in the different forms of IBD.
Rodent colitis models can be classified into four major groups that are discriminated on basis of the trigger to induce disease: (i) chemically induced colitis, (ii) bacterially induced colitis, (iii) spontaneous colitis (including congenital and genetically engi-neered [GE] colitis) and (iv) adoptive cell transfer colitis. Here, we present an overview of the advantages and limits of the murine colitis models that are currently employed to test probi-otic strains, and review the results obtained with traditional and NGPs using these models. Our aim is to highlight the importance of selecting the appropriate murine model when investigating candidate probiotic strains as well as the value of the knowledge generated by such in vivo tests to translate to humans.
MURINE COLITIS MODELS COMMONLY
USED TO TEST CANDIDATE PROBIOTICS
Chemically induced colitisThere are multiple etiological factors involved in the devel-opment of colitis, and not all of them have been identified. Nonetheless, different models use chemically induced colitis to yield greater insight into the onset and progression of intesti-nal inflammation. They can also be used to test for possible beneficial effects provided by treatment agents such as drugs (Zeeff et al.2016) and candidate probiotics. In this sense, these models are still being used to test peptides and other molecules with therapeutically potential (Cobos Caceres et al.2017; Nagata, Yamasaki and Kitamura2017). Moreover, they can enable the characterization of the mechanisms underlying host–probiotic interactions. The models strive to recreate the morphological, histopathological and clinical features of the different forms of human IBD including dysbiosis (BOX 1) (Randhawa et al.2014). Several of these models are widely used in laboratories to test the efficacy of candidate probiotics to suppress intestinal in-flammation. Two general modes for inducing colitis are used that are distinguished by the form of chemical administration: rectal and oral.
Rectally induced colitis
Acute or chronic colitis can by induced by rectally injecting a haptenating agent dissolved in ethanol. The ethanol allows the agent to pass through the mucosal barrier, where the agent is then thought to develop its hapten activity, it means that it acts on colonic autologous or microbiotic proteins, rendering them immunogenic to the host immune system (Wirtz et al.2007). The most commonly used haptenating agents are trinitroben-zene sulfonic acid (TNBS), dinitrobentrinitroben-zene sulfonic acid (DNBS) and oxazolone. All three produce an isolated point of inflamma-tion and necrosis as well as self-antigens that provoke immune responses (Fig.1) (Elson et al.2005). In some cases, prior to the rectal injection, the animal is exposed to the haptenating agent topically to induce an allergic reaction (Lamprecht et al.2005). Wirtz et al. (2007) published complete protocols for inducing col-itis using TNBS and oxazolone.
Although the models are similar, they are not identical, and careful considerations should be taken along in model se-lection. For instance, TNBS and DNBS are considered to elicit a Th1-mediated immune response, whereas oxazolone elic-its a Th2-mediated response (Randhawa et al.2014). However, model functionality also varies depending on the host species and its genetic background (Mizoguchi and Mizoguchi 2008; Mizoguchi 2012). TNBS-induced colitis in mice was initially described in SJL/J mice, which are highly susceptible to chronic colitis, and chemical treatment induces a major Th1-mediated response, and includes prominent mucosal infiltration of lym-phocytes/macrophages and thickening of the colon wall (Neu-rath et al.1995,1996). In contrast, IFN-γ−/-mice with a Balb/c background elicit a Th2-mediated response when treated with these chemicals (Dohi et al.1999). Importantly, use of these mod-els requires accurate optimization of the TNBS (or DNBS) con-centrations to be administered, particularly when the model is intended to mimic chronic colitis (Wirtz et al.2007). These mod-els have been employed to test several probiotic strains and strain mixtures (Table1). Disease severity during colitis devel-opment as well as probiotic effectiveness in ameliorating them can be determined using changes in body mass, clinical symp-toms (e.g. diarrhea, constipation and bloody feces), changes in colon morphology, histological structure, colonic cytokine con-centrations and myeloperoxidase (MPO) activity (an indicator of neutrophil infiltration that reflects the local immune response (Fig.2) (Wirtz et al.2007; Martin et al.2014b)). In addition, the degree of inflammation can be characterized using biochemical markers, such as levels of glutathione (GSH), lipid peroxidation (TBARS) and nitric oxide (NO) as well as systemic inflammation indicators such as C-reactive protein and lipocalin- C (Martin et al.2015; Satish Kumar et al.2015; Munyaka et al.2016).
In TNBS-induced colitis models, colitis symptoms were alle-viated following treatment with a variety of probiotics, includ-ing Lactobacillus plantarum TN8 (Trabelsi et al.2016), Lb. helveticus NS8 (Rong et al.2015), Bifidobacterium infantis (Javed, Alsahly and Khubchandani 2016), CitogenexR (a commercial mix) (Traina
et al.2016), Lb. salivarius Ls33 (Daniel et al.2006), Lb. fermentum CECT5716 (Rodriguez-Nogales et al.2015), B. bifidum 231 (Satish Kumar et al.2017), Lb. sakei K 17 (Eun et al.2016) and Lb. plantarum 21 (Satish Kumar et al. 2015). More specifically, Lb. plantarum 21 significantly decreased levels of TBARS and NO, increased GS H concentrations, downregulated IL-1β and TNF-α expres-sion and upregulated IL-10 expresexpres-sion in TNBS-challenged mice (Satish Kumar et al.2015). Lactobacillus sakei K 17 administra-tion could suppress colon shortening and the activity of MPO, nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases, which was proposed to be achieved by the bacterially induced increase of expression of tight junction proteins and IL-10 (Eun et al.2016). Escherichia coli Nissle 19 157 could reduce the disease activity index, colonic MPO activity and TNF-α lev-els, and increased IL-10 expression (Sha et al.2014). However, this effect was found to be dose dependent, as low doses of the strain (107 CFU/day) were able to protect rats from colitis better than high doses (109CFU/day) (Sha et al.2014). Bifidobac-terium bifidum 231 administration in TNBS-challenged rats re-sulted in both macroscopic and histological anti-inflammatory effects, combined with lower levels of TBARS, NO and IL-1β, and higher levels of GSH and IL-10 (Satish Kumar et al.2017). In a mouse model of chronic DNBS-induced colitis, Lb. sanfrancis-censis LBH1068 was reported to improve health by reducing the loss of body mass and significantly decreasing gut permeabil-ity, and modulating cytokine production (Torres-Maravilla et al.
2016). Finally, in an oxazolone-induced colitis model, Clostridium
Figure 1. Effect of a haptenating substance at the colon. After intrarectal injection, the compound arrives into the colon dissolved in ethanol. The ethanol damages the epithelial barrier allowing the substance to pass through it unchaining inflammatory responses such as immune activation and oxidative stress. On the other way round, the gut barrier is disturbed (epithelial and mucosa) with the consequence increase in permeability. The microbiota also suffers alterations (dysbiosis) as a consequence of this altered ecosystem.
butyricum CGMCC313, a strain that has been used for improv-ing gastrointestinal function in mice (Zhang et al.2009; Shang, Sun and Chen2016), promoted the repair of colonic mucosa and recovery and normalization of the microbiota, whereas treat-ment with Lb. acidophilus in a similar model led to reduced dis-ease activity index and lowered C-reactive protein concentra-tion in serum (Abdin and Saeid2008; Zhang et al.2009). Taken together, a variety of parameters support the protective effects of different probiotic strains in these rodent colitis models. No-tably, many studies report probiotic induction of tolerogenic re-sponses, which is highlighted by the elevated production of IL-10, as an important element in the protective effects observed (Zoumpopoulou et al.2008).
Besides these protective effects, it has been reported that not all candidate probiotics have displayed beneficial effects using these models. For example, Lb. acidophilus NCFM (Daniel et al.
2006), Streptococcus macedonicus ACA-DC 198 (Zoumpopoulou
et al.2008), Lb. plantarum NCIMB8826 (Grangette et al.2005) and Lb. plantarum Lp115 (Daniel et al.2006) were shown to elicit low IL-10/IL-12 production ratios in in vitro PBMC stimulation assays, and had no effects in these animal models of colitis. Moreover, for some probiotic strains the results have been ambiguous. For example, treatment with S. faecalis 129 BIO 3B (SF3B) failed to improve body mass, limit diarrhea duration or increase either the macroscopic colitis score or the colonic weight/length ratio, but did restore enteric neurotransmission as measured due to the reduction of the appearance of nontachykininergic and non-cholinergic excitatory components in the colon of SF3B-treated mice (Shiina et al.2015). Similarly, a yeast strain of the species Saccharomyces boulardii did not significantly improve pathology scores or cytokine production levels, but did decrease NO levels in colonic mucosal tissues (Soyturk et al.2012).
Models of TNBS-induced colitis have also been useful in char-acterizing the role of T-helper cell populations in health and
Table 1. Some of the probiotic strains tested in TNBS-induced colitis (non-exhaustive list).
Probiotic strain Animal Outcome Major mechanisms Reference
Bacillus polyfermenticus CD1 + Suppresses apoptosis and
promote epithelial cell proliferation and migration
Im et al. (2009)
Bifidobacterium animalis
subsp. lactis BI07
BALB/c; C57/BL6 + ND Foligne et al. (2007b)
Bifidobacterium animalis
subsp. lactis BL04
BALB/c; C57/BL6 + ND Foligne et al. (2007b)
Bifidobacterium bifidum 231 Wistar rats + Lowers TBARS, nitric oxide and
increases GSH levels
Satish Kumar et al. (2015)
Bifidobacterium bifidum S17 C57/BL6 + ND Preising et al. (2010)
Bifidobacterium infantis Albino Lewis rats + ND Javed, Alsahly and Khubchandani
(2016)
Bifidobacterium infantis BALB/c + Decreases Th1 and Th17 responses
and increases Foxp3(+) Treg response in the colonic mucosa
Zuo et al. (2014)
Bifidobacterium lactis LA 303, Lb. acidophilus LA 201, Lb. plantarum LA 301 and salivarius LA 302
BALB/c + ND Drouault-Holowacz et al. (2006)
Bifidobacterium longum
HY8004
ICR + Inhibits lipid peroxidation, TLR-4 expression, and NF-κB activation
in the colon
Lee et al. (2009)
Citogenex (Lb. casei and B.
lactis)
CD1 + ND Traina et al. (2016)
Escherichia coli strain Nissle
1917
Sprague-Dawley rats
+ Up-regulates ZO-1 expression Sha et al. (2014)
Faecalibacterium prausnitzii BALB/c + Increases colonic IL-10 Sokol et al. (2008); Miquel et al.
(2015b)
Lactobacillus acidophilus Albino rats + ND Abdin and Saeid (2008)
Lactobacillus acidophilus and B. longum
BALB/c + Expands T regulatory (Treg) cells of IELs
Roselli et al. (2009)
Lactobacillus acidophilus
IPL908
BALB/c; C57/BL6 + ND Foligne et al. (2007b)
Lactobacillus acidophilus
NCFM
BALB/c; C57/BL6 – ND Daniel et al. (2006); Foligne et al. (2007b)
Lactobacillus casei BL23 BALB/c + ND Foligne et al. (2007b)
Lactobacillus fermentum
ACA-DC 179
BALB/c + Increases colonic IL-10 Zoumpopoulou et al. (2008)
Lactobacillus fermentum
CECT5716
BALB/c + Decreases IL-6 production and increased MyD88
Mane et al. (2009)
Lactobacillus helveticus NS8 BALB/c + ND Rong et al. (2015)
Lactobacillus plantarum 21 Wistar rats + Lowers TBARS, nitric oxide and
increases GSH levels
Satish Kumar et al. (2017)
Lactobacillus plantarum
AK8–4
ICR + Inhibits TLR-4-linked NF-kB and intestinal bacterial GAG degradation
Lee et al. (2009)
Lactobacillus plantarum
Lp115
BALB/c +/– ND Foligne et al. (2007b)
Lactobacillus plantarum
NCIMB 8826
BALB/c; C57/BL6 +/– ND Pavan, Desreumaux and Mercenier (2003); Grangette et al. (2005); Foligne et al. (2007)
Lactobacillus plantarum TN8 Wistar rats + ND Trabelsi et al. (2016)
Lactobacillus plantarum, S. thermophilus, and B. animalis
subsp. lactis
BALB/c + Expands T regulatory (Treg) cells of IELs
Roselli et al. (2009)
Lactobacillus reuteri BALB/c + Activates H2R, and H2R signaling Gao et al. (2015)
Lactobacillus rhamnosus Lcr35
Sprague Dawley rats
+ Regulates the local IL-23/Th17 immune activation
Darbaky et al. (2017)
Lactobacillus rhamnosus LR32 BALB/c; C57/BL6 + ND Foligne et al. (2007b)
Lactobacillus rhamnosus RC007
BALB/c + Increases the phagocytic activity of peritoneal macrophage
Dogi et al. (2016)
Lactobacillus sakei K17 Mice + Upregulates the expression of
IL-10 and tight junction proteins and inhibits NF-κB activation
Eun et al. (2016)
Table 1. (Continued )
Probiotic strain Animal Outcome Major mechanisms Reference
Lactobacillus salivarius Ls33 BALB/c + Favors the development of
CD103(+) DCs and CD4(+)Foxp3(+) regulatory T cells
Macho Fernandez et al. (2011)
Oenococcus oeni IOEB9115 BALB/c + ND Foligne et al. (2010)
Saccharomyces boulardii BALB/c + ND Foligne et al. (2010)
Sacharomyces boulardii Wistar albino
rats
+/– Increases NO levels Soyturk et al. (2012)
Enterococcus faecalis 129 BIO 3B
Wistar rats –/+ Repairs disruptions of enteric neurotransmissions
Shiina et al. (2015)
Streptococcus macedonicus
ACA-DC 198
BALB/c + ND Zoumpopoulou et al. (2008)
VSL#33 SJL/J + Induces IL-10 and IL-10-dependent
TGF-β bearing regulatory cells
Di Giacinto et al. (2005)
Outcome:+ significant improvement,—no differences, –/+ mixed results. ND: Mechanisms not described in vivo.
Figure 2. DNBS administration and consecuences. Panel A corresponds to DNBS intra-rectal injection. Panel B corresponds to the colon of a mice injected with vehicle (right) or DNBS (left), DNBS injected mice suffer from constipation due to the blockage of the colon at the injection site and consequent development of inflammation and ulcer Panels C and D correspond to the histological analysis of the colon of mice injected with vehicle or DNBS, respectively; in the second one, we can appreciate the presence of immune cell infiltration, ulcer and goblet cell depletion.
disease, where CD4+T cells are a crucial part of the immune response (Wirtz et al.2007). Various studies have observed the effects of candidate probiotic strains on different types of T-helper cells. For example, a strain of B. infantis was found to at-tenuate TNBS-induced colitis by decreasing Th1 and Th17 re-sponses while increasing the Foxp3+ T
reg response in colonic mucosa (Zuo et al.2014). Likewise, oral administration of VSL#3 (a commercial probiotic mixture composed of Lb. casei, Lb. plan-tarum, Lb. acidophilus, Lb. delbrueckii subsp bulgaricus, B. longum, B. breve, B. infantis and S. salivarius ssp thermophilus) limited the recurrence of chronic Th1-driven colitis by inducing IL-10 and increasing the number of TGF-β-positive regulatory CD4+T cells (Di Giacinto et al.2005). Highly similar results were obtained for other (non-commercial) mixtures of bacteria that contained Lb. acidophilus, S. thermophilus, B. animalis subsp lactis and either B. longum or Lb. plantarum (Roselli et al.2009).
As indicated above, tolerogenic responses, including in-creased IL-10 production, appears to be a prominent mechanism underlying the beneficial effects of candidate probiotics. How-ever, other mechanisms have also been suggested (Claes et al.
2011). These alternative or additional mechanisms include sup-pression of apoptosis and promotion of cell proliferation by Bacil-lus polyfermenticus (Im et al.2009), protection of goblet cell pop-ulations by B. infantis (Javed, Alsahly and Khubchandani2016) and the inhibition of degradation of extracellular-matrix gly-cosaminoglycans by both B. longum HY8004 and Lb. plantarum AK8–4 (Lee et al.2009).
Despite the common use of DNBS models, some researchers consider these models not appropriate for studying IBD since they reproduce situations of intestinal hypersensitivity, which are more in line with symptoms associated with food allergies or IBS (Bailon et al. 2011). Based on this notion, we recently
developed a murine model mimicking an IBS subtype that is characterized by low-grade induced inflammation, which can induced by the administration of low doses of DNBS (Martin et al.2015). In this model, chronic low-grade inflammation and gut dysfunction are induced by two injections of the haptenat-ing agent. The model allows the determination of inflammation markers, histological alterations, gut permeability and immune functions. This model was employed to explore the potential of Lb. rhamnosus CNCM-I3690 and B. animalis ssp. lactis CNCM-I2494 to prevent disorders associated with increased barrier perme-ability (Laval et al.2015; Martin et al.2016). Lactobacillus rham-nosus CNCM-I3690 partially re-established barrier function and increased the levels of the tight junction proteins occludin and E-cadherin (Laval et al.2015). Bifidobacterium animalis ssp. lactis CNCM-I2494 restored barrier function, goblet cells populations and cytokines levels. Furthermore, the latter strain also restored the level of expression of several tight junction proteins (espe-cially claudin-4) as well as the Th1/Th2 ratio within the CD4+ T-cell populations in both spleen and mesenteric lymph nodes. Specifically, this Bifidobacterium strain increased Th2 responses, which was reflected by the increased production of the repre-sentative cytokines IL-4, IL-5 and IL-10 (Martin et al.2016). Fi-nally, administration of two active pharmaceutical ingredients derived from Lb. rhamnosus Lrc35 (Lcr Lenio and Lcr RestituoR )R in a TNBS-induced model was reported to reduce hypersensitiv-ity, which may imply that this strain could be considered as a candidate probiotic for treating IBS (Darbaky et al.2017). Orally induced colitis
Colitis can be also induced by providing dextran sodium sul-phate (DSS) containing drinking water for several days to ro-dents (Wirtz et al.2007). DSS is toxic to colonic epithelial cells and causes the complete loss of the surface epithelium in the intestine (Randhawa et al.2014). As a consequence, the integrity of the mucosal barrier is severely compromised, allowing large molecules to pass through (Ni, Chen and Hollander1996). The onset of DSS-induced colitis is independent of the adaptive im-mune system (B and T cells) given that immunodeficient mice also develop colitis when treated with DSS (Dieleman et al.1994). Therefore, DSS models are particularly useful for studying the role of the innate immune system and gut barrier in colitis de-velopment, and how these functions can be affected by candi-date probiotics.
In these models, colitis symptoms include bloody diarrhea, hyperaemia, ulcerations, submucosal edema and histopatholog-ical changes (Dharmani, Leung and Chadee2011). The disease severity depends on DSS type, concentration and treatment fre-quency (Okayasu et al.1990). Although it has been suggested that DSS mainly affects the large intestine, it has been reported to also affect the small intestine (Ohtsuka and Sanderson2003; Perse and Cerar2012).
The DSS-induced colitis model can be applied in all mouse backgrounds (Dieleman et al.1997). It is one of the most widely used models because DSS is inexpensive and highly available (Randhawa et al.2014) and can also be applied in GE mice. It has been used to test several probiotic strains and strain mixtures (Table2). Analogous to the rectally induced colitis, disease de-velopment and probiotic effectiveness can be evaluated using changes in body mass, disease symptoms, colon morphology, histological structure as well as with immunological and bio-chemical markers (Wirtz et al.2007).
Some of the most extensively studied probiotic bacteria have also been tested using the DSS model. For instance, E. coli Nissle 1917 was found to alleviate disease symptoms by reducing the
production of the proinflammatory cytokines IL-6 and IFN-γ , via a TLR-2 and TLR-4-dependent mechanism as confirmed in TLR-2 or TLR-4 knockout mice (Schultz et al.2004; Grabig et al.
2006; Kokesova et al.2006; Ukena et al.2007; Kamada et al.2008). Furthermore, preventive oral administration of E. coli Nissle or transfer of fecal microbiota containing E. coli Nissle modified the inflammatory response to DSS (Souza et al.2016). Analogously, a TLR-4-dependent mechanism was also reported to be involved in the capacity of Lb. casei DN-114 001 (=CNCM-I3689) to pre-vent the development of acute DSS-induced colitis (Chung et al.
2008). Other bacterial strains, including Lb. casei Shirota (Herias et al.2005), Lb. casei BL.23 (Rochat et al.2007), Lb. crispatus M247 (Castagliuolo et al.2005) and E. coli M-17 (Fitzpatrick et al.2008), have also been reported to be health-promoting probiotics in DSS-induced mouse models being able to improve clinical pa-rameters and reduce the severity of DSS colitis. The VSL#3 that was effective in reducing disease symptoms in TNBS-challenged mice (see above) also elicited beneficial effects in DSS models. It was shown to be effective in both preventive and curative treat-ments for acute or chronic DSS colitis, and decreased the dis-ease activity index and MPO activity, and improved the histo-logical scores, via a mechanism that was proposed to involve TLR-9-dependent signaling (Rachmilewitz et al.2004; Reid et al.
2004; Loren et al.2016). The VSL#3 treatment was specifically shown to protect the epithelial barrier by maintaining levels of tight junction proteins and by blocking epithelial cell apopto-sis (Mennigen et al.2009). Analogously, other mixtures of strains were also found to be able to relieve the symptoms linked to DSS-induced colitis, including the G17 LAB mixture (contain-ing Lb. acidophilus, Lb. plantarum, Lb. rhamnosus, Lactococcus lac-tis, B. bifidum, B. breve and S. thermophilus) that reduced the loss of body mass, colon shortening, MPO activity, intestinal bleed-ing and histological damage (Kim et al.2016); the I3.1 mixture (composed of Lb. plantarum CECT7484, Lb. plantarum CECT7485 and Pediococcus acidilactici CECT7483) that reduced colitis sever-ity and IFN-γ levels (Loren et al.2016) Finally, the UltrabiotiqueR mixture (containing Lb. acidophilus, Lb. plantarum, B. lactis and B. breve) stimulated a rapid recovery after acute DSS colitis, and al-leviated colitis symptoms and improved histological scores. It reduced NO and IFNγ production in the plasma, as well as TLR4, iNOS and NF-κB expression in colonic tissue (Toumi et al.2014). However, some extensively studied probiotic strains appeared to have no impact in the DSS colitis models. For example, Lb. rham-nosus GG (Claes et al.2010), Lb. acidophilus NCFM (Mohamadzadeh et al.2011) and B. longum ssp. longum CCDM 372 (Srutkova et al.
2015) did not attenuate symptoms. Furthermore, some probi-otics such as Lb. rhamnosus GG (Mileti et al.2009), Lb. plantarum NCIMB8826 (Mileti et al.2009) and Lb. crispatus CCTCC M206119 (Cui et al.2016) have been found to aggravate DSS-induced coli-tis.
Model-specific effects of different probiotic strains or strain-specific effects within a single bacterial species are not surprising because most probiotic properties are strain specific. For example, while B. longum ssp. longum CCDM 372 confers no protection against acute DSS-induced colitis, its close relative B. longum CCM 7952 improves epithelial barrier functioning and prevents disease development (Srutkova et al.2015). However, a strain’s efficacy in ameliorating symptoms in these models can also depend on the probiotic preparation and timing of its ad-ministration as in acute active inflammatory phases the symp-toms are too strong to allow a potential probiotic strain to show protective effects. In consequence, preventive treatments and the administration during the recovery phase are normally more effective. For example, while preventive treatment with B. breve
Table 2. Some of the probiotic strains tested in DSS-induced colitis (non-exhaustive list).
Probiotic strain Animal Outcome Major mechanisms Reference
Anaerostipes hadrus BPB5 C57BL/6J – ND Zhang et al. (2016)
Bacillus polyfermenticus CD-1 + Suppresses apoptosis and promotes
epithelial cell proliferation and migration
Im et al. (2009)
Bacillus subtilisis C57BL/6J + Balances gut microbiota Zhang et al. (2016)
Bifidobacterium bifidum
S17/pMGC
C57BL/6 + ND Grimm, Radulovic and
Riedel (2015)
Bifidobacterium animalis
subsp. lactis
C57BL/6J + Increases regulatory T cells in mesenteric lymphoid nodes
Hidalgo-Cantabrana et al. (2016)
Bifidobacterium breve M-16V F344/N rats + ND Izumi et al. (2015)
Bifidobacterium breve NutRes 204 C57BL/6 +(resolution
phase) ND Zheng et al. (2016) Bifidobacterium longum ssp. longum CCDM 372 (Bl 372) C57BL/6 – ND Srutkova et al. (2015) Bifidobacterium longum ssp. longum CCM 7952 (Bl 7952)
C57BL/6 + Improves intestinal barrier function Srutkova et al. (2015)
Enterococcus durans TN-3 BALB/c + Induces Treg cells and restores
the diversity of the gut microbiota
Kanda et al. (2016)
Escherichia coli strain Nissle 1917 Balb/c + Increases the number of
regulatory T cells in Peyer’s patches
Souza et al. (2016) I3.1 probiotic formula (Lb.
plantarum (CECT7484, CECT7485)
and Pediococcus acidilactici (CECT7483))
Balb/c + ND Loren et al. (2016)
LAB cocktail GI7 (Lb. acidophilus, Lb.
plantarum, Lb. rhamnosus, L. lactis, B. bifidum, B. breve and S. thermophilus)
Mice + ND Kim et al. (2016)
Lactobacillus brevis KY21 Balb/c + ND Kim et al. (2015)
Lactobacillus casei BL23e Balb/c – Inhibits TLR-4-linked NF-kB and
intestinal bacterial GAG degradation
Lee et al. (2009)
Lactobacillus casei BL23 with Dairy
Delivery Matrix
Balb/c + Inhibits TLR-4-linked NF-kB and intestinal bacterial GAG degradation
Lee et al. (2009)
Lactobacillus crispatus CCTCC
M206119
Balb/c – ND Cui et al. (2016)
Lactobacillus curvatus WiKim38 Mice + ND Jo et al. (2016)
Lactobacillus fermentum CCTCC
M206110
Balb/c + ND Cui et al. (2016)
Lactobacillus helveticus (R0052) C57BL/6 + ND Emge et al. (2016)
Lactobacillus plantarum LS/07 Sprague-Dawley rats
+ Decreasesβ-glucuronidase
activity
Hijova et al. (2015)
Lactobacillus plantarum NCIMB8826 Balb/c +/– ND Cui et al. (2016)
Lactobacillus reuteri R2LC or 4659 ? + Increases tight junction proteins
occludin and ZO-1
Ahl et al. (2016)
Lactobacillus rhamnosus (R0011) C57BL/6 + ND Emge et al. (2016)
Lactobacillus rhamnosus NutRes 1 C57BL/6 +(resolution
phase)
ND Zheng et al. (2016)
Probiotic cocktail UltrabiotiqueR (Lb. acidophilus, Lb. plantarum, B.
lactis and B. breve)
Balb/c + Reduces TLR4, iNOS and NF-kB expression in colonic tissue
Toumi et al. (2014)
Saccharomyces cerevisiae strain
UFMG A-905
Balb/c + ND Tiago et al. (2015)
VLS#3 Balb/c + ND Loren et al. (2016)
Outcome:+ significant improvement,—no differences, –/+ mixed results.
NCC2950 effectively reduced DSS colitis severity, MPO activity and cell damage and increased the IL-10/IL-12 production ratio, the administration of the same strain during active DSS colitis was not effective (Hayes et al.2014). Similarly, Lb. rhamnosus Nu-tRes 1 and B. breve NuNu-tRes 204 were only effective when
adminis-tered during the recovery phase following active DSS colitis dis-ease (Zheng et al.2016).
In humans, anxiety, depression and altered memory are as-sociated to intestinal disease and colitis including IBD (Filipovic and Filipovic2014). Intriguingly, during peaking and declining
inflammation status in DSS colitis models, mice were reported to have increased anxiety behavior and impaired recognition memory, indicating that changes in mood and behavior are as-sociated to acute inflammation and dysbiosis (Emge et al.2016). Notably, such changes can be prevented by administering pro-biotics, as has been demonstrated for Lb. rhamnosus (R0011) and Lb. helveticus (R0052) that were found to relieve apparent anxi-ety and improve recognition memory during acute DSS colitis (Emge et al.2016). These findings suggest that these models that are canonically used to study intestinal inflammation may also be useful for behavioral changes associated with inflammatory stress responses.
Pathogen-related colitis
Pathogens reduction is important in the new EFSA guidelines on substantiation of health claims relevant for probiotics (EFSA Panel on Dietetic Products2016) underling the necessity the de-velop colitis model associated to specific pathogens. As an ex-ample, although E. coli belongs to the typical members of the normal microbiota, some strains are pathogenic or opportunis-tically pathogenic and associated with intestinal disorders. Dif-ferent pathovars can be distinguished: enteroaggregative E. coli that may or may not produce Shiga toxin, enterohemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enteroinvasive E. coli, enterotoxigenic E. coli, diffusely adherent E. coli and adherent-invasive E. coli (AIEC). EHEC and AIEC may cause severe colitis and bloody diarrhea, and may contribute to additional compli-cations such as haemolytic-uraemic syndrome (for EHEC) and Crohn’s disease (CD; for AIEC). Mouse models to study EHEC colonization have been developed, although they have failed to fully reproduce the infection physiopathology. Nevertheless, a few studies showed that some probiotic strains help prevent the loss of body mass, the accumulation of cecal luminal fluid and renal tubular necrosis in EHEC or other pathogen challenge mod-els in mice. When germ-free Swiss Webster mice were monocol-onized with EHEC 86–24 and treated daily with Lb. reuteri ATCC PTA 6475, EHEC colonization was suppressed and mice were sig-nificantly protected from the disease’s progression (Eaton et al.
2011). Another example is the use Sc. cerevisiae to prevent and limit colonization by EHEC O157:H7, thereby minimizing disease symptoms (Th ´evenot et al.2015). In vitro research has shown that this yeast strain significantly decreases the transcription (mRNA) levels of Shiga toxin gene (stx). Subsequent ex vivo exper-iments using murine ileal loops revealed that this yeast strain inhibited the pathogen’s interactions with Peyer’s patches and could thereby prevent the development of hemorrhagic lesions. Such probiotic properties could also support health-promoting effects with other enterohemorrhagic bacteria, including the in-vasive pathogen Shigella. A major hurdle in testing the efficacy of probiotics in this context is the absence of suitable animal models for acute rectocolitis that adequately mimics Shigella-induced dysentery in humans (Singer and Sansonetti2004). Re-cently, a short-term challenge-based mouse model suggested that an Aloe vera-supplemented probiotic yogurt drink (contain-ing Lb. lactis ssp. lactis biovar diacetylactis and Lb. paracasei ssp. paracasei [NCDC 627]) could prevent Shigella infection, which was likely associated with modulation of innate immune responses (Hussain et al.2017).
In CD patients, AIEC strains have been proposed to be the most likely candidate bacteria to cause specific damage in peo-ple who are genetically predisposed for this disease. AIEC strains were present in 21.7% of chronic CD ileal lesions but in only 6.2%
of control lesions (Darfeuille-Michaud et al.2004). This notion led to the development of a mouse model to explore how AIEC could potentiate intestinal inflammation after DSS induction (Car-valho et al.2008) and induce intestinal inflammation in trans-genic mice expressing human CEACAM (Carvalho et al.2009). In the latter study system, the human glycoprotein CEACAM6, which is upregulated in CD patients’ ileal mucosa (Barnich et al.
2007), serves as a preferential receptor for the FimH (type 1 pili adhesin) variant found in AIEC strains (Dreux et al.2013). Inter-estingly, a low-methyl diet led to epigenetic modifications that resulted in increased expression of CEACAM6 in the gut, which favored AIEC colonization and subsequent inflammation (Deni-zot et al.2015). To date, no evidence exists that probiotics could help clinical remission in CD patients, with the possible excep-tion of Sc. boulardii in certain populaexcep-tions (e.g. non-smokers) (Lichtenstein, Avni-Biron and Ben-Bassat2016). Recent results from in vitro studies and CEACAM10 transgenic mouse models have shed light on the potential mechanisms involved. In par-ticular, the cell wall of yeast strains can contain high amounts of manose residues (mannans) that can serve as a adhesion target for type 1 pili adhesin of AIEC, thereby reducing their adherence to the CEACAM6 receptor by competitive exclusion (Sivignon et al.2015a,b). The study proposes that this probiotic property could be particularly effective in CD patients who have abnormally high expression of CEACAM6 receptors on their ileal mucosa, and consequently are more susceptible to AIEC colo-nization. An alternative treatment that has been proposed aims to specifically target AIEC in CD patients using viruses that in-fect bacteria (i.e. bacteriophages) (Galtier et al.2017). In both CE-ABAC10 mice and control mice given antibiotics, a cocktail of bacteriophages (LF82 P2, LF82 P6 and LF82 P8, which have com-plementary AIEC-host ranges) strongly decreased colonization by the AIEC reference strain LF82 and suggested that this highly selective elimination approach could support the restoration of a normal microbiota and homeostasis (i.e. eubiosis) in CD pa-tients. In fact, the intestinal microbiota is essential to host func-tions, development and protection. Microbiota aberrations and dysbiosis are also associated to IBD colitis, and IgA coating ap-proach identifies inflammatory commensals that preferentially drive intestinal disease (Palm et al.2014). One of the particularity of CD is that patients have a significantly lower abundance of the anti-inflammatory bacterium Faecalibacterium prausnitzii, which makes this species an attractive potential NGP (Miquel et al.
2013). Moreover, this bacterium is the first anti-inflammatory commensal bacterium tested in vitro and in vivo (Sokol et al.2008; Martin et al. 2014b) identified on the basis of human clinical data (Sokol et al.2008). Faecalibacterium prausnitzii is able to pro-tect and treat mice against chemical-induced colitis, and was shown to excrete effectors and metabolic compounds that drove this response (Sokol et al.2008; Miquel et al.2015b; Rossi et al.
2015). However, F. prausnitzii effect remains to be tested using CEABAC10 transgenic/AIEC-associated mice model.
Even though considerable knowledge is available on the molecular pathogenesis of Enterobacteriaceae and the taxon’s role in intestinal inflammation, most mouse models are inca-pable of producing stable bacterial infections (i.e. bacterial clear-ance occurs after 3–4 weeks) or characteristic epithelial damage (i.e. effacement of microvilli by EPEC or EHEC). A growing num-ber of studies have used the natural murine bacterial pathogen, Citrobacter rodentium, whose dynamics mimic those of human EPEC and EHEC infections (for review, see Borenshtein, McBee and Schauer 2008). This model has enabled the evaluation of probiotic efficiency in preventing bacterially induced colitis, and allows to distinguish between the two proposed mechanisms
namely (i) direct interference with pathogen effectors and (ii) host-response modulation. For instance, when infected mice were given a daily dose of Sc. boulardii (MFI Pharma, Richmond Hill, ON, Canada), the numbers of C. rodentium adhering to the mucosa dropped significantly, which reduced epithelial barrier dysfunction and inflammation (Wu et al.2008). This beneficial ef-fect correlated with a marked decline in the expression of the C. rodentium type III secretion system (as measured by the marker gene espB) and protein secretion. Moreover, in these experi-ments, the C. rodentium Tir protein, which allows bacterial ad-hesion, was less secreted and translocated into mouse colono-cytes. Other mechanisms of protection by probiotic strains in the C. rodentium colitis model have been reported associated with Lactobacillus properties. For example, several Lactobacillus preparations (i.e. Lb. acidophilus NCFM or a mixture containing Lb. rhamnosus strain R0011 and Lb. helveticus strain R0052 [Lacid-ofil; Institut Rosell-Lallemand]) could stimulate dendritic cell functions, enhancing the mucosal immune system responses (Chen et al.2009), or could modulate Th1 and Th17 responses to promote levels of anti-inflammatory cytokines (Rodrigues et al.
2012). Moreover, in studies that included modulation of envi-ronmental factors (social stress) and genetic background (TLR expression), Lb. reuteri ATCC23272 and Lb. rhamnosus GG could protect against C. rodentium-induced colitis, via CCL2- or TLR2-dependent mechanisms, respectively (Mackos et al.2016; Ryu et al.2016). The C. rodentium model also enables the investiga-tion of intestinal dysbiosis, in particular, the increasing rela-tive abundance of the Enterobacteriaceae and parallel decreasing relative abundance of the members of the Lactobacillales order during C. rodentium challenge can be prevented by prophylac-tic treatment with probioprophylac-tic Lactobacillus strains (Rodrigues et al.
2012). Interestingly, treatments containing lactobacilli-enriched enteric bacterial cultures have similar effects, and particularly can restore Gammaproteobacteria and Actinobacteria populations and intestinal barrier function, exemplifying that probiotic and commensal bacteria can act synergistically in protecting the in-testine against dysbiosis and enterocolitis (Vong et al.2015).
The use of antibiotics can lead to microbiota disruption and infections by enteric pathogens, with C. difficile as the most prominent example. Clostridium difficile infections vary in sever-ity, ranging from mild diarrhea to pseudomembranous colitis and toxic megacolon. Several murine models have been devel-oped to study this pathogen (for review, see Hutton et al.2014), and beneficial effects of probiotics in Cl. difficile infections have been reviewed previously (Fitzpatrick2013; Hell et al.2013). How-ever, in mouse models, only a few probiotic species have been tested, namely Lb. rhamnosus and Lb. acidophilus (Fitzpatrick2013; Yun et al.2014); Ba. coagulans, Ba. amyloquifaciens and Ba. sub-tilis (Colenutt and Cutting2014; Fitzpatrick2013; Geeraerts et al.
2015); and Sc. boulardii and Sc. cerevisiae (Fitzpatrick2013). In-terestingly, a recent study has highlighted that murine models may be limited in their ability to represent human clinical tri-als. Strikingly, the Lifeway kefir probiotic supplement (whichR contains a mixture of 12 probiotic species) shows promise for treating severe and recurring Cl. difficile infections in humans, which displayed a disease exacerbated effect in a Clyndamycine mouse model for Cl. difficile infection (Spinler, Ross and Savidge
2016).
To test candidate probiotics, developing useful in vivo murine models of bacterially induced colitis is a challenge because of in-fection physiopathology specificities. However, such models are crucial since they will reveal how probiotics behave during fections with specific pathogens, commensal microbiota or in-teract with host physiology. Moreover, the economic relevance
of development of probiotic-associated therapy for bacterial as-sociated infections needs to be kept in mind to adapt the better model. For example, probiotics targeting EHEC infections are of interest to the cattle industry, while probiotics for treating AIEC or Cl. difficile are of greater interest to the healthcare industry. Spontaneous colitis
To study IBD, a major effort has been made to develop vari-ous complimentary models that either selectively breed strains of mice with an abnormal incidence of spontaneous colitis (C3H/HeJBir mice; Sundberg et al.1994) or employ GE mice. The objective here was to avoid the use of an exogenous trigger to induce the inflammation, in order to get as close as possible to real situation.
C3H/HeJBir mice are more sensitive to the antigens produced by its endogenous intestinal microbiota, and respond by over-producing B and T cells, leading to the development of colitis (Brandwein et al.1997; Elson, Cong and Sundberg2000). This model has helped to underpin the crucial role of intestinal mi-crobiota in IBD. However, as is often the case in mimi-crobiota- microbiota-triggered spontaneous colitis, the disease’s manifestation is highly dependent on rearing conditions. As a result, this model is rarely used to study host–probiotic interactions.
More than 70 different GE murine models have been scribed for the study of IBD. These GE mice spontaneously de-velop colitis and/or ileitis (Mizoguchi et al.2016). Furthermore, there are almost GE murine 800 models in which animals display an altered susceptibility to chemically induced colitis and/or barrier dysfunction. In these GE mouse lines, a broad range of genes have been targeted, enabling disease onset to vary across time, tissues and organs. They are not yet systematically used to study host–probiotic interactions because of their specificity. These models were generally developed to study the effects of limited sets of genes, and can only be effectively used in probi-otic research if there is a reason to suspect a role of these tar-geted genes in the probiotic effect, thereby requiring a better understanding of the mechanisms underlying the effects of spe-cific probiotic strains.
At present, despite its drawbacks, the most common GE mouse line used for in vivo probiotics research is the IL-10-deficient mouse. IL-10 plays a key role in intestinal homeosta-sis, and it is known that many IBD patients have defective al-leles of the corresponding gene (Tagore et al.1999; Begue et al.
2011). Kuhn et al. (1993) developed IL-10-deficient mice in 1993, and found that they shared physiological similarities with hu-man IBD patients, including inflammatory lesions, cell infiltra-tion into the lamina propria and submucosa, mucin depleinfiltra-tion, ulcers, thickening of the intestinal tissue and epithelial hyper-plasia (see Keubler et al.2015for more details). However, as men-tioned above, this model has its drawbacks. The onset of colitis development, which can be monitored by the appearance of a rectal prolapsus (Fig.3), varies among mice as a result of fac-tors such as age or the local microbial environment. To over-come this, some laboratories synchronize symptom appearance using non-steroidal anti-inflammatory drugs or small amounts of chemicals such as DSS (Berg et al.2002; Holgersen et al.2014). With such adaptations, the IL-10-deficient mouse model has been successfully used to study several probiotic strains and al-lowed the gathering of valuable data on probiotic efficiency and mechanisms (see below).
Lactobacillus plantarum 299v was demonstrated to alleviate symptoms intensity by reducing the production of IL-12, IFN-γ and IgG2a (Schultz et al. 2002). Lactobacillus salivarius subsp.
Figure 3. Representative aspects of rectal prolapsus in IL10 KO mice. Panels A, B, C and D correspond to control, mild symptome, moderate symptom and severe symptom, respectively.
salivarius 433 118 (=UCC118) and B. infantis 35 624 were found to reduce macroscopic damage in the colon in general and in the cecum in particular. Furthermore, splenocytes or Peyer’s patch cells produced lower levels of the cytokines IFN-γ , TNF-α and IL-12, whereas TGF-β levels were unaffected by Lb. salivarius and were reduced by B. infantis (McCarthy et al.2003; Sheil et al.2006). Lactobacillus plantarum CGMCC 1258 significantly reduced histo-logical damage in IL-10-deficient mice, and was proposed to act via its capacity to reduce expression of adhesion molecules in the mucosa that play an essential role in regulating leukocyte in-filtration, including mucosal addressing cell adhesion molecule 1 (MAdCAM-1), the intercellular adhesion molecule 1 (ICAM-1), CD3 and a4b7 integrin (Chu et al.2010). Notably, an experiment using Lb. salivarius UCC118 revealed that probiotic benefits were not necessarily dependent on oral administration, since subcu-taneous injection of the strain could reduce macroscopic dam-age as well as Il-12 and TNF-α production by splenocytes, despite the administration at a site distant from the site of inflammation (Sheil et al.2004).
The VSL#3 mixture (see also above) was also tested in the IL-10-deficient mouse model and elicited interesting effects on epithelial barrier function (Madsen et al.2001), which paralleled those observed in the DSS model. Colon samples were tested in Ussing chamber-based assays, a specific instrument enabling measurements of transepithelial electric resistance or flux of specific molecules. Results indicated that VSL#3 restored barrier function. The activity of this probiotic mixture was then investi-gated in more detail using a transcriptome approach (Reiff et al.
2009), which revealed that VSL#3 regulates immune responses by activating Th1 transcription factors and upregulating poten-tial NF-κB antagonists such as PPARs and proteins involved in xenobiotic and lipid signaling. Another probiotic mixture named Bifico (containing B. longum, Lb. acidophilus and Enterococcus fae-calis) was also evaluated in the IL-10-deficient mouse model, and
elicited a modest but significant reduction in macroscopic dam-age, as well as an improvement in barrier function (Shi et al.
2014).
Interestingly, the effects of VSL#3 were also analyzed using the Muc2-deficient mouse colitis model (Kumar et al.2017). Muc2 encodes the major component of the intestinal mucus layer that serves as a lubricant and protects the intestinal epithelium. Muc2-deficient mice develop spontaneous colitis, most likely due to extensive exposure of their mucosa to the intestinal mi-crobiota (Van der Sluis et al.2006). VSL#3 was able to reduce the inflammatory response in the Muc2-deficient mice and acetate was identified as one of mediators of this beneficial effect.
In summary, identifying and validating the mechanisms by which probiotic bacteria act remains a challenge that could bet-ter harness the possibilities offered through GE animals. When testing hypothetical mechanisms, it is clearly an advantage to overexpress or knock out specific target genes in the animal model used. At present, the IL-10-deficient mouse model is the most common GE model for studying host–probiotic interac-tions and has yielded information on the different mechanisms employed by probiotic strains. However, it is clear that any strain that acts through IL-10-dependent mechanisms (which has been proposed for many protective effects; see above) cannot be ade-quately evaluated in this model. Furthermore, it is important to note that a given probiotic strain could act via different media-tors, which likely vary in efficiency, and the resultant beneficial effects might be a multifactorial process that involves interac-tion with multiple host pathways and control mechanisms. Adoptive cell transfer colitis
Of the many models of colitis that have been developed trying to reproduce the features of IBD, some of them have yielded insight in the crucial role of circulating immune cells (Jurjus, Khoury
and Reimund2004). However, in spontaneous models, the on-set and severity of colitis are highly variable, while in inducible models of colitis, factors other than immune cells contribute to disease development. To overcome this and enable immune cell subset-specific investigations, adaptive cell transfer models have been developed in mice (Ostanin et al.2009). The adaptive transfer of CD4+CD45RBhighT cells (naive T cells) from healthy wild-type donors into syngeneic recipients lacking T and B cells (SCID or Rag−/−mice) is the prototypical and best-characterized cell-transfer model for chronic colitis. Inflammation of the en-tire intestinal tract, from the small intestine to the colon, occurs 5 to 8 weeks after T-cell transfer (Powrie et al.1994; Powrie1995). Transmural inflammation (i.e. inflammation that spans the en-tire depth of the intestinal wall), epithelial cell hyperplasia, crypt abscesses, epithelial cell erosion, and infiltration of neutrophils and mononuclear cells are also observed in this model (Jurjus, Khoury and Reimund2004).
Other models have also been used. First, SCID mice have been reconstituted with CD62L+CD4+ T cells (Wirtz et al.2002), re-sulting in chronic transmural colitis, with IFN-gamma and IL-18 playing prominent roles. Second, TCR−/−or SCID mice have been the recipients of hsp60-specific CD8+T lymphocyte clones that were pre-activated by bacterial hsp60. In this model, dis-ease development is dependent on interactions between hsp60 and MHC class I molecules, with TNF-α playing a major role. In-terestingly, colitis develops even in the absence of microbiota in this mouse model (Steinhoff et al.1999).
This general class of models is ideal for examining the ear-liest immunological events associated with colitis development and for studying the specific roles played by immune cell pop-ulations, including regulatory T cells, dendritic cells and mono-cytes, in suppressing or limiting inflammation onset or duration. Therefore, these models have been used to test the effects of probiotics, with the aim of pinpointing their specific impact on immune cells. For instance, when colitis was induced in RAG2−/− mice via the intraperitoneal administration of CD4+CD45RBhigh and CD4+CD45RBlowT cells, B. lactis oral treatments reduced in-flammation, tissue damage and weight loss (Philippe et al.2011). In the same model, oral administration of Lb. paracasei also re-sulted in intestinal protection (Oliveira et al.2011). Immunocom-promised mice that were reconstituted with na¨ıve CD4+T cells could be protected from colitis development by oral administra-tion of B. breve, via an IL-10-dependent pathway (Jeon et al.2012). These models have also been used to test effects of novel pro-biotics, such as LAB that have been genetically engineered to express protective molecules. Motta et al. (2012) described that L. lactis and Lb. casei engineered to express the protease inhibitor Elafin could protect mice against tissue damage and cell infiltra-tion, while the parental strain did not confer a benefit. It should be noted that to test the effects of a probiotics, adoptive trans-fer models are frequently used in addition to other models (i.e. involving chemical induction or GE animals) to illustrate that probiotics have protective properties even in immunity-focused models.
Adoptive transfer models can also be employed to demon-strate the role of specific immune cell types in probiotic-induced protection or the transferable nature of that protection. Di Gi-acinto et al. (2005) showed that when lamina propria-derived monocytic cells harvested from VSL#3-treated mice were trans-ferred to na¨ıve mice, these mice became protected against TNBS-induced colitis and mortality. This result clearly demon-strates that the protective effect of VSL#3 resides within the mononuclear cell population, rendering mice resistant to col-itis induction by TNBS challenge. Other studies have indicated
that some lactobacilli (e.g. Lb. salivarius Ls33), but not others (e.g. Lb. acidophilus), can partially activate dendritic cells, which then upon transfer, can confer protection against TNBS-induced coli-tis (Foligne et al.2007b). Interestingly, this protection was shown to be NOD2 dependent and was reproduced by the peptidogly-can of the Ls33 strain (Macho Fernandez et al.2011). Tregcells also play an important role in the transfer of probiotic-induced protection. Indeed, adoptive transfer of CD4+CD62Llo+intT cells from mice treated with Lb. casei Lbs2 could induce protection against TNBS colitis, which displayed lower granulocyte infil-tration, higher tissue structural protection and limited weight loss compared to untreated control animals (Thakur et al.2016). Adoptive transfer of dendritic cells from mice orally treated with Lb. acidophilus could be used to protect and treat mice infected with C. rodentium (Chen et al.2009).
Adoptive transfer models have also been used to study the mechanisms of action of probiotic treatments on extra-intestinal diseases. For instance, the protective effects of oral administration of Lb. rhamnosus on asthma have been shown to be transferable by dendritic cells (Jang et al.2012). Similarly, the protective effects of a probiotic mixture including Lb. acidophilus, Lb. casei, Lb. reuteri, B. bifidum and S. thermophilus on atopic der-matitis model was transferred by CD4+Foxp3+Tregs(Kwon et al.
2010).
Finally, adoptive transfer models have been used to in-vestigate the tolerogenic properties of recombinant probiotics. Achieving antigen-specific immunosuppression is a major goal in the development of strategies to treat autoimmune, inflam-matory and allergic diseases. Using probiotics to actively deliver recombinant autoantigens or allergens to the intestinal mucosa has been proposed as a novel therapeutic approach for inducing tolerance. Allergy-susceptible mice were indeed protected from delayed-type hypersensitivity when they received CD4+CD25−T cells from mice that had been treated with an L. lactis genetically modified to secrete ovalbumin (OVA) (Huibregtse et al.2007), and OVA-specific immunotolerance was shown to be mediated by CD4+CD25−T cells in these mice.
In conclusion, adoptive transfer models are particularly useful for studying host–probiotic interactions. These models complement results generated by chemical and genetic mod-els, because they can decipher mechanisms underlying probi-otic benefits. Adoptive transfer models have highlighted that specific signals probiotics can induce on the immune system, and identify specific cell types involved in transferable probiotic effects.
