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. 2006 May;144(2):273–280. doi: 10.1111/j.1365-2249.2006.03051.x

Role of interleukin (IL-10) in probiotic-mediated immune modulation: an assessment in wild-type and IL-10 knock-out mice

B Sheil *, J MacSharry †, L O'Callaghan †, A O'Riordan †, A Waters †, J Morgan †, J K Collins *,†, L O'Mahony †, F Shanahan *
PMCID: PMC1809667  PMID: 16634801

Abstract

While the impact of Bifidobacterium infantis 35624 and other probiotics on cytokines has been shown in established colitis, the effects of B. infantis consumption in pre-inflammation of interleukin (IL)-10 knock-out (KO) mice and on the wild-type (WT) C57Bl/6 mice have not been well demonstrated. The objective of this study was to examine cytokine responses in mucosal and systemic lymphoid compartments of IL-10 KO mice early in disease and to compare with control WT mice. Mice were fed B. infantis or placebo for 5 weeks and culled prior to the onset of chronic intestinal inflammation (12–14 weeks). The spleen, Peyer's patches and intestinal mucosa were removed and stimulated with various bacterial stimuli. Cytokine levels were measured by enzyme-linked immunosorbent assay. While basal intestinal and systemic cytokine profiles of WT and IL-10 KO mice were similar, transforming growth factor (TGF)-β was reduced in the spleen of IL-10 KO mice. Following probiotic consumption, interferon (IFN)-γ was reduced in the Peyer's patch of both WT and IL-10 KO mice. Alterations in IFN-γ in the Peyer's patches of WT mice (enhancement) versus IL-10 KO (reduction) were observed following in vitro stimulation with salmonella. Differential IL-12p40, CCL2 and CCL5 responses were also observed in IL-10 KO mice and WT mice. The cytokine profile of IL-10 KO mice in early disease was similar to that of WT mice. The most pronounced changes occurred in the Peyer's patch of IL-10 KO mice, suggesting a probiotic mechanism of action independent of IL-10. This study provides a rationale for the use of B. infantis 35624 for the treatment of gastrointestinal inflammation.

Keywords: colitis, cytokines, probiotics

Introduction

Probiotic bacteria have modest but consistent prophylactic efficacy in animal models of enterocolitis [1–6], and beneficial effects have been reported in ulcerative colitis and pouchitis [7–13]. Conclusive evidence for efficacy in Crohn's disease, despite amelioration of disease in some Crohn’s-like animal models, is lacking. The mechanisms of action of probiotic bacteria have not been elucidated completely and are likely to be multi-factorial and strain-specific. Anti-inflammatory activity appears to involve signalling with the gastrointestinal epithelium and interaction with mucosal regulatory T cells and dendritic cells, resulting in the modulation of locally produced proinflammatory cytokines [14,15]. Dendritic cells determine the balance of effector T cell versus regulatory T cell responses and different lactobacilli have been shown to induce distinct patterns in murine dendritic cell activation in vitro [16]. Competition for ecological niches within the gut, inhibition of nuclear factor (NF) κB signalling, anti-microbial effects and immune modulation are also proposed to be probiotic mechanisms of action (for review see [17–23])

The initiation and pathogenesis of inflammatory bowel disease involve interactions among genetic, environmental and immune factors [24]. Crohn's disease and ulcerative colitis patients exhibit loss of immune tolerance to enteric bacterial species ([25–28]) and data from experimental models implicate the enteric flora as a common factor driving the inflammatory process, irrespective of the genetic underlying predisposition and immunological effector mechanism [29–33]).

Interleukin (IL)-10 knock-out (KO) mice develop a chronic enterocolitis associated with apparently dysregulated production of Th1-type proinflammatory cytokines, similar to that of Crohn's disease [31,34]. Anti-IL-12 monoclonal antibodies completely prevented disease development in IL-10 KO mice while anti-interferon (IFN) monoclonal antibodies were also shown to have a protective effect in young mice [35].

Bifidobacterium infantis 35624 has been shown previously to attenuate colitis in this model [3]. Reduced histological inflammatory scores were associated with a decreased production in proinflammatory cytokines, while transforming growth factor (TGF)-β levels were maintained. Attenuation of colitis in this model suggests that mediators other than IL-10 are involved in the demonstrable probiotic efficacy and that the mechanism may occur at the level of the controlling factors over the balance of proinflammatory and anti-inflammatory or regulatory cytokines.

The purpose of this study was to investigate B. infantis 35624 consumption on colitis in IL-10 KO mice prior to onset of chronic inflammation, because the molecular mechanisms initiating inflammation have been shown to be distinct from those involved in maintaining chronic inflammation [36]. We report the intestinal and systemic cytokine and chemokine profiles under basal and stimulated conditions in wild-type (WT) and IL-10 KO mice before establishment of the intestinal inflammation and assess the effect of B. infantis 35624 on these profiles.

Materials and methods

Preliminary assessment of disease progression in IL-10 KO mice

To ensure that IL-10 KO mice were in early disease, a preliminary longitudinal exploration of colitis was undertaken. Mice were observed over a period of 42 weeks and assessed for change in growth, behaviour and health. To calculate the disease activity index 41 mice were killed at 4 weeks (n = 4), 10 weeks (n = 1), 16/17 weeks (n = 5), 24/26 weeks n = (9), 31/33 weeks (n = 3), 39/40/41 weeks (n = 7) and 42/43 weeks (n = 10) and awarded points according to Spencer et al. [36]. One point was awarded each of the following for inflamed colon: rectal prolapse of <1 mm: soft stool with an additional point were awarded for diarrhoea and severe rectal prolapse >1 mm. For histological analysis 24 mice were killed at six time-points: week 10 (n = 4), week 16/17 (n = 6), weeks 24/26 (n = 3), weeks 29/31/33 (n = 3), weeks 39/40/41 (n = 4) and weeks 42/43 (n = 4). Dysplasia was scored according to the method of Dieleman et al. [37].

Probiotic feeding trial

Healthy C57Bl mice

Female C57 WT mice (n = 50) were obtained from B&K Universal Laboratories (Grimston, Aldbrough, Hull, UK). Mice were divided into two groups of 25 mice and fed B. infantis 35624 or placebo product daily for 4–5 weeks, from 8 to 9 weeks of age until time of culling. Placebo product was comprised of freeze-dried sterile reconstituted skimmed milk with 5% ascorbic acid.

IL-10 KO mice

Female 129 Ola × C57BL/6 IL-10 KO mice (n = 50) were used in this study (B&K Universal Laboratories). In this specific pathogen-free (SPF) environment, KO mice of this genetic background do not develop colitis until ≥20 weeks. Mice were divided into two groups of 25 mice and fed B. infantis 35624 or placebo product daily for 4–5 weeks, from 8 to 9 weeks of age until time of culling.

Mice were maintained on a homozygous background and were housed under specific pathogen-free conditions in a 12-h light/dark cycle. Following initiation of this study, all mice consumed a standard non-sterile diet. Each mouse consumed approximately 5 × 108 live microorganisms per day. This was achieved by resuspending appropriate quantities of freeze-dried powder in water that the mice consumed ad libitum. At the time of killing (weeks 12–14), Peyer's patches, intestinal mucosa and spleen cells were removed and single cell suspensions generated. Cytokine production in response to defined stimuli, in vitro, was measured using enzyme-linked immunosorbent assay (ELISA).

Source and maintenance of bacterial strains

The probiotic bacterial strain B. infantis UCC35624 was obtained from the University College Cork (UCC) culture collection. This strain was isolated originally from sections of the healthy human gastrointestinal tract [38]. The bifidobacterium strain was grown in Man–Ragosa–Sharpe (MRS) broth supplemented with 0·05% (v/v) l-cysteine hydrochloride (Sigma C-1276, Sigma-Aldrich Ireland Ltd., Dublin, Ireland). B. infantis 35624 was incubated at 37°C under anaerobic conditions (BBL Anaerobic Jars and Anaerocult A Gas Pak system, Merck, Darmstadt, Germany) for approximately 40 h. The enteric pathogen Salmonella typhimurium strain UK1 (kindly donated by Roy Curtis, Washington University, USA), was cultured aerobically routinely in tryptic soya broth for 18–24 h. Enumeration of the bacterial cultures was performed via spread plating 10-fold serial dilutions of the cultures on appropriate media; bifidobacterium–MRS agar supplemented with 0·05% (v/v) l-cysteine hydrochloride and salmonella–tryptic soya agar (TSA) (Difco, BD Biosciences, Oxford, UK) under incubation conditions as stated above. Stocks of all bacterial strains were maintained at −20°C and −80°C in the appropriate medium with 40% (v/v) glycerol (Sigma-Aldrich Ireland Ltd., Dublin, Ireland).

Isolation of Peyer's patch single cell suspensions

The small intestine of mice was removed by dissection and the lymphoid follicles of the Peyer's patches were removed carefully from the intestinal serosal side with curved scissors. Seven to 10 Peyer's patches were usually obtained, collected in 5 ml of phosphate-buffered saline (PB) containing 1 mM ethylenediamine tetraacetic acid (EDTA) and 1·2 U/ml dispase 1 (Roche Diagnostics, Lewes, UK) and placed in a shaking oven at 37°C for 20 min. The collected patches were then placed between two sterile glass slides and crushed. Cells from the follicles were released into the medium. This cell suspension was centrifuged (100 g for 10 min) and the pellet was resuspended and diluted in Dulbecco's essential Eagle's medium (DMEM), 25 mM glucose supplemented with 1% penicillin–streptomycin and 50 µg/ml gentamicin (Gibco brl, Paisley, Scotland, UK) to a concentration of 1 × 106 cells/ml.

Isolation of gut mucosal single cell suspensions

Sections of small intestine were also digested in 1 mM EDTA and 1·20 U/ml dispase 1 in a shaking oven at 37°C for 20 min. The resultant cell suspension was centrifuged (200 g for 10 min) and gut lymphocytes were resuspended and diluted in DMEM, 25 mM glucose supplemented with 1% penicillin–streptomycin and 50 µg/ml gentamycin to a concentration of 1 × 106 cells/ml.

Isolation of splenocytes

Spleens were removed from mice and ruptured using a forceps and wire mesh in 0·5% ammonium chloride. Splenocytes were centrifuged twice at 200 g for 10 min and resuspended and diluted in DMEM, 25 mM glucose supplemented with 1% penicillin–streptomycin to a concentration of 1 × 106 cells/ml.

In vitro cytokine production

Peyer's patch, mucosal and splenic single cell suspensions were seeded in duplicate conditions in 24-well tissue culture plates (Costar, Corning, Sigma-Aldrich) at 1 × 106 cells per well. Single cell suspensions were stimulated for 72 h with B. infantis 35624 and S. typhimurium UK1 at 1 × 105 colony-forming units (cfu)/ml and faecal flora lysates. Non-stimulated cells were also present to assess background cytokine secretion. Following a 72-h incubation period (5% CO2 and 37°C humidified atmosphere) all supernatants were harvested for cytokine analysis. Supernatants were aliquoted and stored at −70°C for analysis of cytokine production in batches by ELISA (R&D Systems Europe Ltd, Oxon, UK).

Faecal flora lysates were obtained by plating murine-derived faecal samples on thioglycolate media (Difco) for 2 days anaerobically at 37°C. The bacterial lawn was scraped off the plates using a glass rod and washed by centrifugation and resuspended in phosphate-buffered saline (PBS). Cell lysis was accomplished using glass beads. Following centrifugation, supernatants were removed and filtered through a 2 µm filter. Protein concentration was assessed using the BioRad assay and aliquots were stored at −70°C (2000 µg/ml) until required. All bacterial cultures were harvested by centrifugation (3000 g for 15 min), washed with PBS and diluted subsequently to final cell densities of 1 × 105 cfu/ml in DMEM.

Cytokine production by single cell suspensions, following in vitro stimulation, was measured by ELISA (R&D Systems and BD Pharmingen). Chemokines measured included CCL2, CCL3 and CCL5. Proinflammatory cytokines examined in this study include tumour necrosis factor alpha (TNF-α), IL-6 and IL-1β. Th1 cytokines measured include IL-2, IL-12 and IFN-γ, while the Th2 cytokine IL-4 was also measured. Regulatory cytokines measured in this study include IL-10 and TGF-β1.

Statistical analysis

Results were analysed using unpaired t-test. Cytokine levels are illustrated as the mean value per group of mice (n = 8–12 mice group). Results are expressed as mean ± standard error mean. A P-value < 0·05 was considered to be statistically significant.

Results

Confirmation of early disease in IL-10 KO mice

IL-10 KO mice were culled at various time-points and disease activity assessed using the disease activity index and histological inflammation [39]. At 12–14 weeks mice had a disease activity index of 1 (Fig. 1a) and a low histology score (Fig. 1b), indicating minimal inflammation at this time. This is consistent with a study by our group, in which IL-10 mice of this genetic background, kept in specific pathogen-free conditions, did not develop colitis until ≥20 weeks [3].

Fig. 1.

Fig. 1

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Confirmation of early disease in interleukin (IL)-10 knock-out (KO) mice. IL-10 KO mice were investigated over 43 weeks and scores for disease activity and histology taken. At 12–14 weeks mice had a disease activity index of 1 (a) and a low histology score (b), indicating minimal inflammation at this time.

Basal cytokine levels of IL-10 KO and WT mice

As expected, IL-10 was not detected in IL-10 KO mice at any immune compartment. The cytokine profiles of WT and IL-10 KO mice were similar at all anatomical sites investigated (Fig. 2). However, site-specific differences were observed in IL-10 KO mice, most notably TGF-β, which was reduced significantly in the spleen following IL-10 gene deletion (Fig. 2c). IL-12p40 was decreased in the Peyer's patch and CCL5 increased in the gut mucosa of IL-10 KO mice compared to WT.

Fig. 2.

Fig. 2

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Spontaneous cytokine production by supernatants of cells isolated from the Peyer's patch, gut mucosa and spleens of wild-type (WT) and interleukin (IL)-10 knock-out (KO) mice. The level of (a) IL-12p40 was decreased significantly in the Peyer's patch, (b) CCL5 increased significantly in the gut mucosa and (c) transforming growth factor (TGF)-β1 attenuated significantly in the spleen of IL-10 KO compared to WT mice. *P< 0·05. Results expressed as mean ± standard error, n = 8–12 mice.

Basal cytokine levels following B. infantis 35624 consumption

B. infantis 35624 consumption resulted in altered cytokine responses in the intestine and spleen with a reduction in most cytokines, particularly in IL-10 KO mice (Fig. 3). Proinflammatory IFN-γ was attenuated in the Peyer's patch of WT and IL-10 KO mice. To a lesser extent, IL-4 was reduced in WT mice and TNF-α in IL-10 KO mice following probiotic consumption (Fig. 3a).

Fig. 3.

Fig. 3

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Cytokine production by supernatants of cells isolated from the Peyer's patch of (a) wild-type (WT) and (b) interleukin (IL)-10 knock-out (KO) mice following Bifidobacterium infantis 35624 and placebo feeding. Interferon (IFN)-γ was attenuated significantly in WT and IL-10 KO mice following probiotic consumption. IL-4 was reduced significantly in WT mice only and tumour necrosis factor (TNF)-α was reduced significantly in IL-10 KO mice only. *P< 0·05. Results expressed as mean ± standard error, n = 8–12 mice.

In contrast to the Peyer's patch, cytokine differences in the gut mucosa and spleen after probiotic consumption were less striking. B. infantis 35624 consumption had no effect on cytokine production in gut mucosal cells of WT mice, whereas IL-4 and IL-12p40 were increased significantly in IL-10 KO mice. IL-2 and CCL3 were attenuated in the spleen of WT mice (data not shown, available from the author).

In vitro production of cytokines after bacterial stimulation of Peyer's patch cells from mice fed probiotics

Results show marked changes in cytokines and chemokines in the Peyer's patch after probiotic feeding and particularly after in vitro stimulation. In addition, results differ from WT and IL-10 KO with the opposing changes in CCL2 and IFN-γ being particularly striking. Basal levels of IFN-γ were attenuated significantly in IL-10 KO and WT mice receiving B. infantis 35624 (Fig. 4a). IFN-γ was also reduced significantly in probiotic-consuming IL-10 KO mice following in vitro stimulation with all bacterial stimuli. Contrastingly, in WT mice, IFN-γ production was enhanced in probiotic-fed mice following in vitro stimulation with S. typhimurium UK1.

Fig. 4.

Fig. 4

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Change in cytokine production from Peyer's patch cells isolated from of wild-type (WT) and interleukin (IL)-10 knock-out (KO) mice fed Bifidobacterium infantis 35624 following in vitro stimulation with bacterial stimuli. The levels of (a) interferon (IFN)-γ, (b) IL-12p40, (c) CCL5 and (d) CCL2 production by Peyer's patch cells of WT and IL-10 KO mice following placebo and B. infantis 35624 feeding and in vitro challenge with bacterial stimuli. IFN-γ was attenuated significantly in all mice following probiotic consumption. However, a striking difference between IL-10 KO and WT mice following probiotic consumption and in vitro stimulation was noted, particularly with Salmonella typhimurium UK1 challenge, where IFN-γ was attenuated significantly in IL-10 KO mice but enhanced significantly in WT. IL-12p40 and CCL5 levels were enhanced significantly in IL-10 KO mice following probiotic feeding. CCL2 was decreased significantly in IL-10 KO mice fed B. infantis 35624 following in vitro challenge. In contrast, there was no significant change in IL-12p40, CCL5 and CCL2 levels in WT mice following B. infantis 35624 consumption. Results are expressed as percentage change [(B. infantis 35624 fed − control fed/control fed) × 100] for WT and IL-10 KO mice. *P< 0·05, n = 8–12 mice. Statistical comparison of control-fed versus probiotic-fed in WT and IL-10 KO mice.

In IL-10 KO mice basal CCL2 levels, and levels following in vitro stimulation with S. typhimurium UK1, B. infantis 35624 and faecal lysates were attenuated significantly. Contrastingly, CCL2 levels were enhanced in WT C57Bl/6 mice receiving probiotic and following in vitro stimulation with B. infantis 35624 and faecal lysate, although these levels did not reach statistical significance (Fig. 4d). IL-12p40 and CCL5 levels were also affected differentially in WT and IL-10 KO mice (Fig. 4b,c) with significantly increased levels in IL-10 KO mice following probiotic consumption and in vitro stimulation with S. typhimurium, B. infantis 35624 and faecal lysates.

Discussion

The results show that probiotics have immunomodulatory effects that are evident outside the context of established inflammation and at the pre-inflammatory or early stage of disease in IL-KO mice. Cytokine changes were particularly evident in the intestinal Peyer's patch, in contrast to the spleen and lamina propria. In addition, probiotic-induced immunomodulation was not IL-10-dependent, as a significant reduction in IFN-γ occurred in both WT and KO mice after probiotic feeding. This is consistent with what we have reported previously ([3] and O’Mahony et al. submitted). However, the contribution of IL-10 to some probiotic-induced cytokine changes was shown by differences between WT and KO mice after salmonella stimulation in vitro using Peyer's patch cells isolated from mice pre-fed the B. infantis probiotic.

To ensure that IL-10 KO mice were in early disease, longitudinal exploration of colitis was undertaken to establish the time of early disease in our hands before chronic inflammation complicated the cytokine environment. A time of sacrifice was chosen at 12–14 weeks, when inflammation is minimal, contrasting to previous studies where mice were killed at >25 weeks of age.

In early disease, basal cytokine profiles without probiotic consumption of WT and IL-10 KO mice were comparable. Evidence of cytokine imbalance and a shift towards inflammation were becoming evident in IL-10 KO mice; however, CCL5 was increased significantly in the intestine and TGFβ1 was attenuated in the spleen. The reduction of IL-12p40 in the Peyer's patch may be an indication of early events in this model.

Colonic extracts from IL-10 KO mice with established colitis have been shown previously to produce high levels of proinflammatory cytokines while WT colonic explants produced low or undetectable amounts of these cytokines. IL-4 was also undetectable, suggesting that the colon has a polarized Th1 phenotype [40]. We did detect IL-4, and no conclusive TH1 or Th2 polarization was apparent at any site. Accumulating evidence suggests that it is the effector T cell/regulatory T cell balance that is likely to be the crucial factor in immunoregulatory disorders [20] and modulation of Teffector/T regulatory (Treg) balance is likely to be a probiotic mechanism of action, perhaps explaining probiotic efficacy in Th1- and Th2-mediated inflammatory disorders.

Evidence implicating the gut flora in the induction and perpetuation of colitis provide a rationale for the use of probiotic bacteria in treating inflammatory bowel disease. Oral consumption of B. infantis 35624 modulated cytokine production significantly, particularly in IL-10 KO mice, suggesting that probiotic-mediated immune modulation, while dependent on the inflammatory state of the host, is not dependent on IL-10. The Peyer's patch of IL-10 KO mice was predominantly affected, with B. infantis 35624 consumption resulting in significant amelioration of proinflammatory IFN-γ and TNF-α. This was consistent with our previous study in established colitis [3] and in probiotic trials using a IL-10 KO murine model. Treatment of IL-10 KO mice with VSL#3, Lactobacillus plantarum 299v and Escherichia coli ssp. laves demonstrated improvements in inflammation and histological disease in conjunction with significantly decreased mucosal secretions of TNF-α and IFN-γ [41–43]. Release of TNF-α by inflamed Crohn's disease mucosa has also been shown previously to be decreased significantly following co-culture with probiotic bacteria [15].

While subtle changes in cytokine and chemokine responses following B. infantis 35624 consumption are evident, more pronounced changes in mice receiving the probiotic are seen when cells are stimulated in vitro. Several bacterial stimuli were chosen as in vitro challenges to investigate the immune responses in WT and IL-10 KO mice following probiotic consumption. Particularly striking was the opposing IFN-γ responses in the Peyer's patches of WT mice (enhancement) versus IL-10 KO (reduction) following B. infantis 35624 consumption and in vitro stimulation with salmonella. IFN-γ is involved in the initiation and perpetuation of mucosal inflammation in the early phase of disease [36]. These results demonstrate distinct host-dependent cytokine responses following probiotic consumption. Healthy WT mice can differentiate between stimuli, reducing IFN-γ levels in response to in vitro challenge with non-pathogenic probiotic bacteria while mounting an appropriate Th1-mediated immune response to pathogenic salmonella. This effect was also seen by our group in Balb/c mice, where mice fed B. infantis 35624 exhibited enhanced production of IFN-γ following in vitro stimulation with salmonella (O’Mahony et al. submitted). This was not the case in IL-10 KO mice, where IFN-γ was attenuated following in vitro challenge, with probiotic bacteria and pathogenic bacteria initiating the same response. In a study by Braat et al. [44], the probiotic bacterium Lactobacillus rhamnosus was shown to induce T cell hyporesponsiveness in healthy subjects through modification of dendritic cell function.

The recruitment and activation of leucocytes at sites of intestinal inflammation is mediated by chemokines and is the hallmark of inflammatory bowel disease (IBD). CCL2 and CCL5 are down-regulated by IL-10 and elevated levels of CCL2 are seen in the inflamed mucosa of IBD patients [45]. In this study chemokine responses were modulated differentially in WT and IL-10 KO mice. CCL2 levels were decreased in IL-10 KO mice but enhanced in WT mice following B. infantis 35624 consumption and in vitro stimuli. Additional work investigating differential CCL5 and CCL2 as well as IFN-γ and IL-12p40 responses to in vitro challenge is warranted and may demonstrate further divergence with different challenges. Further studies are also warranted to investigate the effect of this probiotic bacterium on chemokine and chemokine receptors in Crohn's disease.

Not withstanding the different responses in IL-10 KO and WT mice, the more unexpected finding of this study is the ability of in vivo exposure to microorganisms, whether commensal or pathogenic, to influence a cytokine response following subsequent exposure to the same or different organisms. Microbial imprinting, where microbial exposure to a primary organism (e.g. commensal) may modulate the cytokine–chemokine environment, thereby indirectly influencing the response to a subsequent challenge, has been put forward as a potential alternative or adjunct to pharmaceutical strategies for a variety of inflammatory conditions [46]. Our findings are consistent with this hypothesis and also add valuable information should the database or periodic table of microbes be constructed as suggested. This would catalogue systematically the effects of various microbes on the cytokine and chemokine network, which will aid in the selection of microbial preparations to treat disease.

In conclusion, the results confirm that the probiotic B. infantis 35624 has immunomodulatory effects in IL-KO mice prior to the onset of chronic inflammation and also on control mice of the same genetic background. The effects are not dependent exclusively on IL-10 and are most prominent within the Peyer's patch.

Acknowledgments

This study was supported in part by Science Foundation Ireland in the form of a centre grant (Alimentary Pharmabiotic Centre), the Health Research Board of Ireland and the Higher Education Authority of Ireland. Fergus Shanahan, J. K. Collins, Liam O’Mahony and John MacSharry are affiliated with a multi-departmental university campus company (Alimentary Health Ltd) that investigates host–flora interactions and the therapeutic manipulation of these interactions in various human and animal disorders. The content of this report was neither influenced nor constrained by this fact.

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