Probiotics and ileitis

Abstract

Probiotics have gained tremendous popularity amongst individuals searching for alternative and “natural” means to promote intestinal health. It has been suggested that the probiotic formulation VSL#3 promotes several aspects of intestinal health including attenuation of inflammatory bowel diseases (IBD). Although a definitive mechanism of action has not been clearly identified, it is generally accepted that probiotics suppress development of chronic inflammation by inhibiting activation of various inflammatory signaling pathways. This concept however needs to be revisited in light of a recent publication by Pagnini et al. showing that VSL#3 prevents development of ileitis through activation of NFκB and production of the prototypical inflammatory cytokine TNFα.

Inflammatory bowel diseases (IBD) consist of two main manifestations, ulcerative colitis (UC) and Crohn's disease (CD). The combined prevalence of UC and CD is ∼300–400 cases/100,000 individuals in western countries. IBD treatment is significant with annual costs in the United States estimated to be ∼$6 billion. Although the etiology of IBD is unknown, it is generally recognized that abnormal inflammatory responses observed in patients involve a complex interaction between host genetic factors and the intestinal microbiota. Numerous animal models of colitis fail to develop intestinal inflammation when reared under germ-free conditions, but rapidly develop disease when returned to specific pathogen free-(SPF) otherwise known as conventionalized conditions.¹ These observations clearly show that the microbiota plays a central part in the etiology of IBD. Therapeutic strategies currently available for the management of colitis are numerous and generally include administration of 5-aminosalicylates or sulfasalazine, antibiotics, glucocorticoids, immunosuppressive agents (6-mercaptopurine, azathioprine, methotrexate, cyclosporine, tacrolimus) and biologics such as anti-TNF agents (infliximab, adalimumab, certolizumab). However, despite their efficacy, some patients are unresponsive to these therapies and often suffer from numerous side effects that preclude the continuation of the treatment. Consequently, an increasing number of IBD patients are seeking treatment outside the network of allopathic medicine. Complementary and alternative medicine^1a (CAM) group diagnostic and therapeutic approaches not associated with allopathic medicine. It is estimated that Americans spend ∼$34 billion out-of-pocket on CAM products and materials in 2007.² Natural products encompass components or complex mixtures obtained from plant, microbe or animal sources and represent the most popular form of CAM in the United States. Probiotics belong to the class of natural products/dietary supplements and represent an increasingly popular CAM approach among people seeking to maintain healthy “gut function” and/or searching for a therapeutic alternative to mainstream medications.

As mentioned above, the microbiota has taken the center stage in IBD research and a vast effort is currently underway way to determine the composition of disease-associated microbiota. The human colon plays host to over 100 trillion bacteria, comprising of ∼1,000 bacterial species.^3–5 The microbiota and its associated prokaryotic-genome (over 3 million genes) is an integral part of the host and uniquely contributes to various biological processes such as maturation and development of the mucosal immune system, metabolic capacity and intestinal epithelial cell proliferation/differentiation.^4,6 An international effort is currently underway to catalogue the repertoire of microorganisms present in the intestines of healthy individuals as well as in those with pathological conditions.^7,8 Already, these investigations have provided an early peek into this fascinating microbial world. In particular, the microbial consortia present in the human and mouse intestinal tract are quite similar and composed predominantly of two phyla; the Firmicutes (64%) and Bacteroides (23%) followed by Proteobacteria (8%) and Actinobacteria (3%).^3,9 Another important observation is that a core human microbiome exists at the genus level, with a large number of microbial genes and pathways shared among individuals.⁵ This important observation suggests that deviation from this core microbiome could potentially affect normal intestinal function as well as disease process. Finally, it has been reported that the stool and colonic mucosa of IBD patients showed a reduction in total gut microbial concentration and a decrease in richness/diversity accompanied by changes in the proportion of the Firmicutes and Bacteroidetes compared to healthy subjects.^3,5,10–12 These changes in the microbiota appear to directly impact host homeostasis as indicated by fecal transplant studies. For example, microbiota transplanted from colitis-prone TRUC mice (“Tbx21^-/-; Rag2^-/- ulcerative colitis”) confer disease to colitis-resistant wild type mice.¹³ Additionally, microbiota transplantation from obese mice into lean germ-free recipients induces weight gain and adipose deposition, demonstrating that these traits are conferred through a microbial dependent mechanism.^6,14 Toll-like receptors (TLRs) are critical innate sensors that detect the presence of various bacteria and bacterial-associated structural molecules. Tlr5^-/- mice develop spontaneous hyperphagia and metabolic syndrome comprised of hyperlipidemia, hypertension, insulin resistance and enhanced adiposity.¹⁵ Interestingly, hyperphagia and metabolic syndrome can be induced in WT mice upon fecal transplantation of microbiota obtained from Tlr5^-/- mice. These findings strongly suggest that the microbiota influences the health status of the host.

Since colitis is associated with microbial dysbiosis, the concept of fighting “fire with fire” by using “healthy” microorganisms to correct for the actions of harmful ones has been entertained by the field of medicine for quite some time. In fact, it was at the beginning of the 20^th century that the visionary and Nobel laureate Eli Metchnikoff suggested that it would be possible to modulate the microbiota by replacing harmful microorganisms with beneficial ones, a thought that gave birth to the field of probiotics research. Probiotics are defined as “live microorganisms which when administered in adequate amounts confer a health benefit to the host”. Probiotics are readily available to the general public as dietary supplements and are present in various food products such as miso soup, soft cheeses and yogurt. Among the various probiotics, VSL#3 formulation has been the subject of intense research both in experimental models and human diseases. This probiotic is a mixture of 8 strains of lactic acid-producing bacteria (Lactobacillus plantarum (L. plantarum), L. delbrueckii subsp. Bulgaricus, L. casei, L. acidophilus, Bifidobacterium breve (B. breve), B. longum, B. infantis and Streptococcus salivarius subsp. thermophilus). Probiotics including VSL#3 have been shown to prevent the development of, as well as treat established colitis in the Il10^-/- murine model.^16,17 Moreover, VSL#3 is well tolerated in IBD patients and has been shown to induce remission in patients with UC.^18–22 The exact mechanism of probiotic action is unclear although the beneficial activity appears to be mediated by a wide range of effects including competitive exclusion of bacterial adherence and/or translocation, release of bacteriocidin and lactic acid, production of butyric acid, antioxidative effects, enhancement of barrier function, modulation of immune cell responses and an inhibition of the transcription factor NFκB activation.^16,23–32

A recent report has shed new light on the mechanism underlying the beneficial effects of VSL#3.³³ In this study, the authors demonstrated that VSL#3 prevents the development of CD-like ileitis in the SAMP1/YitFc (SAMP) murine model. The finding that VSL#3 attenuates experimental ileitis in SAMP mice is not as unexpected as its mechanism of action, which appears to be dependent on activation of intestinal epithelial cell (IEC)-derived NFκB and production of TNFα. This finding is counter-intuitive in the light of the fact that anti-TNFα therapy is used to treat CD patients. Indeed, the thought of activating NFκB and inducing TNFα secretion is not forthcoming when the goal is to alleviate chronic inflammation.

Since its discovery by Dr. David Baltimore's research group in 1986,³⁴ NFκB has been widely viewed as a therapeutic target for various inflammatory conditions including IBD.^35–37 NFκB, in combination with various nuclear proteins regulates the expression of numerous pro-inflammatory cytokines, adhesion molecules, growth factors, proliferation—and survival genes that impact both the extent and duration of intestinal inflammation. Additionally, NFκB is activated by a wide array of agents found in the intestinal milieu including bacteria, bacterial products, viruses, cytokines and growth factors.^38,39 Finally, IBD involves the dysregulated production of pro- inflammatory cytokines, many of which lie downstream in the activated NFκB pathway. For example, the blockade of pro-inflammatory cytokines such as TNFα and IL-12p40 with neutralizing antibodies are efficient modalities for the treatment of IBD.⁴⁰ How could these compelling observations be reconciled with the current report showing that VSL#3-mediated beneficial effects require activation of NFκB and TNFα production? The authors argue that the site of NFκB activation (i.e., within the IEC) is a key factor in the protective effect of VSL#3 on intestinal inflammation. This concept has strong support from the literature. For example, deletion of the NFκB activator, IkappaB kinase β gene (IKKβ) in IEC (IKKβ^IEC-/-) did not prevent dextran sulfate sodium (DSS)-induced colitis in mice, but rather exacerbated the inflammatory response.⁴¹ In addition, RelA^IEC-/- mice exhibit enhanced susceptibility to DSS-induced colitis.⁴² Mice selectively defective in IEC for the main IKK subunit, IKKγ (IKKγ^IEC-/-) spontaneously develop colitis.⁴³ Other experimental models also point to the key role of NFκB signaling in maintaining intestinal homeostasis. For example, ionizing radiation-induced intestinal injury worsens in IKKβ^IEC-/- mice.⁴⁴ Similarly, intestinal ischemia reperfusion- induced injury is exacerbated in IKKβ^IEC-/- mice.⁴⁵ The protective function of NFκB in these models appears to be mediated by an anti-apoptotic response that protects the epithelium and promotes a wound healing response, two key biological processes in the case of intestinal injury.^39,46 Therefore, the concept put forward by Pagnini et al. that VSL#3 prevents the development of ileitis by activating IEC- derived NFκB signaling appears to be in line with these reports. However, it is important to emphasize that these protective responses were mainly observed in response to overt epithelium damage induced by radiation, ischemia or chemical exposure. In contrast, colitis developed spontaneously in SAMP mice and was not the result of overt intestinal damage as observed in the aforementioned injury models. Interestingly, pharmacological blockade of NFκB signaling using either Bay 11-7085 or the nemo-binding peptide prevented bacterial-induced colitis in Il10^-/- mice, a model of spontaneous chronic inflammation.^46,47 Attenuation of colitis was associated with downregulated transcription of NFκB-dependent genes Il12p40 and Tnfα. Moreover, a previous report using Il10^-/-; Ikkβ^IEC-/- and Il10^-/-; Ikkβ^Mye-/- mice indicated that myeloid-, and not IEC-derived NFκB signaling is important for disease development in these mice.⁴⁸ Finally, VSL#3 was shown to decrease bacterial-induced colitis in Il10^-/- mice, an effect associated with reduced TNFα production.¹⁶ The fact that VSL#3 attenuates inflammation in both SAMP and Il10^-/- mice indicates that the role of NFκB/TNFα appears different in these models. Intestinal inflammation in Il10^-/- mice develops almost exclusively in the colon whereas the ileum is the primary site of inflammation in SAMP mice. It is possible that IEC-derived NFκB activity is protective in the context of ileitis whereas myeloid-derived NFκB activity drives colitis in Il10^-/- mice. It will be important to directly investigate the role of NFκB signaling in SAMP mice using either a pharmacological or genetic-based approach. Likely, the location, duration and threshold level of NFκB activity directly impact on the biological function of this transcription factor, ranging from protective to pathological effect. Another possibility is that the main mechanism by which VSL#3 functions is through enhancing barrier function and reduction of intestinal permeability. Indeed, VSL#3 was found to reduce intestinal permeability in both Il10^-/- and SAMP mice, an effect that is expected to prevent bacterial translocation and uncontrolled activation of lamina propria immune cells. In this context, activation of NFκB and TNFα secretion may be secondary to the effect of VSL#3 on barrier function in the SAMP model. It would be useful to evaluate intestinal barrier function in SAMP mice treated with VSL#3 followed by administration of an anti-TNFα antibody.

Another important question relates to the impact of VSL#3 on the composition of the microbiota. As described above, IBD is associated with the presence of a dysbiotic microbial community consisting of a reduction in Firmicutes and Bacteroidetes. It would be interesting to establish the composition of the microbial communities present at the surface of the intestinal mucosa or in the stool of VSL#3-fed mice. Interestingly, a recent report showed that administration of VSL#3 cause an overall decreased in bacterial richness and diversity, which associated with an attenuation of trinitrobenzene sulfonic acid-induced colitis in rats.⁴⁹ Although Pangini et al. demonstrated a direct effect of VSL#3 on NFκB signaling using a colonic explant cell culture system, the possibility that the probiotic impacts disease progression through a multifactorial process such as prevention of dysbiotic microbiota formation could not be formally excluded. For example, the proportion of mucosal- associated, anti-inflammatory commensal bacterium Faecalibacterium prausnitzii significantly decreased in the ileium of CD patients.^50,51 Although the role of this bacterium in the development of ileitis in SAMP mice has yet to be investigated, one could speculate that VSL#3 cause changes to the microbiota (composition and/or metabolic capacity), which then affect intestinal permeability, NFκB signaling and TNFα production. Since the microbial community causing ileitis in SAMP mice is likely different than the one promoting colitis in the Il10^-/- mice, it is possible that the effect of VSL#3 on these microbial communities differently affect down-stream signaling targets such as NFκB/TNFα. High-throughput sequencing and metagenomics approaches would be necessary to address this possibility.

In conclusion, the study of Pagnini et al. elegantly demonstrates the complex relationship between the VSL#3 probiotic, intestinal inflammation and activation of NFκB/TNF in enterocytes. Further studies are needed to clarify the impact of VSL#3 in additional animal models of colitis before a final verdict is rendered on the concept that NFκB activation is beneficial in chronic intestinal inflammation. The continued characterization of beneficial effects conferred by probiotics will likely lead to the identification of novel mechanisms as well as compounds capable of mediating these effects. Since VSL#3, at least in vitro appears to mediate its effect through a secreted compound,³³ it would be interesting to identify this product(s) and directly test its effect on intestinal barrier function and NFκB activity. For example, secreted proteins from Lactobacillus rhamnosus GG (LGG) have been identified and showed to prevent cytokine- induced apoptosis through activation of AKT.⁵² Identification/purification of probiotic-associated molecule(s) could represent a novel means by which to modulate intestinal inflammation.