We studied the effect of administration of a probiotic preparation on composition of stool microbiota, as also on various measures of immune responses in a cohort of healthy women. Contrary to our expectations, probiotic administration failed to induce any change in the diversity and composition of gut microbiota. Furthermore, the changes in immune responses were limited to a reduction in in vitro production of cytokines which are produced predominantly by monocytes in response to stimulation with LPS, and a decrease in the frequency of Th17 cells, with no change in the frequency of Th1, Th2 or Treg cells, plasma cytokine levels, or cytokine production in response to stimulation of T cells.

The microbiota is believed to play an intimate role in regulation and maintenance of normal intestinal physiology, metabolism and immune functions. The main bacterial phyla in baseline specimens in our subjects, in the decreasing order of abundance, were Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria. This was similar to the pattern reported from developed countries [26], other Asian countries [27, 28] and India [29].

Probiotics are believed to restore the ‘dysbiotic’ gut microbiota to a state closer to normal [30] and to modulate the host immune responses. VSL#3 is a commonly used probiotic preparation that contains eight bacterial species considered to be beneficial to humans. In some studies, its use in patients with ulcerative colitis [31], pouchitis [32] and irritable bowel syndrome [33] was found to lead to changes in gut microbiota. These changes included an increase in the abundance of lactobacilli and bifidobacteria, species that are present in this preparation [32], and specific detection in stool or tissue biopsies of the bacterial species contained in this preparation [33]. In addition, in one study, there was a decline in the abundance of Bacteriodes [33], and another study showed an increase in bacterial diversity [32]. However, in our study, administration of VSL#3 did not lead to any change in either the composition, i.e. relative abundance of various taxa, or in the diversity of gut microbiota. There could be several explanations for this lack of change. First, the gut microbial community is known to be quite stable, and is rapidly restored on removal of an external influence, such as acute inflammation or antibiotic administration. It is possible that the gut microbiota in our volunteers changed on administration of VSL#3, but that this change was transient and was reversed despite its continued administration for 4 weeks, when the follow-up specimens were collected. Second, our study included healthy volunteers with normal microbiota and not patients. It is possible that probiotics can induce change in the abnormal gut microbiota of diseased persons, but not in normal microbiota of healthy persons.

In a previous study, VSL#3 administration to healthy persons led to an increase in Lactobacillus, Bifidobacterium, and Bacteroides species; however, the dose used in that study was four-fold higher than that used in our study [33]. In another study among healthy elderly subjects (aged 65–85 years), VSL#3 supplementation in a dose similar to our study, but for 8 weeks, failed to show any change in the abundance of various Bifidobacteria [34]. Also, in patients with juvenile arthritis, VSL#3 supplementation in a dose similar to ours for 3 months did not lead to any significant change in gut microbiota [35]. Whether these differences between various studies are related to the dose of probiotic used remains unclear.

Administration of VSL#3 in our study led to a decrease in the production of various cytokines (IL-6, TNF, IL-10) in ex-vivo LPS-stimulated whole-blood cultures. In a recent study, ingestion for 4 weeks of a probiotic that contained Lactobacillus casei led to a reduction in the frequency of IL-6-producing monocytes on ex-vivo LPS stimulation [36]. However, in some older studies, supplementation with lactobacilli in healthy adults had no effect on IL-6 production on LPS stimulation [37, 38]. Administration of Lactobacillus rhamnosus GG or of Bifidobacterium animalis subspecies lactis to healthy adults has also been shown to reduce TNF-α production in whole-blood cultures [37,38,39]. In in vitro co-culture experiments, VSL#3 led to an increase in the frequency of colonic and blood dendritic cells that produced IL-10 [40]. Bifidobacteria are also known to induce IL-10 production [41]. However, in contrast, we found a significant reduction in LPS-induced IL-10 production in whole-blood cultures after VSL#3 administration. This may be because of an opposing effect of Lactobacillus casei, another constituent of VSL#3, intake of which has previously been shown to reduce IL-10 production in whole-blood cultures from healthy adults [42].

Lack of effect of probiotic administration on plasma cytokine levels in our study is similar to that observed in several previous studies [43, 44]. Moreover, the cytokine responses may vary depending on the probiotic preparation used [45]. With preparations such as VSL#3, which contain a mixture of organisms, the stimulatory effect of some organisms may be cancelled out by the inhibitory effect of the other constituents [38]. Thus, future studies on effect of probiotics on immune responses may need to focus on individual probiotics, rather than their mixtures. Further, it is possible that the effect of probiotics on immune responses in healthy persons differs from that in various disease states. Such a difference could explain why we failed to find a change in immune responses following VSL#3, though some previous clinical studies have shown this preparation to be useful in some inflammatory conditions.

Probiotic administration did not lead to a change in the frequencies of Th1, Th2 or Treg cells in our subjects. In mouse, VSL#3 preparation has been shown to shift a Th2-polarized response to a Th1/Treg type response [46]. It is possible that normal Th1/Th2 homeostasis is not amenable to a change by probiotic administration; by contrast, in disease states such as IBD, where this ratio is disturbed, probiotics may help restore this balance towards normal.

A reduction in Th17 cells observed after probiotic usage, similar to that observed in our study, has also been reported in animal models and in colonic tissue from patients with IBD [47]. Intestine is rich in Th17 cells, which are known to secrete pro-inflammatory cytokines such as IL-6, TNF-α, IL-22 and IL-23 and play a role in the pathogenesis of IBD [47]. An imbalance in gut microbiota may lead to immune activation through an expansion of Th17 cells [48]. For instance, gut dysbiosis may provide an increased amount of TLR ligands, leading to activation of dendritic cells or monocytes to produce IL-6 and TGF-β, which may in turn mediate differentiation of Th17 cells.

We did not observe any change in the frequency of Tregs after VSL#3 administration. By contrast, in patients with IBD, the use of probiotics that contain either one (such as Lactobacillus casei BL23 or Bifidobacterium infantis) or several (e.g. VSL#3) bacterial species, or of prebiotics (e.g. lactic acid) has been shown to increase the frequency of Treg cells [49, 50]. This difference could be related to the fact that probiotics restore the immune milieu towards normal in disease states, but have no effect when the milieu is healthy.

Our study has some limitations. First, gut microbiota are known to vary across geographical regions, and with dietary habits, etc. Since we studied subjects in only one population group, it may be difficult to extrapolate our data to other population groups. Thus, further similar studies in other populations may be warranted. Further, our study included only young women. This was done to ensure homogeneity of the study subjects, and it is unlikely that this by itself would have influenced the effect of probiotic administration.