Enterococcus faecium; a Suitable Probiotic Candidate for Modulation of Immune Responses Against Pathogens

Probiotics are sets of nonpathogenic microorganisms without virulence which inhibit pathogen growth in animals. Enterococcus faecium has been introduced as a probiotic and its probiotic characteristics have been evaluated in several investigations. Evidence suggests that probiotics may modulate the immune system of the host to improve responses against pathogens. Thus, this review aimed to present recent studies regarding the effects of the E. faecium probiotic strains on the host immune responses. It seems that E. faecium AL41, CGMCC, NCIMB, SF68, Strain 26, JWS 833 and EF55 strains improve beneficially the immune responses of the host. However, most studies have been performed on animal models, therefore, clinical trials on humans are required to understand the beneficial mechanisms ofE. faecium on the human immune system.


Introduction
Probiotics are nonpathogenic microorganisms which have several benefits on human health or physiology. 1 Probiotic is a Greek word 'pro bios' which means 'for life' as opposed to antibiotics which means 'against life.' Accordingly, it has been proposed that there are beneficial effects of probiotics on human health by a Russian researcher Ellie Metchnikoff.Probiotics have several positive and negative effects on the gut microflora and the microbial toxic activities, respectively. 2,3Both yeast and bacteria, especially lactic acid bacteria, are the main members of probiotics. 1 Probiotics modulate endogenous flora, pathogenic micro-organisms and also the immune responses of hosts in either a direct or indirect manner. 4Accordingly, it has been hypothesized that probiotics can be considered as potential agents to prevent or help to cure human infectious diseases, especially intestinal disturbances. 5nterococcus faecium is a strain of gram positive bacteria Firmicutes phylum, ba-cilli class, Lactobacillales order, Enterococcaceae family, Enterococcus genus, the E. faecium species, which exhibits some probiotic properties.6 Therefore, it has been proposed as a probiotic and several studies have evaluated the bacteria for treatment of several animal and human models.[7][8][9] The following sections describe the bacteria in details.It has been hypothesized that probiotics, including E. faecium, fight the pathogenic microbes via several mechanisms.7 It appears that modulation of the immune responses against the pathogens is a plausible mechanism to fight against pathogens.10 Several probiotic strains including E. faecium SF68, AL41, EF55, JWS 833, Strain 26, NCIMB 10415 and CGMCC 2516 have been used to investigate the immunological modulation with focus on safety, anti-pathogenic and immunomodulatory properties which are listed in Table 1.Although, there are several research and review articles related to E. faecium and its benefits for human, the interaction of the bacterium with the immune system has not been discussed previously.Additionally, E. faecium is now recognized as a probiotic, and hence, this review article collates recent information regarding the roles of E. faecium in modulation of the immune responses.

Enterococcus faecium and its Effects on the Immune Responses
Enterococcus faecium has been considered as a probiotic and several investigations have evaluated its effects on the immune responses in the human and animal models. 11The probiotic may modulate innate and adaptive immunity to either increase responses to infectious disease to clear them or modulate immune responses in inflammatory conditions to decrease their complications.Accordingly, investigations on the immunomodulatory effects of E. faecium have been categorized into three sections including (1) The effects of E. faecium on the innate immunity, (2) The effects of E. faecium on the adaptive immunity, and (3) the effects of E. faecium on the modulation of the immune responses during pro-inflammatory diseases.
The Effects of Enterococcus faecium on the Innate Immunity Based on the probiotic effects of E. faecium, it seems that the bacterium not only considered as a foreign particle for immune system, but also can modulate innate immune system.Accordingly, Dvoroznakova et al have reported the effects of E. faecium on the innate immunity (monocytes, polymorphonuclear leukocytes, phagocytosis and oxidative burst) of Trichinella spiralis infected mice 8 and have demonstrated that the administration of E. faecium to mice prior to T. spiralis infection led to increased innate immunity against the parasite and elevated prolonged phagocytic activities of the host leukocytes.The administration of E. faecium also increases the ingestion capacity of the innate immune cells, including macrophages. 8The E. faecium also plays key roles in the induction of an appropriate mucosal immunity against the pathogenic bacteria such as Salmonella typhimurium. 12Lauková et al also reported the positive effects of the E. faecium probiotic on the mucosal immunity of rabbits. 9Based on these results, Tiantong et alalso showed the beneficial effects of

Piglet
Reduction in the cell density counts of α/βCD8+ intraepithelial and percentage of α/βCD8+ circulating T cells -37

Dog
No differences in the IgA production SF68 38

Dog
No alteration in the immune responses during the chronic enteropathy NCIMB the E. faecium on the mucosal immunity and reported that the probiotic elevated the matrix metalloproteinase 9 (MMP9) and also improved the neutrophil infiltration to the infected glands of Holstein cows. 13In addition, Siepert et alshowed that pre-treatment of the animals by the E. faecium NCIMB 10415 before S. typhimurium challenge led to significantly increased expression levels of IL-8 in the mesenteric lymph nodes. 14IL-8 is a pro-inflammatory cytokine which recruits neutrophils to the infected sites. 15Therefore, it may be concluded that E. faecium fights against pathogens via the induction of the immune responses at the site of infection.The E. faecium also affects the immune responses in a strain of fishes (Paralichthys olivaceus).Accordingly, Lee et alrevealed that the citrus by-products (CB) of fermentation with E. faecium enhanced the innate immunity of fish.Their results demonstrated that probiotic treatment led to increased myeloperoxidase and lysozyme activities in a dose-dependent manner. 16Another study on fish using rainbow trout, Oncorhynchus mykiss, as a model revealed that E. faecium enhanced leukocytes superoxide anion production and serum complement activities through the alternative pathway. 17The study also showed that expression levels of interleukin 1 alpha (IL-1α), Tumor necrosis factor-alpha (TNF-α) and transforming growth factor beta (TGF-β) were increased significantly in the spleen and the kidney. 17Choi et alused E. faecium JWS 833 isolated from the duck intestineand reported that the heat-killed JWS833 enhanced production of nitric oxide (NO), IL-1β and TNF-α by mouse peritoneal macrophages in response to Listeria monocytogenes. 18sing the E. faecium AL41 in hens also resulted in improvement of the intestine integrity, which is a part of the innate immunity against microbes. 19The probiotic also enhanced phagocytosis by neutrophils and production of antibodies in humans. 20Schmitz et al studied the dog's duodenal biopsies and whole blood and demonstrated that E. faecium stimulated the expression of pro-inflammatory cytokines including IL-17A, IL-22, Interferon gamma (IFN-γ) and TNF-α in the duodenal biopsies and serum and subsequently enhanced the immune responses against pathogens. 21The probiotic form of E. faecium (NCIMB 10415) also increased the production of NO by the phagocyte cells of pigs. 22Based on the results, it appears that E. faecium modulates the innate immunity to fight against pathogens.
The Effects of Enterococcus faecium on the Adaptive Immunity Enterococcus faecium also modulates the adaptive immunity.Rieger et al demonstrated that E. faecium treatment led to the improvement of the mucosal immunity against S. typhimurium by increasing the number of mucosal intraepithelial lymphocytes. 23Intraepithelial lymphocytes are the first line of cellular immunity against microbes and play key roles in the mucosal immunity. 24hmed et aldemonstrated that the administration of Citrus junos probiotics, a by-product of fermentation with E. faecium, to chicks showed that the product led to increased serum levels of immunoglobulin (Ig)M. 25Wang et al also demonstrated the immunomodulatory effects of the E. faecium reporting that the E. faecium NCIMB 10415 in combination with zinc oxide stimulated the humoral immune responses against trivalent influenza vaccination and also led to an increased numbers of activated T helper and cytotoxic T cells in pigs. 26Another study on Holstein calves using the E. faecium NCIMB 10415 showed that probiotic treatment led to increased numbers of the peripheral blood helper, cytotoxic and γδ T cells. 27Their study also demonstrated that the numbers of CD14(+), CD21(+) and TLR2(+) cells as well as the expression of IL-6, and IFN-γ were increased significantly in the probiotic treated animals on day 7. 28 Treatment of chickens with E. faecium prior to challenge with the Escherichia coli K88 also led to up-regulation of IL-4, TNF-α and IgA in the mucosal immunity which eventually assisted in the eradication of the E. coli. 29nother study on chicks identified that the administration of E. faecium EF55 was associated with the elevated numbers of the peripheral blood lymphocytes and CD3, CD4, CD8, and IgM positive cells in response to Salmonella enterica serotype enteritidis phage type 4 (SE140 PT4). 29Talebi et al reported that the administration of E. faecium to chicken improved the humoral immunity responses to vaccination with the Newcastle disease virus and infectious bursal disease. 30Interestingly, the administration of E. faecium led to decreased numbers of intestinal CD8+ cytotoxic T cells and their results revealed that this was associated with suppression of the E. coli infection. 31It suggested that the probiotic enhances the immune responses against E. coli in a CD8+ cytotoxic T cells independent manner.E. faecium SF68 also increased fecal IgA and specific circulating IgG and IgA against the canine distemper virus (CDV) vaccine. 32n sum, it may be hypothesized that E. faecium modulate the mucosal adaptive immunity to fight against pathogens.
The Effects of Enterococcus faecium on the Modulation of the Immune Responses During Pro-inflammatory Diseases Although, E. faecium plays key roles in induction of appropriate immune responses against pathogens, there are some evidences which confirm the roles played by E. faecium to modulate immune responses during pro-inflammatory based diseases.Another study investigated the roles of E. faecium on the liver functions of broiler chickens which revealed that the probiotic decreased the inflammatory responses ,damage associated with molecular patterns (PAMPs), and induced homeostasis. 33iepert et al also used the E. faecium NCIMB 10415 strain to treat the pigs prior to the infection with S. typhimurium. 14They reported that treatment of healthy pigs with the E. faecium NCIMB 10415 led to reduced expression of IL-8, IL-10 and CD86/B7-2 (a co-stimulatory molecule present on activated antigen presenting cells, and interaction of CTLA-4 [cytotoxic T-lymphocyte antigen-4 or CD152] with CD86 inhibits human T-cell activation) (a co-stimulatory molecule) in the ileal Peyer's patches and elevated serum levels of TGF-β. 23TGF-β is an anti-proliferative cytokine with angiogenesis properties and its strong chemotactic capacity. 34,35It is noteworthy to know that this cytokine is essential for the maintenance of mucosal integrity. 36Thus, it seems that E. faecium could play crucial roles in mucosal health programs.Thus, it seems that E. faecium may be considered as a therapeutic target for further investigations, especially on human subjects.Figure 1 summarizes the immunoregulatory effects of E. faecium.
In contrast with the aforementioned investigations, Mafamane et al showed that the administration of E. faecium was associated with a reduction in the cell density counts of CD8+α/β intraepithelial and percentage of circulating T cells, and did not influence the control or proliferation of Salmonella growth in the piglets. 37n addition, short-term treatment of dogs suffering from giardiasis did not have any correlation with the associated response of IgA production against the worms. 38Furthermore, Schmitz et al, 39 showed that E. faecium was unable to alter the immune responses during the chronic enteropathy in dogs.

Conclusion
The strong point of this review is that some strains of E. faecium can be considered as probiotics and can be used for the treatment against pathogens in the humans.Some E. faecium strains can be used in vivo in a safe manner and either induce the innate and adaptive immune responses or inhibit the inflammatory related diseases.Thus, they may be useful for the immunomodulation of the human immune related diseases such as hypersensitivities, autoimmunity, transplantation and so on.However, some limited investigations revealed that the use of E. faecium was not associated with the improved immune responses and suggested negative results regarding the roles played by E. faecium against Salmonella growth in the piglets.Thus, it may be hypothesized that using shortterm treatments, low doses of E. faecium or the use of the probiotic in chronic based disorders such as enteropathy, which is caused by a low grade chronic inflammation, are the main reasons for the negative results of E. faecium administration.
In sum, it seems that Enterococcus faecium AL41, CG-MCC, NCIMB, SF68, Strain 26, JWS 833 and EF55 strains may use common mechanisms of action that are seen in other probiotics.

Figure 1 .
Figure 1.Immunoregulatory Effects of Enterococcus faecium.E. faecium enhances mucosal immunity by increasing numbers of intraepithelial T lymphocytes (A) and production of IgA (B).It also stimulates macrophages and dendritic cells (DCs) to produce NO and digest microbes (C).The probiotic also increases infiltration of neutrophils to the breast and milk as well as milk levels of pro-inflammatory cytokines (D).

Table 1 .
The Major Effects of Enterococcus faecium Probiotic Strains on the Immune System of Hosts