Probiotic Molecules That Inhibit Inﬂammatory Diseases

Featured Application: Exopolysaccharide from Bacillus subtilis induces an anti-inﬂammatory response that protects mice from several inﬂammatory diseases, including enteric and blood-borne pathogens, allergic eosinophilia, and graft versus host disease. This EPS, designated EPS Bs , has potential as a therapeutic for inﬂammatory diseases in humans. Abstract: Consumption of probiotics for health purposes has increased vastly in the past few decades, and yet the scientiﬁc evidence to support health beneﬁts from probiotics is only beginning to emerge. As more probiotics are studied, we are beginning to understand the mechanisms of action by which they beneﬁt human health, as well as to identify the bacterial molecules responsible for these beneﬁts. A new era of therapeutics is on the horizon in which puriﬁed molecules from probiotics will be used to prevent and treat diseases. In this review, we summarize the active molecules from probiotic bacteria that have been shown to affect innate and adaptive immunity and have health beneﬁts in experimental settings. We focus particularly on the cellular and molecular mechanisms of the probiotic Bacillus subtilis and its active molecule, exopolysaccharide (ESP Bs ).


Introduction Mod
The World Health Organization defines probiotics as "live microorganisms that when administered in adequate amounts, confer a health benefit on the host." (FAO/WHO 2002). Consumption of live organisms for maintaining good health is a concept accepted by the general population, and in fact, The National Health Interview Survey showed that probiotics were the third most used non-vitamin, non-mineral dietary supplement in 2012 [1]. Despite their popular use, probiotics have yet to be approved for any medical indication by the US Food Drug Administration [2], in part because results from clinical trials on the efficacy of probiotics in the treatment or prevention of disease have been equivocal [3]. The lack of clarity emanating from the clinical trials has created a state of confusion regarding the clinical use of probiotics. Some of the confusion may arise from the use of different probiotic strains, or combinations thereof, batch preparation, and the outcomes readout. Furthermore, humans vary widely in their diets, genetic backgrounds, and gut microbiome composition, all of which likely influence the efficacy of probiotics.
The probiotic bacteria most widely studied belong to the Lactobacillus sp., Bacterioidetes sp., Bifidobacteria sp., and Bacillus sp. These bacteria have been used to treat diarrhea, irritable bowel syndrome [4], and atopic dermatitis [5], among other diseases. Probiotic bacteria can be beneficial to human health, for example, by inhibiting growth of pathogenic bacteria in the intestine and by secreting bioactive metabolites, such as short-chain fatty acids [6]. The use of intact bacteria for clinical interventions can be challenging, however, in part because the administration of bacteria may alter the endogenous microbiome, Table 1. Probiotic bacterial molecules with anti-inflammatory activity.

Organism
Molecule Results Reference

EPS Bs
LTA activates a TLR2-dependent inflammatory response and concomitantly induces activation of MerTK signaling to counteract the inflammation in vitro. EPS Bs reduces Citrobacter rodentium infection and generates peritoneal anti-inflammatory macrophages. EPS Bs prevents allergic eosinophilia. EPS Bs ameliorates GvHD and can generate tolerogenic DCs in vitro.

EPS Bs protects against systemic infection of
Staphylococcus aureus. [14] [9] [10,12] [8] [7] [ 11,13] Bacteroides fragilis Polysaccharide A (PSA) Glycosphingolipids PSA is protective and therapeutic in murine models of colitis and multiple sclerosis via the induction of IL-10 secreting Tregs. PSA activates colonic DCs and produces IFN-β that enhances resistance to viral infection in murine models. This protection is dependent on TLR4. Glycosphingolipids decrease the number of invariant natural killer T cells in the colonic lamina propria leading to improved outcomes in a murine colitis model. [15][16][17][18][19] [20] [21,22] Bifidobacterium adolescentis EPS Ba EPS Ba induces IL-10 production, protects from colitis by activation of DCs and macrophages, and increases the Treg/Th17 cell ratio in mice. [23] Bifidobacterium breve UCC2003 EPS Bb EPS Bb reduces Citrobacter rodentium infection in mice. EPS Bb prevents the maturation of DCs and activation of antigen-specific CD4+ T cells. EPS Bb reduces the rate of small epithelial cell shedding in a mouse model of pathological cell shedding. [24] [25] [26] Bifidobacterium breve WBBR04 EPS Bb EPS Bb enhances the intestinal barrier integrity to prevent allergen infiltration and food allergy in mice. [27] Bifidobacterium longum Serpin Serpin inhibits eukaryotic elastase-like serine proteases, which are dysregulated in inflammatory disorders. [28,29]  EPS Bl dampens pro-inflammatory cytokines and reduces inflammatory symptoms in a T cell transfer colitis model. EPS Bl stimulates the release of IL-10 in a TLR2-dependent manner and reduces eosinophil recruitment in the lungs in a respiratory inflammation mouse model. [30] [31] Bifidobacterium Longum YS108R EPS Bl EPS Bl reduces the pro-inflammatory cytokines IL-6 and IL-17A, alleviating inflammation in a colitis murine model. [32] Bifidobacterium sp. Fimbriae Fimbriae facilitate gut colonization and stimulation of macrophage cytokine production, TNF-α, IL-6, and Il-10.

Surface layer protein A (SlpA)
SlpA binds the lectin-receptor DC-SIGN and increases IL-10 and reduces IL-12p70 production from DCs. [37] Lactobacillus casei Lactoceptin Lactoceptin can selectively hydrolyze pro-inflammatory chemokine IP-10 leading to reduced lymphocyte recruitment in an ileitis murine model. [38] Lactobacillus casei BL23 p40 and p75 Secreted proteins p40 and p75 stimulate Akt activation, display anti-apoptotic activity, and prevent epithelial barrier damage in colitis murine models.

Lipoteichoic acid (LTA) Supernatants
LTA improves colitis in a murine model. Administration of culture supernatants reduces eosinophil numbers, goblet cells, and lung inflammation in murine allergy model. [53,54]

EPS Lr
LTA EPS Lr reduces hydrogen peroxide-induced intestinal oxidative damage and apoptosis by Keap1/Nrf2 and Bax/Bcl-2 pathways in vitro. LTA protects intestinal epithelial cells from radiation injury through the activation of pericryptal macrophages. These macrophages release CXCL12 that binds to CXCR4 on COX-2 expressing mesenchymal stem cells and stimulate the release of PGE, which protects epithelial stem cells from radiation. [60] [61] Lactobacillus rhamnosus KL37 EPS Lr EPS Lr inhibits T cell-dependent immune response reducing the arthritogenic antibodies in an arthritis murine model. [62] Propionibacterium freudenreichii Guanidine surface protein extract Treatment of human peripheral blood with guanidine surface protein extract releases IL-10 and IL-6, while having no effect on IL-12, TNF-α, and IFNγ. [63]

Cell Envelope Molecules
Probiotics' cell envelope molecules have immunomodulatory properties that can reduce pro-inflammatory cytokines, increase production of anti-inflammatory IL-10, generate T regulatory cells (Tregs), and help protect from radiation damage. One of the best characterized anti-inflammatory probiotic molecules is the capsular polysaccharide A (PSA) from Bacteroides fragilis. Oral administration of PSA, a zwitterionic polysaccharide, is protective and therapeutic in murine models of colitis and multiple sclerosis by inducing IL-10-secreting Tregs. Protection by PSA is TLR2 and MHCII-dependent, likely due to TLR2 signaling in plasmacytoid dendritic cells to induce Tregs and IL-10 production [15][16][17][18][19]64]. Furthermore, B. fragilis produces glycosphingolipids that decrease the number of invariant natural killer T cells in the colonic lamina propria, leading to improved outcomes in a murine colitis model [21,22]. Recently, B. fragilis PSA was shown to activate colonic DCs and produce IFN-β in a TLR4-dependent manner that enhances resistance to viral infection in murine models [20].
A polysaccharide peptidoglycan complex on L. casei Shirota was shown to improve ileitis and inhibit IL-6/STAT3 signaling in a murine colitis model using bacterial mutants in vivo and purified peptidoglycan in vitro [65]. The oral administration of probiotic L. rhamnosus GG mutant with modified lipoteichoic acid (LTA) molecules improved colitis in a murine model, correlating with decreased TLR expression and pro-inflammatory cytokine secretion [53]. Further, oral gavage with WT L. rhamnosus GG or intraperitoneal (i.p.) injection with L. rhamnosus LTA protected intestinal epithelial cells from radiation injury through the activation of pericryptal macrophages. These macrophages release CXCL12 that binds to CXCR4 on COX-2 expressing mesenchymal stem cells and stimulates the release of PGE, which protects epithelial stem cells from radiation [61]. Supernatants from L. rhamnosus GG cultures also reduced eosinophil numbers, goblet cell numbers, and lung inflammation in an allergy inflammation murine model [54]. Peritoneal administration of isolated peptidoglycan from L. salivarius Ls33 protects mice from chemically induced colitis in a NOD2-IL-10-dependent manner [56]. Other cell wall components from probiotics have been identified as listed in Table 1. As suggested above, these examples illustrate the potential for using purified active components of probiotics to treat human diseases prophylactically and therapeutically.

Secreted Molecules
A. Proteins. Numerous extracellular and secreted bacterial proteins are known to diminish inflammation. These include serpin from Bifidobacterium longum, which inhibits eukaryotic elastase-like serine proteases that are dysregulated in inflammatory disorders, such as celiac disease [28,29], and p40 and p75, molecules from Lactobacillus casei BL23 that in vitro protect from disruption of epithelial cell tight junctions induced by hydrogen peroxide in a PKC and MAP kinase-dependent manner [39]. A p40 secreted molecule from L. rhamnosus GG activated EGFR in vitro and, after oral administration, prevented DSSinduced intestinal epithelial damage in mice [40,41]. Moreover, L. casei secretes lactoceptin, which hydrolyzes the pro-inflammatory chemokine IP-10, and its i.p. administration leads to reduced lymphocyte recruitment in an ileitis murine model [38]. Another extracellular molecule that moderates the immune response is flagellin from Escherichia coli Nissle 1917, which induces the release of the antimicrobial peptide β-defensin 2 in epithelial cells in vitro [36]. Other secreted probiotic proteins that can modulate immune responses are summarized in Table 1. These examples illustrate the potential of using extracellular and secreted peptides from probiotics for therapeutic treatments of immune-mediated diseases.
B. Exopolysaccharides. Many probiotics secrete polysaccharides that have anti-inflammatory properties, which can reduce the production of pro-inflammatory cytokines, increase anti-inflammatory cytokines, enhance the intestinal epithelial barrier, and inhibit T celldependent immune responses. These polysaccharides are usually obtained by alcohol precipitation from culture supernatants, and the solubilized precipitate is designated as exopolysaccharide (EPS). As discussed below, EPS molecules of different origins vary widely in the anti-inflammatory effects and the mechanism of protection. Although the term "EPS" is used for all of these samples, the composition and structure of them are generally not known. Likely, EPS from different microorganisms have different compositions and structures, leading to diverse functions and mechanisms of immune regulation. A defined classification of EPS from different organisms will require the structure and structure/function relationship of these polysaccharides. In this review, we distinguish the EPS preparations by using superscripts to symbolize the bacterium from which a specific EPS is isolated, e.g., EPS Bs from Bacillus subtilis.
Using bacterial mutants, EPS Bb from Bifidobacterium breve has been implicated in reducing colitis during Citrobacter rodentium infection in mice [24] and in reducing rates of epithelial cell shedding [26]. Oral administration of EPS Bb from B. breve has also been shown to enhance the intestinal barrier integrity, thereby preventing allergen infiltration and food allergy [27]. Another species of Bifidobacterium, the non-aggregating strain IF1-03 B. adolescentis, protects from colitis by inducing IL-10 production, activating dendritic cells (DCs) and macrophages, and increasing the ratio of Treg/Th17 cells in mice [23]. Based on data from a bacterial mutant, EPS Bl from still another Bifidobacterium species, B. Longum 35624, dampens pro-inflammatory cytokines and reduces inflammatory symptoms in the T cell transfer colitis model [30], and in an allergy model, EPS Bl stimulates the release of IL-10 in a TLR2-dependent manner that reduces recruitment of eosinophils to the lungs [31].
For Lactobacillus, numerous immunomodulatory effects of EPS Lh have been identified and are reviewed in Laiño et al. [66]. Oral administration of EPS Lh isolated from L. helveticus KLDS1.8701 reduces intestinal inflammation and improves mucosal barrier function in a colitis model [44], whereas EPS Lr from L. rhamnosus KL37 inhibits T cell-dependent immune responses and reduces the arthritogenic antibodies in an arthritis murine model [62].
Several other examples of the anti-inflammatory effects of EPS from probiotics are listed in Table 1. While the molecular mechanisms by which EPS induces anti-inflammatory responses are not well understood, it is noteworthy that EPS Lp from L. plantarum N-14 reduces TLR4-mediated pro-inflammatory cytokine production by porcine intestinal epithelial cells. This anti-inflammatory response by EPS Lp is due to the induction of negative regulators of TLR signaling, especially RP105, a type I transmembrane molecule, considered part of the TLR family [51]. RP105 complexes with the accessory protein, MD1, and together inhibit LPS signaling through the TLR4/MD2 complex [67]. These data suggest that EPS from some probiotic species may dampen an inflammatory response or induce an anti-inflammatory response by binding to receptors that negatively regulate TLR4 signaling. EPS is easily extractable from bacteria, and as the structures and functions become better defined, EPS from numerous microorganisms may become widely used for therapeutic purposes.

EPS Bs as Probiotic
The EPS (EPS Bs ) that we study is derived from Bacillus subtilis and induces antiinflammatory responses that protect mice from several inflammatory diseases. Below, we discuss these diseases, the cells and molecules that promote protection, and the potential of EPS Bs as a therapeutic for humans.
A. C. rodentium-induced colitis. Oral administration of a single dose of B. subtilis spores was first shown to reduce colitis in mice after infection with the enteric pathogen, Citrobacter rodentium [9]. In this model, B. subtilis does not function by reducing the colonization of C. rodentium, but instead alters the inflammatory disease process, as indicated by reduced epithelial hyperplasia, diarrhea, and goblet cell loss (Figure 1). Analysis of B. subtilis mutants revealed that a mutation in epsH, which regulates biofilm synthesis [68], did not protect from disease caused by C. rodentium, suggesting that biofilm-associated carbohydrate exopolysaccharide (EPS Bs ) was required for protection. EPS Bs was isolated and purified by treatment with DNase, RNase, proteinase K, and gel filtration [10,12], and indeed, it protected from disease. In fact, a single intraperitoneal injection of EPS Bs (2.5 mg/kg) administered one day prior to or as much as 3 days after infection with C. rodentium, was sufficient to reduce epithelial hyperplasia, diarrhea, and goblet cell loss. Protection by EPS Bs is mediated by anti-inflammatory macrophages, sometimes designated as M2 macrophages. Intraperitoneal administration of EPS Bs results in the accumulation of macrophages with M2 macrophage markers, IL4Ra, CD206, arginase, and PD-L1 in the peritoneum, and adoptive transfer of these cells to untreated mice protects them from colitis after infection with C. rodentium [10,12]. These findings demonstrate the anti-inflammatory potential of EPS Bs , and of the anti-inflammatory macrophages induced by EPS Bs .
Appl. Sci. 2022, 12, x FOR PEER REVIEW 7 of 15 signaling. EPS is easily extractable from bacteria, and as the structures and functions become better defined, EPS from numerous microorganisms may become widely used for therapeutic purposes.

EPS Bs as Probiotic
The EPS (EPS Bs ) that we study is derived from Bacillus subtilis and induces anti-inflammatory responses that protect mice from several inflammatory diseases. Below, we discuss these diseases, the cells and molecules that promote protection, and the potential of EPS Bs as a therapeutic for humans.
A. C. rodentium-induced colitis. Oral administration of a single dose of B. subtilis spores was first shown to reduce colitis in mice after infection with the enteric pathogen, Citrobacter rodentium [9]. In this model, B. subtilis does not function by reducing the colonization of C. rodentium, but instead alters the inflammatory disease process, as indicated by reduced epithelial hyperplasia, diarrhea, and goblet cell loss (Figure 1). Analysis of B. subtilis mutants revealed that a mutation in epsH, which regulates biofilm synthesis [68], did not protect from disease caused by C. rodentium, suggesting that biofilm-associated carbohydrate exopolysaccharide (EPS Bs ) was required for protection. EPS Bs was isolated and purified by treatment with DNase, RNase, proteinase K, and gel filtration [10,12], and indeed, it protected from disease. In fact, a single intraperitoneal injection of EPS Bs (2.5 mg/kg) administered one day prior to or as much as 3 days after infection with C. rodentium, was sufficient to reduce epithelial hyperplasia, diarrhea, and goblet cell loss. Protection by EPS Bs is mediated by antiinflammatory macrophages, sometimes designated as M2 macrophages. Intraperitoneal administration of EPS Bs results in the accumulation of macrophages with M2 macrophage markers, IL4Ra, CD206, arginase, and PD-L1 in the peritoneum, and adoptive transfer of these cells to untreated mice protects them from colitis after infection with C. rodentium [10,12]. These findings demonstrate the anti-inflammatory potential of EPS Bs , and of the anti-inflammatory macrophages induced by EPS Bs . B. Systemic infection with Staphylococcus aureus. Similar to infection with the enteric pathogen, C. rodentium, EPS Bs also moderates disease caused by infection with bloodborne S. aureus [11]. In this case, EPS Bs increases survival by reducing weight loss and systemic inflammation, as evidenced by decreased levels of inflammatory cytokines and chemokines in blood and bacterial burden [11]. EPS Bs induced hybrid-like M1/M2 macrophages, which not only inhibited T cell activation, characteristic of M2 B. Systemic infection with Staphylococcus aureus. Similar to infection with the enteric pathogen, C. rodentium, EPS Bs also moderates disease caused by infection with bloodborne S. aureus [11]. In this case, EPS Bs increases survival by reducing weight loss and systemic inflammation, as evidenced by decreased levels of inflammatory cytokines and chemokines in blood and bacterial burden [11]. EPS Bs induced hybrid-like M1/M2 macrophages, which not only inhibited T cell activation, characteristic of M2 macrophages but also inhibited S. aureus growth through reactive oxygen species (ROS), characteristic of M1 macrophages [69]. Together, data from infection by C. rodentium and S. aureus show that EPS Bs from B. subtilis induces an anti-inflammatory environment with decreased inflammatory cytokines and increased anti-inflammatory macrophages that limit T cell activation, as well as macrophages that restrict the growth of bacteria. C. Allergic eosinophilia. The association of changes in microbiota to allergic disease is well known, not only because of the hygiene hypothesis [70] but also because of a landmark study by Stein et al., who showed that children who grow up in a farm environment with close proximity to farm animals develop considerably fewer allergies than children that grow up without much interaction with farm animals [71]. This "farm effect" is likely explained by the interaction of children with microbes of the farm animals [72]. Swartzendruber et al. orally administered B. subtilis spores to mice and showed that they prevented the development of allergic eosinophilia in response to intranasal administration of house dust mite (HDM) antigen [8]. The infiltration of eosinophils is due in part to cytokines secreted by T cells [73]. Because DCs are also crucial for the activation of T cells and the development of eosinophilia, Swartzendruber et al. hypothesized that EPS Bs -treated DCs could mitigate the allergic eosinophilia caused by an allergy to HDM. Intranasal adoptive transfer of EPS Bstreated bone marrow-derived DCs (BMDCs) prevented eosinophilia induced by HDMpulsed DCs, indicating that EPS Bs induces anti-inflammatory DCs, which can prevent an allergic response, as might be predicted by previous studies [70,72]. D. Graft versus host disease (GvHD). Another T cell-mediated disease attenuated by EPS Bs is GvHD, a severe and often lethal complication of hematopoietic stem cell transplantation, which is frequently used to treat leukemia. The devastating effects of GvHD are mediated by alloreactive donor T cells that recognize host antigens as foreign, become activated, and destroy host tissues and organs. Intraperitoneal injection of EPS Bs (2.5 mg/kg) administered several times, 7, 5, and 3 days prior to induction of GvHD, increased survival of mice 80 days post GvHD from 10% to 70% ( Figure 2). Kalinina et al. assessed inflammation in live mice during GvHD using a caspase-1 reporter mouse to measure inflammasome activation [7]. With this biosensor mouse model, they found that the administration of EPS Bs prevented the activation of alloreactive donor T cells, explaining the increased survival of mice. The results showed that EPS Bs did not directly affect alloreactive T cells. In mixed lymphocyte reactions (MLR) in vitro, EPS Bs -treated BMDCs potently inhibited alloreactive T cells, suggesting that in vivo, EPS Bs induces DCs or other innate cells to become inhibitory and prevent the activation of alloreactive T cells, thereby reducing GvHD.
caspase-1 reporter mouse to measure inflammasome activation [7]. With this biosensor mouse model, they found that the administration of EPS Bs prevented the activation of alloreactive donor T cells, explaining the increased survival of mice. The results showed that EPS Bs did not directly affect alloreactive T cells. In mixed lymphocyte reactions (MLR) in vitro, EPS Bs -treated BMDCs potently inhibited alloreactive T cells, suggesting that in vivo, EPS Bs induces DCs or other innate cells to become inhibitory and prevent the activation of alloreactive T cells, thereby reducing GvHD.

Mechanism by Which EPS Bs Inhibits Inflammation
The mechanisms by which EPS from different bacteria ameliorate disease are likely to be highly variable, depending on the structure of the EPS. As indicated above, EPS Bs affects innate cells, such as macrophages and DCs, converting them into anti-inflammatory cells. EPS Bs does not directly affect T cells, either CD4 + or CD8 + , even though inflammation in many diseases is caused by both pathogenic T cells. Instead, the effect of EPS Bs on T cells occurs primarily through EPS Bs -induced anti-inflammatory innate cells. Molecules, thus far, known to be associated with these processes include TLR4, TGF-β, PD-L1, and indoleamine 2,3 dioxygenase (IDO), as discussed below and are shown in Figure 3. Adapted from [7].

Mechanism by Which EPS Bs Inhibits Inflammation
The mechanisms by which EPS from different bacteria ameliorate disease are likely to be highly variable, depending on the structure of the EPS. As indicated above, EPS Bs affects innate cells, such as macrophages and DCs, converting them into anti-inflammatory cells. EPS Bs does not directly affect T cells, either CD4 + or CD8 + , even though inflammation in many diseases is caused by both pathogenic T cells. Instead, the effect of EPS Bs on T cells occurs primarily through EPS Bs -induced anti-inflammatory innate cells. Molecules, thus far, known to be associated with these processes include TLR4, TGF-β, PD-L1, and indoleamine 2,3 dioxygenase (IDO), as discussed below and are shown in Figure 3. In all of the model systems tested, EPS Bs inhibits the activation of T cells, but it does not directly affect them [7,10]. Instead, EPS Bs induces anti-inflammatory M2-like macrophages and anti-inflammatory DCs, both of which have the capacity to inhibit T cell pro-

Cells
In all of the model systems tested, EPS Bs inhibits the activation of T cells, but it does not directly affect them [7,10]. Instead, EPS Bs induces anti-inflammatory M2-like macrophages and anti-inflammatory DCs, both of which have the capacity to inhibit T cell proliferation. Intraperitoneal injection of EPS Bs leads to the induction of peritoneal M2-like macrophages that inhibit proliferation of activated T cells in the C. rodentium colitis model [10,12], as well as hybrid M1-M2-like macrophages in mice infected with S. aureus [11]. These M1-M2-like macrophages not only inhibit T cell activation by S. aureus superantigen but also upregulate ROS and have the capacity to growth arrest S. aureus. For DCs, in vitro stimulation of BMDCs with EPS Bs results in anti-inflammatory cells that inhibit alloreactive T cells in MLR and presumably in GvHD [7]. Other cells affected by EPS Bs include NK cells, although the mechanism by which this occurs remains a mystery [13].

Immune Regulator Molecules
1. TLR4. The anti-inflammatory effect of EPS Bs requires TLR4, as shown in the disease models for colitis [12], bacterial sepsis [11], and GvHD [7]. The requirement for TLR4 resides in the macrophages and DCs [7,10,12], but TLR4 on other cell types, e.g., epithelial cells, mesenchymal, and NK cells, may also be required for, or contribute to, the protection by EPS Bs . Experiments with cell-specific knockout mice will establish the identity of TLR4 + cells required for the anti-inflammatory activity of EPS Bs . The finding that EPS Bs induces an anti-inflammatory effect through TLR4 is, of course surprising, because TLR4 signaling is associated with an inflammatory response due to LPS signaling. Although LPS is generally associated with pro-inflammatory responses, LPS can also induce tolerance, known as low dose tolerance or endotoxin tolerance. In this case, prolonged administration of LPS [74], or a single low dose of LPS, can dampen inflammation and induce tolerance. The mechanism by which this occurs remains under investigation [75]. Other TLR4-mediated anti-inflammatory responses have also been described and in these cases, the immunoregulatory effect is often mediated by other receptors. For example, the toll-like receptor family protein RP105/MD1 complex is involved in the immunoregulatory effect of EPS Lp from Lactobacillus plantarum N14, which inhibits inflammatory TLR4 signaling [51]. Furthermore, Horvatinovich et al. showed that soluble CD83 inhibits T cell activation by binding to the TLR4/MD-2 complex on CD14 + monocytes and altering the signaling cascade to induce the production of anti-inflammatory molecules IDO and IL-10 [76]. Gringhuis et al. showed that the lectin DC-SIGN modulates TLR signaling via Raf-1 kinase-dependent acetylation of the transcription factor NF-κB, which increased the anti-inflammatory response by increasing the transcription of IL-10 [77]. Furthermore, Yao et al. showed that leukadherin-1-mediated activation of CD11b inhibits LPS-induced pro-inflammatory responses in macrophages and protects mice from endotoxic shock by blocking LPS-TLR4 interaction [78]. Lastly, Li et al. showed that galectin-3 is a negative regulator of LPS-mediated inflammation [79]. We hypothesize that EPS Bs also engages a receptor that negatively regulates TLR4-mediated signaling.
2. TGF-β and PD-L1. Paynich et al. showed that EPS Bs -induced peritoneal M2 macrophages inhibit proliferation and activation of both CD4 + and CD8 + T cells activated with anti-CD3 and anti-CD28 antibodies, in vitro [10]. M2 macrophages are known to mediate an anti-inflammatory response by several molecules, including TGF-β, Arg-1, IL-10, PD-L1, and PD-L2. EPS Bs -induced peritoneal M2 macrophages inhibited T cells in a contact-dependent manner, and this inhibition was dependent on TGF-β in the case of CD4 + T cells and on TGF-β and PD-L1 in the case of CD8 + T cells [10]. EPS Bs -induced peritoneal M2 macrophages also upregulated Arg-1 and secretion of IL-10, and although these molecules were not required for the inhibition of T cell proliferation and activation in vitro, we predict that they are involved in the EPS Bs -induced anti-inflammatory response in vivo.
3. IDO. Kalinina et al. showed that EPS Bs induces inhibitory BMDCs that completely inhibited alloreactive T cell proliferation in an MLR [7]. This inhibition was neither depen-dent on TGF-β or PD-L1, nor on other inhibitory molecules, such as Arg-1, IL-10, CTLA4, and PD-L2. Instead, the EPS Bs -induced BMDCs inhibited alloreactive T cells in the MLR through the inhibitory molecule, IDO [7]. EPS Bs -treated ido1 −/− BMDCs do not inhibit T cell proliferation, and the addition of the IDO inhibitor 1-methyl-L-tryptophan to the MLR cultures with EPS Bs -treated WT BMDCs restored T cell proliferation. IDO inhibition of T cell proliferation is due to degradation of the essential amino acid, tryptophan, and the production of tryptophan metabolites, kynurenines [80].

Translational Potential of EPS Bs
B. subtilis has anti-inflammatory properties and is protective in several T cell-mediated diseases [7][8][9][10][11][12][13]. One molecule of B. subtilis that confers the anti-inflammatory responses is EPS Bs , secreted as a part of a biofilm. Using B. subtilis mutants that overexpress and secrete EPS, large quantities of EPS Bs can be easily purified for prophylactic or therapeutic administration. Injection of EPS Bs confers protection similar to that shown by oral administration of B. subtilis spores [9], and currently, in collaboration with colleagues in Food Science at the University of Massachusetts, we plan to formulate capsules to optimize oral administration of EPS Bs . Using an active molecule instead of live bacteria has many advantages: it is not dangerous for immunocompromised patients, it allows for more accurate dosage, its effects are temporary, and it will not alter the intestinal microbiome. Although there is no evidence that EPS Bs is toxic to mice, clinical studies will be needed to confirm that it is also not toxic for humans.
Thus far, EPS Bs has been used primarily as a prophylactic [7][8][9][10][11][12][13]. However, in studies with C. rodentium-induced colitis, EPS Bs injected 3 days post infection also inhibited disease [10], indicating that EPS Bs has potential as a therapeutic, especially for patients suffering from acute diarrhea, currently treated with fluid and electrolyte replacement.
Another potential use of EPS Bs is for inflammatory bowel disease (IBD), where, in remission, most patients relapse and require treatment [81]. EPS Bs could be used in combination with other anti-inflammatory agents not only to achieve remission but also could be used continuously during remission to prevent future relapses. Similarly, seasonal allergies and asthma induce inflammation after contact with allergens or molecular triggers. Because intranasal administration of EPS Bs reduces allergic eosinophilia in a murine model [8], it could be used as a preventative medication to prevent allergic and asthma attacks in a prophylactic manner during allergy seasons or when exposure to allergens is unavoidable. This idea is in line with Strachan's hygiene hypothesis [70], which states that as human settings become free from bacteria, the incidence of allergies rises. By dosing individuals who suffer from allergies with EPS Bs , the allergic inflammation flares may be reduced.
EPS Bs also shows exciting promise to prevent GvHD where alloreactive T cells become activated and can initiate severe inflammation. In an in vitro MLR, human cells from different MHC types are cultured together, and alloreactive T cells become activated, as in GvHD. If BMDCs treated with EPS Bs are added to the MLR cultures, T cell activation is inhibited, whereas with untreated BMDCs no inhibition is observed [7]. It may well be that the administration of EPS to the donor and/or recipient of the graft may limit inflammation and the resultant GvHD. Another possibility is to treat BMDCs in vitro with EPS Bs , and then administer them along with the graft. This may be especially helpful for bone marrow transplants in patients with a hematologic malignancy, many of which require bone marrow transplants. Other disease candidates to test the clinical efficacy of EPS Bs are chronic diseases, including atopic dermatitis, psoriasis, chronic sinusitis, and eosinophilic esophagitis. These are inflammatory disorders that cycle from remissions to flares or remain at low continuous inflammation. EPS Bs can be easily administered for symptom relief.

Concluding Remarks
Since Strachan's [70] hygiene hypothesis, numerous studies have demonstrated the potential of bacteria, especially commensal bacteria to provide health benefits (reviewed in [3]). Such bacteria are now referred to as probiotics and are readily ingested by millions of people in over-the-shelf supplements. As discussed above, most studies with probiotics utilize intact bacteria instead of identifying bioactive bacterial molecules and determining how they can provide health benefits. The advantage to identifying bioactive molecules, such as cell envelope and secreted molecules, is that they can be administered locally, systemically, and by intranasal or oral administration without the potential of developing sepsis, as might occur after the administration of live bacteria.
B. subtilis is one of the probiotics for which a bioactive molecule has been identified. Structural analysis for this and other probiotic molecules will provide the opportunity to synthesize molecules and chemically optimize them for biologic longevity, oral administration, and minimal toxicity. It is quite possible that other compounds, either secreted or derived from bacterial cell envelopes that modulate immune responses are yet to be discovered. We are optimistic that probiotic molecules will provide a new source of bioactive molecules that can prevent or treat numerous inflammatory diseases.

Conflicts of Interest:
The authors have no conflict of interest.