# Probiotic Molecules That Inhib Inflammatory Diseases
## 4 EPS**Bs** as Probiotic  
The EPS (EPSB) 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 EPSB as a therapeutic for humans. 1. _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 _eps_H, which regulates biofilm synthesis [68], did not protect from disease caused by _C. rodentium_, suggesting that biofilm-associated carbohydrate exopolysaccharide (EPSB) was required for protection. EPSB 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 EPSB (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 EPSB is mediated by anti-inflammatory macrophages, sometimes designated as M2 macrophages. Intraperitoneal administration of EPSB results in the accumulation of macrophages with M2 macrophage markers, IL4 Ra, 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 EPSB, and of the anti-inflammatory macrophages induced by EPSB. Figure 1: Colonic histological analysis of the effect of _B. subtilis_ spores in _C. rodentium_ disease 10 days post infection. Note goblet cells present in normal tissue (arrows), but reduced in colon infected with _C. rodentium_. Reprinted from [9]. Systemic infection with _Staphylococcus aureus_. Similar to infection with the enteric pathogen, _C. rodentium_, EPS\({}^{\text{Bs}}\) also moderates disease caused by infection with blood-borne _S. aureus_[11]. In this case, EPS\({}^{\text{Bs}}\) 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\({}^{\text{Bs}}\) 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\({}^{\text{Bs}}\) 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. * Allergic eosinophil. 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 [PERSON] 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]. [PERSON] et al. orally administered _B. subtilis_ spores to mice and showed that they prevented the development of allergic eosinophil 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].