In these studies, we examine the effects of LGG on both baseline cytokine levels and on cytokine signaling responses to intestinal injury in the developing murine intestine. In initial studies, we showed that even in the absence of exogenous inflammatory stimuli, the baseline expression of key cytokines mediating both promotion and resolution of intestinal inflammation in models of NEC-like injury are developmentally regulated during the postnatal period. Initial profiling identified TNF-α, MIP-2, and IL-12p40, and IL-10 as developmentally regulated prior to weaning in the immature intestine. While expression of proinflammatory cytokines, MIP-2, TNF-α and IL-12p40 was highest during the first two weeks of life, then declined, IL-10 expression was initially low and increased between two and three weeks of life. Therefore, 2 week-old mice had increased basal expression of proinflammatory cytokines, at a time when IL-10 expression remained low.
Enteral feeding with the probiotic LGG
was not associated with significant changes in IL-10 production. However, LGG
did upregulate the R2 subunit of the IL-10 receptor, as well as IL-10 receptor-dependent phosphorylation of STAT3, and downstream expression of SOCS3. Furthermore, LGG
-dependent changes in IL-10 receptor expression were associated with a reduction in the expression of the proinflammatory cytokines MIP-2 and TNF-α and protection from intestinal injury. These results suggest that LGG
may increase sensitivity to IL-10, and implicate IL-10 signaling as a key pathway mediating the anti-inflammatory effects of LGG
. Of interest, IL-10 is increased in models of NEC and in patients with severe or surgical NEC 
we found that IL-10 was in fact increased after intestinal injury in both LGG-
and vehicle-treated mice. Thus, increases in IL-10 cytokine production might be a response to counterbalance the pro-inflammatory response of NEC/intestinal injury. Alternatively, increased IL-10 production by itself may play a pathophysiological role by inhibiting T-cell effector function 
. However, in the PAF/LPS model of intestinal injury, the protective effects of LGG
were more specifically associated with changes in IL-10R2 expression, implying that this protective mechanism, based on receptor rather than cytokine expression has the potential to be regulated in a cell-type specific manner. Therefore, the role of the IL-10 receptor in this background may be crucial for enhancement of effective downstream anti-inflammatory responses, and in our studies LGG
-dependent induction of IL-10R2 and SOCS3 was associated with protection from intestinal injury.
Imbalances in inflammatory signaling have been implicated in the pathogenesis of NEC 
. Walker and colleagues have reported that fetal intestinal cells mount an exaggerated proinflammatory cytokine response to inflammatory stimuli and bacterial antigens 
. Additional evidence argues that decreased capacity for IL-10-dependent signaling may contribute to increased inflammatory responses in the immature intestine. Specifically, IL-10 is present in human breast milk, and Fituch et al
. have demonstrated that premature infants who developed NEC despite receiving maternal milk feedings had mothers with lower concentrations of IL-10 in their breast milk 
The findings in this study demonstrating developmental regulation of IL-10 and its receptor in the murine intestine are intriguing given the experimental evidence implicating protective roles for the IL-10 pathway both during colonization of the developing intestine and in infants with NEC. Our study is focused on the effect on the developing colon since as the site of maximal bacterial colonization, however, it should be noted that probiotic effects may differ in large and small intestine, both at baseline and under conditions of inflammation such as in NEC. The effect of LGG
may also not be limited to epithelial cells but may also involve other populations of lamina propria cells. We can speculate, however, that lower levels of IL-10 production early in intestinal development may predispose the immature intestine to increased or sustained inflammatory responses to luminal bacteria. Thus, part of the protective influence of maternal milk may reside in its provision of exogenous IL-10 
. Furthermore, probiotic induction of the IL-10 receptor may offer some protection by increasing the capacity for IL-10 receptor signaling in response to IL-10 present in maternal milk.
The IL-10 pathway appears to have the potential for regulation through two temporally distinct mechanisms in the developing intestine, initially by flora-dependent receptor induction, followed later by increased baseline capacity for IL-10 secretion. Our findings indicate the potential for probiotic-dependent induction of the IL-10 receptor promoting activation of the IL-10 signaling pathway prior to the induction of a robust cytokine response. This early receptor-based mechanism of augmenting sensitivity to IL-10 through induction of IL-10R2 may represent a cell-type specific mode of regulation of cytokine signaling in the developing intestine.
Probiotics provide protective effects in animal models of NEC 
and in initial human trials 
. However, the safety and long-term consequences of probiotic administration have yet to be established. Furthermore, probiotics have been shown in case reports to cause sepsis in immunocompromised patients including premature infants 
. Our study identifies a potential mechanism by which probiotic bacteria protect the developing intestine through increased potential for IL-10 receptor signaling in response to low levels of endogenous cytokine as well as exogenous IL-10 derived from maternal milk. While safety concerns remain, the availability of probiotic bacteria which have been genetically engineered to secrete human IL-10 
offers a candidate alternative therapy for the prevention of NEC, particularly in cases where maternal milk is not available. SOCS3-signaling still needs to be carefully evaluated in future clinical trials with probiotics as well, due to its additional role in negatively regulating fetal liver hematopoiesis by attenuating erythropoietin signaling 
. However, specific targeting of probiotic-dependent signaling pathways such as IL-10 receptor-mediated activation of SOCS3 may provide pharmacologic alternatives to the administration of live bacteria to our most vulnerable patients.