Probiotics and Toll-like Receptors

The innate immune response is the first line of defense against infectious diseases. The main challenge for the host is to detect the pathogen and create a rapid defensive response (78). Sensing of bacterial products by the innate immune system is mediated by Toll-like receptors (TLRs). TLR2, TLR4, and TLR9, highly conserved through evolution and expressed on both enterocytes and immune cells, recognize specific microbial components, such as surface determinants and DNA sequences, and induce the production of Th1 cytokines through a ·process dependent on NFKB (54), which is activated as a response to pathogen-associated molecular patterns such as lipopolysaccharide (LPS). TLR4 is required for the recognition of LPS (79).

Da Silva et al. (80) described that LPS initiates its biological activities through a heteromeric receptor complex containing CD14, TLR4, and at least one other protein, MD-2. LPS binds directly to CD14 but whether LPS then binds to TLR4 and/or MD-2 is unknown. The researchers used transient transfection to express human TLRs, MD-2, or CD14 alone or in different combinations in HEK 293 cells. Interactions between LPS and these proteins were studied using a chemically modified, radio iodinated LPS containing a covalently linked, UV light-activated cross­linking group.

They showed that LPS is cross-linked specifically to TLR4 and MD-2 only when co-expressed with CD14. These data support that LPS is in close proximity to the three known proteins of its membrane receptor complex. Thus, LPS binds directly to each of the members of the tripartite LPS receptor complex (80).

Jiang et al. (81) found that CD14, a GPI-linked protein, plays a pivotal role in LPS-mediated signaling by potentiating leukocyte adherence, activation, and cytokine production. Re­cent studies have identified TLR4 as a membrane cofactor in LPS-mediated transmembrane signal­ing in cytokine induction, although the mechanism responsible for this cooperation is unknown. It was demonstrated that LPS triggers a physical associa­tion between CD14 and TLR4. Since LPS stimula­tion up-regulates CD14 and TLR4 expression, it was necessary to consider the possibility that these newly expressed molecules were associated with another independent of LPSstimulation. Although calcium ionophore A23187 increased the expression of CD14 and TLR4, there was no energy transfer.

However, following A23187 treatment, LPS pro­moted physical proximity between CD14 and TLR4. Therefore, they suggested that a close interaction between CD14 and TLR4 participates in LPS signaling, leading to nuclear translocation of NFKB (81).

Abreu et al. (82) have described that intestinal epithelial cells express low levels of TLR4 and are unresponsive to LPS. The luminal surface of the colonic epithelium is continually exposed to Gram­negative commensal bacteria and LPS. The recognition of LPS by TLR4 results in proinflammatory gene expression in several cell types. Normally, commensal bacteria and their components do not elicit an inflammatory response from intestinal epithelial cells.

In their study they explained the molecular mechanisms by which intestinal epithelial cells limit chronic activation in the presence of LPS. Three intestinal epithelial cell lines (Caco-2, T84, HT-29) were tested for their ability to activate an NFKB reporter gene in response to purified, protein-free LPS. No intestinal epithelial cell lines responded to LPS, whereas human dermal micro­vessel endothelial cells did respond to LPS.

Intestinal epithelial cells responded vigorously to IL -1 ~ demonstrating that the IL-1 receptor signaling pathway shared by TLRs was intact. Therefore, probiotics may interact with these sensors and contribute to their immunomodulation. Lactobacillus GG has been shown to induce such a NFKB-mediated interaction. Although this effect alone might suggest a direct link with decreased Th2-mediated allergy, it has become clear that the Th1/Th2 paradigm is inadequate to explain mucos~lI immune re­sponses.

Specific immunological unresponsiveness to dietary components (oral tolerance) and to commensal bacteria is critically dependent on inhibition of potential lymphocyte reactivity, and two recently recognized suppressor-cell populations are central in this process. In murine models Th3 and T-regulator 1 (Tr1) cells, that produce TGF and IL-10, respectively, down-regulate mucosal inflammatory responses through "bystander suppression" of near­by lymphocytes of various specificities: deficiency of either cytokine or cell type leads to mucosal inflammation as a consequence of unchecked response to the enteric flora (83).