Probiotics, Genetics and the Immune System
DNA from bacteria has stimulatory effects on mammalian immune cells, which depend on the presence of un methylated CpG dinucleotides in the bacterial DNA. CpG DNA induces a strong T-helper-like inflammatory response (84). Data suggest that CpG-DNA initiates signaling via the TLR/ IL-1 R pathway in androgen-presenting cells (APCs) similar to LPS and to T helper cell-mediated CD40 ligation. Activation of the TLR/IL-1 R signaling pathway by foreign bacterial DNA may be one way to initiate innate defense mechanisms against infectious pathogens in vivo (85).
Accumulating evidence has revealed the therapeutic potential of CpG DNA as adjuvants for vaccination strategies for cancer, allergy and infectious diseases. Hemmi et al. (84) showed in their study that cellular response to CpG DNA was mediated by a Toll-like receptor, TLR9. TLR9-deficient mice did not show any response to CpG DNA, including proliferation of splenocytes, inflammatory cytokine production from macrophages and maturation of dendritic cells. These experimental models showed resistance to the lethal effect of CpG DNA without any elevation of serum proinflammatory cytokine levels. The in vivo CpG-DNA-mediated T-helper type-1 response was also abolished in TLR9-deficient mice. Thus, vertebrate immune systems appear to have evolved a specific Toll-like receptor that distinguishes bacterial DNA from self-DNA.
In this regard, Chu et al. (86) have also described the synthetic oligodeoxynucleotides (ODNs) that contain unmethylated CpG motifs (CpG-ODN) that induce macrophages to secrete IL-12, which in turn induce interferon IFN-y secretion by NK cells. These cytokines may induce Th1 differentiation.
Chu et al. (86) examined the effects of coadministered CpG ODN on the differentiation of Th responses to hen egg lysozyme (HEL). In both BALB/c (Th2-biased) and B10.D2 (Th1-biased) mice, immunization with HEL in incomplete Freund's adjuvant (IFA) resulted in Th2-dominated immune responses characterized by HEL-specific secretion of IL-5 but not IFN-y. In contrast, immunization with IFA-HEL plus CpG-ODN switched the immune response to a Th 1-dominated cytokine pattern, with high levels of HEL-specific IFN-y secretion and decreased HEL-specific IL-5 production. IFA-HEL plus CpG ODN also induced antiHEL IgG2a (a Th1-associated isotype), not induced by IFA-HEL alone. Control non-CpG ODN did not induce IFN-y or IgG2a, excepting lesser increases in B10.D2 (Th1-biased) mice. Thus, CpGODN provide a signal to switch on Th 1-dominated responses to coadministered antigen and are potential adjuvants for human vaccines to cause a protective Th1 immune response. Roman et al. (87) also observed that the Th1 responses are potently stimulated in gene-vaccinated animals by non coding immunostimulatory DNA sequences (ISS) or ISS oligodeoxynucleotides. The mechanisms of action of ISS-DNAs, all of which foster Th1 responses and enhance cell-mediated immunity are listed in Table III (87).
According to Martin-Orozco et al. (88) bacterial genomic DNA, plasmid DNA and CpG containing ISS stimulate a Th1 response via the release of type-1 cytokines from macrophages, dendritic cells, NK cells and B-cells. They showed that ISS-enriched DNA up-regulates a distinct profile of cell surface molecules on macrophages and B-cells in vitro and in vivo. ISS-ODN and ISS-containing pDNA enhanced the expression of antigen presentation molecules (major histocompatibility complex [MHC] class I and II), co-stimulatory molecules (B7-1, B72 and CD40), cytokine receptors (IFN-y receptor and IL-2 receptor), an adhesion molecule (ICAM-1) and an (Fc-y receptor) on murine B-cells or bone marrow-derived macrophages. The increased expression of these surface molecules was found in purified cell populations and was largely independent of the effects of type-1 cytokines. Splenic antigen-presenting cells stimulated with ISS-ODN in vivo efficiently activated naive T-cells and bias their differentiation toward a Th1 phenotype in vitro.
Table III: The mechanisms of action of ISS-DNAs (63).
Thus, the induction of both type-1 cytokines and a distinct profile of cell surface molecules contribute to the powerful immunostimulatory effects of ISS-containing DNA on innate and adaptive immunity. These strong immunostimulatory effects explain mucosal immunity (89). Wagner et al. (90) also described the immunostimulatory effects as enhances of the host's defence against invading pathogens.
Rachmilewitz et al. (91 ) have hypothesized a role for ISS-DNA in experimental colitis. They described that the cell survival-promoting effects of ISS-DNA and its immunostimulatory properties on innate immunity could inhibit damage to the colonic mucosa and limit exposure of the involved mucosal tissue to commensal bacteria and their inflammatory products. These combined effects might thereby result in the inhibition of intestinal inflammation. Based on the observations described above they thought that the beneficial effects of probiotics might be derived from its DNA and, therefore, they evaluated the effect of DNA-free methylated and unmethylated genomic DNA (gDNA) isolated from the probiotic VSL#3 in experimental models of colitis.
In this study groups of Balb/C mice were treated s.c. with ISS oligonuceotides (ODN, 10 I-1g) or with one of the genomic DNA preparations (50 I-1g) 2 hours prior to administration of 2.5% dextran sulfate sodium (DSS) to the drinking water, or to rectal installation of 0.5 mg trinitrobenzene sulfonic acid (TNBS) dissolved in 0.1 ml 50% ethanol. Mice were weighed and inspected for rectal bleeding and diarrhea and sacrificed after 1 week. The disease activity index (DAI) is the combined score of weight loss and rectal bleeding.
The colon was weighed, sections obtained for histology and mucosal myeloperoxidase (MPO) activity was determined. The results of this study showed that the amelioration of DSS- and TNBS-induced colitis by VSL-gDNA reflected by DAI, MPO activity and the histological score was similar to that induced by ISS-ODN. VSL-M-gDNA, VSL-gDNA and control calf thymus gDNA did not affect the severity of DSSor TNBS-induced colitis. Therefore, it was concluded that VSL#3 attenuation of experimental colitis was caused by the immunostimulatory and antiinflammatory properties of its genomic DNA sequences.
The recent identification of NOD2/CARD15 as a susceptibility gene for Crohn's disease has provided another link between the immune response to enteric bacteria and the development of mucosal inflammation. The leucin-rich-repeat domain of NOD2/CARD15 has binding activity for bacterial peptidoglycans and its deletion stimulates the NFKB pathway. The most frequent variants of NOD2/CARD15 observed in Crohn's disease tend to cluster in the leucin-richrepeat domain and its adjacent regions. This suggests that the leucin-rich-repeat domain of CD-associated variants is likely to be impaired in its recognition of microbial components. Continuing studies are investigating the pathophysiological mechanisms induced by NOD2/CARD15 variants in the intestinal mucosa (68) (Figs. 4 and 5).