Bacterial CpG Motifs Research Articles

Dissecting the metabolic signaling pathways by which microbial molecules drive the differentiation of regulatory B cells

The host-microbiome axis has been implicated in promoting anti-inflammatory immune responses. Yet, the underlying molecular mechanisms of commensal-mediated IL-10 production by regulatory B cells (Bregs) are not fully elucidated. Here, we demonstrate that bacterial CpG motifs trigger the signaling downstream of TLR9 promoting IκBNS-mediated expression of Blimp-1, a transcription regulator of IL-10. Surprisingly, this effect was counteracted by the NF-κB transcription factor c-Rel. A functional screen for intestinal bacterial species identified the commensal Clostridium sporogenes, secreting high amounts of short-chain fatty acids (SCFAs) and branched-chain fatty acids (BCFAs), as an amplifier of IL-10 production by promoting sustained mTOR signaling in B cells. Consequently, enhanced Breg functionality was achieved by combining CpG with the SCFA butyrate or the BCFA isovalerate thereby synergizing TLR- and mTOR-mediated pathways. Collectively, Bregs required two bacterial signals (butyrate and CpG) to elicit their full suppressive capacity and ameliorate T cell-mediated intestinal inflammation. Our study has dissected the molecular pathways induced by bacterial factors, which might contribute not only to better understanding of host-microbiome interactions, but also to exploration of new strategies for improvement of anti-inflammatory cellular therapy.

A New CpG ODN Induces a Fine-Tuning of Innate Response Resulting in Mycobacterium tuberculosis Containment

Synthetic oligodeoxynucleotides containing bacterial CpG motifs trigger an immunomodulatory response that correlates with both the CpG hhhexamers and their flanking regions. In this study, four new phosphodiester backbone CpG ODNs were studied in the contest of the innate immune response to Mycobacterium tuberculosis (MTB) infection. Two out of the four CpG ODNs (CpG2 and CpG3) displayed significant and opposite immunomodulatory effects: CpG2 enhanced MTB containment by human Monocyte-Derived-Macrophages (MDM), while CpG3 promoted an increased pathogen growth, higher ROS and Labile Iron Pool (LIP) levels. Accordingly, for iron homeostasis genes transcription, CpG2 and CpG3 induced, respectively, an iron retention and iron release phenotype. Moreover, CpG2 induced NLRC4 and TRAF6 gene expression and repressed IKK alpha and TREM1 while CpG3 induced PPBP and IL-36 RN and repressed TRAF6, IL-1B, IL-1R2 and NF-kB2. After MTB infection, CpG2 increased the release of soluble TREM1 protein among many others as compared with fewer innate response-associated proteins induced by CpG3. We suggest that CpG2 helps the containment of MTB infection, by inducing an early tight balance of MDM activation players, including LIP, chemokines, ROS and TREM1 receptors. Oppositely, CpG3 induces in MDM an excessive and unregulated inflammatory response unable to contain MTB infection.

Diminazene aceturate (Berenil), a new use for an old compound?

Diminazene aceturate or Berenil has been the drug of choice for treatment of animal trypanosomiasis. Although the compound has been in the market since 1955, its mechanisms of action have remained poorly understood. While some earlier reports show that Berenil possesses trypanolytic and trypanostatic properties, some studies show it may also indirectly affect the host immune system. Our recent extensive studies show that treatment with Berenil reduces pro-inflammatory cytokine (IL-6, IL-12 and TNF) production in macrophages in vivo and in vitro following stimulation with Trypanosoma congolense, lipopolysaccharide (LPS), unmethylated bacterial CpG motifs and Poly I:C. This global effect was not due to downregulation of Toll-like receptor (TLR) expression on innate immune cells. Instead, Berenil significantly downregulated phosphorylation of mitogen activated protein kinases (MAPKs, including ERK, p38 and JNK), signal transducer and activator of transcription (STAT) proteins (including STAT1 and STAT3) and NFκB p65 subunit, key signaling molecules and transcription factors involved in the production of proinflammatory cytokines. The ability of Berenil to downregulate major intracellular signaling pathways that lead to proinflammatory cytokine production suggests that it could be used to treat conditions caused by excessive production of inflammatory cytokines.

CpG DNA is a Potent Enhancer of Humoral and Cell-Mediated Immune Responses against Orientia tsutsugamushi in C3H/HeN Mice

Background: Synthetic oligodeoxynucleotide (ODNs) encoding CpG motifs mimic the activity of bacterial CpG motifs that trigger innate immune responses, and their use as an adjuvant has been demonstrated to significantly improve humoral immune response. Methods: CpG ODNs were evaluated for their immunostimulatory activities on mice vaccinated with recombinant outer membrane protein P56 (rP56Δ) of Orientia tsutsugamushi. Results: CpG ODNs significantly enhanced immune responses of mice against rP56Δ post three doses of immunization, with IgG titer ranging from 80,000 to more than 640,000. Furthermore, they also improved the immunization efficiency of rP56Δ by shortening the immunization schedule. Only two doses of immunization of rP56Δ plus CpG ODNs were needed to induce equally high antibody level as the three-dose immunization scheme of rP56Δ plus Freund's adjuvant. Isotypic analysis of antibodies revealed that rP56Δ plus CpG ODNs enhanced mice humoral immune responses as rP56Δ plus Freund's adjuvant did. IgG1, IgG2a, IgG2b and k light chain were induced significantly. Interestingly, rP56Δ stimulated spleen cells of vaccinated BALB/c mice to produce more IL-12 and gamma interferon than IL-4. In contrast, spleen cells stimulated with ConA, IL-4 was dominantly induced. Furthermore, CpG ODNs could cooperatively elicit memory immune responses in rP56Δ-immunized mice, as demonstrated by quick IgM induction boost shot. Conclusion: Results from this study revealed that rP56Δ could stimulate C3H/HeN mice to produce immune responses; and CpG ODN adjuvant enhanced its immunization effect and shortened the immunization time.

Newly identified CpG ODNs, M5-30 and M6-395, stimulate mouse immune cells to secrete TNF-α and enhance Th1-mediated immunity

Bacterial CpG motifs are known to induce both innate and adaptive immunity in infected hosts via toll-like receptor 9 (TLR9). Because small oligonucleotides (ODNs) mimicking bacterial CpG motifs are easily synthesized, they have found use as immunomodulatory agents in a number of disease models. We have developed a novel bioinformatics approach to identify effective CpG ODN sequences and evaluate their function as TLR9 ligands in a murine system. Among the CpG ODNs we identified, M5-30 and M6-395 showed significant ability to stimulate TNF-alpha and IFN-gamma production in a mouse macrophage cell line and mouse splenocytes, respectively. We also found that these CpG ODNs activated cells through the canonical NF-kappa B signaling pathway. Moreover, both CpG ODNs were able to induce Th1-mediated immunity in Mycobacterium tuberculosis (Mtb)-infected mice. Our results demonstrate that M5-30 and M6-395 function as TLR9-specific ligands, making them useful in the study of TLR9 functionality and signaling in mice.

Immunostimulatory Activity of Specific CpG Oligonucleotides from Bifidobacterium longum Genome on RAW 264.7 Macrophage Cells

Bacterial CpG motifs have attracted considerable interests because of their immunomodulatory activities. Since Bifidobacterium is an important probiotic bacteria with characteristically high G+C content of 60 to 67%, investigation on the immune regulatory activity of CpG oligodeoxynucleotides (ODNs) from Bifidobacterium is warranted. The ODNs containing repeated TCG motifs were designed and synthesized based upon B. longum genomic sequence. Four 21-mer ODNs (ODNBL1∼4) and three 25-mer ODNs (CpG ODN1∼2, ODNBL5) induced macrophages to secrete cytokines such as IL-6, IL-12p40 and TNF-α. In particular, ODNBL5 induced macrophages to secret high amount of IL-6 and IL-12 compared with positive control PE2080 which was reported as a strong immune activating CpG ODN. This indicated that the specific functional sequences in the genome of B. longum could activate murine macrophage RAW 264.7 possibly acting as an immunostimulatory modulator for immune response.

Comparative Proteomics Study Reveals That Bacterial CpG Motifs Induce Tumor Cell Autophagy in Vitro and in Vivo

Unmethylated CpG dinucleotides, present in bacterial DNA, are recognized in vertebrates via the Toll-like receptor 9 (TLR9) and are known to act as an anticancer agent by stimulating immune cells to induce a proinflammatory response. Although the effects of CpG-oligodeoxynucleotides (CpG-ODNs) in immune cells have been widely studied, little is known regarding their molecular effects in TLR9-positive tumor cells. To better understand the role of these bacterial motifs in cancer cells, we analyzed proteome modifications induced in TLR9-positive tumor cells in vitro and in vivo after CpG-ODN treatment in a rat colon carcinoma model. Proteomics analysis of tumor cells by two-dimensional gel electrophoresis followed by mass spectrometry identified several proteins modulated by bacterial CpG motifs. Among them, several are related to autophagy including potential autophagic substrates. In addition, we observed an increased glyceraldehyde-3-phosphate dehydrogenase expression, which has been shown to be sufficient to trigger an autophagic process. Autophagy is a self-digestion pathway whereby cytoplasmic material is sequestered by a structure termed the autophagosome for subsequent degradation and recycling. As bacteria are known to trigger autophagy, we assessed whether bacterial CpG motifs might induce autophagy in TLR9-positive tumor cells. We showed that CpG-ODN can induce autophagy in rodent and human tumor cell lines and was TLR9-dependent. In addition, an increase in the number of autophagosomes can also be observed in vivo after CpG motif intratumoral injection. Our findings bring new insights on the effect of bacterial CpG motifs in tumor cells and may be relevant for cancer treatment and more generally for gene therapy approaches in TLR9-positive tissues.

IKK-i/IKKϵ Controls Constitutive, Cancer Cell-associated NF-κB Activity via Regulation of Ser-536 p65/RelA Phosphorylation

Nuclear factor kappaB (NF-kappaB) has been studied extensively as an inducible transcriptional regulator of the immune and inflammatory response. NF-kappaB activation downstream of lipopolysaccharide or cytokine stimulation is controlled by the IkappaB kinase complex, which contains IKKalpha and IKKbeta. Significantly, the constitutive activity of NF-kappaB has been implicated as an important aspect of many cancer cells, but mechanisms associated with this activity are poorly understood. An inducible kinase, IKK-i/IKKepsilon, related to the catalytic forms of the IkappaB kinase, has been studied as an anti-viral, innate immune regulator through its ability to control the activity of the transcription factors IRF-3 and IRF-7. Here, we demonstrate that IKK-i/IKKepsilon is expressed in a number of cancer cells and is involved in regulating NF-kappaB activity through its ability to control basal/constitutive, but not cytokine-induced, p65/RelA phosphorylation at Ser-536, a modification proposed to contribute to the transactivation function of NF-kappaB. Knockdown of IKK-i/IKKepsilon or expression of a S536A mutant form of p65 suppresses HeLa cell proliferation. The data indicate a role for IKK-i/IKKepsilon in controlling proliferation of certain cancer cells through regulation of constitutive NF-kappaB activity.

Enhancement of rat tear IgA antibody responses following intranasal immunization with antigen and CpG ODN

Purpose. To determine the effect of unmethylated oligodeoxynucleotides containing bacterial CpG motifs (CpG ODN) on the induction of rat tear IgA antibody responses. Methods. Rats were immunized intranasally with either soluble dinitrophenylated bovine serum albumin (DNP-BSA) or poly(lactide-co-glycolide) (PLG) encapsulated DNP-BSA in combination with CpG ODN. The animals received two immunizations 21 days apart. Following the second immunization, tear, saliva and serum samples were collected for 28 days and analyzed for antigen specific antibodies. Tear IgA, saliva IgA and serum IgG antibody concentrations were determined by ELISA. Results. Co-administration of CpG ODN with either soluble or encapsulated antigen resulted in significantly elevated levels of both tear and salivary IgA antibodies as well as levels of serum IgG antibodies. Microencapsulated DNP-BSA plus CpG ODN elicited higher levels of IgA antibodies in tears than did soluble antigen plus CpG ODN. Conclusions. CpG ODN is an effective mucosal immune modulator for enhancing rat tear IgA antibody responses to both soluble and microencapsulated antigens.