Co-administration Of CpG Oligodeoxynucleotides Research Articles

Conjugation of human serum albumin and flucloxacillin provokes specific immune response in HLA-B*57:01 transgenic mice

Flucloxacillin (FLX) induces adverse liver reactions, which has been reported to be related to human leukocyte antigen (HLA)-B*57:01. In a previous study, abacavir-induced hypersensitivity was induced in HLA-B*57:01-transgenic mice (B*57:01-Tg), originally constructed by our group (Susukida et al., 2021). In this study, B*57:01-Tg mice were used to reproduce FLX-induced liver injury. However, treatment of B*57:01-Tg mice with FLX alone did not increase serum ALT levels. Immune-deficient B*57:01-Tg/PD-1-/-mice were produced by mating B*57:01-Tg with PD-1-/- mice. The immune response of B*57:01-Tg/PD-1-/- mice was further modulated by co-administration of CpG-oligodeoxynucleotides and anti-CD4 mAb. Nevertheless, immune regulation in B*57:01-Tg mice did not contribute to the onset of FLX-induced liver injury or immune activation. Moreover, we generated an FLX-human serum albumin (HSA) conjugate and showed that FLX covalently bound to HSA in a time-dependent manner. The FLX-HSA conjugate was administered to the B*57:01-Tg mice. The immune response was investigated using flow cytometry, revealing the phenotype of CD44highCD62Llow in CD8+ T cells (TEM cells). Administration of the FLX-HSA conjugate resulted in an HLA-B*57:01 restricted immune response as shown by the stimulation of TEM cells in the draining lymph nodes. In conclusion, administration of FLX alone to B*57:01-Tg mice did not induce liver injury or immune activation. Immune system sensitivity does not play a decisive role in this process. The conjugation of FLX and HSA results in specific TEM cell stimulation, which suggests that HLA-B*57:01 drives a stronger interaction with CD8+ T cells. These results suggest that patients carrying HLA-B*57:01 could be more susceptible to a conjugate of FLX and albumin and drive CD8+ T cell activation, which may be a vital risk factor for FLX-induced liver injury. In addition, the application of the FLX-HSA adduct may be an effective method for the construction of FLX-induced idiosyncratic liver injury in mice.

Role of Kupffer cells in liver injury induced by CpG oligodeoxynucleotide and flucloxacillin in mice.

CpG oligodeoxynucleotide (CpG-ODN) is a Toll-like receptor 9 (TLR9) agonist that can induce innate immune responses. In a previous study, flucloxacillin (FLUX; 100 mg/kg, gavage)-induced liver injury in mice was enhanced by co-administration of CpG-ODN (40 μg/mouse, intraperitoneally). In this study, the mechanism of CpG-ODN sensitization to FLUX-induced liver injury was further investigated in mice inhibited of Kupffer cells (KCs) function by gadolinium chloride (GdCl3; 10 mg/kg, intravenously). GdCl3-treated mice administrated with CpG-ODN and FLUX showed lower liver injury than wild-type (WT) mice treated with CpG-ODN and FLUX. Upregulation of Fas and FasL by CpG-ODN was also inhibited in GdCl3-treated mice and mitochondrial swelling in response to FLUX failed to occur regardless of pre-treatment with CpG-ODN. When FasL-mutant gld/gld mice were treated with CpG-ODN, mitochondrial swelling in response to FLUX was also inhibited. These results suggest that KCs play an essential role in liver injury induced by CpG-ODN and FLUX. CpG-ODN may activate KCs, resulting in induction of Fas/FasL-mediated apoptosis of hepatocytes. The Fas/FasL pathway may also be an upstream regulator of CpG-ODN- and FLUX-induced changes in mitochondrial permeability transition. These results enhance our understanding of the mechanism of the adjuvant effect of CpG-ODN in this mouse model of liver injury.

CpG-ODNs and Budesonide Act Synergistically to Improve Allergic Responses in Combined Allergic Rhinitis and Asthma Syndrome Induced by Chronic Exposure to Ovalbumin by Modulating the TSLP-DC-OX40L Axis.

The experimental model of combined allergic rhinitis and asthma syndrome (CARAS) has shown that CpG oligodeoxynucleotides (CpG-ODNs) are potential inhibitors of type 2 helper cell-driven inflammatory responses. Currently available CpG-ODNs modestly inhibit allergic responses in CARAS, while a combination strategy for upper airway treatment by co-administration of CpG-ODNs and glucocorticoids may show good efficacy. This study aimed to assess the therapeutic effects of CpG-ODNs combined with budesonide (BUD) on upper and lower-airway inflammation and remodeling in mice with CARAS induced by chronic exposure to ovalbumin (OVA), exploring the possible underlying molecular mechanisms. A BALB/c mouse model of chronic CARAS was established by systemic sensitization and repeated challenge with OVA. Treatment with CpG-ODNs or BUD by intranasal administration was started 1h after OVA challenge. Then, nasal mucosa and lung tissues were fixed and stained for pathologic analysis. The resulting immunologic variables and TSLP-DC-OX40L axis parameters were evaluated. Both CpG-ODNs and BUD intranasal administration are effective on reducing Th2-type airway inflammation and tissue remodeling. Co-administration of CpG-ODNs and BUD was more effective than each monotherapy in attenuating upper and lower-airway inflammation as well as airway remodeling in chronic CARAS. Notably, combination of CpG-ODNs with BUD modulated the TSLP-DC-OX40L axis, as demonstrated by decreased TSLP production in the nose and lung, alongside decreased TSLPR and OX40L in DC. Intranasal co-administration of CpG-ODNs and BUD synergistically alleviates airway inflammation and tissue remodeling in experimental chronic CARAS, through shared cellular pathways, as a potent antagonist of the TSLP-DC-OX40L axis.

Effect of CpG oligodeoxynucleotide on the immune response of 28 kDa outer membrane protein of Brucella

In order to develop an effective new generation vaccine against brucellosis, it is important to identify and evaluate the immune response of protein antigens of Brucella. It has been demonstrated that 28 kDa outer membrane protein (OMP28) of Brucella induced Th2 type of immune response with predominant IgG1 and suppressed IL-2 production. However, coadministration of CpG oligodeoxynucleotide caused a shift in the immune response of antigen towards Th1 type with predominant IgG2a and upregulated IL-2 production. This suggested that the OMP28 of Brucella may act as a B-cell antigen and may induce Th1 type of immune response on addition of suitable adjuvant.

CpG Used as an Adjuvant for an Adenovirus-Based Venezuelan Equine Encephalitis Virus Vaccine Increases the Immune Response to the Vector, But Not to the Transgene Product

An adenovirus-based (ad-based) vaccine delivering antigens from the Alphavirus Venezuelan equine encephalitis virus (VEEV) is a strategy that offers clinical potential. A vaccine against VEEV is desirable because of the re-emerging nature of this virus, and also the potential that it may be used as a biological weapon. This study was designed to investigate whether the co-administration of CpG oligodeoxynucleotides (ODNs) with an ad-based VEEV vaccine could enhance the protective efficacy of the vaccine. We report that the co-administration of CpG ODN was unable to increase VEEV-specific antibody responses in mice, and was unable to increase the protective efficacy of the vaccine against aerosol challenge with virulent VEEV. However, it was noted that antibody responses directed against the adenovirus vaccine vector were increased, which may be detrimental, particularly in the context of homologous boosting.

CpG ODN mediated prevention from ovalbumin-induced anaphylaxis in mouse through B cell pathway

Allergic inflammation is induced by type 2 T helper cell (Th2) and Th2 cytokines such as interleukin (IL)-4, IL-5 and IL-13. These cytokines induce the production of allergen-specific immunoglobulin (Ig)E by B cells, and the ensuing degranulation of mast cells via IgE cross-linking leads to most clinical manifestations of allergic diseases. We examined the ability of immunomodulatory unmethylated CpG oligodeoxynucleotides (ODN), which are potent inducers of Th1 cytokines, to prevent allergic symptoms in mice immunized and sensitized with allergen. Coadministration of CpG ODN with ovalbumin (OVA) before OVA sensitization substantially prevented mice from allergic anaphylaxis representing enhanced circulating concentrations of OVA-specific IgE and histamine, and decreased body temperature. Although CpG ODN provokes an abundance of Th1-skewing cytokines, including IL-12, interferon (IFN)-α and IFN-γ, administration of CpG ODN in IFN-γ deficient mice inhibited IgE production and prevented from OVA-induced anaphylaxis, indicating a dispensable role of IFN-γ in mediating these protective effects. In vitro analysis revealed that CpG ODN inhibited class switching from IgM to IgE and IgG1 in response to CD40 and IL-4 in B cells, and this effect did not correlate with up-regulation of IFN-α production. These results imply a B cell-intrinsic, T cell-independent mechanism by which CpG ODN directly acts on B cells and inhibits IgE and IgG1 production leading to cause prevention from allergic symptoms.

Decreased cytotoxic T cell activity generated by co-administration of PSA vaccine and CpG ODN is associated with increased tumor protection in a mouse model of prostate cancer

Immunization with an adenovirus-PSA (Ad5-PSA) vaccine alone strongly induces the expansion of CD8+ T cells with enhanced cytotoxic T lymphocyte (CTL) activity against the antigen-bearing tumor cells in vitro as well as in vivo in a mouse model of prostate cancer. However, in an attempt to enhance the anti-tumor immunity induced by the vaccine, co-administration of CpG oligodeoxynucleotides (CpG ODN) with Ad5-PSA vaccine dramatically reduces the immune responses measured by in vitro CTL activity and the number of IFN-γ producing cells. Surprisingly, in vivo experiments showed that mice immunized with the combined approach of Ad5-PSA and CpG had enhanced protection against the subsequent tumor challenge as compared to mice immunized with vaccine alone. These data demonstrate an unexpected dichotomous relationship between in vitro CTL activity and in vivo tumor protection suggesting that an alternative mechanism of tumor destruction was invoked after co-administration of the CpG ODN with the vaccine.

Changes in immune responses to antigen applied to tape-stripped skin with CpG-oligodeoxynucleotide in mice

CpG-oligodeoxynucleotide (CpG-ODN) plays a critical role in immunity via the augmentation of Th1 and suppression of Th2 responses. We examined here the effect of CpG-ODN on the immune response to an antigen applied to tape-stripped mouse skin by evaluating the production of cytokines and Ig isotypes. Confocal laser scanning microscopy revealed that the model antigen, OVA, and CpG-ODN easily penetrated the tape-stripped skin. Co-administration of CpG-ODN and OVA to the disrupted skin elicited an antigen-specific Th1-predominant immune response and enhanced the production of Th1-type cytokines, IL-12 and IFN-γ. On the other hand, the production of a Th2-type cytokine, IL-4, was drastically suppressed. Cytokine production was supported by the expression of mRNA in the draining lymph node. In terms of antigen-specific antibody production, the level of IgG2a which is regulated by IFN-γ was increased by CpG-ODN, but IgE production regulated by IL-4 was suppressed. Furthermore, administration of CpG-ODN via the skin drastically attenuated the production of IgE in mice undergoing IgE-type immune response. Administration of CpG-ODN through the skin may shift the immune response from Th2 to Th1-like response. These results suggested that administration of CpG-ODN via skin is a simple strategy for patients with diseases like AD, which is characterized by Th2-dominated inflammation.

Changes in Immune Responses to Antigen Applied to Tape-Stripped Skin with CpG-Oligodeoxynucleotide in NC/Nga Mice

CpG-oligodeoxynucleotide (CpG-ODN) plays a critical role in immunity via the augmentation of Th1 and the suppression of Th2 responses. We examined here the effect of CpG-ODN on the immune response to an antigen applied to a tape-stripped skin of NC/Nga mouse, a human atopic dermatitis (AD) model, by evaluating the production of cytokines and immunoglobulin isotypes. Model antigen, ovalbumin (OVA), and CpG-ODN were applied on to the shaved skin. The penetration of OVA and CpG-ODN was evaluated using confocal laser scanning microscopy (CLSM). Secretion of cytokine from splenocytes and changes in immunoglobulin isotype levels were determined by enzyme-linked immunosorbent assay (ELISA). Through CLSM it was revealed that the model antigen, OVA, and CpG-ODN easily penetrated the tape-stripped skin. Coadministration of CpG-ODN and OVA to the skin elicited an antigen-specific, Th1-predominant immune response and enhanced the production of IFN-gamma. On the other hand, the production of a Th2-type cytokine, IL-4, was drastically suppressed. In terms of antigen-specific antibody production, the level of IgG2a regulated by IFN-gamma was increased by CpG-ODN, but IgE production regulated by IL-4 was suppressed. Administration of CpG-ODN with antigen through the skin may shift the immune response from a Th2- to Th1-like response. These results suggested that administration of CpG-ODN via skin is a simple strategy for patients with diseases such as AD, which is characterized by Th2-dominated inflammation.

Toll-like receptor 4 signaling by intestinal microbes influences susceptibility to food allergy.

The mechanisms by which signaling by the innate immune system controls susceptibility to allergy are poorly understood. In this report, we show that intragastric administration of a food allergen with a mucosal adjuvant induces allergen-specific IgE, elevated plasma histamine levels, and anaphylactic symptoms in three different strains of mice lacking a functional receptor for bacterial LPS (Toll-like receptor 4 (TLR4)), but not in MHC-matched or congenic controls. Susceptibility to allergy correlates with a Th2-biased cytokine response in both the mucosal (mesenteric lymph node and Peyer's patch) and systemic (spleen) tissues of TLR4-mutant or -deficient mice. TLR4-mutant mice are not inherently impaired in their ability to regulate Th1 cytokine production because they respond to stimulation via TLR9. Coadministration of CpG oligodeoxynucleotides during sensitization of TLR4-mutant mice with allergen plus CT abrogates anaphylactic symptoms and Ag-specific IgE, and results in a Th1-polarized cytokine response. When the composition of the bacterial flora is reduced and altered by antibiotic administration (beginning at 2 wk of age), TLR4 wild-type mice become as susceptible to the induction of allergy as their TLR4-mutant counterparts. Both allergen-specific IgE and Th2 cytokine responses are reduced in antibiotic-treated mice in which the flora has been allowed to repopulate. Taken together, our results suggest that TLR4-dependent signals provided by the intestinal commensal flora inhibit the development of allergic responses to food Ags.

CpG oligodeoxynucleotide vaccination suppresses IgE induction but may fail to down-regulate ongoing IgE responses in mice.

Antigen-specific IgE plays an important role in the pathogenesis of allergic disorders. Immunostimulatory CpG motifs (CpG) in bacterial DNA or synthesized oligodeoxynucleotides (ODN) are gaining recognition as potential immunomodulators for switching on protectiveT(h)1-mediated immunity and preventing or potentially inhibiting T(h)2-dependent allergic responses. To date, allergic models used in CpG ODN studies have been established by immunization of mice with allergen in the presence of adjuvant. This, in addition to failure to assess specific IgE production in most of the studies, has limited understanding of the role of CpG ODN vaccination in allergic responses. Here, we examine the effects of synthesized CpG ODN on both developing and ongoing IgE responses in mice sensitized using a recombinant mosquito salivary antigen (rAed a 2) without adjuvant. Pretreatment of mice with CpG ODN mixed with rAed a 2 successfully inhibited subsequent induction of serum rAed a 2-specific IgE (but not IgG1) and antigen-induced IL-4 and IL-5 production in spleen cells. This was associated with an increase of serum IgG2a and IL-12, and increased IFN-gamma and IL-12 production by spleen cells. In this model, however, co-administration of CpG ODN with rAed a 2 to presensitized mice failed to down-regulate ongoing IgE responses despite significant up-regulation of serum IL-12 and specific IgG2a. Strikingly, a transient skin delayed-type hypersensitivity reaction occurred in CpG ODN-treated mice. These observations provide a new insight into the potential therapeutic application of CpG ODN to allergic disorders.