OK-432 Acts as Adjuvant to Modulate T Helper 2 Inflammatory Responses in a Murine Model of Asthma

Abstract

Enhanced type 2 helper T (Th2) cell responses to inhaled harmless allergens are strongly associated with the development of allergic diseases. Antigen formulated with an appropriate adjuvant can elicit suitable systemic immunity to protect individuals from disease. Although much has been learned about Th1-favored immunomodulation of OK-432, a streptococcal preparation with antineoplastic activity, little is known about its adjuvant effect for allergic diseases. Herein, we demonstrate that OK-432 acts as an adjuvant to favor a systemic Th1 polarization with an elevation in interferon- (IFN-) γ and ovalbumin- (OVA-) immunoglobulin (Ig) G2a. Prior vaccination with OK-432 formulated against OVA attenuated lung eosinophilic inflammation and Th2 cytokine responses that were caused by challenging with OVA through the airway. This vaccination with OK-432 augmented the ratios of IFN-γ/interleukin- (IL-) 4 cytokine and IgG2a/IgG1 antibody compared to the formulation with Th2 adjuvant aluminum hydroxide (Alum) or antigen only. The results obtained in this study lead us to propose a potential novel adjuvant for clinical use such as prophylactic vaccination for pathogens and immunotherapy in atopic diseases.

Figure 1

OK-432 is a strong Th1 adjuvant. (a) Schematic diagram depicting protocols for immunization protocol. The immune responses of B6 mice immunized with different formulations were determined. The levels of IFN-γ (b) and IL-4 (c) in culture supernatants, in which spleen cells were stimulated by 100 μg/ml OVA for 6 days, and the titers of OVA-specific IgG2a (e) and IgG1 (f) in sera were measured by ELISA. The ratios of Th1/Th2 cytokine (d) and immunoglobulin (g) are shown. Data are represented as the mean ± SEM from two independent experiments (n = 3–6 per group). ^∗p < 0.05; ^∗∗p < 0.01.

Figure 2

Pulmonary inflammatory responses induced by OVA challenge were diminished in mice preimmunized with OK-432 adjuvant. (a) Schematic diagram depicting protocols for immunization and challenge protocol. (b) Pulmonary function test was determined 24 hours after the last challenge. Mice were stimulated by increasing concentrations of methacholine (0, 10, 30, and 100 mg/ml), and airway resistances of different groups are shown. Cells in BAL fluid were stained by Liu's stain, and the number and percentage of leukocyte were calculated. Total cell number (c) and absolute number (d) and percentage of eosinophils (e) in BAL are shown. Data are represented as the mean ± SEM (n = 8–12 per group) from three independent experiments. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.

Figure 3

Histopathology shows less lung eosinophilia in mice preimmunized with OVA/OK-432 formula. (a) Formaldehyde-fixed, paraffin-embedded lung tissues from each group were sliced and stained with hematoxylin and eosin. One representative section at 200X is shown for each experimental group. Data are representatives of three independent experiments (n = 8–12). (b) Quantitative results of eosinophil infiltration (defined as inflammatory index) were analyzed by MetaMorph and expressed as percentage (mean ± SEM), compared to the OVA control mice. The mRNA levels of CCL11 (c), IL-4 (d), IL-5 (e), IL-13 (f), and IFN-γ (g) in lung tissues were determined by real-time PCR. Data are represented as the mean ± SEM (n = 8–12 per group) from three independent experiments. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.

Figure 4

OVA/OK-432 formula induces more Th1 responses but controls Th2 reactions. The levels of IFN-γ (a), IL-4 (b), and IL-5 (g) in culture supernatants, in which spleen cells were restimulated by 100 μg/ml OVA for 6 days in vitro, are shown. (c) IFN-γ/IL-4 ratios were calculated and depicted. The levels of OVA-IgG2a (d) and OVA-IgG1 (e) in sera were determined by ELISA. (f) OVA-IgG2a/OVA-IgG1 ratio from indicated mice was calculated and depicted. The titers of OVA-IgE (h) and total IgE (i) in sera from each group are shown. Data are represented as the mean ± SEM (n = 8–12 per group) from three independent experiments. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.