Oral clarithromycin enhances airway immunoglobulin A (IgA) immunity through induction of IgA class switching recombination and B-cell-activating factor of the tumor necrosis factor family molecule on mucosal dendritic cells in mice infected with influenza A virus

Abstract

We previously reported that the macrolide antibiotic clarithromycin (CAM) enhanced the mucosal immune response in pediatric influenza, particularly in children treated with the antiviral neuraminidase inhibitor oseltamivir (OSV) with low production of mucosal antiviral secretory IgA (S-IgA). The aims of the present study were to confirm the effects of CAM on S-IgA immune responses, by using influenza A virus (IAV) H1N1-infected mice treated with or without OSV, and to determine the molecular mechanisms responsible for the induction of mucosal IgA class switching recombination in IAV-infected CAM-treated mice. The anti-IAV S-IgA responses and expression levels of IgA class switching recombination-associated molecules were examined in bronchus-lymphoid tissues and spleens of infected mice. We also assessed neutralization activities of S-IgA against IAV. Data show that CAM enhanced anti-IAV S-IgA induction in the airway of infected mice and restored the attenuated antiviral S-IgA levels in OSV-treated mice to the levels in the vehicle-treated mice. The expression levels of B-cell-activating factor of the tumor necrosis factor family (BAFF) molecule on mucosal dendritic cells as well as those of activation-induced cytidine deaminase and Iμ-Cα transcripts on B cells were enhanced by CAM, compared with the levels without CAM treatment, but CAM had no effect on the expression of the BAFF receptor on B cells. Enhancement by CAM of neutralization activities of airway S-IgA against IAV in vitro and reinfected mice was observed. This study identifies that CAM enhances S-IgA production and neutralizing activities through the induction of IgA class switching recombination and upregulation of BAFF molecules in mucosal dendritic cells in IAV-infected mice.

Fig 1

Experimental protocol for infection with IAV/PR8/34 virus. (A) At 20 h after infection with 25 PFU of IAV, mice were treated orally with 150 μg of CAM, 50 μg of OSV, OSV (50 μg) plus CAM (150 μg) in 100 μl of sterilized MC (n = 10), or 100 μl of MC as a control (n = 10). The dose was given twice daily for 5 days. (B) Changes in body weight in the four treatment groups. Data are means ± SEM (from day 0 to 7, n = 10; from day 8 to 12, n = 5).

Fig 2

Anti-IAV-specific S-IgA immune response in external secretions and mucosal lymphoid tissues. Mice infected with IAV at day 0 were treated on five (from day 1 to 5) consecutive days with CAM, OSV, OSV-CAM, or MC. (A) At days 8 and 12, IAV-specific S-IgA levels were determined in nasal washings (NWs) and bronchoalveolar lavage fluid (BALF) by ELISA. Data are means ± SEM (n = 5). *, P < 0.05, versus MC; # and ##, P < 0.05 and P < 0.01, respectively, versus OSV. (B) At days 8 and 12, mononuclear cells isolated from the nasal passages (NPs), mediastinal lymph nodes (MeLNs), and lung were subjected to IAV-specific ELISPOT assay to determine the numbers of IgA AFCs. Data are means ± SEM (n = 5). *, P < 0.05 versus MC; # and ##, P < 0.05 and P < 0.01, respectively, versus OSV.

Fig 3

Systemic IAV-specific immune responses in mice after infection. Mice infected at day 0 were treated for five consecutive days (from days 1 to 5) with CAM, OSV, OSV-CAM, or MC. (A) IAV-specific IgA and IgG levels in plasma determined by ELISA. Data are means ± SEM (n = 5). There were no significant differences between CAM and MC and between OSV and OSV-CAM. (B) Mononuclear cells isolated from the spleen were subjected to the ELISPOT assay to determine the numbers of IgA and IgG AFCs. Data are means ± SEM (n = 5). There were no significant differences between CAM and MC and between OSV and OSV-CAM.

Fig 4

Preferential presence of AID, Iμ-Cα transcripts in B cells from the nasopharyngeal-tracheal effector sites in IAV-infected-mice treated with CAM, OSV, OSV-CAM, or MC. Total RNA extracted from the mediastinal lymph nodes (MeLNs), lung, nasal passages (NPs), and spleen of IAV-infected mice at day 6 was subjected to RT-PCR analysis. The left panels show representative results, and the graphs on the right show the expression level of the respective CSR-associated molecules relative to the density of GAPDH expression, calculated as 100 with ChemiDoc XRS Quantity One Analysis software (Bio-Rad). Data are means ± SEM from 5 mice for each group and represent a total of five separate experiments. *, P < 0.05 versus MC; # and ##, P < 0.05 and P < 0.01, respectively, versus OSV.

Fig 5

Expression of B-cell-activating factor (BAFF) and proliferation-inducing ligand (APRIL) in CD11c⁺ DCs on mucosal effector sites. The day after the last dose of CAM, OSV, OSV-CAM, and MC (i.e., at day 6), mononuclear cells were isolated from the mediastinal lymph nodes (MeLNs), lung, and nasal passages (NPs) of IAV-infected mice, and mucosal DCs were purified with AutoMacs using anti-CD11c MAb-labeled microbeads, as described in Materials and Methods. Total RNA extracted from CD11C⁺ DCs was subjected to RT-PCR analysis. The left panels show representative results, and the graphs on the right show the results of quantitative analysis. Data are means ± SEM from 5 mice for each group and represent a total of five separate experiments. *, P < 0.05 versus MC; #, P < 0.05 versus OSV.

Fig 6

Expression of B-cell-activating factor (BAFF) and proliferation-inducing ligand (APRIL) receptor molecules (BAFF-R, transmembrane activator and calcium modulator cyclophilin ligand interactor [TACI], and B-cell maturation antigen [BCMA]) in mucosal B220⁺ B cells. The day after the last dose of CAM, OSV, OSV-CAM, or MC, mononuclear cells were isolated from the mediastinal lymph nodes (MeLNs), lung, and nasal passages (NPs) of IAV-infected mice. The mucosal B cells were purified with AutoMacs using anti-B220 MAb-conjugated microbeads. Total RNA extracted from B220⁺ B cells was subjected to RT-PCR analysis. The left panels show representative results, and the graphs on the right show the results of quantitative analysis. Data are means ± SEM of 5 mice for each group and represent a total of five separate experiments.

Fig 7

Neutralization activities of S-IgA in nasal washings (NWs) and bronchoalveolar lavage fluid (BALF) against IAV/PR8/34(H1N1) infection in MDCK cells. At day 12 postinfection, S-IgA was purified from NWs and BALF of mice treated with CAM, OSV, OSV-CAM, or MC or from naïve mice (uninfected), and 2 μg of purified S-IgA from each group was then preincubated with IAV (1,450 PFU, 100 μl) for 1 h at 37°C. Each mixture was subsequently incubated with confluent monolayers of MDCK cells at 37°C for 1 h for infection. Sixteen hours after infection, the cells were fixed with 4% paraformaldehyde–phosphate-buffered saline (PBS), permeabilized with 0.3% Triton X-100–PBS, and then visualized by TrueBlue peroxidase substrate. NWs and BALF from uninfected mice were used as the control. Original magnification, ×40. The values in the graph are mean numbers of infected cells ± SEM (n = 4). *, P < 0.01 versus uninfected; # and ##, P < 0.05 and P < 0.01, respectively, versus MC or CAM; †, P < 0.05 versus OSV-CAM.

Fig 8

Reinforcement of protective immunity by CAM in reinfected mice. (A) At 20 h after infection with 5 PFU of IAV, mice were treated orally with CAM, OSV, OSV-CAM, or MC as described in the legend to Fig. 1. (A) Changes in body weight in the four treatment groups (n = 10). Data are means ± SEM. (B) Hematoxylin-and-eosin-staining sections in the lungs of four groups of mice at day 14 after reinfection and noninfection control mice. Each result is representative of 10 animals in each group. Bar, 100 μm.