Clarithromycin expands CD11b⁺Gr-1⁺ cells via the STAT3/Bv8 axis to ameliorate lethal endotoxic shock and post-influenza bacterial pneumonia

<div><p>Macrolides are used to treat various inflammatory diseases owing to their immunomodulatory properties; however, little is known about their precise mechanism of action. In this study, we investigated the functional significance of the expansion of myeloid-derived suppressor cell (MDSC)-like CD11b⁺Gr-1⁺ cells in response to the macrolide antibiotic clarithromycin (CAM) in mouse models of shock and post-influenza pneumococcal pneumonia as well as in humans. Intraperitoneal administration of CAM markedly expanded splenic and lung CD11b⁺Gr-1⁺ cell populations in naïve mice. Notably, CAM pretreatment enhanced survival in a mouse model of lipopolysaccharide (LPS)-induced shock. In addition, adoptive transfer of CAM-treated CD11b⁺Gr-1⁺ cells protected mice against LPS-induced lethality via increased IL-10 expression. CAM also improved survival in post-influenza, CAM-resistant pneumococcal pneumonia, with improved lung pathology as well as decreased interferon (IFN)-γ and increased IL-10 levels. Adoptive transfer of CAM-treated CD11b⁺Gr-1⁺ cells protected mice from post-influenza pneumococcal pneumonia. Further analysis revealed that the CAM-induced CD11b⁺Gr-1⁺ cell expansion was dependent on STAT3-mediated Bv8 production and may be facilitated by the presence of gut commensal microbiota. Lastly, an analysis of peripheral blood obtained from healthy volunteers following oral CAM administration showed a trend toward the expansion of human MDSC-like cells (Lineage⁻HLA-DR⁻CD11b⁺CD33⁺) with increased arginase 1 mRNA expression. Thus, CAM promoted the expansion of a unique population of immunosuppressive CD11b⁺Gr-1⁺ cells essential for the immunomodulatory properties of macrolides.</p></div

CAM-treated CD11b⁺Gr-1⁺ cells exhibit an immunosuppressive phenotype.

<p>(A) Top 25 upregulated and downregulated genes determined by a microarray analysis in splenic CD11b⁺Gr-1⁺ cells sorted from vehicle- and CAM-treated mice. *<i>Stxbp6</i> (chromosome 12:45956210–46175345). **<i>Stxbp6</i> (chromosome 12:45953470–45956090). Results are presented as fold changes relative to the expression levels of each gene in vehicle-treated CD11b⁺Gr-1⁺ cells. (B) Arginase activity in the spleen of vehicle- and CAM-treated mice (n = 4 per group). N.D., not detected. (C) Immunofluorescence staining of Gr-1 and arginase-1 in the lungs of mice treated with CAM daily for three consecutive days (n = 4 per group). Scale bar, 200 μm. (D) The concentration of nitric oxide (NO) in spleen extracts of vehicle- and CAM-treated mice (n = 4 per group) ***<i>p</i> < 0.001 by the Mann–Whitney U-test. (E) Expression of the surface marker CD244 on splenic CD11b⁺Ly-6G⁺ cells determined by flow cytometry (n = 4 per group). (F–H) Cytokine profile of the culture supernatant from bone marrow-derived macrophages (BMDMs) with or without equal numbers of vehicle-treated or CAM-treated CD11b⁺Gr-1⁺ cells (5 × 10⁵ cells) in the spleen: TNF-α (F), IFN-γ (G), and IL-10 (H). Representative data for three independent experiments are shown. Data are expressed as the mean ± SEM. ***<i>p</i> < 0.001 by a one-way ANOVA with Tukey’s multiple comparison tests.</p

CAM improves survival in post-influenza pneumococcal pneumonia via an essential contribution of CD11b⁺Gr-1⁺ cells.

<p>(A) Survival rate of post-influenza pneumococcal pneumonia mice treated with vehicle, ampicillin (ABPC) (100 mg/kg), or clarithromycin (CAM) (100 mg/kg) (n = 37 per group). *<i>p</i> < 0.05, ***<i>p</i> < 0.001 by the log-rank test. (B) Cell counts in bronchoalveolar lavage fluid (BALF) obtained from mice with post-influenza pneumococcal pneumonia treated with vehicle, ABPC (100 mg/kg), or CAM (100 mg/kg) (n = 7–8 per group). Data are presented as the mean ± SEM. *<i>p</i> < 0.05. **<i>p</i> < 0.01, ***<i>p</i> < 0.001 by a two-way ANOVA with Tukey’s multiple comparison tests. (C and D) Bacterial load in the lungs (C) and blood (D) of post-influenza pneumococcal pneumonia mice treated with vehicle, ABPC (100 mg/kg), or CAM (100 mg/kg) at 18 and 36 h after pneumococcal infection (n = 15–16 per group). Data are presented as the mean ± SEM. *<i>p</i> < 0.05 by a two-way ANOVA with Tukey’s multiple comparison tests. (E) Lung H&E staining at 48 h after pneumococcal infection. Representative data for 5 mice per group are shown. Scale bar, 100 μm. (F–I) Levels of IFN-γ (F) and IL-10 (G) in BALF were measured by ELISA. The levels of IFN-γ (H) and IL-10 (I) in serum were measured by ELISA (n = 7–8 per group). Data are presented as the mean ± SEM. *<i>p</i> < 0.05. **<i>p</i> < 0.01. ***<i>p</i> < 0.001 by a two-way ANOVA with Tukey’s multiple comparison tests. (J) Survival rate of mice following adoptive transfer of CD11b⁺Gr-1⁺ cells treated with vehicle, ABPC (100 mg/kg), or CAM (100 mg/kg) in post-influenza pneumococcal pneumonia mice (n = 34 per group). Combined data for two independent experiments are shown. *<i>p</i> < 0.05. **<i>p</i> < 0.01 by the log-rank test. (K) Survival rate of vehicle-, ABPC-, and CAM-treated mice intranasally inoculated with recombinant IFN-γ (16 μg/kg) or PBS at 30 min and 24 h after pneumococcal infection (n = 20 per group). (L) Survival rate of vehicle- or CAM-treated WT and <i>Ifng</i>^<i>-/-</i> mice with post-influenza pneumococcal pneumonia (n = 7–16 per group).</p

CAM ameliorates LPS-endotoxin shock via the essential contribution of CD11b⁺Gr-1⁺ cells.

<p>(A) Survival rate for LPS (50 mg/kg)-endotoxin shock in mice pretreated with vehicle or CAM (100 mg/day) daily for three consecutive days (n = 36 per group). *<i>p</i> = 0.0009 by the log-rank test. (B–D) Cytokine profiles in serum 12 h after LPS challenge in vehicle- or CAM-treated mice: TNF-α (B), IFN-γ (C), and IL-10 (D). (n = 5–6 per group). Data are represented as the mean ± SEM. **<i>p</i> < 0.01. ***<i>p</i> < 0.001 by the Mann–Whitney U-tests. (E and F) Representative two-parameter dot plots of CD11b⁺Gr-1⁺ cells in the spleen (E) and lungs (F) of mice intraperitoneally treated with vehicle or CAM (100 mg/day) daily for three consecutive days, followed by intraperitoneal injection with PBS or LPS (50 mg/kg) (n = 4 per group). (G) Quantification of CD11b⁺Gr-1⁺ cells in the spleen and lungs sorted from intraperitoneally vehicle- and CAM-treated (once a day for 3 days), followed by intraperitoneally LPS-treated mice (n = 4 per group). *<i>p</i> < 0.05, **<i>p</i> < 0.01 by Mann–Whitney U-tests. (H) Survival rate for LPS-endotoxin shock in vehicle- and CAM-injected mice pretreated with either anti-Gr-1 antibody (250 μg/mouse) or control IgG (n = 20–21 per group) 24 h before LPS challenge. *<i>p</i> = 0.0128 by the log-rank test. (I) Survival rate for LPS-endotoxin shock in vehicle- and CAM-injected mice pretreated with either anti-Gr-1 antibody (250 μg/mouse) or control IgG (n = 25–26 per group) 1 h before initiation of CAM treatment (i.e., 73 h before LPS challenge). Combined data for two independent experiments are shown. ***<i>p</i> < 0.001 by the log-rank test. (J) Adoptive transfer of CAM-treated CD11b⁺Gr-1⁺ cells improved the survival rate in LPS endotoxin shock (n = 24 per group). *<i>p</i> = 0.0023 by the log-rank test. (K-M) TNF-α (K), IFN-γ (L), and IL-10 (M) levels in serum at 12 h after intraperitoneal LPS injection (n = 5–6 per group). Data are presented as the mean ± SEM. *<i>p</i> < 0.05. **<i>p</i> < 0.01. ***<i>p</i> < 0.001 by the Mann–Whitney U-tests.</p

IL-10 production by CAM-treated CD11b⁺Gr-1⁺ cells is beneficial for LPS endotoxin shock.

<p>(A) CAM-treated or vehicle-treated CD11b⁺Gr-1⁺ cells in the spleen sorted from either IL-10 knockout or WT mice were adoptively transferred into LPS endotoxin-treated recipients (n = 25–30 per group). Combined data for two independent experiments are shown. <i>p</i> = 0.0137 by the log-rank test. (B and C) Levels of the pro-inflammatory cytokines TNF-α (B) and IFN-γ (C) in the supernatant of WT BMDMs co-cultured with either WT or IL-10 knockout CD11b⁺Gr-1⁺ cells in the spleen treated with vehicle or CAM (n = 3 per group). Data are presented as the mean ± SEM. **<i>p</i> < 0.01 by a one-way ANOVA with Tukey’s multiple comparison tests.</p

CAM expands the CD11b⁺Gr-1⁺ cell population via the STAT3-Bv8 axis.

<p>(A and B) Representative immunoblotting of Bv8 (A) and phosphorylated (p)-STAT3 (B). Beta-actin and total (t) STAT3 were used as loading controls. Relative band intensities ware quantified by densitometry. (C and D) Representative two-parameter dot plots of CD11b⁺Gr-1⁺ cells in the spleen (C) and lungs (D) sorted from mice intraperitoneally treated with vehicle or CAM (100 mg/day) daily for three consecutive days. Mice were intraperitoneally injected with an anti-Bv8 antibody (5 mg/kg) or control IgG at 4 days and 1 day before CAM treatment and daily for three consecutive days during CAM treatment (n = 4 per group). (E and F) Representative two-parameter dot plots of CD11b⁺Gr-1⁺ cells in the spleen (E) and lungs (F) sorted from poly(I:C)-treated <i>Stat3</i>^{<i>flox/flox</i>}/Mx1-Cre (<i>Stat3</i>^Δ<i>Mx1</i>) mice and control <i>Stat3</i>^{<i>flox/flox</i>} mice that were intraperitoneally treated with vehicle or CAM (100 mg/day) daily for three consecutive days (n = 4 per group). (G) Bv8 (<i>Prok2</i>) mRNA expression in CD11b⁺Gr-1⁺ cells sorted from <i>Stat3</i>^Δ<i>Mx1</i> mice and control <i>Stat3</i>^{<i>flox/flox</i>} mice (n = 4 per group). Data are presented as the mean ± SEM. ***<i>p</i> < 0.001 by the Mann–Whitney U-test.</p