Correlation between the number of bacteria and bacterial PI <i>in vitro</i> and <i>in vivo</i>.

<p>BLI sensitivity was assessed using a CCD-based macroscopic detector to quantify the bacterial photon intensity (PI  =  photons/sec/cm²/steradian) for various numbers of bacteria (1.25×10⁵ to 2.0×10⁶ CFU per well). (A) <i>In vitro</i> study: the bacterial PI from colonies of bioluminescent MRSA was significantly correlated with the number of bacterial CFUs (<i>R</i>² = 0.9912). (B) <i>In vivo</i> study: various numbers of bacteria (1.25×10⁵–2.0×10⁶ CFU per inoculation) were inoculated into the SGM, and bioluminescence in the region of interest (ROI) was monitored by BLI. There was a significant correlation between the number of inoculated bacteria and the bacterial PI <i>in vivo</i> (<i>R</i>² = 0.9882).</p

Histological findings.

<p>Changes in histology over time in the SGM from infected mice. Axial sections of the infected SGM were prepared at 7 (A and B), 28 (C and D), and 42 (E and F) days after bacterial inoculation and stained with hematoxylin and eosin. Lower images: magnified views of the boxed regions in the upper images. On day 7, marked neutrophil infiltration and muscle destruction were detected at the injection sites. On day 28, an abscess that contained necrotic materials (asterisk) and covered by fibrous tissue was observed in the SGM. On day 42, scaring tissue (asterisk) observed in the subcutaneous tissue. Bars  = 200 µm (A, C, E), 10 µm (B, D, F).</p

Bacterial quantification.

<p>To evaluate the correlation between the bacterial PI and the number of bacteria in the infectious process <i>in vivo</i>, the positive area of gram-stained MRSA on the each tissue section from the different time-point animals (3, 7, and 28 days after inoculation) were calculated. Six axial sections which cover whole abscess in SGM were obtained from each animal. The total numbers of pixels in gram-positive area (blue stains) were measured in five fields from six sections (A). Image obtained from software (B) shows that gram-positive blue area has been replaced to red pixel dots (C).</p

Dual optical imaging in the mouse SGM model.

<p>(A) Bioluminescence and (B) fluorescence images in the same animal, 10 days after bacterial inoculation, are shown. (A) MRSA inoculated into the left SGM was sufficient to produce an observable bioluminescence signal. (B) A Cy5.5-conjugated inflammation probe accumulated at the inoculation site only 5 minutes after it was injected into the tail vein, and was monitored for over 2 weeks in the same animal. (C) In the merged image, the accumulation of the inflammation probe (yellow) appeared as an area of fluorescence that completely covered the entire region of bacterial bioluminescence signal (multi-color).</p

Changes in bacterial PI in the mouse SGM model and correlation between the number of inoculated bacteria and bacterial PI in the infection process.

<p>We inoculated MRSA Xen31 (3×10⁷ CFU/µl) in 5µl of medium into the SGM, and measured the bacterial PI in the ROI immediately after inoculation, and on days 1, 3, 7, 14, 21, and 28 (N = 6). (A) The mean bacterial PI in the SGM peaked immediately after inoculation (2.486×10⁴ PI) and remained high for at least 28 days (2.016×10⁴ PI). Means and SEM are shown. (B) There was a significant correlation between the pixels number of gram-positive area and the bacterial PI during 28 days <i>in vivo</i> (<i>R²</i> = 0.9788).</p

Serological data.

<p>Mean serum levels of IL-6, IL-1β, and CRP in MRSA-inoculated animals at various time points are shown (N = 4 each). Compared to the pre-inoculation level, the IL-6 level was significantly higher 12 hours after inoculation (<i>P</i><0.05). The IL-6 level gradually decreased, becoming normal by day 21. IL-1β was significantly elevated after 12 hours, and remained high for 7 days (P<0.05). CRP was significantly higher than the pre-inoculation level 0.5, 1, 3, and 21 days after inoculation (<i>P</i><0.05). The means and SEM are shown.</p

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

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 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

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