Azithromycin alters macrophage phenotype and pulmonary compartmentalization during lung infection with Pseudomonas

Abstract

Infection with mucoid strains of Pseudomonas aeruginosa in chronic inflammatory diseases of the airway is difficult to eradicate and can cause excessive inflammation. The roles of alternatively activated and regulatory subsets of macrophages in this pathophysiological process are not well characterized. We previously demonstrated that azithromycin induces an alternatively activated macrophage-like phenotype in vitro. In the present study, we tested whether azithromycin affects the macrophage activation status and migration in the lungs of P. aeruginosa-infected mice. C57BL/6 mice received daily doses of oral azithromycin and were infected intratracheally with a mucoid strain of P. aeruginosa. The properties of macrophage activation, immune cell infiltration, and markers of pulmonary inflammation in the lung interstitial and alveolar compartments were evaluated postinfection. Markers of alternative macrophage activation were induced by azithromycin treatment, including the surface expression of the mannose receptor, the upregulation of arginase 1, and a decrease in the production of proinflammatory cytokines. Additionally, azithromycin increased the number of CD11b(+) monocytes and CD4(+) T cells that infiltrated the alveolar compartment. A predominant subset of CD11b(+) cells was Gr-1 positive (Gr-1(+)), indicative of a subset of cells that has been shown to be immunoregulatory. These differences corresponded to decreases in neutrophil influx into the lung parenchyma and alteration of the characteristics of peribronchiolar inflammation without any change in the clearance of the organism. These results suggest that the immunomodulatory effects of azithromycin are associated with the induction of alternative and regulatory macrophage activation characteristics and alteration of cellular compartmentalization during infection.

FIG. 1.

Weight loss, survival, and infection burden over time postinfection with P. aeruginosa in C57BL/6 mice treated with AZM. (A) Weight loss over time postinfection expressed as the percent weight loss from the baseline value at day 0 through day 14 after infection with 1.5 × 10⁵ CFU P. aeruginosa-laden agarose beads or sham beads given intratracheally under light isoflurane anesthesia. Mice received either AZM via oral gavage daily or vehicle only (control). (B) Percent survival in mice receiving approximately 1× LD₅₀ (1.3 × 10⁶ CFU). (C) CFU counts from harvested lungs of infected mice determined over time postinfection. Statistically significant differences (P < 0.05) are indicated (*) and are representative of the results for multiple experimental replicates.

FIG. 2.

Immune cell infiltration into the lungs of P. aeruginosa-infected mice are influenced by AZM. C57BL/6 mice were infected with P. aeruginosa intratracheally while receiving either AZM via oral gavage or vehicle only (control). Immune cell subtypes were quantified by flow cytometry in lung digest (A) and lung lavage fluid (B) compartments, including total cells, CD11b⁺ cells (nonlymphocyte gate), CD11c⁺ cells (nonlymphocyte gate), neutrophils (CD11b⁻ Gr-1⁺), and CD4⁺ T cells. Mean values ± SDs are depicted, and statistical significance was defined as a P value of <0.05 for differences between groups at the same time point (*) and between groups when all animals were taken into account (†) without consideration of time point effects.

FIG. 3.

Surface expression of CD11b and CD11c among cells after infection with P. aeruginosa. Cells within a nonlymphocyte gate based on forward and side scatter characteristics were analyzed for CD11b and CD11c expression by flow cytometry. Mice received either oral AZM or vehicle only (control) starting 4 days prior to infection. Dot plots depicting CD11b and CD11c expression are shown from representative mice on cells isolated from lung digest (A) and lung lavage fluid (B) preparations on day 0 and on days 3, 7, and 14 postinfection. The percentages of cell subsets for which differences between the AZM-treated and the untreated groups were statistically significant (P < 0.05) are bordered by bold lines. All corresponding percentages are listed in Table 2.

FIG. 4.

Partitioning of macrophage subsets into the airway spaces after infection with P. aeruginosa. C57BL/6 mice were infected with P. aeruginosa intratracheally while receiving either AZM via oral gavage or vehicle only (control). Cells from the lung lavage fluid and lung digest compartments were phenotyped by flow cytometry on the basis of size, granularity, and surface receptor expression. Mean values ± SDs for the percentage of cells of a given phenotype that were found in the airway (lavage fluid) relative to that specific population in the entire lung (lavage fluid plus digest) were compared between groups. One-way ANOVA on ranks and the Student Newman-Keuls post hoc test were used, and P values less than 0.05 (*) were deemed statistically significant.

FIG. 5.

Characteristics of macrophage activation status in AZM-treated mice after lung infection with P. aeruginosa. C57BL/6 mice were infected with P. aeruginosa-laden agarose beads intratracheally while receiving either AZM via oral gavage or vehicle only (control). (A) Surface staining was analyzed by multiparameter flow cytometry to examine the expression of surface markers characteristic of classical (CD80, CCR7, and MHC-IIhi) or alternative (MR and CD23) macrophage activation on cells that were CD11b⁺. (B) Fold increase from the baseline in the arginase 1 level, as measured by arginase's activity in the conversion of arginine to urea. Samples from each mouse group were pooled and run in triplicate. Mean values ± SDs are depicted, and statistical significance (*) was defined as a P value of <0.05.

FIG. 6.

AZM induces a CD11b⁺ Gr-1⁺ subset of granulocytes that efficiently infiltrates the alveolar space after infection with P. aeruginosa. C57BL/6 mice were infected with P. aeruginosa-laden agarose beads intratracheally while receiving either AZM via oral gavage daily or vehicle only (control). The CD11b⁺ Gr-1⁺ subset in both the lung digest and the lung lavage fluid preparations was evaluated for the expression of MHC-II, CD80, and MR. (A) Number of cells exhibiting the upregulation of each activation protein over time postinfection. Mean values ± SDs are depicted, and statistical significance was defined as a P value of <0.05 for differences between groups at the same time point (*) and between groups when all animals were taken into account (†) without the consideration of time point effects. SSC and FSC, side and forward scatter, respectively. (B) Expression of these surface proteins on nonlymphocytic (on the basis of gating upon forward and side scatter characteristics) CD11b⁺ cells that were either Gr-1⁺ or Gr-1⁻. Dot plots of CD11b⁺ cells obtained from representative mice on day 7 postinfection are shown for comparison of the levels of MHC-II, CD80, and MR expression in both Gr-1⁺ and Gr-1⁻ subsets, with the percentage of the total CD11b⁺ cells in each gate being displayed.

FIG. 7.

Lung sections from day 7 postinfection with P. aeruginosa of mice receiving AZM or vehicle only (control). The mice were humanely killed after infection, and lung tissue sections were examined for inflammatory changes. Hematoxylin-eosin staining of the lung sections from day 7 postinfection from AZM-treated (A) and control (B) mice are shown. As the magnification increases from top to bottom, the scale bars represent distances of 50, 25, and 12.5 μm, respectively. Representative pictures show a predominantly monocytic cellular infiltration into the lung parenchyma in the AZM-treated animals (triangular pointers), while the inflammatory response in the control mice on day 7 postinfection is dominated by neutrophils (arrows). Br, bronchiole; V, venule.

FIG. 8.

Cytokine production from CD11b⁺ cells in lung digests and cytokine concentrations in lavage fluid after lung infection with P. aeruginosa in mice treated with AZM. C57BL/6 mice were infected with P. aeruginosa-laden agarose beads intratracheally while receiving either AZM via oral gavage or vehicle only (control). (A) The numbers of macrophages in the lung digests that were actively secreting TNF-α and IL-10 were measured by intracellular cytokine staining and flow cytometry. (B) Cytokine concentrations in the first 1 ml wash of lavage fluid were measured by cytometric bead array analysis. Mean values ± SDs are shown, and statistical significance (*) was defined as a P value of <0.05.