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. 2015 Jul;83(7):2627-35.
doi: 10.1128/IAI.00369-15. Epub 2015 Apr 13.

Macrophage Polarization during Murine Lyme Borreliosis

Carrie E Lasky  1 , Rachel M Olson  1 , Charles R Brown  2
Affiliations

Macrophage Polarization during Murine Lyme Borreliosis

Carrie E Lasky et al. Infect Immun. 2015 Jul.

Abstract

Infection of C3H mice with Borrelia burgdorferi, the causative agent of Lyme disease, reliably produces an infectious arthritis and carditis that peak around 3 weeks postinfection and then spontaneously resolve. Macrophage polarization has been suggested to drive inflammation, the clearance of bacteria, and tissue repair and resolution in a variety of infectious disease models. During Lyme disease it is clear that macrophages are capable of clearing Borrelia spirochetes and exhausted neutrophils; however, the role of macrophage phenotype in disease development or resolution has not been studied. Using classical (NOS2) and alternative (CD206) macrophage subset-specific markers, we determined the phenotype of F4/80(+) macrophages within the joints and heart throughout the infection time course. Within the joint, CD206(+) macrophages dominated throughout the course of infection, and NOS2(+) macrophage numbers became elevated only during the peak of inflammation. We also found dual NOS2(+) CD206(+) macrophages which increased during resolution. In contrast to findings for the ankle joints, numbers of NOS2(+) and CD206(+) macrophages in the heart were similar at the peak of inflammation. 5-Lipoxygenase-deficient (5-LOX(-/-)) mice, which display a failure of Lyme arthritis resolution, recruited fewer F4/80(+) cells to the infected joints and heart, but macrophage subset populations were unchanged. These results highlight differences in the inflammatory infiltrates during Lyme arthritis and carditis and demonstrate the coexistence of multiple macrophage subsets within a single inflammatory site.

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Figures

FIG 1
FIG 1
In vitro characterization of BMDM from C3H mice. BMDM were cultured with (infected) or without (uninfected) B. burgdorferi spirochetes (MOI, 10:1) for 24 h. Cells were gated on live cells and F4/80+ and then assessed for expression of CD206 and NOS2. Representative flow plots are shown, and percentages of F4/80+ cells expressing NOS2 (M1), CD206 (M2), or both NOS2 and CD206 (M1/M2) are presented (A). Levels of nitrite were determined in supernatants of uninfected (control) or infected BMDM (B).
FIG 2
FIG 2
Flow cytometric characterization of macrophage subsets from ankle joints of mice infected with B. burgdorferi. Representative flow cytometry plots of cells isolated from mouse ankle joints were generated (A). Arthritis development was monitored by measuring ankle swelling over time (B). Cell number plots from joints of control (uninfected) or mice infected for various days with B. burgdorferi and gated for CD45.2+ hematopoietic cells (C), F4/80+ macrophages (D), M1 (NOS2+ CD206−) (E), M2 (NOS2− CD206+) (F), or rM (NOS2+ CD206+) (G) were generated. n = 4 for each time point and the experiment was performed twice. Asterisks indicate data are significantly different from control data at a P value of <0.05 as determined by ANOVA followed by Dunnett's test. Swelling curve differences were determined by comparing infected versus uninfected animals at each time point via Student's t test.
FIG 3
FIG 3
Flow cytometric characterization of macrophage subsets from hearts of mice infected with B. burgdorferi. Hearts were perfused and digested, and single-cell suspensions were isolated as described in Materials and Methods. Cell number plots from hearts of control (uninfected) or mice infected for various days with B. burgdorferi and gated for CD45.2+ hematopoietic cells (A), F4/80+ macrophages (B), M1 (NOS2+ CD206−) (C), M2 (NOS2− CD206+) (D), or rM (NOS2+ CD206+) (E) were generated. n = 4 for each time point, and the experiment was performed twice. Asterisks indicate data are significantly different from control data at a P value of <0.05 as determined by ANOVA followed by Dunnett's test.
FIG 4
FIG 4
In vitro polarization and characterization of BMDM from wild-type (WT) or 5-LOX−/− mice. Following treatment with polarizing cytokines M(LPS+IFN-γ) or M(IL-4) for 24 h, supernatant nitrite (A) and cell lysate arginase activity (B) were determined. Cells were gated on live cells and F4/80+ and then assessed for percentage of expression of NOS2 (C), CD206 (D), or both NOS2 and CD206 (E) by flow cytometry. Asterisks indicate statistically significant differences between 5-LOX cells compared with WT controls from the same group at a P value of >0.05.
FIG 5
FIG 5
Macrophage phenotype and numbers were analyzed from the joints of Borrelia-infected WT or 5-LOX-deficient mice on days 21 and 56 postinfection. Numbers of F4/80+ macrophages (A), M1 (NOS2+ CD206−) (B), M2 (NOS2− CD206+) (C), or rM (NOS2+ CD206+) (D) were determined. n = 4 for each time point, and the experiment was performed twice. Asterisks indicate data that are significantly different from control data at a P value of <0.05 as determined by ANOVA followed by Dunnett's test.
FIG 6
FIG 6
Macrophage phenotype and numbers were analyzed from the hearts of Borrelia-infected WT or 5-LOX-deficient mice on days 21 and 56 postinfection. Numbers of F4/80+ macrophages (A), M1 (NOS2+ CD206−) (B), M2 (NOS2− CD206+) (C), or rM (NOS2+ CD206+) (D) were determined. n = 4 for each time point, and the experiment was performed twice. Asterisks indicate data that are significantly different from control data at a P value of <0.05 as determined by ANOVA followed by Dunnett's test.
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