Article Figures & Data
Figures
- FIG 1
Macrophage polarization is dynamically altered during infection with C. neoformans in vivo. BALB/c mice were infected intratracheally with 104 live C. neoformans cells. Lung macrophages isolated at the indicated time points postinfection were analyzed by qPCR for mRNA expression of iNOS and Arg-1 genes, marker genes for M1 and M2 polarization, respectively. (A) A plot comparing the kinetics of Arg-1 and iNOS expression by pulmonary macrophages. Symbols indicate expression as average percent GAPDH. (B) A plot of the average ratio of Arg-1 expression to iNOS expression. The dashed line indicates the baseline expression ratio. In both plots, the symbols depict data from at least 4 mice per time point, and error bars denote SEMs. Note that the polarization of these macrophages evolves over time.
- FIG 2
Interaction with encapsulated C. neoformans cells is insufficient to drive M1-type polarization of RAW macrophages. C. neoformans yeast cells were rinsed thoroughly and opsonized with normal mouse serum (NMS) or left unopsonized. Wild-type C. neoformans cells were then fed to RAW macrophages (A) or BMM (B) at 1, 5, or 10 yeast cells per macrophage, as indicated. Twenty-four hours later, macrophage mRNA was harvested and subsequently analyzed for the indicated M1 or M2 marker genes by RT-PCR and gel electrophoresis (A) or qPCR (B). Data in panel A are representative images from three independent experiments. Data in panel B are data combined from 6 experiments. *, P < 0.05, and **, P < 0.005, in comparisons between the indicated data and the unstimulated control. Note that the macrophages stimulated with C. neoformans only weakly upregulate polarization genes.
- FIG 3
Cryptococcus-mediated polarization is superseded by exogenous cytokine signals in vitro. RAW macrophages were first pulsed with NMS-opsonized (right two lanes of each panel) or unopsonized C. neoformans (middle two lanes) or no particle controls (left two lanes) and incubated for 24 h and then stimulated with IL-4 (A) or IFN-γ (B) for a second 24-h period. The mRNA expression for iNOS (M1) or Arg-1 (M2) was analyzed by RT-PCR. Data are representative images from three independent experiments. Note that C. neoformans-mediated polarization can be superseded by subsequent IL-4 signaling.
- FIG 4
IFN-γ and IL-4 stimulation of RAW macrophages in vitro results in gene transcription, protein expression, and surface antigen expression consistent with polarized M1 versus M2 profiles. RAW macrophages were stimulated with 100 ng/ml IFN-γ or 20 ng/ml IL-4 or cultured in control medium (no stim) for 24 h. The expression of the indicated genes was measured by RT-PCR (A) or flow cytometry (C), while TNF-α secretion of the RAW cells was measured by ELISA using the culture supernatants (B). Note that “MR” in panel A refers to expression of mannose receptor (CD206), and panels A and C are representative data from three independent experiments. The data in panel B are cumulative, with n > 20 experimental replicates. *, P < 0.05; **, P < 0.005.
- FIG 5
Cytokine stimulation induces similar changes in M1 and M2 gene induction in RAW murine macrophage cell line and primary macrophage cultures. (A) RAW 264.7 cell line macrophages (RAW 264.7), M-CSF-differentiated bone marrow-derived macrophages (BMM M-CSF), and primary alveolar macrophages (AM) were stimulated with 100 ng/ml IFN-γ or 20 ng/ml IL-4 or left unstimulated (−) for 24 h. mRNA was harvested from cells and analyzed by RT-PCR. Note that all these macrophage types responded to IFN-γ by increased iNOS expression and to IL-4 by inducing Arg-1 and Ym-2, consistent with M1 and M2 polarization, respectively. Data are representative images from three independent experiments. (B) M-CSF BMM were stimulated, and mRNA was harvested. Gene expression was analyzed quantitatively using qPCR and expressed as fold change. Error bars in panel B represent SEMs from data pooled from 6 experiments. *, P < 0.05; **, P < 0.005.
- FIG 6
Cytokine-induced M1 versus M2 macrophage polarization profiles are plastic and thus reflect the most recent stimulus. RAW macrophages were incubated with the indicated primary stimulus, either IL-4 or IFN-γ, or no stimulus for 24 h. Cultures were then split and stimulated with the indicated secondary stimulus, IL-4 or IFN-γ. After 24 h of secondary stimulation, cells were harvested and mRNA was collected and analyzed for the indicated genes by RT-PCR (A) or fixed and stained with antibodies specific for iNOS and Arg-1 (B). Note that gene expression in all the assayed cultures more closely resembled cells stimulated only with the last (second) stimulus than those cultures stimulated only with the first stimulus. Data are representative images of three (A) or two (B) independent experiments.
- FIG 7
M1 versus M2 polarization plasticity in vitro is reflected by fungicidal activity of macrophages. BMM were stimulated for 24 h with IL-4 or IFN-γ or left unstimulated. These cultures were divided into subgroups and restimulated with IL-4 or IFN-γ. After 24 h of secondary stimulation, cells were harvested, and mRNA was collected and analyzed for the indicated genes by qPCR (A to D) or BMM were then incubated with NMS-opsonized C. neoformans. After another 24 h, the culture supernatants were reserved and analyzed for nitric oxide production by Griess reaction E, and the yeast cells were collected by lysing the macrophages and enumerated (F). In panel F, the data on the y axis is the percent inhibition calculated as described in Materials and Methods. Error bars represent SEMs. Graphs in panels A to F are cumulative from 6 experiments. Note that raw CFU data from each independent experiment in panel F displayed statistically significant P values between the comparisons, which are noted in panel F. *, P < 0.05; **, P < 0.005.
