DnaK-treated macrophages enhance tumor growth in murine allogeneic melanoma model.

<p>(A) The murine melanoma cell line B16F10 was co-injected with macrophages exposed to 30 µg/mL DnaK, 30 ng/ml of LPS, untreated macrophages, or no other cells for 24 h as illustrated in experimental design. (B) Cells were subcutaneously injected into BALB/c mice (4 mice per group) and the tumor volume was measured 8 days later as indicated. The values represent means ± SEM. (*) p<0.05 and (***) p<0.001 indicate significant difference between the macrophages exposed to DnaK in relation to B16 group. The data were analyzed by one-way ANOVA with Tuckey post hoc test in each time point. (C) Macroscopically view of tumor size. (D) Tumor weight on day 16 after tumor injection. The values represent means ± SEM. All data representative of three independent experiments.</p

Induction of M2 surface marker CD206 by DnaK treatment.

<p>Representative dot plots of surface (A) CD80 and (B) CD206 expression in BMMs treated with 30 ng/mL of LPS, 40 ng/mL of IL-4, 30 µg/mL or 60 µg/mL of DnaK, or left unstimulated for 24 h. The percentage values of (C) F4/80⁺CD80⁺ and (D) F4/80+CD206+ cells represent means ± SEM from triplicates. (E) and (F) show respective values for MFI analyses. (*) p<0.05, (**) p<0.01 and (***) p<0.001 indicate significant difference treated groups in relation to medium group. All data were analyzed by one-way ANOVA with Tukey post hoc test. Data are representative of three independent experiments.</p

Extracellular DnaK induces the expression of CD206 in peritoneal macrophages.

<p>Peritoneal macrophages were isolated from B6 mice and treated with 30 ng/mL of LPS, 40 ng/mL of IL-4, 30 µg/mL or 60 µg/mL of DnaK, or left unstimulated for 24 h. After that, cells were analyzed by flow cytometry and data presented as representative dot plots of (A) CD80, (B) CD206 expression. Data representative of three independent experiments.</p

Extracellular DnaK induces the expression of M2 markers in bone marrow-derived macrophages.

<p>BMMs were treated with LPS (30 ng/ml), IL-4 (40 ng/mL), DnaK (30 µg/mL or 60 µg/mL), or left unstimulated for 24 h. (A) iNOS activity was determinated by nitrite (NO²⁻) accumulation in the supernatant of macrophages. Data are the mean ± S.D. from triplicates. Data representative of three independent experiments. (***) p<0.001 indicates difference between LPS and other treatment groups. (B) Arginase activity was assessed by measuring the formation of urea from arginine. Data are the mean ± S.D. from triplicates. (**) p<0.01 and (***) p<0.001 indicate difference between treated groups and the medium group. Effect of DnaK on Ym1 (C) and FIZZ1 (D) expression in macrophages were quantified by real time PCR. The total amount of Ym1 and FIZZ1 mRNA were normalized to β-microglobulin signals and expressed as 2^−Δ/ΔCT. The values represent means ± SEM from triplicates. Data representative of three independent experiments. All data were analyzed by one-way ANOVA with Tukey post hoc test.</p

Macrophages release an M2-like cytokine profile upon stimulation with DnaK.

<p>BMMs were treated with 30 ng/mL of LPS, 40 ng/mL of IL-4, 30 µg/mL or 60 µg/mL of DnaK or left unstimulated for 24 h. (A) TNF-α, (B) IL-6, (C) MCP-1 and (D) IL-10 were measured from culture supernatants by flow cytometry. The values represent means ± SEM in pg/ml from triplicates. (**) p<0.01 and (***) p<0.001 indicate significant difference between treated groups and medium group. All data has been by one-way ANOVA with Tukey post hoc test. Data representative of four independent experiments.</p

Extracellular Mycobacterial DnaK Polarizes Macrophages to the M2-Like Phenotype

Macrophages are myeloid cells that play an essential role in inflammation and host defense, regulating immune responses and maintaining tissue homeostasis. Depending on the microenvironment, macrophages can polarize to two distinct phenotypes. The M1 phenotype is activated by IFN-γ and bacterial products, and displays an inflammatory profile, while M2 macrophages are activated by IL-4 and tend to be anti-inflammatory or immunosupressive. It was observed that DnaK from Mycobacterium tuberculosis has immunosuppressive properties, inducing a tolerogenic phenotype in dendritic cells and MDSCs, contributing to graft acceptance and tumor growth. However, its role in macrophage polarization remains to be elucidated. We asked whether DnaK was able to modulate macrophage phenotype. Murine macrophages, derived from bone marrow, or from the peritoneum, were incubated with DnaK and their phenotype compared to M1 or M2 polarized macrophages. Treatment with DnaK leads macrophages to present higher arginase I activity, IL-10 production and FIZZ1 and Ym1 expression. Furthermore, DnaK increased surface levels of CD206. Importantly, DnaK-treated macrophages were able to promote tumor growth in an allogeneic melanoma model. Our results suggest that DnaK polarizes macrophages to the M2-like phenotype and could constitute a virulence factor and is an important immunomodulator of macrophage responses

Differential Macrophage Activation Alters the Expression Profile of NTPDase and Ecto-5′-Nucleotidase

<div><p>Macrophages are key elements in the inflammatory process, whereas depending on the micro-environmental stimulation they exhibit a pro-inflammatory (classical/M1) or an anti-inflammatory/reparatory (alternative/M2) phenotype. Extracellular ATP can act as a danger signal whereas adenosine generally serves as a negative feedback mechanism to limit inflammation. The local increase in nucleotides communication is controlled by ectonucleotidases, such as members of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family and ecto-5′-nucleotidase/CD73 (ecto-5′-NT). In the present work we evaluated the presence of these enzymes in resident mice M1 (macrophages stimulated with LPS), and M2 (macrophages stimulated with IL-4) macrophages. Macrophages were collected by a lavage of the mice (6–8 weeks) peritoneal cavity and treated for 24 h with IL-4 (10 ng/mL) or LPS (10 ng/mL). Nitrite concentrations were measured using the Greiss reaction. Supernatants were harvested to determine cytokines and the ATPase, ADPase and AMPase activities were determined by the malachite green method and HPLC analysis. The expression of selected surface proteins was evaluated by flow cytometry. The results reveal that M1 macrophages presented a decreased ATP and AMP hydrolysis in agreement with a decrease in NTPDase1, -3 and ecto-5′-nucleotidase expression compared to M2. In contrast, M2 macrophages showed a higher ATP and AMP hydrolysis and increased NTPDase1, -3 and ecto-5′-nucleotidase expression compared to M1 macrophages. Therefore, macrophages of the M1 phenotype lead to an accumulation of ATP while macrophages of the M2 phenotype may rapidly convert ATP to adenosine. The results also showed that P1 and P2 purinoreceptors present the same mRNA profile in both phenotypes. In addition, M2 macrophages, which have a higher ATPase activity, were less sensitive to cell death. In conclusion, these changes in ectoenzyme activities might allow macrophages to adjust the outcome of the extracellular purinergic cascade in order to fine-tune their functions during the inflammatory set.</p> </div