Escherichia coli adhesion portion FimH polarizes M2 macrophages to M1 macrophages in tumor microenvironment via toll-like receptor 4

Abstract

Background: Macrophages are key effector cells of innate immunity and play a critical role in the immune balance of disease pathogenesis, especially in the tumor microenvironment. In previous studies, we showed that FimH, an Escherichia coli adhesion portion, promoted dendritic cell activation. However, the effect of FimH in macrophage polarization has yet to be fully examined. In this study, we investigated the potential effect of FimH on macrophages, as well as the polarization from M2 to M1 macrophages, contributing to the overall antitumor effect.

Methods: Mouse bone marrow derived macrophages and peritoneal macrophages were generated to test the effect of FimH in vitro. The expression of costimulatory molecules and production of cytokines were analyzed. The effect of FimH in the tumor-associated macrophages was examine in the B16F10-tumor bearing C57BL/6.

Results: FimH was found to promote M1 macrophage activation. In addition, FimH polarized M2 macrophages, which were induced by interleukin (IL)-4 and IL-13 into M1 macrophages were dependent on toll-like receptor 4 and myeloid differentiation factor 2. Moreover, FimH reprogramed the tumor-associated macrophage (TAM) into M1 macrophages in B16 melanoma tumor-bearing mice and promoted an inflammatory reaction in the tumor microenvironment (TME). Furthermore, FimH promoted M1 macrophage activation, as well as the reversion of M2 macrophages into M1 macrophages in humans. Finally, FimH treatment was found to enhance the anti-cancer immunity of anti-PD-L1 antibody by the induction of M1 polarization from TAM.

Conclusion: This study demonstrated the potential effect of FimH on the activation of macrophages, responsible for the repolarization of M2 macrophages into the M1 phenotype via the TLR4 signaling pathway. Moreover, FimH could also reprogram TAM polarization to the M1 status in the TME, as well as enhance the anti-tumor activity of immune checkpoint blockade.

Keywords: fimH; macrophage polarization; myeloid differentiation factor 2; toll-like receptor 4; tumor-associated macrophage.

Figure 1

Macrophage activation by FimH treatment. Bone marrow cells were incubated with 20 ng/mL murine macrophage colony-stimulating factor (M-CSF) for 6 days to generate bone marrow-derived macrophages (BMDM) and then stimulated with 0.5, 1, 2.5, and 5 μg/mL FimH or LPS for 24 h. (A) Mean fluorescence intensity (MFI) of CD80, CD86, and major histocompatibility complex (MHC) class II in BMDM. BMDM and peritoneal macrophage (pMAC) were stimulated with FimH (5 μg/mL) or LPS (2 μg/mL) for 24 h. (B) MFI of CD80, CD86, and MHC class II in BMDM (left panel) and pMAC (right panel). (C) Mean values of the mRNA expression levels in the BMDM (upper panel) and pMAC (lower panel). (D) Concentrations of interleukin (IL)-6, IL-12, and tumor necrosis factor alpha (TNF-α) in the culture medium of BMDM (upper panel) and pMAC (lower panel). (E) Concentration of nitrite in culture medium of BMDM (left panel) and pMAC (right panel). ( ^*p < 0.05, ^**p < 0.01, ^***p < 0.001).

Figure 2

FimH reverses M2 macrophages toward an M1 profile. Bone marrow-derived macrophages (BMDM) and peritoneal macrophage (pMAC) were obtained from C57BL/6 mice and treated with 20 ng/mL murine interleukin (IL)-4 and 20 ng/mL IL-13 for 24 h, and then stimulated with FimH (5 μg/mL) or LPS (2 μg/mL) for an additional 24 h. (A) Representative flow cytometry gating of macrophages (left panel) and quantification of the CD206⁺ macrophages (right panel). (B) Mean fluorescence intensity (MFI) of CD80, CD86, and major histocompatibility complex (MHC) class II in BMDM (left panel) and pMAC (right panel). (C, D) Mean values of the mRNA expression levels in the BMDM (upper panel) and pMAC (lower panel). (E) Concentrations of IL-6, IL-12, and tumor necrosis factor alpha (TNF-α) in the culture medium of BMDM (left panel) and pMAC (right panel). ( ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, n.s., none significant).

Figure 3

FimH reprograms macrophages in TLR4-dependent manner. Bone marrow-derived macrophages (BMDM) were obtained from toll-like receptor 4 (TLR4)-knockout (KO) and myeloid differentiation protein 2 (MD2)-KO mice and treated with 20 ng/mL murine interleukin (IL)-4 and 20 ng/mL IL-13 for 24 h, and then stimulated with FimH (5 μg/mL) or LPS (2 μg/mL) for an additional 24 h. (A) Representative flow cytometry gating of macrophages (left panel) and quantification of the CD206⁺ macrophages (right panel). (B) Mean values of the mRNA expression levels for M2 profile in the BMDM. (C) Mean values of the mRNA expression levels for M1 profile in the BMDM. (D) Concentrations of IL-6, IL-12, and tumor necrosis factor alpha (TNF-α) in the culture medium of BMDM. ( ^*p < 0.05, ^**p < 0.01, ^***p < 0.001).

Figure 4

FimH promotes TAM polarization to M1 status. C57BL/6 mice were subcutaneously (s.c.) inoculated with 1 × 10⁶ B16F10 cells. After 7 days of tumor-cell injection, the mice were further divided in three groups and s.c. injected with PBS, FimH (2.5 mg/Kg), or LPS (1 mg/Kg). (A) Representative flow cytometry gating of tumor-associated macrophage (TAM) in tumor. (B) Percentage of CD206⁺ F4/80⁺ macrophages. (C) Immunofluorescence detection of TAMs in the tumor sections by sequential staining with anti-F4/80 and anti-CD206 antibodies, and nuclei counterstained by DAPI. Red and green signals indicate the detected F4/80⁺ and CD206⁺ cells, respectively. Magnification: 200×. (D) Mean fluorescence intensity (MFI) of CD80, CD86, and MHC class II in TAM. (E) Tumor infiltration T cells were measured in the mouse tumor in vivo. Dot plots showed the percentages of CD3⁺ and CD8α⁺ T cells. (F) Mean percentages of CD3 (left panel) and CD8α (right panel) cells. (G) Intracellular perforin and granzyme B producing CD8α⁺ T cells in the tumor. (n = 6 mice, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001).

Figure 5

FimH promotes human monocyte-derived macrophages (hMDM) toward M1 phenotype. Peripheral blood mononuclear cells (PBMCs) were collected from healthy volunteers. The CD14⁺ monocytes from PBMCs were positively selected and cultured with 20 ng/mL human M-CSF for 7 days to derive macrophages. The hMDM were then stimulated with FimH (5 μg/mL) or LPS (2 μg/mL) for 24 h. (A) Gating of hMDM by flow cytometry. (B) Mean fluorescence intensity (MFI) of CD80, CD86, HLA-DR, and CD274 in hMDM. M2-like polarization was achieved by treatment with human 20 ng/mL IL-4 and 20 ng/mL IL-13 on day 7 for 24 h. (C) Representative flow cytometry gating of hMDM (left panel) and quantification of the CD206⁺ macrophages (right panel). (D) MFI of CD80, CD86, and HLA-DR in hMDM. ( ^*p < 0.05, ^**p < 0.01, ^***p < 0.001).

Figure 6

FimH enhances anti-cancer immunity of anti-PD-L1 antibody via the induction of M1 polarization from TAM. C57BL/6 mice were subcutaneously (s.c.) injected with 1 × 10⁶ B16F10 cells. After 5 days of tumor-cell injection, the mice were treated with anti-PD-L1 Abs (10 mg/Kg), FimH (2.5 mg/Kg) or a combination of anti-PD-L1 Abs and FimH for every 5 days. (A) Representative flow cytometry gating of tumor-associated macrophage (TAM) in tumor. (B) Mean percentage of CD206⁺ F4/80⁺ macrophages. (C) Representative flow cytometry dot plots of CD3⁺ and CD8α⁺ T cells in the mouse tumor. (D) Mean percentages of CD3 (left panel) and CD8α (right panel) cells. (E) Intracellular perforin and granzyme B producing CD8α⁺ T cells in the tumor. (n = 6 mice, ^**p < 0.01, ^***p < 0.001).