Ficolin-2 defends against virulent Mycobacteria tuberculosis infection in vivo, and its insufficiency is associated with infection in humans

Abstract

Human ficolin-2 (ficolin-2/P35) is a lectin complement pathway activator that is present in normal human plasma and is associated with infectious diseases; however, little is known regarding the roles and mechanisms of ficolin-2 during Mycobacterium tuberculosis (Mtb) infection. Here, we describe our novel findings that the ficolin-2 serum levels of 107 pulmonary tuberculosis (TB) patients were much lower compared with 107 healthy controls. In vitro analysis showed that ficolin-2 bound to the virulent Mtb H37Rv strain much more strongly than to the non-virulent M. bovis BCG and M. smegmatis. Ficolin-2 bound to the surface glycolipid portion of H37Rv and blocked H37Rv infection in human lung A549 cells. Opsonophagocytosis was also promoted by ficolin-2. Importantly, we found that administration of exogenous ficolin-2 had a remarkable protective effect against virulent Mtb H37Rv infection in both C57BL/6J and BALB/c mice. Ficolin-A (a ficolin-2-like molecule in mouse) knockout mice exhibited increased susceptibility to H37Rv infection. We further demonstrated that ficolin-2 could defend against virulent Mtb H37Rv infection at least partially by activating JNK phosphorylation and stimulating the secretion of interferon (IFN)-γ, interleukin (IL)-17, IL-6, tumor necrosis factor (TNF)-α, and nitric oxide (NO) production by macrophages. Our data provide a new immunotherapeutic strategy against TB based on the innate immune molecule ficolin-2 and indicate that ficolin-2 insufficiency is associated with higher susceptibility to infection in humans.

Figure 1. Comparisons of serum ficolin-2 levels in tuberculosis patients and healthy donors.

Serum ficolin-2 concentrations in the TB patients (n=107) vs. those in the healthy donors (n=107), * p < 0.05. The data shown are the mean values of three experiments. All data were analyzed by one-way ANOVA with Bonferroni post-test.

Figure 2. Ficolin-2 recognizes and binds to a surface glycolipid of the virulent Mtb H37Rv.

(A) Analysis of the purified recombinant GST-ficolin-2 protein by SDS-PAGE and western blotting using anti-GST mAb. (B) Analysis of the recombinant GST-ficolin-2 protein by native PAGE. (C) Flow cytometry analysis of the binding abilities of GST-ficolin-2/GST (20 µg) with 1×10⁸ CFU Mtb H37Rv in the presence of 10 µg of ManLAM in TBS-Ca²⁺ buffer or in the presence of 10 mM EDTA. PE-anti-GST mAb was used. (D) Examination of the binding responses of different Mtb strains and ficolin-2 protein, in parallel, using Octet streptavidin (SA) sensors. Biotinylated GST-ficolin-2 (biotin-GST-ficolin-2) or biotin-GST proteins were loaded onto SA sensors. The SA sensors were incubated with 10⁹ CFU/mL of H37Rv, BCG, or M. smegmatis for 300 s. All of the binding traces and curves were used after dual deduction of GST binding and buffer binding for each type of bacteria. The experiments were repeated three times.

Figure 3. Ficolin-2 inhibits Mtb H37Rv infection and stimulates opsonization of macrophages.

(A) Ficolin-2/ficolin A inhibited Mtb H37Rv infection of human lung A549 cells in vitro. pcDNA3.1-ficolin-2 or pVAX-1-ficolin-A plasmids were transfected into A549 cells for 48 h. Rhodamine B-labeled Mtb H37Rv (RhoB-H37Rv) was mixed with ficolin-2- or ficolin A-transfected human lung A549 cells with or without ManLAM, and the cells were analyzed using flow cytometry. (B) Ficolin-2 stimulated opsonization of macrophages. RhoB-H37Rv bacteria were incubated with 20 µg of GST, GST-ficolin-2, GST-ficolin-2 plus CytB, or CytB, respectively, at 4°C for 30 min. The uptake of the H37Rv strain (detected as Rho-H37Rv) by macrophages (detected as FITC-F4/80) was analyzed using flow cytometry. The results shown are representative of three independent experiments. (C) The binding of ficolin-2 with the H37Rv strain leads to the activation of the lectin complement pathway. GST-ficolin-2 or GST proteins were incubated with Mtb H37Rv in the presence of human serum and C4 protein, and C4c deposits on the bacteria were measured as described in the Materials and Methods. The data shown are the mean ± SEM of three independent experiments GST-ficolin-2 group vs. GST group and control group, *p < 0.05. The data were analyzed by ANOVA method.

Figure 4. Ficolin-2 prolongs the survival time of both WT and FCNA KO mice infected with Mtb H37Rv.

(A) pcDNA3.1-ficolin-2 or empty vector pcDNA3.1 plasmids were transfected into murine carcinogen-induced colon tumor cells (CT26) (H-2^d), and the expression of ficolin-2 protein in the supernatants at 72 h post-transfection was detected by western blotting with anti-ficolin-2 mAb GN5. β-actin, which is a housekeeping gene with constant expression, was used as an internal control. Lane 1: pcDNA3.1; Lane 2: pcDNA3.1-ficolin-2. (B) Mice were transformed with the pcDNA3.1 empty vector or the pcDNA3.1-ficolin-2 vector (10 µg DNA/mouse) through intramuscular electroporation, and ficolin-2 protein expression in the muscle and spleen tissues were detected on post-injection days 4, 7, and 10 by western blot analysis. Lanes 1, 2, 3: pcDNA3.1-ficolin-2; lane 4 (10^th day): pcDNA3.1 empty vector. (C) Ficolin-2 concentrations in sera obtained from mice injected with pcDNA3.1-ficolin-2 (day 0 group vs. other groups, *p < 0.05). Data were analyzed by one-way ANOVA. (D) C57BL/6J mice were challenged with 1×10⁶ CFU Mtb H37Rv per mouse, which was administered on the same day as the vehicles (NS and pcDNA3.1) and pcDNA3.1-ficolin-2. Each group contained eight mice. The survival times of H37Rv-infected mice after administration of pcDNA3.1-ficolin-2 or empty vector through i.m. electroporation on the day of intravenous challenge with virulent Mtb H37Rv were determined. The data shown are the means of five independent experiments with eight mice per group. Ficolin-2-treated group and SM-treated group vs. vehicle (saline or pcDNA3.1)-treated group, * p < 0.05. (E) The survival time of H37Rv-infected BALB/C mice challenged with virulent Mtb H37Rv after administration of lentivirus-ficolin-2. Lentivirus-ficolin-2-treated group vs. lentivirus (mock) control-treated and vehicle (saline)-treated groups, * p < 0.05. (F) The survival times of H37Rv-infected FCNA KO mice after administration of pcDNA3.1-ficolin-2, pVAX-1-ficolin A, or empty vector through i.m. electroporation on the day of intravenous challenge with virulent Mtb H37Rv. The ficolin-2-treated group and ficolin A-treated group vs. vehicle (saline or pcDNA3.1)-treated group, * p < 0.05. The Kaplan-Meier method was used to plot survival curves for each group. (G) Spleens and lungs of Mtb H37Rv-infected C57BL/6 mice were harvested at post-infection day 30, and Mtb H37Rv bacteria were enumerated. The ficolin-2-treated group or SM-treated group (in lung) vs. vehicle (saline or pcDNA3.1)-treated groups, * p < 0.05. Data were analyzed by one-way ANOVA. (H) Lung tissues of Mtb H37Rv-infected C57BL/6 mice were harvested at 4 weeks post-infection. The number of Mtb H37Rv bacteria and lung pathological changes were analyzed with Ziehi-Neelsen acid-fast bacillus staining and H&E staining, respectively.

Figure 5. Ficolin-2 significantly stimulated macrophages to produce IFN-γ, IL-17A, TNF-α, IL-6, and NO in vitro.

(A) For in vitro analysis, 1×10⁶ isolated macrophages, neutrophils, CD8⁺ T, and CD4⁺ T cells from untreated BALB/c mice were incubated with 20 µg/ml of GST-ficolin-2 or GST at 37°C for 48 h. The cells were collected and lysed, and the supernatants were collected for analysis of IFN-γ production using cytokine-specific ELISA methods. For neutrophils and macrophages, GST-ficolin-2 group vs. GST group or control group, * p < 0.05. (B) Isolated macrophages or neutrophils were cultured in 96-well plates (1×10⁴ cells/well) and stimulated with GST-ficolin2 or GST at final concentration of 20 µg/ml for 48 h. The supernatants were harvested, and the concentrations of NO were measured by ELISA. For macrophages, GST-ficolin-2 group vs. GST group or control group, * p < 0.05. (C, D) The indicated concentrations of GST-ficolin2 (C), GST-ficolin-A (D), or GST proteins were incubated with the isolated macrophages (1×10⁴ cells/well) at 37°C for 0, 6, 12, 24, or 48 h, respectively. The supernatants were harvested, and the concentration of NO was measured by ELISA. GST-ficolin-2 group, GST-ficolinA group vs. GST group or control group, * p < 0.05. (E) A total of 1×10⁶ macrophages isolated from untreated BALB/c mice were incubated with 20 µg/ml of GST-ficolin-2 or GST at 37°C for 48 h. IL-4, IL-12, IL-17A, TNF-α, IFN-γ, and IL-6 production in the supernatants was measured by cytokine-specific ELISA methods. The data shown are the means ± SEM of at least four independent experiments. All data were analyzed by one-way ANOVA.

Figure 6. Ficolin-2 stimulated JNK phosphorylation.

(A) Murine macrophages (2×10⁶) were stimulated with 20 µg/ml of GST (0.8 µM), GST-ficolin-2 (0.4 µM) or LPS (0.4 µM) at 37°C for 0, 15, 30, or 60 min, respectively. The cells were collected and lysed, and the phosphorylated-JNK (p-JNK) (46/54 kDa) and total JNK were detected by western blotting using the corresponding mAbs. (B) Densities of p-JNK protein shown in (A) were normalized based on the density of the house keeping protein β-actin. The data shown are the means ± SEM of three independent experiments. The data were analyzed by ANOVA. (C) Murine macrophages were cultured in 6-well plates (2×10⁶ cells/well) and subsequently stimulated with 20 µg/ml of GST, GST-ficolin-2, or GST-ficolin-2 plus JNK inhibitor (SP600125, 40 µM, Sigma) at 37°C for 0, 6, 14, 24, 48, or 64 h, respectively. The supernatants were collected, and the concentrations of TNF-α and IL-17A were detected by ELISA. GST-ficolin-2 group vs. GST-ficolin-2 plus SP600125 group, GST group or cell alone control group, * p < 0.05. The data were analyzed by one-way ANOVA. (D) In vivo analysis of LPS-stimulated TNF-α and IL-17A production. Wild-type C57BL/6 mice (n=8) or FCNA KO C57BL/6 mice (n=8) were injected with LPS (0.15 mg/mouse) via the tail vain. The blood sample (100 µl) was collected from the orbital vein of each mouse at 0, 3, 6 or 10 h. Subsequently, the serum TNF-α and IL-17 levels were examined by ELISA. The data shown are means ± SEM of at least three independent experiments. C57BL/6 group vs. FCNA KO C57BL/6 group, * p < 0.05. The data were analyzed by Student’s t test.

Figure 7. Hypothetical model of the mechanisms mediated by ficolin-2 during Mtb infection.