Forty PTB patients who received treatment in the Hospital and 40 healthy volunteers were included in this study. The inclusion criteria for PTB group were: (1) patients receiving anti-tuberculosis treatment in the hospital; (2) pathological confirmation of PTB; (3) no history of other tuberculosis treatment. Inclusion criteria for the normal volunteer group: (1) no history of tuberculosis; (2) no history of major diseases; (3) health examination to confirm good health. The exclusion criteria were: (1) history of mental illness; (2) severe hepatic or renal dysfunction; and (3) coexistence of other types of diseases. All patients or their families/legal guardians as well as healthy volunteers voluntarily signed an informed consent form. The experiment has been approved by the Ningbo Ninth Hospital Medical Health Group Ethics Review Committee (No. NNHM-20230201).

All PTB patients received anti-tuberculosis treatment and the mean age was (45.3 $\pm$ 10.7) years. The 40 individuals in the healthy volunteer group were healthy adults with a mean age of (46.5 $\pm$ 9.4) years without any history of tuberculosis or any other major disease. All volunteers underwent a detailed health examination to confirm their health status before inclusion in the study. Venous blood samples were obtained from all PTB patients and healthy volunteers. Blood collection tubes were placed at room temperature for 30 minutes to promote blood coagulation. Serum was collected by centrifugation at 3000 g for 10 minutes. The serum samples collected were stored in a freezer at –80 °C for subsequent testing. This study was conducted in accordance with the Declaration of Helsinki.

1 $\times$ 10⁴ mouse monocyte macrophage RAW264.7 (SNL-112, Sunncell, Wuhan, China) were cultured in RPMI 1640 medium (R8758, Sigma Aldrich, St. Louis, MO, USA) containing 10% fetal bovine serum (16010142, Thermo fisher, Waltham, MA, USA), 1% penicillin-streptomycin (15140122, Thermo fisher, MA, USA) in RPMI 1640 medium (R8758, Sigma Aldrich, St. Louis, USA) incubated at 37 °C in 5% CO₂ [16]. M2 polarization was induced by 20 ng/mL interleukin-4 (IL-4) (214-14, Proteintech, Chicago, IL, USA) was induced [17]. All cell lines were validated by short tandem repeat (STR) profiling and tested negative for mycoplasma. Cells were all cultured in a humidified incubator at 37 °C and 5% CO₂.

The coding sequences (CDS) of TNFR2 and GRN was cloned into the pcDNA3.1 vector (V79020, Thermo fisher, MA, USA) to construct oe-TNFR2 and oe-GRN, respectively. The oe-TNFR2, oe-GRN, oe-negative control (NC) plasmids, or si-GRN (5^′-GCCUGACGUCACCAUGACCUG-3^′) and si-NC (5^′-UUCUCCGAACGUGUCACGUUU-3^′, SIC001, Sigma Aldrich, St. Louis, USA) were transfected with Lipofectamine 3000 transfection reagent (L3000075, Thermo fisher, MA, USA) for transfection into RAW264.7 cells. The procedure was as follows: the RAW264.7 cells were inoculated into 96-well plates at a density of 1 $\times$ 10⁴ cells per well. When the cells grew to 90% fusion, Lipofectamine 3000 transfection reagent and GRN and TNFR2 overexpression or TNFR2 knockdown plasmids were diluted with Opti-MEM medium (51985091, Thermo fisher, MA, USA), respectively, and cultured at 37 °C for 20 minutes. The cells were then incubated with the Lipofectamine 3000 transfection reagent and GRN and TNFR2 overexpression or TNFR2 knockdown plasmids, respectively. The incubated mixture was then transferred into cell culture wells and incubation was continued at 37 °C for 48 hours.

Total RNA was extracted from peripheral serum and RAW264.7 cells of patients in each group using MagMAX Pathogen Nucleic Acid Isolation Kit (A42352, Thermo fisher, MA, USA), and then reversed to cDNA using Reverse Transcription Kit (K1691, Thermo fisher, MA, USA). GAPDH was used as an endogenous reference gene, and the primer sequences were given in Table 1. The PCR experiments were repeated three times with three replicate wells each time, and the absolute relative mRNA content was finally determined by the qPCR algorithm (relative quantification, 2^{-Δ⁢Δ⁢Ct} method).

Mononuclear cells in blood were first isolated by density gradient centrifugation. The anticoagulated blood samples were diluted 1:1 in phosphate buffered saline (PBS) and then carefully layered on Ficoll-Paque solution (F4375, Sigma Aldrich, St. Louis, USA) allowed to centrifuge at 400 g for 30–40 minutes. After centrifugation, the interleukocyte layer (monocyte layer) was aspirated, placed in a new centrifuge tube and washed 2–3 times with PBS to remove residues. The isolation process was completed by finally resuspending the monocytes in PBS. After isolation, cells were washed and incubated with human Trustain Fc XTM (422302; BioLegend, San Diego, CA, USA) for 10 min at room temperature to capture the Fc receptor and cells were treated with anti-F4/80-Fluorescein isothiocyanate (FITC) (11-4801-82, Thermo fisher, MA, USA), anti-CD11b-PE-Cy7 (101215, Biolegend, CA, USA) and FVD-eFluor 506 (65-0866-14, Thermo fisher, MA, USA) staining at 4 °C for 30 min. After fixation and permeabilisation, intracellular staining was performed with anti-CD206-PE (12-2061-82, Thermo fisher, MA, USA) and anti-CD86-PE (ab77226, Abcam, Cambridge, UK). FVD-positive cells were considered to be live cells; F4/80 and CD11b-positive cells were identified as macrophages; F4/80-positive, CD11b-positive, and CD206-positive cells were defined as M2-type macrophages; and F4/80-positive, CD11b-positive, and CD86-positive cells were identified as M1-type macrophages [18]. Cells were analysed using a flow cytometer (LSRII; BD Bioscience, Franklin Lake, NJ, USA) and data were analysed using FlowJo software V10 (Tree Star, Ashland, OR, USA).

RAW264.7 cells were lysed using radio immunoprecipitation assay (RIPA) lysis buffer (ab170197, Abcam, Cambridge, UK) and total protein was collected. Total protein was determined using the bicinchoninic acid (BCA) Protein Quantification Kit (P0010, Beyotime, Shanghai, China). The same amount of protein samples was separated by SDS-PAGE electrophoresis and transferred to PVDF membranes using a wet transfer method (ab133411, Abcam, Cambridge, UK). Subsequently, membranes were incubated overnight at 4 °C with the following primary antibodies: TNFR2 (72337, Cellsignal, Danvers, MA, USA), tumor necrosis factor-$\alpha$ (TNF-$\alpha$) (1:1000, ab183218, Abcam, Cambridge, UK), inducible nitric oxide synthase (iNOS) (1:1000, ab178945, Abcam, Cambridge, UK), interleukin 6 (IL-6) (1:1000, ab233706, Abcam, Cambridge, UK), CD206 (1:5000, ab64693, Abcam, Cambridge, UK), IL-6 (1:1000, ab233706, Abcam, Cambridge, UK), CD206 (1:5000, ab64693, Abcam, Cambridge, UK), IL-10 (1:1000, ab133575, Abcam, Cambridge, UK), and Arginase 1 (Arg1) (1:2000, ab133543, Abcam, Cambridge, UK), GRN (1:1000, ab266738, Abcam, Cambridge, UK), GAPDH (1:2500, ab9485, Abcam, Cambridge, UK) were washed from the membranes and then the membranes were washed with secondary antibody (1:1000, ab6702, Abcam, Cambridge, UK) incubated for 2 hours. The membranes were developed using ECL (A38554, Thermo fisher, MA, USA) and the bands were analysed using ImageJ software (V1.8.0.112, NIH, Madison, WI, USA).

First, RAW264.7 cells were cultured to a density of 1 $\times$ 10⁴, and then the cells were fixed with an appropriate amount of 4% paraformaldehyde (MA0192, Melun, Dalian, China). Permeabilize the cell membrane using 0.1% Triton X-100 (BL934B, Biosharp, Hefei, China) washing solution. Next, incubate the cells overnight with anti-CD86 (1:100, ab239075, Abcam, Cambridge, UK) and anti-CD206 antibodies (1:1000, ab64693, Abcam, Cambridge, UK). Then, incubate the cells with the corresponding secondary antibody (1:200, ab150077, Abcam, Cambridge, UK) at room temperature (22–25 °C) for 1 hour. Incubate the cells with DAPI (P-CA-008, Procell, Wuhan, China) at room temperature for 5 minutes. Finally, observe and capture images using a confocal fluorescence microscope (Olympus, Tokyo, Japan).

RAW264.7 cells were lysed using immunoprecipitation buffer (S7705, Sigma Aldrich, St. Louis, USA), then cell lysates were incubated with agarose beads coupled with immunoprecipitating antibodies GRN (1:70, ab208777, Abcam, Cambridge, UK) and TNFR2 (1:100, ab7369, Abcam, Cambridge, UK) were incubated on agarose beads with IgG (ab172730, Abcam, Cambridge, UK) as a negative control. Unbound proteins were subsequently removed by washing the beads and the immunoprecipitated complexes were analyzed by WB. Bands were developed using ECL (A38554, Thermo fisher, MA, USA) and analyzed using ImageJ software (V1.8.0.112, NIH, Madison, WI, USA).

Statistical analysis was performed using GraphPad Prism9 (Dotmatics, Boston, MA, USA) software. Comparisons between groups were made using the Student’s t-test, and one-way ANOVA was used only for three or more groups, with p 0.05 being considered a statistical difference, and each experiment was repeated at least three times.

To determine the TNFR2 expression in PTB patients and the polarization status of macrophages. The expression level of TNFR2 in patients was detected by RT-qPCR and M1 and M2 type macrophages were analyzed using FC. The results showed that TNFR2 mRNA expression was significantly elevated in PTB patients compared with Control group (Fig. 1A, p 0.0001). Furthermore, we found that both M1 and M2 macrophages were increased in PTB patients, and a higher proportion of M2 macrophages were observed (Fig. 1B, p 0.05). This suggests that PTB occurs with elevated expression of TNFR2 and that macrophages are predominantly M2 polarized.

Fig. 1.

TNFR2 expression and macrophage phenotype in PTB patients and healthy volunteers. (A) TNFR2 mRNA expression detected by RT-qPCR. (B) Using FC to analyze the proportions of M1 and M2 macrophages in the peripheral blood of PTB patients and healthy volunteers. Specific markers were used to distinguish between M1 and M2 phenotypes. The data are presented as percentages of total macrophages. N = 40; *, p 0.05; ****, p 0.0001. TNFR2, tumor necrosis factor receptor 2; FC, flow cytometry; PTB, pulmonary tuberculosis; RT-qPCR, Quantitative Reverse Transcription Polymerase Chain Reaction; FITC-H, Fluorescein Isothiocyanate-Hyaluronic; UL, up left; UR, up right; LL, lower left; LR, lower right.

After confirming the expression of TNFR2 in the serum of PTB patients with M2-polarised macrophages, we performed in vitro experiments. We induced RAW264.7 into M2 macrophages using IL-4 and examined the expression level of TNFR2. The results showed that the mRNA expression of Tnfr2 was up-regulated (Fig. 2A, p 0.0001) and the expression of TNFR2 protein level was significantly increased (Fig. 2B, p 0.001) in IL-4-induced RAW264.7 cells compared to normal RAW264.7 cells. The results suggest that TNFR2 is highly expressed in M2-polarised macrophages.

Fig. 2.

TNFR2 levels in RAW264.7 cells and M2 macrophages. (A) RT-qPCR for Tnfr2 mRNA expression in M2 macrophages, (B) WB for TNFR2 protein expression in M2 macrophages. N = 3; ***, p 0.001; ****, p 0.0001. WB, Western blot.

A study has shown that the activity of TNFR2 correlates with changes in GRN [13]. We found that the expression of GRN was significantly elevated in the serum of PTB patients (Fig. 3A, p 0.001). To investigate the effect of GRN on macrophage polarization, GRN expression was knocked down in IL-4-induced RAW264.7 cells, and macrophage polarization was subsequently measured. Results showed that the level of GRN in the cells of the si-GRN group was significantly reduced compared with that of the si-NC group (Fig. 3B, p 0.001), indicating that the plasmid transfection was successful. Additionally, compared with the si-NC group, the expression of M1 polarization markers TNF-$\alpha$, iNOS and IL-6 was elevated in the si-GRN group (Fig. 3B,C, p 0.01), and the levels of M2 polarization-associated factors CD206, IL-10 and Arg1 were lowered (Fig. 3B,C, p 0.01). The results of IF detection showed that the fluorescence intensity of CD86 in the si-GRN group was significantly increased (Fig. 3D, p 0.001) and the fluorescence intensity of CD206 was decreased in the si-GRN group compared with the si-NC group (Fig. 3D, p 0.05). The results of FC (F4/80 is a macrophage surface marker in Mus musculus) showed that compared with si-NC, the macrophage type of si-GRN increased in M1 type (Fig. 3E, p 0.05) and decreased in type M2 (Fig. 3E, p 0.01). These results indicated that GRN could inhibit macrophage polarization to M1 polarization and promote macrophage metastasis to M2 polarization.

Fig. 3.

Macrophage status after GRN knockdown. (A) GRN mRNA Expression in Serum: RT-qPCR was used to detect the expression levels of GRN mRNA in the serum of pulmonary tuberculosis (PTB) patients and healthy volunteers. (B) RT-qPCR to detect the expression of Grn mRNA and M1 and M2 polarization-related markers in macrophages. Helps to gain insight into the changes in macrophage polarization state after GRN knockdown. (C) WB detection of the expression of GRN protein and M1 and M2 polarization related marker proteins in macrophages. Gain a detailed understanding of the impact of GRN knockout at the protein level. (D) The fluorescence intensities of CD86 and CD206 were detected by IF. Measurement of the fluorescence intensity of these markers provides a visual and quantitative assessment of the polarization status of macrophages. Magnification 400$\times$, scale bars, 100 µm. (E) FC analysis of macrophage polarization status. Data are expressed as a percentage of the total number of macrophages, highlighting the shift in polarization induced by GRN knockdown. N = 3; *, p 0.05; **, p 0.01; ***, p 0.001; ****, p 0.0001. NC, negative control; GRN, Granulin; IF, Immunofluorescence.

To further explore the interaction of GRN and TNFR2 in macrophage M2 polarization, we examined whether GRN binds to TNFR2 by Co-IP assay. Results demonstrated that TNFR2 was detected after immunoprecipitation with GRN antibody and GRN after immunoprecipitation with TNFR2 antibody, but the expression of both was not detected in the control IgG group, respectively (Fig. 4A), suggesting that there is a reciprocal binding effect of GRN and TNFR2. Furthermore, we transfected GRN or TNFR2 overexpressed plasmid into RAW264.7 cells, respectively. The increased expression of GRN and TNFR2 in the cells indicates successful transfection (Fig. 4B, p 0.05). Additionally, the OE-GRN2 and the OE-TNFR2-2 plasmids were used in the following studies. Subsequently, we found that the expression of TNFR2 was elevated when GRN was overexpressed (Fig. 4C, p 0.001), whereas there was no significant change in GRN expression when TNFR2 was overexpressed (Fig. 4D, p $>$ 0.05). These data suggest that GRN and TNFR2 combine in macrophages and that GRN positively regulates TNFR2 expression.

Fig. 4.

Validation of the relationship between GRN and TNFR2. (A) Co-IP combined with WB to detect the interaction between GRN and TNFR2. Protein lysates from macrophages are immunoprecipitated using antibodies specific for GRN or TNFR2. The precipitated complexes are then analyzed by WB to detect the presence of interacting proteins (TNFR2 is detected when GRN is immunoprecipitated and vice versa). (B) WB and RT-qPCR were performed to detect the expression of GRN and TNFR2 in RAW264.7 cells. (C) WB were used to detect the expression of TNFR2 in macrophages after GRN overexpression. This experiment was designed to determine whether increased levels of GRN affect TNFR2 expression, thereby suggesting a regulatory relationship between the two proteins. (D) WB were used to detect the expression of GRN in macrophages after TNFR2 overexpression. This analysis was designed to understand whether upregulation of TNFR2 affects GRN expression, thus further elucidating the interaction between these proteins. N = 3; ns, p $>$ 0.05; *, p 0.05; **, p 0.01; ***, p 0.001. Co-IP, Co-immunoprecipitation; ns, not significant.

We further overexpressed TNFR2 while knocked down GRN in IL-4-induced RAW264.7 cells and measured the macrophage M2 polarization level. The results showed that GRN and TNFR2 protein and mRNA expression was reduced in the si-GRN group compared to the si-NC group (Fig. 5A,B, p 0.0001), whereas there was no significant difference in GRN changes between the si-GRN+OE-NC and si-GRN+OE-TNFR2 groups (Fig. 5A,B, p $>$ 0.05), the expression of TNFR2 was increased (Fig. 5A,B, p 0.0001). In addition, TNF-$\alpha$, iNOS and IL-6 were elevated and CD206, IL-10 and Arg1 were significantly lower in the si-GRN group compared to the si-NC group (Fig. 5A,B, p 0.001). However, these effects were inversed by TNFR2 overexpression (Fig. 5A,B, p 0.01). Meanwhile, the immunofluorescence results showed that the CD86 expression was increased (Fig. 5C, p 0.01) while the CD206 expression was diminished (Fig. 5C, p 0.001). Nevertheless, the CD86 and CD206 expression altered by GRN silencing were reversed by TNFR2 overexpression (Fig. 5C, p 0.05). In addition, FC detection results showed that compared with si-NC cells, the number of M1 macrophages in si-GRN cells increased (Fig. 5D, p 0.0001), while the number of M2 macrophages decreased (Fig. 5D, p 0.0001). However, these effects were overturned by TNFR2 overexpression (Fig. 5D, p 0.01). These results suggest that inhibition of GRN expression can promote macrophage M1 polarization by down-regulating TNFR2 and that high expression of TNFR2 reverses this change and promotes macrophage transition to the M2 phenotype.

Fig. 5.

Expression of indicators and macrophage polarisation when knocking down GRN and overexpressing TNFR2. (A) RT-qPCR for mRNA expression of Grn, Tnfr2, and M1 and M2 polarization markers in macrophages. (B) WB detection of protein expression of GRN, TNFR2, and M1 and M2 polarization markers in macrophages. (C) The fluorescence intensities of CD86 and CD206 were detected by IF. Fluorescently labeled antibodies were used to visualize these markers, and the fluorescence intensity indicates the expression levels and spatial distribution of the M1 and M2 phenotypes. Magnification 400$\times$, scale bars, 100 µm. (D) FC analysis of macrophage phenotype. Data are expressed as a percentage of the total number of macrophages, providing a quantitative assessment of polarization status. N = 3; ns, p $>$ 0.05; *, p 0.05; **, p 0.01; ***, p 0.001; ****, p 0.0001.