Progranulin inhibits LPS-induced macrophage M1 polarization via NF-кB and MAPK pathways

After stimulated by 100 <ng/ml LPS or normal medium for 24 <h, LPS-stimulated RAW264.7 cells showed significantly higher expression of CD86, the special surface phenotype marker of M1, than negative control (Fig. 1a, b). In addition, mRNA and protein expression of iNOS and TNF-α, the special functional markers for M1, and secretion of TNF-α also significantly increased (Fig. 1c, d, e, f). Interestingly, compared to the control, LPS stimulation for 24 or 48 <h significantly down-regulated PGRN expression both in gene and protein expression (Fig. 1g, i, j) and its secretion was also significantly decreased after 48 <h stimulation (Fig. 1h). This suggests that PGRN may be involved in LPS-induced macrophage M1 polarization.

CCK-8 assays revealed that at concentrations below 80 <ng/ml, rPGRN significantly promoted cell proliferation in dose dependent fashion after cultured for 24 and 48 <h (Fig. 2a, b, d). Nevertheless, proliferative capacity of cells cultured at 160 and 320 <ng/ml rPGRN approached to flat, and even declined. Furthermore, the proliferative capacity of RAW264.7 cells treated with different concentrations of rPGRN was descending as processing time increases (Fig. 2a, b, c, d).

RAW264.7 cells were stimulated by LPS plus 0, 5, 10, 20, 40 and 80 <ng/ml rPGRN and normal medium containing equal amount of PBS to rPGRN and LPS groups was used as negative control. Their phenotype and function status were characterized by flow cytometry, q-PCR, Western blot and ELISA. The results showed that rPGRN at concentrations of 5, 10 and 20 <ng/ml significantly reversed LPS-promoted CD86/CD206 expression ratio, of which 20 <ng/ml rPGRN presented most obvious effect (Fig. 3a, b). Similarly, rPGRN significantly reversed LPS-enhanced mRNA of TNF-α and iNOS at concentrations from 5 to 80 <ng/ml, among which 10 <ng/ml rPGRN presented most obvious effect (Fig. 3c, d). It also inhibited LPS-enhanced protein expression of iNOS (at concentrations from 5 to 40 <ng/ml) (Fig. 3e, f) and TNF-α (at 5 and 10 <ng/ml) (Fig. 3e, g), as well as secretion of TNF-α (at 5 and 10 ng/ml) (Fig. 3h). This implies that PGRN can inhibit LPS-induced M1 polarization in RAW264.7 cells.

Previous research has identified that autocrine TNF-α plays a key role in apoptosis in LPS-induced macrophages [33]. It is also reported that PGRN exerts its anti-inflammatary action mainly via binding to TNFR1 to antagonize TNF-α pro-inflammation action [23]. To explore the key role of TNF-α and observe and conjecture whether PGRN anti-M1 polarization is related to TNFRs in LPS-induced macrophage M1 polarization, we stimulate RAW264.7 cells by rTNF-α, P.g-LPS and P.g-LPS plus anti-TNF-α. Results showed that no significant difference in CD86 expression existed between 20 to 40 ng/ml rTNF-α and the control groups and anti-TNF-α exerted no significant influence on P.g-LPS promoted CD86 expression (Fig. 4a, b). Nevertheless, 40 ng/ml rTNF-α significantly enhanced mRNA and protein expression of TNF-α and iNOS (Fig. 4c-g). More interestingly, anti-TNF-α significantly down-regulated LPS-stimulated expression of iNOS and intracellular TNF-α (Fig. 4c-g). This implies that secondary TNF-α expression followed by LPS stimulation plays a crucial role in LPS-activated M1 macrophage functional status.

To clarify the mechanism of PGRN inhibition of LPS-induced M1 polarization in RAW264.7, the change of NF-кB and MAPK signaling molecules was detected by Western blot. As shown in Fig. 5, rPGRN significantly reduced LPS-induced phosphorylation of IKKα/β (Fig. 5a-c), IкBα (Fig. 5d, e), p65 (Fig. 5f, g), JNK (Fig. 5h, i) and p38 (Fig. 5j, k) and NF-кB p65 translocation from the cytoplasm to the nucleus (Fig. 6), though without LPS stimulation it was able of activating the phosphorylation of IKKα/β (Fig. 5a-c), JNK (Fig. 5h, i) and p38 (Fig. 5j, k) vs negative control group. In addition, rPGRN significantly down-regulated phosphorylation of ERK though LPS had no effect on it (Fig. 5l, m).

To further demonstrate above results in RAW.264.7 cells, BMDMs and THP-1 cell line were used to verify the effect of PGRN on functional change induced by LPS. As in RAW264.7 cells, rPGRN at 10 ng/ml significantly reversed LPS-enhanced mRNA and protein expression of TNF-α and iNOS (Fig. 7a-f) and secretion of TNF-α (Fig. 7g).

RAW264.7 cells were obtained from Stem Cell Bank, Chinese Academy of Sciences. Mouse BMDMs were isolated from femur and tibia of C57BL/6 mice which were purchased from Institute of Shandong University Animal Experimental Center and differentiated into M0 macrophage by 25 ng/ml recombinant macrophage colony-stimulating factor (M-CSF) (R&D Systems, Minneapolis, MN, USA) treatment for 4 days. THP-1 cells (Stem Cell Bank, Chinese Academy of Sciences) were differentiated into M0 by 100 ng/ml PMA (Sigma, USA) treatment for 48 h. All cells were cultured in DMEM (Hyclone, Logan, UT, USA) containing 10% foetal bovine serum (FBS) (BioInd, Kibbutz, Israel) at 37 °C with 5% CO₂. When reached 80% confluence, cells were scraped, dissociated and counted and then plated in 6-well plates at a concentration of 2 × 10⁵/ml (in RAW264.7 cells) or 2 × 10^6/ml(in BMDMs and THP-1 cells). When reaching 60% confluence, cells were stimulated by 100 ng/ml P.g-LPS (InvivoGen, San Diego, CA, USA) with or without different doses of rPGRN (Sino Biological, Beijing, China) and TNF-α antibody (5 μg/ml; Abcam, Cambridge, UK) or a variety of concentrations of rTNF-α (Peprotech, Rocky Hill, NJ, USA) for 24 or 48 h, depending on different experimental goals. The normal medium containing equal amount of PBS to rPGRN and LPS groups was used as negative control.

The surface markers of stimulated cells were detected by flow cytometry. Briefly, RAW264.7 cells were collected from 6-well plates after stimulation for 24 h and washed three times with PBS. The cell suspension respectively containing 1 × 10⁶ M0-unpolarized or M1-polarized cells was then divided into 1.5 ml EP tubes and incubated with blocking antibody CD16/32 (Biolegend, San Diego, CA, USA) on ice for 10 min. After washed twice, cells were incubated in PBS plus Intrapore Permeabilization reagent and the following antibodies (PE anti-mouse CD86 and APC anti-mouse CD206 (both from Biolegend)) on ice for 30 min in the dark. After washed twice, cells was suspended in 500 μl PBS with 3% FBS and then detected by flow cytometry (BD Biosciences, San Diego, CA, USA).

RAW264.7 cells were counted and seeded in a 96-well plate (3000/well) and cultured in medium plus rPGRN at variable concentrations (5, 10, 20, 40, 80, 160, 320 ng/ml) or normal medium for 24, 48 and 72 h. Then, according to the instruction, the culture medium was replaced by 100 μl DMEM medium plus 10 μl cck-8 reagent (MCE, Shanghai, China) and the cells were incubated for another 2 h at 37 °C in 5% CO₂ incubator. Absorbance at 450 nm was read by a microplate reader (SPECTRO star Nano) and cell viability was verified by the percent of the absorbance of various concentrations versus control group.

Real-time reverse transcriptional polymerase chain reaction was performed as follows. Total cellular RNA was isolated from RAW264.7, THP-1 and BMDM cells with TRIzol reagent (Takara, Kusatsu, Japan) and then reverse-transcribed into cDNA with PrimeScript® RT reagent kit with gDNA Eraser (Takara) according to the concentration. Afterwards, Real-time PCR was performed with SYBR® Premix Ex Taq™ II (Takara). Analysis was performed on Light Cycler 96 Real-Time PCR System (Roche, Basel, Switzerland). The housekeeping gene GAPDH was used for normalization. The primers used in this study are shown in Table 1.

Cells were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer (Solarbio, Beijing, China) on ice for 30 min and decomposed with ultrasonic. Afterwards, the mixture was centrifugated at 12,000 rpm at 4 °C for 15 min to eliminate the dead cell debris. Protein concentration was detected by BCA protein assay kit (KeyGEN BioTECH, Nanjing, China). After denaturation with loading buffer at 100 °C, 20 μg protein samples were separated in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Membranes were blocked in 5% milk for 1 h, then covered with the primary antibodies overnight at 4 °C and incubated with anti-mouse or anti-rabbit secondary antibodies (1:10000; Proteintech, Chicago, IN, USA) for 1 h at room temperature on shaker. The protein bands were visualized with chemiluminescent HRP reagents (Millipore, Darmstadt, Germany). Image J 1.44 was used to analyze the protein expression. The primary antibodies were as follows: TNF-α (1:1000; CST, Danvers, MA, USA), iNOS (1:1000; Abcam, Cambridge, UK), Arg-1 (1:500; Santa Cruz, CA, USA), PGRN (1:1000; Abcam), NF-кB p65 (1:1000; CST), phospho-NF-кB p65 (1:1000; CST), IкBα (1:1000; CST), phospho-IкBα (1:1000; CST), IKKα (1:500; Santa Cruz), rabbit anti-IKKβ (1:1000; CST), phospho-IKKα/β (1:1000; CST), p38 (1:1000; Abcam), phospho-p38 (1:1000; Abcam), JNK (1:1000; Abcam), phospho-JNK (1:1000; Abcam), ERK1/2 (1:1000; Abcam), phospho-ERK1/2 (1:1000; Abcam), GAPDH (1:10000; Proteintech).

Cell supernatant was collected from RAW264.7, THP-1 and BMDM cells, centrifuged at 12,000 rpm at 4 °C for 10 min and the concentrations of TNF-α and PGRN were measured with ELISA kits (Novus Biologicals, CO, USA or Abcam, Cambridge, UK). All samples were assayed in triplicate and measured at 450 nm wavelength.

RAW264.7 cells plated in 24-wells plate were fixed with 4% paraformaldehyde for 10 min in a fume hood, permeabilized using 0.5% Triton X-100 (Solarbio, Beijing, China) for 10 min and rinsed three times with cold PBS for 5 min for each. After being blocked with 5% goat serum for 1 h, cells were incubated with an anti-NF-κB p65 primary antibody (1:500; CST) overnight at 4 °C. After washed with cold PBS twice, 1‰ Tween PBS once and incubated with Alexa Fluor 594-conjugated goat anti-rabbit IgG secondary antibody (1:500; Proteintech) for 1 h in the dark, nuclei were stained with DAPI (Proteintech). Images were detected with fluorescence microscope (OLYMPUS, Tokyo, Japan).

Experiment results were expressed as the mean ± SD of at least 3 independent experiments and GraphPad Prism 7 software (San Diego, CA) was used for statistical analysis. Difference between groups was assessed by Unpaired two-tailed Student’s t-test and one-way ANOVA. Statistical significance was expressed as P < 0.05 (*), P < 0.01 (**), P < 0.001 (***) or p < 0.0001(****).