INCB24360 reduces iNOS expression in RAW264.7 and BV-2
IFN-γ polarizes macrophage to M1 type, increases IDO-1 expression (20). To test the IDO-1 inhibitor's effect on the macrophage polarization, we have treated RAW264.7 and BV-2 with IFN-γ at 20ng/mL to increase IDO-1 expression and used 20μM 1-MT and 20μM INCB24360 to inhibit IDO-1 activity. After 24h treatment, we have detected CD206, an M2 marker, iNOS, an M1 marker, IDO-1 by western blotting. Consistent with previous findings (21, 22), we found that IFN-γ treatment increased IDO-1 and iNOS expression in RAW264.7 and BV-2 (Fig.1A, B, C, Si-Fig.1A). INCB24360 treatment reduced IDO-1 expression in RAW264.7 but not in BV-2 (Fig.1B, Si-Fig.1A), INCB24360 treatment reduced iNOS expression in RAW264.7 and BV-2(Fig.1C, Si-Fig.1A). The decreases of iNOS induced by INCB24360 but not 1-MT treatment are statistically significant in RAW264.7 (Fig.1C). 1-MT and INCB24360 treatment did not affect CD206 expression in RAW264.7 (Fig.1D). We also detected the transcription of iNOS, TNF-α, CD206, and Arginase 1 (Arg1) genes by RT-PCR. Our data showed that INCB24360 significantly decreased the transcriptions of iNOS, TNF-α in RAW264.7 and BV-2 compared to the control and IFN-γ treated RAW264.7 (Fig.1E, F) and BV-2 (Si-Fig.1B, C); 1-MT treatment only decreased the transcriptions of iNOS, TNF-α in BV2 cells but not in RAW264.7 (Fig.1E, F, Si-Fig.1B, C). The CD206 gene transcriptions were increased in INCB24360 treated RAW264.7 and BV-2 compared to the control group (Fig.1G, Si-Fig.1D); The transcriptions of Arg 1 gene also were increased in INCB24360 treated RAW264.7 but not BV-2 (Fig.1H, Si-Fig.1E). However, 1-MT treatment did not change the transcriptions of Arg 1 gene in RAW264.7 and BV-2 compared to the control group (Fig.1H, Si-Fig.1E).
Consistent with western blotting, immunofluorescent staining of RAW264.7 and BV-2 treated with INCB24360 and I-MT showed that iNOS expression significantly decreased INCB24360 groups but not CD206 compared to the control group (Fig.1I-K, Si-Fig.1F-H). The data indicate that INCB24360 might preferentially inhibit M1-like RAW264.7 and BV-2 formation.
INCB24360 suppresses the increase of cellular body and membrane filopodia formation of RAW264.7 and BV-2
The cellular morphological changes were correlated with different functional status (23). Non-polarized RAW264.7 is a small and round cell with few processes (20). If polarized, RAW264.7cells form a large round pancake-like cell, representing M1, or a slimmer cell with longer processes, representing the M2 stage (20). To see whether the decrease of iNOS expression in RAW264.7 and BV-2 after INCB24360 treatment parallel with the cellular morphological changes and filopodia formation, we have assessed the morphology of RAW264.7 and BV-2 after IFN-γ, 1-MT, and INCB24360 treatment (Fig.2A, Si-Fig. 2A). IFN-γ treatment dramatically increased the total polarized RAW264.7 and BV-2, while 1-MT or INCB24360 treatment did not significantly change the total polarized RAW264.7 count (Fig.2B) and BV-2 (Data not shown). We divided the polarized macrophage into M1 or M2 macrophage based on their morphology. Consistent with other observations(20), our results showed that IFN-γ treatment preferentially increased M1 RAW264.7 and BV-2 and slightly decreased M2 macrophage(Fig.2C, D, Si-Fig. 2B, C); INCB24360 treatment significantly decreased the proportion of M1 RAW264.7 and BV-2 compared to the control and IFN-γ treated group (Fig.2C, Si-Fig.2B). 1-MT treatment did not change the proportion of M1 and M2 in RAW264.7 (Fig.2C, D). However, both 1-MT and INCB24360 slightly increased the proportion of M2 BV-2 compared to the control group (Si-Fig.2C). These observations are consistent with the formation of an M1-like macrophage.
M1 macrophage showed multiple filopodia that facilitate the phagocytizing of fluid or foreign substances, including inorganic particles (24). We have treated RAW264.7 and BV-2 cells with IFN-γ, 1-MT, and INCB24360 for 24h, fixed the cells and stained with phalloidin Alexa-488, an actin-binding dye, and DAPI. We have scanned cells with high-resolution confocal microscopy and observed that macrophages induced by IFN-γ formed a ruffled border decorated with abundant filopodia (Fig.2E, Si-Fig.2D). Some RAW264.7 in IFN-γ treated group formed multi-nucleus cells that resemble osteoclasts (Data not shown). INCB24360 treated macrophages showed fewer filopodia than the control, IFN-γ and 1-MT groups in RAW264.7 and BV-2 (Fig.2E, Si-Fig.2D). We further measured the cellular perimeter and counted the density of filopodia on the cellular membrane. The data showed that 1-MT and INCB24360 treatment did not change the cellular perimeter and decreased filopodia density on the cellular membrane (Fig.2F, G, Si-Fig 2E, F). Collectively, these findings implied that INCB24360 and 1-MT preferentially block the formation of M1-like macrophages.
INCB24360 reduces the TMR-dextran uptake, macropinocytosis, and phagocytic capacity of RAW264.7 and BV-2
To see the decrease of iNOS expression and reduction of filopodia and ruffled border by INCB24360 treatment also decrease the phagocytic ability in RAW264.7 and BV-2, we have administrated TMR-dextran into RAW264.7 and BV-2 after treating IFN-γ, 1-MT, and INCB24360 for 24h. After 1 hour of TMR-dextran treatment, we fixed the cells and counter-stained by phalloidin-Alex-488 and DAPI. After scanning under microscopy with Z-stack, we have analyzed the total amount of dextran in four group cells by ImageJ. The results showed that INCB24360 treatment significantly reduced DTR-dextran uptake compared to the control and IFN-γ treated group in RAW264.7 but not in BV-2 (Fig.3A, 3B, 3D, 3E).
Macropinocytosis is a way by which macrophage uptakes large amounts of extracellular fluid (25). Macropinosome formation was dependent on membrane ruffling. The decrease of filopodia on the ruffled border after the INCB24360 treatment implied that INCB24360 might reduce the macropinocytosis of RAW264.7 and BV-2. The macropinosomes' size is heterogeneous; the diameter range from 0.2μm to 5μm (26). The vesicles, which are larger than 0.75µm, were defined as apparent macropinocytic vesicles in DTR-dextran uptake assays by some researchers (27). We have analyzed the macropinocytic vesicles that were larger than 0.75µm by Imaris9.6 software and found that INCB24360 treatment decreased the number of phagocytic vesicles larger than 0.75µm in RAW264.7 and BV-2 (Fig.3A, C, D, F).
Macrophage, especially tissue-resident macrophage, can also phagocytize foreign-derived particulates such as alum, silica et al. M1 macrophage can phagocytize foreign-derived particulates (28, 29). We also treated RAW264.7 and BV-2 with Latex beads, a spherical polymer particle, fluorescence red, and 1µm, after treating with IFN-γ, 1-MT and INCB24360 for 24h. After fixing with 4% PFA, we analyzed the phagocytized Latex beads in both cells and found that RAW264.7 (Data not shown) has a weaker ability to phagocytize Latex beads compared to BV-2. We observed that 1-MT and INCB24360 treatment slightly decrease the Latex beads uptake of BV-2 compared to the control, but the decreases are not statistically significant (Fig.3G-J).
Contrary to M2, M1 is weak in motility (30, 31). We also test 1-MT and INCB24360 effects on the migration and proliferation of macrophages; we have used the Transwell Migration Assay to test the migrating ability and Edu staining to test the proliferative potential. Consistent with the formation of M1 macrophage, the results showed that IFN-γ dramatically decreases the migration ability of RAW264.7 and BV-2(Si-Fig.3A-C) but not effects the proliferation of RAW264.7 and BV-2(Si-Fig.3D-F); 1-MT and INCB24360 do not affect the migration and proliferation of RAW264.7 and BV-2 (Si-Fig.3). Taken together, 1-MT and INCB24360, especially INCB24360, preferentially inhibit the phagocytic ability and macropinocytosis of the M1-like macrophage but not migrating and proliferating capacity.
INCB24360 suppresses IFN-γ-induced iNOS upregulation in RAW264.7
INCB24360 significantly blocks M1 RAW264.7 and BV-2 formation. IFN-γ increased IDO-1 expression (9). Thus, we hypothesized that INCB24360 might suppress IFN-γ induced iNOS increase. To test if 1-MT and INCB24360 suppress IFN-γ-induced iNOS upregulation, we treated RAW264.7 and BV-2 with IFN-γ for 12h, and then added 1-MT and INCB24360 for 24h. We have detected the expression CD206, iNOS, and IDO-1. Both 1-MT and INCB24360 treatment in the IFN-γ treated group significantly decreased iNOS levels but not changed CD206 levels compared to the IFN-γ treatment group in RAW264.7 (Fig.4A-C); more decreases of iNOS were observed in the INCB24360 group (Fig.4B); 1-MT and INCB24360 did not change iNOS and CD206 expression in IFN-γ induced BV-2 (Si-Fig.4A). We also detected the transcription of iNOS, TNF-α, CD206, and Arg1 genes by RT-PCR in four group cells. Our data showed that INCB24360 treatment suppressed the transcriptions of iNOS, TNF-α in IFN-γ induced RAW264.7 and BV-2 (Fig.4E, F, Si-Fig.4B, C); INCB24360 treatment significantly increased the transcriptions of CD206 but not Arg1 genes in IFN-γ treated RAW264.7 (Fig.4H), 1-MT treatment does not significantly affect the transcription of iNOS, TNF-α, CD206, and Arg1 genes in IFN-γ treated RAW264.7(Fig.4E-H). In agreement with western blotting results, immunofluorescent staining of results showed that iNOS expression significantly decreased in IFN-γ induced cells treated with INCB24360 or 1-MT compared to the IFN-γ treatment RAW264.7 and BV-2(Fig.4I, J, Si-Fig.4D, E). 1-MT treatment significantly increased CD206 expression in IFN-γ induced RAW264.7, 1-MT and INCB24360 increased CD206 expression in IFN-γ induced BV-2 (Fig.4I, K, Si-Fig.4D, F).
To test if INCB24360 also inhibits IFN-γ-induced cellular size and filopodia growth, we treated RAW264.7 and BV-2 with IFN-γ for 12h and then added 1-MT or INCB24360 for 24h. After imaging, we divided the macrophage into M1 or M2 based on macrophage morphology and found that INCB24360 treatment not only decreased the total polarized RAW264.7(Fig.5A, B) and BV-2(Data not shown) driven by IFN-γ but INCB24360 treatment preferentially decreased IFN-γ-induced M1 RAW264.7 and BV-2 (Fig.5A, C, Si-Fig.5A, B). Interestingly, INCB24360 treatment significantly increased IFN-γ-induced M2 macrophage proportion (Fig.5A, D, Si-Fig.5A, C). 1-MT treatment decreased the count of total IFN-γ-induced polarized macrophage, and this decrease did not have a preference for M1 or M2 (Fig.5A-D, Si-Fig.5A-C). The findings further support that INCB24360 can reverse IFN-γ driven M1 macrophage formation.
To test if IDO-1 inhibitors block IFN-γ-driven ruffled border formation and filopodia growth, we treated RAW264.7 and BV-2 with IFN-γ for 12h, and then added 1-MT or INCB24360 for 24h, and fixed the cells, and stained with phalloidin Alexa-488 and DAPI. We also scanned the cells with confocal microscopy with Z-stack, analyzed cellular perimeters, and filopodia density on the cellular border. Our data showed that INCB24360 inhibits IFN-γ induced ruffled border and filopodia formation in RAW264.7 and BV-2 (Fig.5E, Si-Fig.5D). The measuring and counting data showed that both 1-MT and INCB24360 treatment significantly inhibited the increase of cellular size induced by IFN-γ in RAW264.7 (Fig.5F) but not in BV-2 (Si-Fig.5E); INCB24360 treatment also inhibits the increase of filopodia density on the cellular membrane induced by IFN-γ (Fig.5G, Si-Fig.5F); 1-MT treatment does not restrict the increase of filopodia density on the cellular membrane induced by IFN-γ (Fig.5G, Si-Fig.5F). Our data showed that INCB24360 inhibits IDO-1 induced cellular size increase and filopodia formation. 1-MT weakly inhibits IDO-1-induced cellular size increase.
INCB24360 suppress IFN-γ induced endocytic, macropinocytic phagocytic ability
IFN-γ increased the phagocytic ability of RAW264.7 and BV-2 (Fig.3A, D). 1-MT and INCB24360 reduce the endocytic, phagocytic, and macropinocytic abilities of RAW264.7 and BV-2. We hypothesized that 1-MT and INCB24360 can decrease the endocytic and macropinocytic ability induced by IFN-γ. To test this hypothesis, we have treated BV-2 and RAW264.7 with IFN-γ for 12 hours and then treated with 1-MT and INCB24360 for another 12 hours. After that, we have administrated DTR-dextran to the culturing medium. After treating with DTR-dextran for 1h, we have fixed the cells and counter-stained by phalloidin Alex-488. After scanning under microscopy with Z-stack, we have analyzed the total amount of dextran in four group cells. The results showed that INCB24360 decreased TMR-dextran uptake ability in both RAW264.7 and BV-2; 1-MT significantly decreased TMR-dextran uptake ability in RAW264.7 but not in BV-2 (Fig.6A, B, D, E). We have analyzed the DTR-dextran phagocytic vesicles that were larger than 0.75µm, which is reflective of macropinocytosis, by Imaris9.6 software and found that INCB24360 treatment decreased the number of phagocytic vesicles larger than 0.75µm in RAW264.7 and BV-2, and 1-MT significantly decreased the number of phagocytic vesicles larger than 0.75µm in RAW264.7 but not in BV-2 (Fig.6A, C, D, F).
To test the effect of 1-MT and INCB24360 on macrophage phagocytic ability, we treated RAW264.7 and BV-2 with IFN-γ for 12h, and then added 1-MT or INCB24360 for 24h, and added Latex beads to the culturing medium to phagocytize. After fixing, we analyzed the phagocytized Latex beads in the two cells and found that INCB24360 and 1-MT treatment significantly decreased the count of BV-2 that phagocytize Latex beads, the number of phagocytic latex beads in each IFN-γ induced BV-2 treated with INCB24360 was significantly lower than that in IFN-γ induced BV-2, the number of phagocytic latex beads in each IFN-γ induced BV-2 treated with 1-MT was not significantly lower than that in IFN-γ induced BV-2 (Fig.6G-J). Collectively, I-MT and INCB24360, especially INCB24360, preferentially inhibit the endocytic, phagocytic, and macropinocytic ability of M1-like macrophages induced by IFN-γ.
1-MT and INCB24360 reduced NLRP3 expression and NLRP3 gene transcription in RAW264.7 and BV-2
NLRP3 inflammasome, a large protein complex, includes NLRP3, ASC, and caspase 1 (32). NLRP3 inflammasome is involved in M1 macrophage formation and increases IL-1β production in inflammation (33). Curcumin, an IDO-1 inhibitor, inhibits chronic unpredictable mild stress (CUMS) induced NLRP3 expression, reduces depressive-like behaviors in rats (34). Thus, we hypothesized that 1-MT and INCB24360 might also suppress NLRP3 expression. To test if the inhibition of IDO-1 with 1-MT and INCB24360 reduce NLRP3 expression, we have treated RAW264.7 and BV-2 with IFN-γ, 1-MT, and INCB24360 for 24h and detected NLRP3 expression by western blotting, and found that both 1-MT and INCB24360 decreased NLRP3 expression in RAW264.7 and BV2 but not caspase-1 (Fig.7A), the decreases of NLRP3 expression in INCB24360 group are significant (Fig.7B), the reduction of NLRP3 expression in the 1-MT group is statistically non-significant (Fig.7B). We also detected NLRP3 and Caspase-1 gene transcription by RT-PCR. The data showed that INCB24360 reduced the transcription of the NLRP3 gene but not Caspase-1 (Fig.7D, E, Si-Fig-6B, C). We also used immunostaining with NLRP3 and iNOS antibodies to detect the changes in NLRP3 and iNOS expression. Consistent with immunoblotting and RT-PCR results, INCB24360 treatment significantly decreases NLRP3 expression in RAW264.7 (Fig.7F, G) and BV-2(Si-Fig.6D, E).
To test if 1-MT and INCB24360 also can decrease IFN-γ induced NLRP3 expression, we also treated RAW264.7 with IFN-γ for 12h, and then added 1-MT or INCB24360 for 24h to inhibit IDO-1 activity. Our data showed that both 1-MT and INCB24360 could inhibit IFN-γ induced NLRP3 expression but not caspase-1 (Fig.7I-K). The RT-PCR data also showed that INCB24360 and 1-MT reduced IFN-γ induced NLRP3 gene transcription in both RAW264.7 and BV-2 but not caspase-1(Fig.7L, M, Si-Fig.6H, I). We have immunostained those cells with NLRP3 and iNOS antibodies, and immunofluorescent staining results in RAW264.7 are consistent with protein blotting results (Fig.7N-P, Si-Fig.6J-L).
1-MT and INCB24360 treatment moderately increase IL-1β secretion in RAW264.7 and BV-2
NLRP3 inflammasome activates caspase 1 that cleaves pro-IL-1β and pro- IL-18 and secretes IL-1β and IL-18 (32). Inhibiting IDO with 1-MT dramatically surge the proinflammatory cytokine secretion of macrophage in influenza virus infection, including IFN-γ, IL-1β, IL-6, and TNF-α (10). To test how the modest decrease of NLRP3 expression by 1-MT and INCB24360 treatment effects IL-1β and IL-18 secretion, we have treated RAW264.7 with IFN-γ at 20ng/mL, 20μM 1-MT, and 20μM INCB24360. After treating for 24h, we have detected IL-1β and IL-18 secretion levels in the medium by ELISA. Our results showed that IL-1β and IL-18 levels in the culturing medium of IFN-γ, 1-MT, and INCB24360 treated RAW264.7 and BV-2 significantly increased compared to the control (Fig.8A, B, Si-Fig.7A, B). It implied that endogenous IDO-1 might can prevent the overactivation of macrophages.
Mammalian Target of Rapamycin (mTORC1) is a nutrient-dependent kinase regulating cellular growth and metabolism (35). Tryptophan deprivation by IFN-γ induced IDO-1 upregulation inhibits mTORC1 kinase, and tryptophan or 1-MT treatment reversed IDO-1 mediated inhibition of mTORC1 kinase; mTORC1 stimulation by 1-MT relies specifically on tryptophan depletion (36, 37). mTORC1 inhibition also decreased glycolysis and suppressed both LPS and ATP induced pro-IL-1β maturation and caspase-1 activation in macrophages (38). Thus, we hypothesized that INCB24360 treatment reduced tryptophan deprivation induced by IFN-γ in the milieu, consequently activating mTORC1. We have used to a phosphorylated (p) S6K (T389) antibody to evaluate mTORC1 activation levels in RAW264.7. Interestingly, we did not observe a direct effect of 1-MT and INCB24630 treatment on pS6K (T389) levels in RAW264.7 (Fig.8C). We observed that inhibiting IFN-γ induced IDO-1 with 1-MT and INCB24360 increases IL-1β and IL-18 secretion in RAW264.7 and BV-2(Fig. 8D, E, Si-Fig.7C, D). The increase of IL-1β secretion in INCB24360 treated RAW264.7 and BV-2 induced by IFN-γ were significantly higher than that in RAW264.7 and BV-2 induced by IFN-γ alone (Fig. 8D, Si-Fig.7C). Interestingly, 1-MT significantly increased IL-18 secretion in RAW2 and BV-2 induced by IFN-γ but not IL-1β (Fig. 8D, E, Si-Fig.7C, D). Also, in agreement with other findings (36), inhibiting IFN-γ induced RAW264.7 with 1-MT and INCB24360 dramatically increased in pS6K (T389) levels (Fig. 8F). Activation of mTORC1 by 1-MT and INCB24360 in IFN-γ induced might partially explain why 1-MT and INCB24360 increase IL-1β secretion in RAW264.7.
Curcumin inhibits CUMS-induced NLRP3 expression (34). NLRP3 deficiency also inhibits the LPS-induced IDO increases in the hippocampal microglia (39). These data indicate that IDO-1 and NLRP3 regulated each other in macrophages. Thus, we hypothesized that inhibiting NLRP3 by its inhibitors might affect IDO-1 expression in macrophages. To test if NLRP3 inhibition affects endogenous IDO-1 levels in RAW264.7, we have inhibited NLRP3 in RAW264.7 with MCC950, an inhibitor of NLRP3 targeting its ATP-hydrolysis (40), and Oridonin, a covalent NLRP3 inhibitor (41). After exposing RAW264.7 to MCC950 and Oridonin for 24h, we have detected NLRP3 and IDO-1 expression (Fig. 8G). Our western blotting and immunofluorescent staining data showed that inhibiting NLRP3 with Oridonin and MCC950 increased IDO-1 expression in RAW264.7 (Fig. 8G-J). Our data indicate that NLRP3 and IDO-1 regulate each other in macrophages.