IRG1/itaconate axis promotes efferocytosis of BMDMs

We treated RAW 264.7 cell line with the itaconate derivative OI (100 µM) or PBS and performed RNA sequencing analysis (Fig. 1A). Enrichment analysis of the Gene Ontology pathway showed that phagocytosis-related pathways, vesicle cytoskeletal trafficking and apoptotic cell clearance pathway were significantly enriched (Fig. 1B). Gene Set Enrichment Analysis also showed significant enrichment of regulatory phagocytics engulfment (Fig. 1C). Then, we treated RAW264.7 with OI (100 µM) and PBS for 12 h, and then co-cultured apoptotic thymocytes with CFSE-labelled apoptotic thymocytes. Compared with the Vehicle group, the efferocytosis of RAW264.7 treated with OI was significantly enhanced (Fig. 1D-E, Supplement Fig. 1B). To explore the optimal concentration and co-culture time of OI to enhance efferocytosis, BMDMs were extracted (Supplement Fig. 1A) from WT mice and co-cultured with CFSE-labelled apoptotic thymocytes as indicated. We found that OI (100 µM) had the strongest effect on promoting efferocytosis(Fig. 1F). Therefore, OI (100 µM) was used as the experimental concentration in subsequent experiments. Simultaneously, OI significantly enhanced the efferocytosis rate of BMDMs. The percentage of efferocytosis did not change with time after cocultivation for 1 h (Fig. 1G).

Fig. 1

IRG1/ itaconate axis promotes the efferocytosis of BMDMs. A RAW 264.7 cells were treated with Vehicle or OI to perform RNA-seq analysis. B The enriched GO pathways from upregulated genes between Vehicle or OI treated RAW 264.7 cells. C A GSEA analysis of phagocytosis engulfment was shown. D-E RAW 264.7 cells were treated as indicated and incubated with apoptotic thymic cells, and then flow cytometry was performed. F BMDMs isolated from C57BL/6 were treated with Vehicle or different concentrations of OI and incubated with apoptotic thymic cells to evaluate the efferocytosis between the groups. G WT BMDMs were exposed to indicated treatments at different time points and the efferocytosis was analyzed using flow cytometry. H The protein level of IRG1 was examined in BMDMs from WT and IRG1-/- mice. I-J Flow cytometry was performed to analysis the efferocytosis between WT and IRG1-/- BMDMs with indicated treatments. K-L WT and IRG1-/- BMDMs were treated as indicated and immunofluorescence staining was performed. Data represent the mean ± SD. n = 3, *p < 0.05, **p < 0.01, ***p < 0.001 vs. Veh. ###p < 0.001 vs. WT BMDMs

To further elucidate the role of IRG1 in macrophage efferocytosis, BMDMs were extracted from IRG1 knockout mice and the absence of IRG1 were verified by western blotting (Fig. 1H). Flow cytometry revealed that efferocytosis of BMDMs extracted from IRG1-/- mice was significantly impaired, and exogenous itaconate rescued this dysfunction (Fig. 1I-J, Supplement Fig. 1C). Next, we verified that OI could significantly enhance efferocytosis of BMDM, IRG1 deficiency significantly attenuated the efferocytosis, and supplementation with exogenous OI restored the dysfunction of BMDM in IRG1 knockout mice by cellular immunofluorescence technology (Fig. 1K-L). The above data suggest that the IRG1/itaconate axis plays a crucial role in modulating BMDM efferocytosis.

IRG1/itaconate axis promotes nuclear translocation of Nrf2 and enhances the expression of TIM4 and TIM4-mediated efferocytosis

Our findings confirmed that OI significantly promoted Nrf2 nuclear translocation and inhibited Keap1 expression in a concentration-dependent manner (Fig. 2A). However, no studies have reported how Nrf2 regulates efferocytosis. We found that OI significantly upregulated the transcription of TIM4 in RAW 264.7 cell line by RNA sequencing (Fig. 2B). Then, to determine the role of TIM4 in the efferocytosis of BMDMs, we screened the common genes -TIM4, MERTK, SIPR1, AXL, CD14, associated with efferocytosis in the treated BMDMs by real-time quantitative PCR. It was showed that OI significantly promote the transcription of TIM4 in BMDMs (Fig. 2C). Flow cytometry analysis revealed that OI significantly promoted TIM4 expression(Fig. 2D-E) and enhanced TIM4-mediated efferocytosis (Fig. 2F-G). Additionally, OI significantly upregulated the levels of Nrf2 and its downstream molecules HO1 and NQO1 in IRG1-/- and WT BMDMs, and promoted the expression of TIM4 in BMDM and the downstream molecules RAC1 and ElMO1 during efferocytosis, thus playing an anti-inflammatory role (Fig. 2H). To verify whether OI enhances efferocytosis by regulating TIM4, we pretreated WT BMDM with Ab-TIM4 or IgA before administering OI or vehicle, and found that Ab-TIM4 inhibited normal efferocytosis. The promotion effect of OI disappeared after the addition of Ab-TIM4(Fig. 2I-J). Previous study has shown that TIM4 and MERTK often mediate efferocytosis synergically [45]. In order to verify that OI promotes efferocytosis by regulating Nrf2-TIM4, rather than MERTK, the MERTK-related efferocytosis was determined by flow cytometry. The results showed that the OI treatment did not promote the expression of MERTK and MERTK-mediated efferocytosis (Supplementary Fig. 1D-F). In addition, OI did not alter the mRNA levels of MERTK in BMDMs as expected (Fig. 2C).These results suggest that OI promotes macrophage efferocytosis by regulating the Nrf2-TIM4 axis.

Fig. 2

IRG1/ itaconate axis promotes Nrf2 nuclear translocation, enhances the expression of TIM4 and TIM4-mediated efferocytosis. A WT BMDMs were treated with Vehicle or OI with different concentrations for 12 h, and Western blotting was performed. B The differential expressed genes are shown with a volcano plot in RAW 264.7 cells treated with indicated treatments detected using RNA-seq. C The mRNA levels of efferocytosis-related genes in WT BMDMs were analyzed after treatment 12 h by using real-time RT-PCR. D-G Expression of TIM4 and TIM4-mediated efferocytosis in each group of BMDMs was detected using flow cytometry. H Expression of relevant proteins in each group of BMDMs was detected using WB. I-J The efferocytosis of macrophages was detected after the addition of Ab-TIM4 and OI. Data represent the mean ± SD. n = 3, *p < 0.05, **p < 0.01, ***p < 0.001 vs. Veh. ##p < 0.01 and ###p < 0.001 vs. IRG1-/- BMDMs treated with OI

Nrf2 inhibition and Nrf2 knockout attenuates the efferocytosis of macrophages

ML385 serves as an inhibitor of Nrf2 which binds to Nrf2 and suppresses the expression of its target genes downstream, displaying anti-inflammatory properties. BMDMs were treated with ML385 2 h before the addition of OI (Fig. 3A). We found that ML385 inhibited efferocytosis and blocked the pro-efferocytotic effect of OI (Fig. 3B-C). In addition, ML385 treatment suppressed the RAC1 and ELMO1 expression in BMDMs(Fig. 3D). The immunofluorescence results were also consistent with the flow cytometry results (Fig. 3E-F). ML385 significantly inhibited TIM4 expression (Fig. 3G-H) and thereby inhibiting TIM4-mediated efferocytosis (Fig. 3I-J). Nrf2 expression and the downstream proteins HO-1 and NQO1 were significantly inhibited after ML385 treatment and that TIM4 was also significantly inhibited (Fig. 3K). BMDMs were extracted from Nrf2-/- mice and the Nrf2 knockout was verified using WB (Fig. 3L). In Nrf2-/- mice, there were no significant differences in efferocytosis, TIM4 expression and TIM4-mediated efferocytosis between the OI and Vehicle treatment groups (Fig. 3N-P).

Fig. 3

Nrf2 inhibition and Nrf2 knockout attenuates the efferocytosis of macrophages. A Schematic procedure of drug treatment. B-C WT BMDMs were exposed to ML385 and OI, the efferocytosis was analyzed using flow cytometry. D Expression of RAC1 and ELMO1 was detected in each group by WB. E–F Immunofluorescence staining was used to verify the efferocytosis of macrophages after indicated treatments. G-J Expression of TIM4 and TIM4-mediated efferocytosis in each group of BMDMs was detected. K The protein levels of Nrf2, Keap1, HO-1,NQO-1, and TIM4 were examined in WT BMDMs treated with ML385 and OI. GAPDH serves as an internal control of the total protein. L Nrf2 gene knockout was verified by WB. M-P The efferocytosis, expression of TIM4 and TIM4-mediated efferocytosis was detected. Data represent the mean ± SD. n = 3, *p < 0.05, **p < 0.01, ***p < 0.001

OI promotes macrophage proliferation and polarization to M2 macrophages

CCK-8 assays were performed to assess the influence of OI on the proliferation of BMDMs in each group. We found that the addition of apoptotic thymic cells promoted the proliferation of macrophages, whereas OI further promoted the proliferation of efferocytotic macrophages. The number of BMDMs pretreated with AC + and AC + OI + significantly increased at 48-h timepoint (Fig. 4A). EdU, a thymidine analogue that can replace thymidine in DNA synthesis process, was used to analyse macrophages proliferation. Consistent with the results of CCK8 experiment, the percentage of EdU + macrophages was higher in the AC + and AC + OI + groups than in the AC − macrophage group at 48-h timepoint, indicating that OI promotes the proliferation of efferocytosis macrophages (Fig. 4B-C). Then, macrophages were pre-labelled with CFSE, a cytoplasmic dye that is used to detect cell proliferation. There is a substantially higher level of CFSE dilution in macrophages with the addition of AC or AC + OI than in macrophages without the addition of ACs at 48-h timepoint (Fig. 4D-E), suggesting that AC + and AC + OI + macrophages underwent cell proliferation. Simultaneously, OI promoted the proliferation of BMDMs that underwent efferocytosis. Using scellular immunofluorescence techniques, we found that the percentage of cells expressing Ki67, a cell proliferation marker, increased significantly in the AC + BMDM and AC + OI + BMDM groups at 48-h timepoint (Fig. 4F-G). Finally, we detected M1 and M2 polarization markers, and found that efferocytosis promoted the transformation of BMDMs into M2 macrophages, and OI further promoted their transformation into M2 macrophages (Fig. 4H-I).

Fig. 4

OI promotes macrophage proliferation and polarization to M2 phenotype macrophages. A Proliferation status of each group of cells was detected using the CCK8 kit. B-C BMDMs were assayed by flow cytometry at the 12 h and 48 h timepoints for the percentage of EdU + macrophages within the AC − , AC + and OI + AC + groups. D-E The percentage of macrophages in each group that doubled, calculated from the decrease in CSFE fluorescence at the 48 h timepoint was assayed by flow cytometry. F-G Immunofluorescence staining images were obtained after treatment 48 h. H-I The percentage of CD206 + CD86- macrophages in each group was analyzed by flow cytometry. Data represent the mean ± SD. n = 3, *p < 0.05, **p < 0.01, ***p < 0.001

IRG1/itaconate axis enhances efferocytosis and alleviates autoimmune liver injury

TUNEL staining is a common technique for detecting DNA fragmentation during apoptosis. The F4/80 and TUNEL assays were used to label macrophages and ACs, respectively, to observe efferocytosis in autoimmune hepatitis. To evaluate the influence of exogenous itaconate on efferocytosis, Con A and Con A + OI were administered to IRG1-/- mice. As expected, exogenous itaconate significantly reduced the infiltration of macrophages in autoimmune hepatitis, enhanced the macrophages efferocytosis, and reduced the number of ACs, thereby effectively eliminating ACs (Fig. 5A-D). However, IRG1 deficiency increased immune liver injury and macrophage infiltration, and significantly attenuated efferocytosis in mice. Compared to the WT mice treated with Con A, ACs were not be cleared in time, and the number of ACs increased in the IRG1-/- mice which received Con A (Fig. 5A-C). We detected the pro-apoptotic proteins BAX and the downstream molecules RAC1 and ELMO1 during efferocytosis in mouse liver tissues. In WT and IRG1-/- mice with immune hepatitis, OI up-regluated key proteins RAC1 and ELMO1 correlated with efferocytosis in the liver and down-regluated pro-apoptotic protein BAX (Fig. 5E). Compared to the WT mice treated with Con A, IRG1 deficiency significantly inhibited RAC1 and ELMO1, and promoted BAX expression (Fig. 5E). Next, using HE staining, we found that OI significantly reduced the liver necrotic area and Ishak score, whereas IRG1 deficiency aggravated autoimmune hepatitis and increased liver necrotic area and Ishak score (Fig. 5F-H). Finally, mouse eyeball blood was collected to assess liver function. Similar results were observed in HE staining, and OI significantly reduces the levels of AST and ALT in the serum, and the deletion of IRG1 aggravated liver injury (Fig. 5I-K). Next, in order to verify whether OI can enhance the efferocytosis of macrophages on apoptotic hepatocytes in vitro, we extracted primary hepatocytes from WT mice. The apoptosis was induced with oxaliplatin, and then apoptotic hepatocytes were co-cultured with BMDMs. Flow cytometry showed that exogenous itaconate could promote the efferocytosis of macrophages on apoptotic hepatocytes, and after IRG1 knockout, the efferocytosis of macrophages on apoptotic hepatocytes was also significantly decreased, and the efferocytosis of macrophages after IRG1 knockout was restored after the supplementation of exogenous itaconate (Supplement Fig. 2A). In addition, OI significantly promoted TIM4 expression and enhanced TIM4-mediated efferocytosis. And there was a significant difference in TIM4 expression and TIM4-mediated efferocytosis between WT and IRG1-/- BMDMs treated with OI (Supplement Fig. 2A). Given that we previously published findings on the hepatoprotective role of 4-OI in autoimmune hepatitis, through the inhibition of hepatocyte apoptosis, IRG1f/fLyz2-cre+ mice were used to verify our hypothesis. Firstly, we isolated hepatocytes and liver Kupffer cells from the control and wild-type ConA groups,, and detected the expression of IRG1 in each group by WB. We found that only the IRG1 of total protein and Kupffer cell in the control group and WT cona group were changed (Supplement Fig. 3A), The expression of IRG1 in hepatocytes was not significantly changed (Supplement Fig. 3A). Then, IRG1f/fLyz2-cre+ mice were used to verify our hypothesis. Compared with WT mice treated with Con A, the liver necrotic area was increased in IRG1f/fLyz2-cre+ mice treated with Con A, and OI attenuated the the liver injury in IRG1f/fLyz2-cre+ mice (Supplement Fig. 3C-D). The levels of AST and ALT in the serum were elevated in IRG1f/fLyz2-cre+ mice treated with Con A. And in contrast, The levels of AST and ALT in the serum were decreased in IRG1f/fLyz2-cre+ mice after the administration of OI (Supplement Fig. 3E-F). Next, we stained the liver tissue of the mice with F480 and TUNEL immunofluorescence to observe efferocytosis. The efferocytosis was significantly attenuated in IRG1f/fLyz2-cre+ mice treated with Con A compared with WT mice treated with Con A (Supplement Fig. 3G-H). Exogenous itaconate promoted the efferocytosis of macrophages in IRG1f/fLyz2-cre+ mice treated with Con A (Supplement Fig. 3G-H).Taken together, IRG1/itaconate axis aggravates or attenuates Con A-induced liver inflammation by regulating efferocytosis.

Fig. 5

IRG1/ itaconate axis enhances efferocytosis and alleviates autoimmune liver injury. A-D Dual immunofluorescence staining for F480 (red) and TUNEL (green) in mice liver tissues. Statistical analysis of F480, TUNEL and F480/TUNEL immunofluorescence were performed. E Expression of BAX, RAC1 and ELMO1 was detected in each group by WB. F–H HE-stained liver sections and Ishak scores of WT or IRG1-/- mice after treating with Con A or OI. I-K Changes in serum ALT, AST, and ALT/AST ratio in mice. Data represent the mean ± SD. n = 5, *p < 0.05, **p < 0.01, ***p < 0.001

IRG1/itaconate axis activates Nrf2-TIM4 signalling pathway to alleviate inflammation and oxidative stress

The results showed that the expression of Nrf2 and TIM4 were increased after OI was administered. In addition, the anti-inflammatory factors (HO-1 and NQO1), downstream molecules of Nrf2, were also promoted. inhibited the expression of Keap1, and inflammatory proteins such as p-P65, p-IκB-α, and p-P38. However, IRG1 deficiency aggravated the inflammatory response and increased p-P65, p-IκB-α, and p-P38 expression. Nrf2 and TIM4, HO-1, and NQO1 were inhibited after IRG1 deletion (Fig. 6A). Immunofluorescence results showed that exogenous itaconate promoted the expression of TIM4 in liver macrophages, whereas IRG1 deficiency resulted in decreased TIM4 expression (Fig. 6B-C). Immunohistochemical results indicated that OI reduced Ly6G + cell infiltration, whereas the number of Ly6G + cells in WT mice received Con A was significantly lower compared to the IRG1-/- mice received Con A (Fig. 6 D-E). Next, ELISA kits was used to detect the expression of inflammation-related cytokine factors in the serum. As esxpected, OI reduced the release of serum cytokine factors such as IFN-γ, IL-1β and IL-6, and increased the expression of IL-10. We also analyzed levels of pro-inflammatory and anti-inflammatory cytokines after IRG1 knockout, including IFN-γ, IL-1β, IL-6 and IL-10. It was worth noting that IRG1 deletion led to the increasing of IFN-γ, IL-1β and IL-6 and the decreasing of IL-10 (Fig. 6F-J). Additionally, the antioxidative ability was assessed by detecting the MDA and total SOD levels in the serum of mice. As expected, MDA levels were decreased, and total SOD levels were increased after OI administration, indicating that antioxidative ability was improved, while the trend of these indexes was opposite in the IRG1-/- mice received Con A compared to the WT mice received Con A (Fig. 6K-L).

Fig. 6

IRG1/ itaconate axis activates Nrf2-TIM4 signaling pathway to alleviate inflammation and oxidative stress. A Western blotting from liver tissues showed that OI promoted the expression of Nrf2, TIM4, HO-1, NQO-1 proteins and decreased the expression of Keap1, p-P65, p-IkB-α, and p-P38 while IRG1-/- reversed these changes. B-C Dual immunofluorescence staining for F480 (red) and TIM4 (green) in mice liver sections. D-E Comparison of immunohistochemistry showing the expression and positivity of Ly6G in the liver tissue of mice in each group. F-J The levels of TNF-α, IFN-γ, IL-1β, IL-10 and IL-6 in serum were detected by ELISA kit. K-L The levels of MDA and total SOD in serum were detected. Data represent the mean ± SD. n = 5, *p < 0.05, **p < 0.01, ***p < 0.001

Inhibiting Nrf2 or Nrf2 deficiency attenuates efferocytosis and aggravates autoimmune hepatitis

ML385 was injected intraperitoneally 2 days before Con A and OI administration [41], and immunofluorescence staining showed that ML385 significantly increased macrophage infiltration and cell apoptosis, and enhanced the expression of BAX. In contrast, ML385 inhibited efferocytosis and decreased the expression of RAC1 and ELMO1 (Fig. 7A-E, supplementary Fig. 1G). ML385 significantly increased the liver necrotic area and Ishak score, aggravated liver damage, and the liver enzyme results were consistent with those of HE staining (Fig. 7F-J). Consistent with liver injury results, ML385 suppressed the expression of Nrf2, TIM4 and anti-inflammatory proteins (HO1 and NQO1), whereas ML385 promoted the p-P65, p-IκB-α, and p-P38 expression (Fig. 7K, supplementary Fig. 1H-I). To verify the role of Nrf2 in regulating TIM4-mediated efferocytosis, we first investigated the lethal concentration of Con A in Nrf2-/- mice, and found that 10 mg/kg did not affect mortality in Nrf2-/- mice(Fig. 7L). Next, Nrf2-/- mice were treated with Con A or Con A + OI. It was found that Nrf2 deletion led to the disappearance of OI promoting efferocytosis, and no significant differences in liver injury or liver enzyme levels were observed(Fig. 7M-N, P). Compared to the WT mice received Con A + OI, Nrf2 deletion inhibited efferocytosis, thereby suppressing TIM4, RAC1, and ELMO1(Fig. 7O, Supplementary Fig. 1 J). Then, we pretreated BMDMs with ML385, and we found that ML385 could also inhibited efferocytosis on apoptotic hepatocytes and blocked the pro-efferocytotic effect of OI, and the expression of TIM4 and TIM4-mediated efferocytosis were also significantly reduced (Supplement Fig. 2A). At the same time, Nrf2-/- BMDMs treated with PBS and OI was co-cultured with apoptotic hepatocytes. The efferocytosis ability of macrophages was significantly reduced after Nrf2 knockout, and the results of TIM4 and TIM4-mediated efferocytosis were also consistent with the previous results (Supplement Fig. 2A). In addition, after Nrf2 knockout, OI could not promote the efferocytosis,the expression of TIM4 and TIM4-mediated efferocytosis (Supplement Fig. 2A).

Fig. 7

Inhibiting Nrf2 or Nrf2 deficiency attenuates efferocytosis and aggravates immune hepatitis. A-D Dual immunofluorescence staining for F480 (red) and TUNEL (green) in mice liver tissues. Statistical analysis of F480, TUNEL and F480/TUNEL positive cells were performed. E Expression of BAX, RAC1 and ELMO1 was detected in each group by WB. F–H HE-stained liver sections and Ishak scores of each group. I-J Changes in serum ALT, AST in mice. K Western blot analysis of Nrf2, TIM4, HO-1, NQO-1, Keap1, p-P65, p-IkB-α, and p-P38 protein levels. L Survival of Nrf2-/- mice after administering different concentrations of Con A. M–N The evaluation of efferocytosis in Nrf2-/- mice treated with Con A and Con A + OI by dual immunofluorescence staining for F480 (red) and TUNEL (green). O Expression of RAC1 and ELMO1 was detected after idicated treamtments. P HE-stained liver sections, Ishak scores, and liver enzyme levels of Nrf2-/- mice treated with Con A and Con A + OI. Data represent the mean ± SD. n = 5, *p < 0.05, **p < 0.01, ***p < 0.001

OI promotes the transformation of macrophages into M2 macrophages, and blocking TIM4 can inhibit this effect and weaken efferocytosis

Compared with the Con A + IgA group, OI promoted the transformation of macrophages to M2 phenotype, and the proportion of M1 macrophages decreased in the Con A + OI + IgA group (Fig. 8A-D). However, the addition of Ab-TIM4 inhibited the effects of OI. After Ab-TIM4 administration, the number of CD206 positive cells was significantly reduced, while the number of CD86 positive cells was significantly increased(Fig. 8A-D), indicating Ab-TIM4 inhibited the transformation of, and the efferocytosis was also weakened in the mice received Con A + OI + Ab-TIM4(Fig. 8E-F). The area of liver necrosis and the Ishak score increased (Fig. 8G-H). The serum levels of TNF-α, IFN-γ, MDA and SOD in the two groups of mice were measured. The Con A + OI + Ab-TIM4 group showed an increased release of inflammatory factors and decreased antioxidant capacity(Fig. 8I-M). Then, in order to verify whether OI enhances The efferocytosis ability of macrophages on hepatocytes by regulating TIM4, we pretreated WT BMDM with Ab-TIM4 or IgA before administering OI or vehicle, and found that Ab-TIM4 inhibited normal efferocytosis. The promotion effect of OI disappeared after the addition of Ab-TIM4 (Supplement Fig. 2E-F).

Fig. 8

OI promotes the transformation of macrophages into M2 phenotype macrophages, and blocking TIM4 can inhibit this effect and weaken efferocytosis. A-B Dual immunofluorescence staining for F480 (red) and CD86 (green) in mice liver tissues. C-D Dual immunofluorescence staining for F480 (red) and CD206 (green) in mice liver tissues. E–F Efferocytosis was attenuated in WT mice after Ab-TIM4 treatment. G-I HE-stained liver sections, Ishak scores of WT mice treated with Ab-TIM4 or Vehicle. Data represent the mean ± SD. n = 5, *p < 0.05, **p < 0.01, ***p < 0.001