Negative Immune Regulator TIPE2 Promotes M2 Macrophage Differentiation through the Activation of PI3K-AKT Signaling Pathway

<div><p>Macrophages play important roles in the regulation of the innate and adaptive immune responses. Classically activated macrophages and alternatively activated macrophages are the two major forms of macrophages and have opposing functionalities. Tumor necrosis factor-α-induced protein 8–2 is expressed primarily by immune cells and negatively regulates type 1 innate and adaptive immune responses to maintain immune tolerance. While previous studies indicate that TIPE2 promotes M2 but inhibits M1 macrophage differentiation, the underlying molecular mechanism by which TIPE2 promotes M2 macrophage differentiation remains unclear. Our current study shows that TIPE2-deficient bone-marrow cells are defective in IL-4 induced M2 macrophage differentiation in vitro. Mechanistic studies revealed that TIPE2 promotes phosphoinositide metabolism and the activation of the down-stream AKT signaling pathway, which in turn leads to the expression of markers specific for M2 macrophages. In addition, our results showed that Tipe2-deficiency does not affect the activation of the JAK-STAT6 signaling pathway that also plays an important role during M2 macrophage differentiation. Taken together, these results indicate that TIPE2 promotes M2 macrophage differentiation through the activation of PI3K-AKT signaling pathway, and may play an important role during the resolution of inflammation, parasite control, as well as tissue repair.</p></div

Inhibition of PI3K reduces the difference of Arg-1 expression between WT and Tipe2-deficient M2 macrophages.

<p>Bone marrow derived macrophages from WT and Tipe2-deficient mice (n = 3) were treated with or without PI3K inhibitor NSC23766 (10 μM) for 30 min before stimulated with IL-4 (10 ng/ml) for 24 h. Expression level of Arg-1 was determined by real time RT-PCR and normalized to the expression level of GAPDH. Data shown are mean±SD of one representative experiment. The experiments were repeated three times with similar results. * p<0.05.</p

Cellular levels of PIP2 and PIP3 were significantly decreased by Tipe2-deficient macrophages.

<p>Bone marrow derived macrophages from WT and Tipe2-deficient mice (n = 3) were treated with IL-4 (10 ng/ml) for 20 min. <b>(A)</b> Cellular level of PIP2 was estimated by dot blot with anti-PIP2 antibody. <b>(B)</b> Relative expression level of PIP2 was determined using β-actin as the control and quantified by densitometry using ImageJ software. <b>(C)</b> Cellular level of PIP3 was estimated by dot blot with anti-PIP3 antibody. <b>(D)</b> Relative expression level of PIP3 was determined using same method as shown in (B). For A and C, results are representative of three independent experiments. For B and D, data shown are mean±SD for cells from three independent experiments. ** p<0.01.</p

Expression levels of M2 macrophage markers regulated by JAK-STAT6 signaling pathway are either increased or not affected by Tipe2-deficient macrophages.

<p>Bo Bone marrow derived macrophages from WT and Tipe2-deficient mice (n = 3) were untreated (M0), treated with IFN-γ (50 ng/ml) and LPS (10 ng/ml) (M1) or IL-4 (10 ng/ml) (M2) for 24 h. Expression levels of Atgl <b>(A),</b> Fabp4 <b>(B)</b> and Cd36 <b>(C)</b> were determined by RT-PCR and normalized to the expression level of GAPDH. Data shown are mean±SD of one representative experiment. The experiments were repeated three times with similar results. * p<0.05.</p

Expression levels of M2 macrophage markers regulated by PI3K-AKT signaling pathway are significantly decreased by Tipe2-deficient macrophages.

<p>Bone marrow derived macrophages from WT and Tipe2-deficient mice (n = 3) were untreated (M0), treated with IFN-γ (50 ng/ml) and LPS (10 ng/ml) (M1) or IL-4 (10 ng/ml) (M2) for 24 h. Expression levels of Fizz1 <b>(A)</b>, Ym1 <b>(B)</b>, Mgl-1 <b>(C)</b> and Mgl-2 <b>(D)</b> were determined by real time RT-PCR and normalized to the expression level of GAPDH. Data shown are mean±SD of one representative experiment. The experiments were repeated three times with similar results. * p<0.05, ** p<0.01.</p

Activation of PI3K-AKT but not JAK-STAT6 signaling pathway is defective in Tipe2-deficient macrophages.

<p>Bone marrow derived macrophages from WT and Tipe2-deficient mice (n = 3) were either untreated (UT) or treated with IL-4 (10 ng/ml) (T) for 20 min. <b>(A)</b> Cells were stained with anti-Phospho-AKT (T308) and analyzed by flow cytometry. <b>(B)</b> The MFI (mean fluorescence intensity) of phosphorylated AKT (pAKT) was determined using FlowJo software. <b>(C)</b> Cells were stained with anti-Phospho-PDK1 (Ser241) and analyzed by flow cytometry. <b>(D)</b> The MFI of phosphorylated PDK1 (pPDK1) was determined using the same method as shown in (B). <b>(E)</b> Cells were stained with anti-Phospho-STAT6 (Tyr641) and analyzed by flow cytometry. <b>(F)</b> The MFI of phosphorylated STAT6 (pSTAT6) was determined using the same method as shown in (B). Results are representative of three independent experiments. For B, D and F, Data shown are mean±SD of one representative experiment. * p<0.05.</p

Table_2_Transcriptomics combined with metabolomics unveiled the key genes and metabolites of mycelium growth in Morchella importuna.XLSX

Morels (Morchella) are one of the most popular edible fungi in the world, especially known for their rich nutrition and delicious taste. Earlier research indicates that the production of fruiting bodies can be affected by the growth of mycelium. To investigate the molecular mechanisms underlying mycelium growth in Morchella importuna, we performed transcriptome analysis and metabolomics analysis of three growth stages of the hypha of M. importuna. As a result, 24 differentially expressed genes, such as transketolase (tktA), glucose-6-phosphate dehydrogenase (G6PDH), fructose-diphosphate aldolase (Fba), and ribose-5-phosphate isomerase (rpiA), as well as 15 differentially accumulated metabolites, including succinate and oxaloacetate, were identified and considered as the key genes and metabolites to mycelium growth in M. importuna. In addition, guanosine 3′,5′-cyclic monophosphate (cGMP), guanosine-5′-monophosphate (GMP), and several small peptides were found to differentially accumulate in different growth stages. Furthermore, five pathways, namely, starch and sucrose metabolism, pentose and glucuronate interconversions, fructose and mannose metabolism, tyrosine metabolism, and purine nucleotides, enriched by most DEGs, existed in the three compared groups and were also recognized as important pathways for the development of mycelium in morels. The comprehensive transcriptomics and metabolomics data generated in our study provided valuable information for understanding the mycelium growth of M. importuna, and these data also unveiled the key genes, metabolites, and pathways involved in mycelium growth. This research provides a great theoretical basis for the stable production and breeding of morels.</p

Table_4_Transcriptomics combined with metabolomics unveiled the key genes and metabolites of mycelium growth in Morchella importuna.XLSX

Morels (Morchella) are one of the most popular edible fungi in the world, especially known for their rich nutrition and delicious taste. Earlier research indicates that the production of fruiting bodies can be affected by the growth of mycelium. To investigate the molecular mechanisms underlying mycelium growth in Morchella importuna, we performed transcriptome analysis and metabolomics analysis of three growth stages of the hypha of M. importuna. As a result, 24 differentially expressed genes, such as transketolase (tktA), glucose-6-phosphate dehydrogenase (G6PDH), fructose-diphosphate aldolase (Fba), and ribose-5-phosphate isomerase (rpiA), as well as 15 differentially accumulated metabolites, including succinate and oxaloacetate, were identified and considered as the key genes and metabolites to mycelium growth in M. importuna. In addition, guanosine 3′,5′-cyclic monophosphate (cGMP), guanosine-5′-monophosphate (GMP), and several small peptides were found to differentially accumulate in different growth stages. Furthermore, five pathways, namely, starch and sucrose metabolism, pentose and glucuronate interconversions, fructose and mannose metabolism, tyrosine metabolism, and purine nucleotides, enriched by most DEGs, existed in the three compared groups and were also recognized as important pathways for the development of mycelium in morels. The comprehensive transcriptomics and metabolomics data generated in our study provided valuable information for understanding the mycelium growth of M. importuna, and these data also unveiled the key genes, metabolites, and pathways involved in mycelium growth. This research provides a great theoretical basis for the stable production and breeding of morels.</p

Table_1_Transcriptomics combined with metabolomics unveiled the key genes and metabolites of mycelium growth in Morchella importuna.XLSX

Morels (Morchella) are one of the most popular edible fungi in the world, especially known for their rich nutrition and delicious taste. Earlier research indicates that the production of fruiting bodies can be affected by the growth of mycelium. To investigate the molecular mechanisms underlying mycelium growth in Morchella importuna, we performed transcriptome analysis and metabolomics analysis of three growth stages of the hypha of M. importuna. As a result, 24 differentially expressed genes, such as transketolase (tktA), glucose-6-phosphate dehydrogenase (G6PDH), fructose-diphosphate aldolase (Fba), and ribose-5-phosphate isomerase (rpiA), as well as 15 differentially accumulated metabolites, including succinate and oxaloacetate, were identified and considered as the key genes and metabolites to mycelium growth in M. importuna. In addition, guanosine 3′,5′-cyclic monophosphate (cGMP), guanosine-5′-monophosphate (GMP), and several small peptides were found to differentially accumulate in different growth stages. Furthermore, five pathways, namely, starch and sucrose metabolism, pentose and glucuronate interconversions, fructose and mannose metabolism, tyrosine metabolism, and purine nucleotides, enriched by most DEGs, existed in the three compared groups and were also recognized as important pathways for the development of mycelium in morels. The comprehensive transcriptomics and metabolomics data generated in our study provided valuable information for understanding the mycelium growth of M. importuna, and these data also unveiled the key genes, metabolites, and pathways involved in mycelium growth. This research provides a great theoretical basis for the stable production and breeding of morels.</p

Data_Sheet_1_Transcriptomics combined with metabolomics unveiled the key genes and metabolites of mycelium growth in Morchella importuna.docx

Morels (Morchella) are one of the most popular edible fungi in the world, especially known for their rich nutrition and delicious taste. Earlier research indicates that the production of fruiting bodies can be affected by the growth of mycelium. To investigate the molecular mechanisms underlying mycelium growth in Morchella importuna, we performed transcriptome analysis and metabolomics analysis of three growth stages of the hypha of M. importuna. As a result, 24 differentially expressed genes, such as transketolase (tktA), glucose-6-phosphate dehydrogenase (G6PDH), fructose-diphosphate aldolase (Fba), and ribose-5-phosphate isomerase (rpiA), as well as 15 differentially accumulated metabolites, including succinate and oxaloacetate, were identified and considered as the key genes and metabolites to mycelium growth in M. importuna. In addition, guanosine 3′,5′-cyclic monophosphate (cGMP), guanosine-5′-monophosphate (GMP), and several small peptides were found to differentially accumulate in different growth stages. Furthermore, five pathways, namely, starch and sucrose metabolism, pentose and glucuronate interconversions, fructose and mannose metabolism, tyrosine metabolism, and purine nucleotides, enriched by most DEGs, existed in the three compared groups and were also recognized as important pathways for the development of mycelium in morels. The comprehensive transcriptomics and metabolomics data generated in our study provided valuable information for understanding the mycelium growth of M. importuna, and these data also unveiled the key genes, metabolites, and pathways involved in mycelium growth. This research provides a great theoretical basis for the stable production and breeding of morels.</p