Polarization of M2 macrophages requires Lamtor1 that integrates cytokine and amino-acid signals

Abstract Macrophages play crucial roles in host defence and tissue homoeostasis, processes in which both environmental stimuli and intracellularly generated metabolites influence activation of macrophages. Activated macrophages are classified into M1 and M2 macrophages. It remains unclear how intracellular nutrition sufficiency, especially for amino acid, influences on macrophage activation. Here we show that a lysosomal adaptor protein Lamtor1, which forms an amino-acid sensing complex with lysosomal vacuolar-type H+-ATPase (v-ATPase), and is the scaffold for amino acid-activated mTORC1 (mechanistic target of rapamycin complex 1), is critically required for M2 polarization. Lamtor1 deficiency, amino-acid starvation, or inhibition of v-ATPase and mTOR result in defective M2 polarization and enhanced M1 polarization. Furthermore, we identified liver X receptor (LXR) as the downstream target of Lamtor1 and mTORC1. Production of 25-hydroxycholesterol is dependent on Lamtor1 and mTORC1. Our findings demonstrate that Lamtor1 plays an essential role in M2 polarization, coupling immunity and metabolism.

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Increased Alternative Macrophage-2 (M2) Polarization with Trib1 Gene over Expression in Myeloma Patients with Progressive Disease and Jak2 Inhibitors Reduce M2 Polarization

Blood ◽

10.1182/blood.v126.23.1011.1011 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1011-1011 ◽

Cited By ~ 1

Author(s):

Haiming Chen ◽

Mingjie Li ◽

Eric Sanchez ◽

Abigail Gillespie ◽

Cathy Wang ◽

...

Keyword(s):

Gene Expression ◽

Tumor Growth ◽

Tumor Cells ◽

Progressive Disease ◽

Scid Mice ◽

Human Monocytes ◽

M2 Macrophages ◽

Jak2 Inhibitor ◽

M2 Polarization ◽

M1 And M2 Macrophages

Abstract Introduction: The bone marrow (BM) microenvironment plays an important role in multiple myeloma (MM). The BM niche is composed of multiple cell types including macrophages. Macrophages polarize into pro-inflammatory macrophage-1 (M1) or alternative M2 states that promote tumor growth and metastasis. We evaluated the proportion of M2 macrophages in BM from MM pts either showing complete response (CR) or progressive disease (PD), the effects of MM cells on M1 and M2 differentiation, and the role of Trib1 in M2 differentiation in MM BM. Since the JAK-STAT signaling pathway plays key roles in macrophages, we also evaluated the effects of the JAK2 inhibitor ruxolitinib (RUX) on M2 polarization in MM. Methods: Using immunofluorescence (IFC), we determined the proportion of M1 and M2 macrophages in BM biopsies and aspirates from MM pts with PD or CR. The BM biopsy samples were stained with antibodies directed against human iNOS and CD86 for M1 and arginase 1(ARG1) and CD36 for M2 cells. MM BM aspirates were also examined using flow cytometric analysis (FCA). Human monocytes isolated from healthy subjects or the THP1 monocyte cell line were co-cultured with MM cell lines (RPMI8226 and U266) or primary MM tumor cells. The effects of RUX at low concentrations (IC20) on M2 polarization were determined. The percentages of M1 and M2 macrophages were determined using FCA. Total RNA was extracted from monocytes. Quantitative PCR was measured with TaqMan technology. For the in vivo studies, human MM tumors (LAGκ-2) were surgically implanted into the left superficial gluteal muscle of SCID mice and tumor volume measured on a weekly basis. Results: The proportion of M2 macrophages (CD36+/ARG1+) was markedly increased in BM biopsies or mononuclear cells from MM pts with PD compared with those in CR using IFC staining. FCA also showed the percentage of M2 macrophages in BM was significantly increased in MM pts with PD (n=25) compared to those in CR (n=10; P=0.005) whereas there was no difference in the percentage of M1 (CD86+/iNOS+) macrophages in BM derived from MM pts with PD compared to those in CR. Trib1 gene mRNA levels were higher among pts with PD compared to those in CR whereas the gene expression of Trib2 and Trib3 was not different. Next, we co-cultured MM cell lines (U266) or fresh MM BMMCs with purified healthy human monocytes for one week. The percentage of M2 cells markedly increased and the proportion of M1 cells decreased. Trib1 gene expression increased during co-culture whereas there was no change in expression of the other two Tribs. When direct cell-to-cell contact occurred between the MM tumor cells and the monocytes, the percentage of M2 macrophages markedly increased. We investigated the effects of the JAK2 inhibitor RUX on M2 differentiation induced with MM tumor cells. After exposure to a low concentration of RUX, the percentage of M2 cells decreased when the monocytes were co-cultured with MM tumor cells. Trib1 gene expression of the monocytes treated with RUX was also notably reduced compared with cells not treated with the JAK2 inhibitor. Using our human MM xenograft model LAGκ-2, RUX (1.5mg/kg) reduced tumor growth and decreased the proportion of M2 macrophages in the tumor tissue of MM tumor-bearing SCID mice. Conclusion: M2 cells are present at high levels in BM derived from MM pts with PD compared to those in CR, MM cells induce monocytes to become M2 macrophages and increase Trib1 gene expression. This induces monocyte differentiation into M2 macrophages that support MM tumor cell growth.. Notably, the JAK2 inhibitor RUX inhibits both M2 macrophage polarization and Trib1 gene expression in MM, and reduces tumor growth in SCID mice bearing human MM. These results suggest that RUX may be effective for treating MM pts. Disclosures No relevant conflicts of interest to declare.

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Chitooligosaccharides alleviate hepatic fibrosis by regulating the polarization of M1 and M2 macrophages

Food & Function ◽

10.1039/d1fo03768d ◽

2022 ◽

Author(s):

Peng Liu ◽

Heng Li ◽

Jinsong Gong ◽

Yan Geng ◽

Min Jiang ◽

...

Keyword(s):

Hepatic Fibrosis ◽

M2 Macrophages ◽

Liver Macrophages ◽

M2 Polarization ◽

M1 And M2 Macrophages

Chitooligosaccharide (COS) ameliorated hepatic fibrosis, possibly by regulating the M1 and M2 polarization of the liver macrophages, which was reflected in the modulation of the JAK1/STAT6 and JAK2/STAT1 pathways.

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Version up-date: The CDKN2B-AS1/ MIR497/TXNIP axis regulated macrophages

10.21203/rs.3.rs-806497/v1 ◽

2021 ◽

Author(s):

JianZhong Xu

Keyword(s):

Rheumatoid Arthritis ◽

Macrophage Polarization ◽

Critical Role ◽

M2 Macrophages ◽

Macrophage Cell Line ◽

Macrophage Cell ◽

Surface Polarization ◽

M2 Polarization ◽

M1 Polarization ◽

Healthy Donors

Abstract The polarization of macrophages plays a critical role in the pathophysiology of rheumatoid arthritis. The macrophages can have pro-inflammatory M1 polarization and various types of alternative anti-inflammatory M2 polarization. Our preliminary results showed that the CDKN2B-AS1/MIR497/TXNIP axis might regulate macrophages of rheumatoid arthritis patients. Therefore, we hypothesized that this axis regulated the polarization of rheumatoid macrophages. Flow cytometry was used to determine the surface polarization markers in M1 or M2 macrophages from healthy donors and rheumatoid arthritis patients. The QPCR and Western Blotting were used to compare the expression of the CDKN2B-AS1/MIR497/TXNIP axis in these macrophages. We Knocked down and overexpressed the axis in the macrophage cell line MD to test its roles in macrophage polarization. Compared to cells from healthy donors, cells from rheumatoid arthritis patients expressed higher levels of CD40 and CD80 and lower levels of CD16, CD163, CD206, and CD200R after polarization, they also expressed higher CDKN2B-AS1, lower MIR497, and higher TXNIP. In macrophages from healthy donors, there was no correlation among CDKN2B-AS1, MIR497, and TXNIP. But in macrophages from patients, there were significant correlations. The CDKN2B-AS1 knockdown, MIR497 mimics suppressed the M1 polarization but promoted the M2 polarization in MD cells, while the MIR497 knockdown and the TXNIP overexpression did the opposite. This study demonstrated that elevated CDKN2B-AS1 in macrophages promotes the M1 polarization and inhibited the M2 polarization of macrophages by the CDKN2B-AS1/ MIR497/TXNIP axis.

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Evidence for the presence in rainbow trout brain of amino acid-sensing systems involved in the control of food intake

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00283.2017 ◽

2018 ◽

Vol 314 (2) ◽

pp. R201-R215 ◽

Cited By ~ 12

Author(s):

Sara Comesaña ◽

Cristina Velasco ◽

Rosa M. Ceinos ◽

Marcos A. López-Patiño ◽

Jesús M. Míguez ◽

...

Keyword(s):

Rainbow Trout ◽

Amino Acid ◽

Food Intake ◽

Glutamine Metabolism ◽

Taste Receptors ◽

General Control ◽

Mechanistic Target Of Rapamycin ◽

Sensing Systems ◽

Branched Chain ◽

Amino Acid Sensing

To assess the hypothesis of central amino acid-sensing systems involved in the control of food intake in fish, we carried out two experiments in rainbow trout. In the first one, we injected intracerebroventricularly two different branched-chain amino acids (BCAAs), leucine and valine, and assessed food intake up to 48 h later. Leucine decreased and valine increased food intake. In a second experiment, 6 h after similar intracerebroventricular treatment we determined changes in parameters related to putative amino acid-sensing systems. Different areas of rainbow trout brain present amino acid-sensing systems responding to leucine (hypothalamus and telencephalon) and valine (telencephalon), while other areas (midbrain and hindbrain) do not respond to these treatments. The decreased food intake observed in fish treated intracerebroventricularly with leucine could relate to changes in mRNA abundance of hypothalamic neuropeptides [proopiomelanocortin (POMC), cocaine- and amphetamine-related transcript (CART), neuropeptide Y (NPY), and agouti-related peptide (AgRP)]. These in turn could relate to amino acid-sensing systems present in the same area, related to BCAA and glutamine metabolism, as well as mechanistic target of rapamycin (mTOR), taste receptors, and general control nonderepressible 2 (GCN2) kinase signaling. The treatment with valine did not affect amino acid-sensing parameters in the hypothalamus. These responses are comparable to those characterized in mammals. However, clear differences arise when comparing rainbow trout and mammals, in particular with respect to the clear orexigenic effect of valine, which could relate to the finding that valine partially stimulated two amino acid-sensing systems in the telencephalon. Another novel result is the clear effect of leucine on telencephalon, in which amino acid-sensing systems, but not neuropeptides, were activated as in the hypothalamus.

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The E3 ubiquitin ligase ZNRF2 is a substrate of mTORC1 and regulates its activation by amino acids

eLife ◽

10.7554/elife.12278 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 8

Author(s):

Gerta Hoxhaj ◽

Edward Caddye ◽

Ayaz Najafov ◽

Vanessa P Houde ◽

Catherine Johnson ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Ubiquitin Ligase ◽

Protein Phosphatase ◽

E3 Ubiquitin Ligase ◽

Mechanistic Target Of Rapamycin ◽

Rag Gtpases ◽

Intracellular Amino Acid ◽

Amino Acid Levels ◽

Amino Acid Sensing

The mechanistic Target of Rapamycin complex 1 (mTORC1) senses intracellular amino acid levels through an intricate machinery, which includes the Rag GTPases, Ragulator and vacuolar ATPase (V-ATPase). The membrane-associated E3 ubiquitin ligase ZNRF2 is released into the cytosol upon its phosphorylation by Akt. In this study, we show that ZNRF2 interacts with mTOR on membranes, promoting the amino acid-stimulated translocation of mTORC1 to lysosomes and its activation in human cells. ZNRF2 also interacts with the V-ATPase and preserves lysosomal acidity. Moreover, knockdown of ZNRF2 decreases cell size and cell proliferation. Upon growth factor and amino acid stimulation, mTORC1 phosphorylates ZNRF2 on Ser145, and this phosphosite is dephosphorylated by protein phosphatase 6. Ser145 phosphorylation stimulates vesicle-to-cytosol translocation of ZNRF2 and forms a novel negative feedback on mTORC1. Our findings uncover ZNRF2 as a component of the amino acid sensing machinery that acts upstream of Rag-GTPases and the V-ATPase to activate mTORC1.

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Amino acid sensing and activation of mechanistic target of rapamycin complex 1

Current Opinion in Clinical Nutrition & Metabolic Care ◽

10.1097/mco.0000000000000240 ◽

2016 ◽

Vol 19 (1) ◽

pp. 67-73 ◽

Cited By ~ 9

Author(s):

Daniel J. Ham ◽

Gordon S. Lynch ◽

René Koopman

Keyword(s):

Amino Acid ◽

Target Of Rapamycin ◽

Mechanistic Target Of Rapamycin ◽

Amino Acid Sensing

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Crystal structure of arginine-bound lysosomal transporter SLC38A9 in the cytosol-open state

10.1101/288233 ◽

2018 ◽

Cited By ~ 1

Author(s):

Hsiang-Ting Lei ◽

Jinming Ma ◽

Silvia Sanchez Martinez ◽

Tamir Gonen

Keyword(s):

Crystal Structure ◽

Amino Acid ◽

Signaling Pathway ◽

Transmembrane Helix ◽

Underlying Mechanism ◽

Transitional State ◽

Mechanistic Target Of Rapamycin ◽

Mtorc1 Signaling ◽

Solute Carrier ◽

Amino Acid Sensing

Amino acid-dependent activation of mechanistic target of rapamycin complex 1 (mTORC1) is essential to reflect nutrient availabilities in cells for cell growth and metabolism1. Solute carrier 38 family A member 9 (SLC38A9) is the lysosomal transporter responsible for amino acid sensing in the mTORC1 signaling pathway2–4. Here we present the first crystal structure of SLC38A9 from Danio rerio in complex with arginine. As captured in the cytosol-open state, the bound arginine was locked in a transitional state stabilized by the transmembrane helix 1 (TM1) of SLC38A9 which was anchored at the grove between transmembrane helix 5 and 7 inside the transporter. The key motif WNTMM on TM1, contributing to the anchoring interactions, is highly conserved in various species. Mutations in WNTMM motif abolished arginine transport by SLC38A9. The underlying mechanism of substrate binding is critical for both sensitizing mTORC1 signaling pathway to amino acids and for maintaining amino acid homeostasis across lysosomal membranes2.

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Increased M2 Macrophages in Multiple Myeloma Patients with Progressive Disease and Down-Regulated Polarization with the JAK2 Inhibitor Ruxolitinib

Blood ◽

10.1182/blood.v124.21.4106.4106 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4106-4106 ◽

Cited By ~ 2

Author(s):

Haiming Chen ◽

Eric Sanchez ◽

Mingjie Li ◽

Cathy Wang ◽

Abby Gillespie ◽

...

Keyword(s):

Gene Expression ◽

Tumor Cells ◽

Progressive Disease ◽

Flow Cytometric Analysis ◽

Human Monocytes ◽

M2 Macrophages ◽

Jak2 Inhibitor ◽

Flow Cytometric ◽

M2 Polarization ◽

M1 And M2 Macrophages

Abstract Introduction: Macrophages polarize into pro-inflammatory M1 or alternative M2 states with distinct phenotypes and physiological functions. M2 cells promote tumor growth and metastasis through secretion of growth factors. However, crosstalk between tumor cells and macrophages in the development of M2 polarization has not been well demonstrated. We evaluated the proportion of M2 macrophages in bone marrow (BM) from patients (pts) with multiple myeloma (MM), the effects of MM cells on M1 and M2 differentiation, and the role of Trib1 in M2 differentiation in MM BM. Since the JAK-STAT signaling pathway plays key roles in the cell growth and differentiation of macrophages, we evaluated the effects of the JAK2 inhibitor ruxolitinib (RUX) on M2 polarization in MM. Methods: Using immunofluorescence (IFC) analysis, we determined the proportion of M1 and M2 macrophages in BM biopsies from MM pts with progressive disease and in remission. The BM biopsy samples were stained with antibodies directed against human iNOS and CD86 for M1 and arginase 1(ARG1) and CD36 for M2 cells, following a standard IFC protocol. Monocyte/macrophage phenotypes for M1 and M2 subtypes in mononuclear cells isolated from MM BM aspirates were also examined using flow cytometric analysis with these same antibodies. Human monocytes isolated from normal subjects or the THP1 monocyte cell line were co-cultured with MM cell lines or primary MM tumor cells with or without exposure to low concentrations (IC20) of ruxolitinib (RUX) using Transwell plates. The percentage of M1 and M2 macrophages was determined using flow cytometric analysis. Total RNA was extracted from monocytes followed the manufacturer’s directions. Quantitation PCR were measured with TaqMan technology performed in an OneStepPlus instrument. Results: IFC demonstrated that the percentage of M2 macrophages (CD36+/ARG1+) was markedly increased in BM sections from MM pts with progressive disease compared with those pts in remission. The flow cytometric data also showed the percentage of M2 (CD36+/ ARG1+) macrophages in BM was significantly increased in MM patients with progressive disease (n=20) compared to those in remission (n=9; P=0.005) whereas there was no significant difference in the percentage of M1 (CD86+/iNOS+) macrophages in BM derived from MM patients with progressive disease compared to those in remission. The results of both RT-PCR and Quantitation PCR showed Trib1 gene expression levels were higher among patients with progressive disease compared to those in remission. In contrast, the gene expression of Trib2 and Trib3 was not related to the MM patient’s clinical status. To determine whether MM tumor cells affected monocyte/macrophage differentiation and Trib gene expression, we co-cultured MM cell lines or fresh MM tumor cells with purified healthy human monocytes using Transwell plates. The percentage of M2 cells markedly increased whereas the proportion of M1 cells decreased. The expression of Trib1 increased during the 7 days of co-culture whereas there was no change in the expression of Trib2 and Trib3. Moreover, when direct cell-to-cell contact occurred between the MM cells and the monocytes, the percentage of M2 macrophages markedly increased after 7 days of incubation. It has been reported that Trib-1 mediated regulation of the MAPK/ERK pathway in a murine model and the Ras/Raf-1/MEK1/ERK cascade culminates in up-regulated expression of the gene encoding STAT3 whereas recruitment and activation of tyrosine kinase JAK-2 phosphorylates it. Thus, we investigated the effects of the JAK2 inhibitor RUX on M2 differentiation induced with MM tumor cells. The percentage of M2 cells was decreased when the monocytes that were co-cultured with MM tumor cells were treated with a low concentration (IC20) of RUX. Trib1 gene expression of the monocytes treated with RUX was also reduced comparing to the cells untreated with JAK2 inhibitor. Conclusion: Our results show that MM cells induce monocytes to become M2 macrophages and increase Trib1 gene expression providing a positive feedback loop on Trib1 expression, monocyte differentiation and tumor cell growth. M2 cells are present at high levels in BM derived from MM pts with progressive disease compared to those in remission. Notably, the JAK2 inhibitor RUX shows inhibition of both M2 macrophage polarization and Trib1 gene expression in MM, and these results suggest this drug may be effective for treating MM. Disclosures No relevant conflicts of interest to declare.

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