Butyrate Prevents the Pathogenic Anemia-Inflammation Circuit by Facilitating Macrophage Iron Export.

Most patients with inflammatory bowel disease (IBD) develop anemia, which is attributed to the dysregulation of iron metabolism. Reciprocally, impaired iron homeostasis also aggravates inflammation. How this iron-mediated, pathogenic anemia-inflammation crosstalk is regulated in the gut remains elusive. Herein, it is for the first time revealed that anemic IBD patients exhibit impaired production of short-chain fatty acids (SCFAs), particularly butyrate. Butyrate supplementation restores iron metabolism in multiple anemia models. Mechanistically, butyrate upregulates ferroportin (FPN) expression in macrophages by reducing the enrichment of histone deacetylase (HDAC) at the Slc40a1 promoter, thereby facilitating iron export. By preventing iron sequestration, butyrate not only mitigates colitis-induced anemia but also reduces TNF-α production in macrophages. Consistently, macrophage-conditional FPN knockout mice exhibit more severe anemia and inflammation. Finally, it is revealed that macrophage iron overload impairs the therapeutic effectiveness of anti-TNF-α antibodies in colitis, which can be reversed by butyrate supplementation. Hence, this study uncovers the pivotal role of butyrate in preventing the pathogenic circuit between anemia and inflammation.

NanoSHP099-Targeted SHP2 Inhibition Boosts Ly6Clow Monocytes/Macrophages Differentiation to Accelerate Thrombolysis.

Tumor-associated thrombus (TAT) accounts for a high proportion of venous thromboembolism. Traditional thrombolysis and anticoagulation methods are not effective due to various complications and contraindications, which can easily lead to patients dying from TAT rather than the tumor itself. These clinical issues demonstrate the need to research diverse pathways for adjuvant thrombolysis in antitumor therapy. Previously, the phenotypic and functional transformation of monocytes/macrophages is widely reported to be involved in intratribal collagen regulation. This study finds that myeloid deficiency of the oncogene SHP2 sensitizes Ly6Clow monocyte/macrophage differentiation and can alleviate thrombus organization by increasing thrombolytic Matrix metalloproteinase (MMP) 2/9 activities. Moreover, pharmacologic inhibition by SHP099, examined in mouse lung metastatic tumor models, reduces tumor and thrombi burden in tumor metastatic lung tissues. Furthermore, SHP099 increases intrathrombus Ly6Clow monocyte/macrophage infiltration and exhibits thrombolytic function at high concentrations. To improve the thrombolytic effect of SHP099, NanoSHP099 is constructed to achieve the specific delivery of SHP099. NanoSHP099 is identified to be simultaneously enriched in tumor and thrombus foci, exerting dual tumor-suppression and thrombolysis effects. NanoSHP099 presents a superior thrombus dissolution effect than that of the same dosage of SHP099 because of the higher Ly6Clow monocyte/macrophage proportion and MMP2/MMP9 collagenolytic activities in organized thrombi.

Protein Tyrosine Phosphatase SHP2 in Macrophages Acts as an Antiatherosclerotic Regulator in Mice.

BACKGROUND: Macrophages have versatile roles in atherosclerosis. SHP2 (Src homology 2 containing protein tyrosine phosphatase 2) has been demonstrated to play a critical role in regulating macrophage activation. However, the mechanism of SHP2 regulation of macrophage function in an atherosclerotic microenvironment remains unknown. METHODS: APOE (apolipoprotein E) or LDLR (low-density lipoprotein receptor) null mice treated with SHP099 were fed a Western diet for 8 weeks, while Shp2MKO:ApoE-/- or Shp2MKO:Ldlr-/- mice and exo-AAV8-SHP2E76K/ApoE-/- mice were fed a Western diet for 12 weeks. In vitro, levels of proinflammatory factors and phagocytic function were then studied in mouse peritoneal macrophages. RNA sequencing was used to identify PPARγ (peroxisome proliferative activated receptor γ) as the key downstream molecule. A PPARγ agonist was used to rescue the phenotypes observed in SHP2-deleted mice. RESULTS: Pharmacological inhibition and selective deletion in macrophages of SHP2 aggravated atherosclerosis in APOE and LDLR null mice with increased plaque macrophages and apoptotic cells. In vitro, SHP2 deficiency in APOE and LDLR null macrophages enhanced proinflammatory polarization and its efferocytosis was dramatically impaired. Conversely, the expression of gain-of-function mutation of SHP2 in mouse macrophages reduced atherosclerosis. The SHP2 agonist lovastatin repressesed macrophage inflammatory activation and enhanced efferocytosis. Mechanistically, RNA sequencing analysis identified PPARγ as a key downstream transcription factor. PPARγ was decreased in macrophages upon SHP2 deletion and inhibition. Importantly, PPARγ agonist decreased atherosclerosis in SHP2 knockout mice, restored efferocytotic defects, and reduced inflammatory activation in SHP2 deleted macrophages. PPARγ was decreased by the ubiquitin-mediated degradation upon SHP2 inhibition or deletion. Finally, we found that SHP2 was downregulated in atherosclerotic vessels. CONCLUSIONS: Overall, SHP2 in macrophages was found to act as an antiatherosclerotic regulator by stabilizing PPARγ in APOE/LDLR null mice.

Cancer-associated fibroblasts facilitate DNA damage repair by promoting the glycolysis in non-small cell lung cancer.

Radiotherapy is an essential treatment modality for the management of non-small cell lung cancer (NSCLC) patients. Tumor radioresistance is the major factor limiting the efficacy of radiotherapy in NSCLC patients. Our study aimed to reveal whether cancer-associated fibroblasts (CAFs), one main component of the tumor microenvironment, regulated DNA damage response of NSCLC cells following irradiation and clarify the involved mechanisms. We found CAFs inhibited irradiation-induced DNA damage while promoted DNA repair of NSCLC cells and caused cell cycle arrest in the radioresistant S phase. CAFs have the ability of up-regulating and stabilizing c-Myc, leading to the transcription activation of HK2 kinase, a key rate-limiting enzyme in glycolysis by activating Wnt/ß-catenin pathway. Attenuation of glycolysis significantly reversed the effect of CAFs on DNA damage response of NSCLC cells. By high-throughput screening of human cytokines/chemokines array, we found CAFs-secreted midkine led to the promotion of glycolysis by activating Wnt/ß-catenin pathway in NSCLC cells. In vivo, CAFs caused the radioresistance of NSCLC cells also by promoting the glycolysis in a ß-catenin signaling-dependent manner. These findings may provide novel strategies for reversing the radioresistance of NSCLC cells.

Epithelial Gab1 calibrates RIPK3-dependent necroptosis to prevent intestinal inflammation.

As a hallmark of inflammatory bowel disease (IBD), elevated intestinal epithelial cell (IEC) death compromises the gut barrier, activating the inflammatory response and triggering more IEC death. However, the precise intracellular machinery that prevents IEC death and breaks this vicious feedback cycle remains largely unknown. Here, we report that Grb2-associated binder 1 (Gab1) expression is decreased in patients with IBD and inversely correlated with IBD severity. Gab1 deficiency in IECs accounted for the exacerbated colitis induced by dextran sodium sulfate owing to sensitizing IECs to receptor-interaction protein kinase 3-mediated (RIPK3-mediated) necroptosis, which irreversibly disrupted the homeostasis of the epithelial barrier and promoted intestinal inflammation. Mechanistically, Gab1 negatively regulated necroptosis signaling through inhibiting the formation of RIPK1/RIPK3 complex in response to TNF-α. Importantly, administration of RIPK3 inhibitor revealed a curative effect in epithelial Gab1-deficient mice. Further analysis indicated mice with Gab1 deletion were prone to inflammation-associated colorectal tumorigenesis. Collectively, our study defines a protective role for Gab1 in colitis and colitis-driven colorectal cancer by negatively regulating RIPK3-dependent necroptosis, which may serve as an important target to address necroptosis and intestinal inflammation-related disease.

SHP2 deneddylation mediates tumor immunosuppression in colon cancer via the CD47/SIRPα axis.

SIPRα on macrophages binds with CD47 to resist proengulfment signals, but how the downstream signal of SIPRα controls tumor-infiltrating macrophages (TIMs) is still poorly clarified. Here, we report that the CD47/signal regulatory protein α (SIRPα) axis requires the deneddylation of tyrosine phosphatase SHP2. Mechanistically, Src homology region 2-containing protein tyrosine phosphatase 2 (SHP2) was constitutively neddylated on K358 and K364 sites; thus, its autoinhibited conformation was maintained. In response to CD47-liganded SIRPα, SHP2 was deneddylated by sentrin-specific protease 8 (SENP8), which led to the dephosphorylation of relevant substrates at the phagocytic cup and subsequent inhibition of macrophage phagocytosis. Furthermore, neddylation inactivated myeloid-SHP2 and greatly boosted the efficacy of colorectal cancer (CRC) immunotherapy. Importantly, we observed that supplementation with SHP2 allosteric inhibitors sensitized immune treatment-resistant CRC to immunotherapy. Our results emphasize that the CRC subtype that is unresponsive to immunotherapy relies on SIRPαhiSHP2hiNEDD8lo TIMs and highlight the need to further explore the strategy of SHP2 targeting in CRC therapy.

Mannose metabolism normalizes gut homeostasis by blocking the TNF-α-mediated proinflammatory circuit.

Mannose is a naturally occurring sugar widely consumed in the daily diet; however, mechanistic insights into how mannose metabolism affects intestinal inflammation remain lacking. Herein, we reported that mannose supplementation ameliorated colitis development and promoted colitis recovery. Macrophage-secreted inflammatory cytokines, particularly TNF-α, induced pathological endoplasmic reticulum stress (ERS) in intestinal epithelial cells (IECs), which was prevented by mannose via normalization of protein N-glycosylation. By preserving epithelial integrity, mannose reduced the inflammatory activation of colonic macrophages. On the other hand, mannose directly suppressed macrophage TNF-α production translationally by reducing the glyceraldehyde 3-phosphate level, thus promoting GAPDH binding to TNF-α mRNA. Additionally, we found dysregulated mannose metabolism in the colonic mucosa of patients with inflammatory bowel disease. Finally, we revealed that activating PMM2 activity with epalrestat, a clinically approved drug for the treatment of diabetic neuropathy, elicited further sensitization to the therapeutic effect of mannose. Therefore, mannose metabolism prevents TNF-α-mediated pathogenic crosstalk between IECs and intestinal macrophages, thereby normalizing aberrant immunometabolism in the gut.

SHP2 inhibition mitigates adaptive resistance to MEK inhibitors in KRAS-mutant gastric cancer through the suppression of KSR1 activity.

Despite the promising antitumor activity of RAF/MEK inhibitors for RAS-driven cancers, not all patients respond to these therapies. Adaptive resistance has been reported as a major culprit in non-responders, which can be reversed by SHP2 inhibitors (SHP2is) in multiple cancer cells; however, the underlying mechanisms remain unknown. In this study, we found that KRAS-mutant gastric cancer cells respond to MEK inhibitors (MEKis) with adaptive resistance. Markedly, SHP2 activation accompanied by ERK signaling restoration in MEKi-treated cells, and a MEKi and SHP2i combination had a synergistic effect on downstream signaling blockade. In vivo, SHP099 combined with AZD6244 (selumetinib) was highly efficacious for the treatment of xenografts. Mechanistically, SHP2 was found to interact with the scaffold protein KSR1 through its protein tyrosine phosphatase domain. KSR1 knockdown sensitized cells to AZD6244, whereas a KSR1 activating mutation (S269A) diminished the synergistic anti-proliferative effect of SHP2i and MEKi. Interestingly, activated SHP2, during adaptive resistance to MEKis, impaired the interaction with KSR1, activating KSR1 to promote MAPK signaling. In conclusion, SHP2 promotes adaptive resistance to MEKis by activating KSR1; selumetinib combined with SHP099 might be an available therapeutic strategy for KRAS-mutant gastric cancers.

GAB1 is upregulated to promote anaplastic thyroid cancer cell migration through AKT-MDR1.

Anaplastic thyroid carcinoma (ATC) represents an undifferentiated, aggressive and highly metastatic form of thyroid cancer with high mortality. GAB1, through direct interaction with the kinase PI3K and phosphatase SHP2, is tightly involved in the activation of oncogenic signals; however, the role of GAB1 in ATC remains unclear. GAB1 was significantly increased in ATC, accompanied with AKT activation. Cell proliferation, migration and invasion were impaired or enhanced by GAB1 knockdown in ATC cells or overexpression in PTC cells. Moreover, GAB1 knockdown in ATC cells inhibited and overexpression in PTC cells promoted the growth of thyroid cancer in nude mice. GAB1 mutation disrupting the interaction between GAB1 and PI3K failed to restore cell migration and invasion in GAB1-knockdown ATC cells. RNA sequencing data showed GAB1-knockdown partially reprogramed gene expression in ATC cells back to that in normal thyroid cells. MDR1 was transcriptionally regulated by GAB1, which was mediated by AKT. MDR1 was upregulated in ATC cells and MDR1 knockdown in ATC cells decreased migration and invasion. In addition, MDR1 overexpression restored cell migration and invasion and lung metastasis of GAB1-knockdown ATC cells. Collectively, GAB1 is upregulated in ATC to promote AKT activation and cellular migration and invasion through regulating MDR1 expression.

Endothelial Shp2 deficiency controls alternative activation of macrophage preventing radiation-induced lung injury through notch signaling.

Radiation-induced lung injury is a common late side effect of thoracic radiotherapy. Endothelial dysfunction following leukocytes infiltration is a prominent feature in this process. Here, we established a clinical-mimicking mouse model of radiation-induced lung injury and found the activity of phosphatase Shp2 was elevated in endothelium after injury. Endothelium-specific Shp2 deletion mice showed relieved collagen deposition along with disrupted radiation-induced Jag1 expression in the endothelium. Furthermore, endothelium-derived Jag1 activated the alternative activation of macrophages in vitro and in vivo by paracrine Notch signaling. Consistently, the Notch pathway was significantly activated by chest irradiation in the peripheral blood leukocytes of patients with cancer. Collectively, our work demonstrates that Shp2 participates in the radiation-induced endothelial dysfunction and subsequently inflammatory microenvironment producing during radiation-induced lung injury. Our findings indicate Shp2 as a potential target for radiation-induced lung injury and provide another way for endothelium to participate in the pathological process of radiation-induced lung injury.

LDLR dysfunction induces LDL accumulation and promotes pulmonary fibrosis.

Treatments for pulmonary fibrosis (PF) are ineffective because its molecular pathogenesis and therapeutic targets are unclear. Here, we show that the expression of low-density lipoprotein receptor (LDLR) was significantly decreased in alveolar type II (ATII) and fibroblast cells, whereas it was increased in endothelial cells from systemic sclerosis-related PF (SSc-PF) patients and idiopathic PF (IPF) patients compared with healthy controls. However, the plasma levels of low-density lipoprotein (LDL) increased in SSc-PF and IPF patients. The disrupted LDL-LDLR metabolism was also observed in four mouse PF models. Upon bleomycin (BLM) treatment, Ldlr-deficient (Ldlr-/-) mice exhibited remarkably higher LDL levels, abundant apoptosis, increased fibroblast-like endothelial and ATII cells and significantly earlier and more severe fibrotic response compared to wild-type mice. In vitro experiments revealed that apoptosis and TGF-ß1 production were induced by LDL, while fibroblast-like cell accumulation and ET-1 expression were induced by LDLR knockdown. Treatment of fibroblasts with LDL or culture medium derived from LDL-pretreated endothelial or epithelial cells led to obvious fibrotic responses in vitro. Similar results were observed after LDLR knockdown operation. These results suggest that disturbed LDL-LDLR metabolism contributes in various ways to the malfunction of endothelial and epithelial cells, and fibroblasts during pulmonary fibrogenesis. In addition, pharmacological restoration of LDLR levels by using a combination of atorvastatin and alirocumab inhibited BLM-induced LDL elevation, apoptosis, fibroblast-like cell accumulation and mitigated PF in mice. Therefore, LDL-LDLR may serve as an important mediator in PF, and LDLR enhancing strategies may have beneficial effects on PF.

Oxidative stress-induced FABP5 S-glutathionylation protects against acute lung injury by suppressing inflammation in macrophages.

Oxidative stress contributes to the pathogenesis of acute lung injury. Protein S-glutathionylation plays an important role in cellular antioxidant defense. Here we report that the expression of deglutathionylation enzyme Grx1 is decreased in the lungs of acute lung injury mice. The acute lung injury induced by hyperoxia or LPS is significantly relieved in Grx1 KO and Grx1fl/flLysMcre mice, confirming the protective role of Grx1-regulated S-glutathionylation in macrophages. Using a quantitative redox proteomics approach, we show that FABP5 is susceptible to S-glutathionylation under oxidative conditions. S-glutathionylation of Cys127 in FABP5 promotes its fatty acid binding ability and nuclear translocation. Further results indicate S-glutathionylation promotes the interaction of FABP5 and PPARß/δ, activates PPARß/δ target genes and suppresses the LPS-induced inflammation in macrophages. Our study reveals a molecular mechanism through which FABP5 S-glutathionylation regulates macrophage inflammation in the pathogenesis of acute lung injury.

Endothelial deletion of SHP2 suppresses tumor angiogenesis and promotes vascular normalization.

SHP2 mediates the activities of multiple receptor tyrosine kinase signaling and its function in endothelial processes has been explored extensively. However, genetic studies on the role of SHP2 in tumor angiogenesis have not been conducted. Here, we show that SHP2 is activated in tumor endothelia. Shp2 deletion and pharmacological inhibition reduce tumor growth and microvascular density in multiple mouse tumor models. Shp2 deletion also leads to tumor vascular normalization, indicated by increased pericyte coverage and vessel perfusion. SHP2 inefficiency impairs endothelial cell proliferation, migration, and tubulogenesis through downregulating the expression of proangiogenic SRY-Box transcription factor 7 (SOX7), whose re-expression restores endothelial function in SHP2-knockdown cells and tumor growth, angiogenesis, and vascular abnormalization in Shp2-deleted mice. SHP2 stabilizes apoptosis signal-regulating kinase 1 (ASK1), which regulates SOX7 expression mediated by c-Jun. Our studies suggest SHP2 in tumor associated endothelial cells is a promising anti-angiogenic target for cancer therapy.

Gremlin2 Activates Fibroblasts to Promote Pulmonary Fibrosis Through the Bone Morphogenic Protein Pathway.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease causing unremitting extracellular matrix deposition. Transforming growth factor-ß (TGF-ß) superfamily involves bone morphogenetic proteins (BMPs) and TGF-ß, and the balance between the activation of TGF-ß-dependent SMADs (Smad2/3) and BMP-dependent SMADs (Smad1/5/8) is essential for fibrosis process. GREM2, initially identified as a TGF-ß-inducible gene, encodes a small secreted glycoprotein belonging to a group of matricellular proteins, its role in lung fibrosis is not clear. Here, we identified Gremlin2 as a key regulator of fibroblast activation. Gremlin2 was highly expressed in the serum and lung tissues in IPF patients. Bleomycin-induced lung fibrosis model exhibited high expression of Gremlin2 in the bronchoalveolar lavage fluid (BALF) and lung tissue. Isolation of primary cells from bleomycin-induced fibrosis lung showed a good correlation of Gremlin2 and Acta2 (α-SMA) expressions. Overexpression of Gremlin2 in human fetal lung fibroblast 1 (HFL-1) cells increased its invasion and migration. Furthermore, Gremlin2 regulates fibrosis functions through mediating TGF-ß/BMP signaling, in which Gremlin2 may activate TGF-ß signaling and inhibit BMP signaling. Therefore, we provided in vivo and in vitro evidence to demonstrate that Gremlin2 may be a potential therapeutic target for the treatment of IPF.

Riok3 inhibits the antiviral immune response by facilitating TRIM40-mediated RIG-I and MDA5 degradation.

The type I interferon (IFN) pathway is a key component of innate immune response upon invasion of foreign pathogens. It is also under precise control to prevent excessive upregulation and undesired inflammation cascade. In the present study, we report that Riok3, an atypical kinase, negatively regulates retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) sensing-induced type I IFN signaling. Riok3 deficiency selectively inhibits RNA viral replication in vitro, resulting from an upregulated type I IFN pathway. Mice with myeloid-specific Riok3 knockout also show a more robust induction of type I IFN upon RNA virus infection and are more resistant to RNA virus-induced pathogenesis. Mechanistically, Riok3 recruits and interacts with the E3 ubiquitin ligase TRIM40, leading to the degradation of RIG-I and melanoma differentiation-associated gene-5 (MDA5) via K48- and K27-linked ubiquitination. Collectively, our data reveal the mechanism that Riok3 employs to be a negative regulator of antiviral innate immunity.

Phosphatase Shp2 regulates biogenesis of small extracellular vesicles by dephosphorylating Syntenin.

As novel mediators of cell-to-cell signalling, small extracellular vesicles (sEVs) play a critical role in physiological and pathophysiological processes. To date, the molecular mechanisms that support sEV generation are incompletely understood. Many kinases are reported for their roles in sEV generation or composition, whereas the involvement of phosphatases remains largely unexplored. Here we reveal that pharmacological inhibition and shRNA-mediated down-regulation of tyrosine phosphatase Shp2 significantly increases the formation of sEVs. By Co-immunoprecipitation (Co-IP) and in vitro dephosphorylation assays, we identified that Shp2 negatively controlled sEV biogenesis by directly dephosphorylating tyrosine 46 of Syntenin, which has been reported as a molecular switch in sEV biogenesis. More importantly, Shp2 dysfunction led to enhanced epithelial sEV generation in vitro and in vivo. The increase of epithelial sEVs caused by shRNA-mediated down-regulation of Shp2 promoted macrophage activation, resulting in strengthened inflammation. Our findings highlight the role of Shp2 in regulating sEV-mediated epithelial-macrophage crosstalk by controlling sEV biogenesis through dephosphorylation of Syntenin Y46. The present study determines the strengthened inflammatory characteristics of alveolar macrophages elicited by epithelial sEVs transferred intercellularly. These findings provide a basis for understanding the mechanism of sEV formation and relevant function in epithelial-macrophage crosstalk.

[Research progress on macrophage in radiation induced lung injury].

Radiation-induced lung injury (RILI), including acute radiation pneumonitis and chronic radiation-induced pulmonary fibrosis (RIPF), is a side effect of radiotherapy for lung cancer and esophageal cancer. Pulmonary macrophages, as a kind of natural immune cells maintaining lung homeostasis, play a key role in the whole pathological process of RILI. In the early stage of RILI, classically activated M1 macrophages secrete proinflammatory cytokines to induce inflammation and produce massive reactive oxygen species (ROS) through ROS-induced cascade to further impair lung tissue. In the later stage of RILI, alternatively activated M2 macrophages secrete profibrotic cytokines to promote the development of RIPF. The roles of macrophage in the pathogenesis of RILI and the related potential clinical applications are summarized in this review.

PDLIM5 inhibits STUB1-mediated degradation of SMAD3 and promotes the migration and invasion of lung cancer cells.

Transforming growth factor ß (TGFß) signaling plays an important role in regulating tumor malignancy, including in non-small cell lung cancer (NSCLC). The major biological responses of TGFß signaling are determined by the effector proteins SMAD2 and SMAD3. However, the regulators of TGFß-SMAD signaling are not completely revealed yet. Here, we showed that the scaffolding protein PDLIM5 (PDZ and LIM domain protein 5, ENH) critically promotes TGFß signaling by maintaining SMAD3 stability in NSCLC. First, PDLIM5 was highly expressed in NSCLC compared with that in adjacent normal tissues, and high PDLIM5 expression was associated with poor outcome. Knockdown of PDLIM5 in NSCLC cells decreased migration and invasion in vitro and lung metastasis in vivo In addition, TGFß signaling and TGFß-induced epithelial-mesenchymal transition was repressed by PDLIM5 knockdown. Mechanistically, PDLIM5 knockdown resulted in a reduction of SMAD3 protein levels. Overexpression of SMAD3 reversed the TGFß-signaling-repressing and anti-migration effects induced by PDLIM5 knockdown. Notably, PDLIM5 interacted with SMAD3 but not SMAD2 and competitively suppressed the interaction between SMAD3 and its E3 ubiquitin ligase STUB1. Therefore, PDLIM5 protected SMAD3 from STUB1-mediated proteasome degradation. STUB1 knockdown restored SMAD3 protein levels, cell migration, and invasion in PDLIM5-knockdown cells. Collectively, our findings indicate that PDLIM5 is a novel regulator of basal SMAD3 stability, with implications for controlling TGFß signaling and NSCLC progression.