The chemokine CCL1 triggers an AMFR-SPRY1 pathway that promotes differentiation of lung fibroblasts into myofibroblasts and drives pulmonary fibrosis.

Recruitment of immune cells to the site of inflammation by the chemokine CCL1 is important in the pathology of inflammatory diseases. Here, we examined the role of CCL1 in pulmonary fibrosis (PF). Bronchoalveolar lavage fluid from PF mouse models contained high amounts of CCL1, as did lung biopsies from PF patients. Immunofluorescence analyses revealed that alveolar macrophages and CD4+ T cells were major producers of CCL1 and targeted deletion of Ccl1 in these cells blunted pathology. Deletion of the CCL1 receptor Ccr8 in fibroblasts limited migration, but not activation, in response to CCL1. Mass spectrometry analyses of CCL1 complexes identified AMFR as a CCL1 receptor, and deletion of Amfr impaired fibroblast activation. Mechanistically, CCL1 binding triggered ubiquitination of the ERK inhibitor Spry1 by AMFR, thus activating Ras-mediated profibrotic protein synthesis. Antibody blockade of CCL1 ameliorated PF pathology, supporting the therapeutic potential of targeting this pathway for treating fibroproliferative lung diseases.

Targeting Degradation of the Transcription Factor C/EBPß Reduces Lung Fibrosis by Restoring Activity of the Ubiquitin-Editing Enzyme A20 in Macrophages.

Although recent progress provides mechanistic insights into the pathogenesis of pulmonary fibrosis (PF), rare anti-PF therapeutics show definitive promise for treating this disease. Repeated lung epithelial injury results in injury-repairing response and inflammation, which drive the development of PF. Here, we report that chronic lung injury inactivated the ubiquitin-editing enzyme A20, causing progressive accumulation of the transcription factor C/EBPß in alveolar macrophages (AMs) from PF patients and mice, which upregulated a number of immunosuppressive and profibrotic factors promoting PF development. In response to chronic lung injury, elevated glycogen synthase kinase-3ß (GSK-3ß) interacted with and phosphorylated A20 to suppress C/EBPß degradation. Ectopic expression of A20 or pharmacological restoration of A20 activity by disturbing the A20-GSK-3ß interaction accelerated C/EBPß degradation and showed potent therapeutic efficacy against experimental PF. Our study indicates that a regulatory mechanism of the GSK-3ß-A20-C/EBPß axis in AMs may be a potential target for treating PF and fibroproliferative lung diseases.

Autophagic Flux Detection: Significance and Methods Involved.

Macroautophagy is an important biological process in eukaryotic cells by which longevity proteins, misfolded proteins, and damaged organelles are degraded. The autophagy process consists of three key steps: (1) the formation of autophagosomes; (2) the fusion of the autophagosomes with lysosomes; and (3) the degradation of the contents of autolysosomes. If any of the three steps is impaired, autophagy will not be able to complete its biological function. Dysfunctional or blocked autophagy is closely involved in the pathogenesis of a variety of diseases. The accurate determination of the autophagy activity in vivo and in vitro has become a challenge in the field of autophagy research. At present, the most widely used detection method to determine autophagy activity in mammalian cells is to quantify LC3B in the cells by Western blot, or to observe the formation and changes of autophagosomes and autolysosomes by immunofluorescence and electron microscopy. However, ignoring the dynamic characteristics of autophagy and only evaluating the number of autophagosomes or the presence of LC3B cannot completely reflect the activation or a blockage of the autophagy system, and objectively analyze its real role in the occurrence and development of a disease. For example, the accumulation of autophagosomes and autolysosomes can occur through an increase in substrate to be degraded after the activation of autophagy, or it may be caused by the partial obstruction or blockage of autophagy. In this chapter, new and familiar ways to detect the autophagic flux are methodically summarized to provide researchers with a multi-angled viewpoint.

TRIB3 Interacts With ß-Catenin and TCF4 to Increase Stem Cell Features of Colorectal Cancer Stem Cells and Tumorigenesis.

BACKGROUND & AIMS: Activation of Wnt signaling to ß-catenin contributes to the development of colorectal cancer (CRC). Expression of tribbles pseudo-kinase 3 (TRIB3) is increased in some colorectal tumors and associated with poor outcome. We investigated whether increased TRIB3 expression promotes stem cell features of CRC cells and tumor progression by interacting with the Wnt signaling pathway. METHODS: We performed studies with C57BL/6J-ApcMin/J mice injected with an adeno-associated virus vector that expresses a small hairpin RNA against Trib3 mRNA (ApcMin/J-Trib3KD) or a control vector (ApcMin/J-Ctrl). We created BALB/c mice that overexpress TRIB3 from an adeno-associated virus vector and mice with small hairpin RNA-mediated knockdown of ß-catenin. The mice were given azoxymethane followed by dextran sodium sulfate to induce colitis-associated cancer. Intestinal tissues were collected and analyzed by histology, gene expression profiling, immunohistochemistry, and immunofluorescence. Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5)-positive (LGR5Pos) and LGR5-negative (LGR5Neg) HCT-8 CRC cells, with or without knockdown or transgenic expression of TRIB3, were sorted and analyzed in sphere-formation assays. We derived organoids from human and mouse colorectal tumors to analyze the function of TRIB3 and test the effect of a peptide inhibitor. Wnt signaling to ß-catenin was analyzed in dual luciferase reporter, chromatin precipitation, immunofluorescence, and immunoblot assays. Proteins that interact with TRIB3 were identified by immunoprecipitation. CRC cell lines were grown in nude mice as xenograft tumors. RESULTS: At 10 weeks of age, more than half the ApcMin/J-Ctrl mice developed intestinal high-grade epithelial neoplasia, whereas ApcMin/J-Trib3KD mice had no intestinal polyps and normal histology. Colon tissues from ApcMin/J-Trib3KD mice expressed lower levels of genes regulated by ß-catenin and genes associated with cancer stem cells. Mice with overexpression of Trib3 developed more tumors after administration of azoxymethane and dextran sodium sulfate than BALB/c mice. Mice with knockdown of ß-catenin had a lower tumor burden after administration of azoxymethane and dextran sodium sulfate, regardless of Trib3 overexpression. Intestinal tissues from mice with overexpression of Trib3 and knockdown of ß-catenin did not have activation of Wnt signaling or expression of genes regulated by ß-catenin. LGR5Pos cells sorted from HCT-8 cells expressed higher levels of TRIB3 than LGR5Neg cells. CRC cells that overexpressed TRIB3 had higher levels of transcription by ß-catenin and formed larger spheroids than control CRC cells; knockdown of ß-catenin prevented the larger organoid size caused by TRIB3 overexpression. TRIB3 interacted physically with ß-catenin and transcription factor 4 (TCF4). TRIB3 overexpression increased, and TRIB3 knockdown decreased, recruitment of TCF4 and ß-catenin to the promoter region of genes regulated by Wnt. Activated ß-catenin increased expression of TRIB3, indicating a positive-feedback loop. A peptide (P2-T3A6) that bound ß-catenin disrupted its interaction with TRIB3 and TCF4. In primary CRC cells and HCT-8 cells, P2-T3A6 decreased expression of genes regulated by ß-catenin and genes associated with cancer stem cells and decreased cell viability and migration. Injection of C57BL/6J-ApcMin/J mice with P2-T3A6 decreased the number and size of tumor nodules and colon expression of genes regulated by ß-catenin. P2-T3A6 increased 5-fluorouracil-induced death of CRC cells and survival times of mice with xenograft tumors. CONCLUSION: TRIB3 interacts with ß-catenin and TCF4 in intestine cells to increase expression of genes associated with cancer stem cells. Knockdown of TRIB3 decreases colon neoplasia in mice, migration of CRC cells, and their growth as xenograft tumors in mice. Strategies to block TRIB3 activity might be developed for treatment of CRC.

Chronic Obstructive Pulmonary Disease and Autophagy.

Chronic Obstructive Pulmonary Disease (COPD) is a classical chronic respiratory disease with the pathological changes involving the bronchi and alveoli. Many of the risk factors of COPD can induce autophagy in different kinds of cells in lung tissue including alveolar epithelial cells, broncho epithelial cells, and fibroblasts. Over-activation of autophagy may cause emphysema by inducing autophagic cell death. However, the bronchitis and fibrosis may be mainly caused by autophagic flux blocking. Thus, understanding the role of autophagy in the pathogenesis of COPD is important for the anti-COPD drug development.

Autophagy and Pulmonary Fibrosis.

Pulmonary fibrosis is a progressive chronic inflammatory disease with a poor clinical outcome. Although pirfenidone and nintedanib have been approved by FDA to treat idiopathic pulmonary fibrosis (IPF), these drugs can only slow the progression of IPF. Autophagy plays an important role in the pathogenesis of pulmonary fibrosis. Whether the autophagic flux is blocked or not is directly related to the development direction of pulmonary fibrosis. Defining how autophagy activity regulates the pathogenesis of pulmonary fibrosis will greatly advance the progression of pulmonary fibrosis therapy.

Asthma and Autophagy.

Asthma is one of the most common diseases of the respiratory system, with typical pathogenesis and pathological changes. The current research shows that autophagy is mainly involved in the pathogenesis of asthma by regulating the body's innate and adaptive immune responses. At the same time, a large number of epidemiological studies have shown that multiple autophagy genes affect the risk of asthma at the level of genetic polymorphism. This chapter will explore the relationship between autophagy and asthma.

Autophagy and Others Respiratory Diseases.

Besides COPD, pulmonary fibrosis, and asthma, autophagy also participates in the development of many other respiratory diseases. Cystic fibrosis is an innate lung disease. Unlike idiopathic pulmonary fibrosis, cystic fibrosis has unique pathogenesis. Autophagy is an essential biological mechanism for the removal of misfolded proteins and damaged organelles in cells. Abnormal autophagy activity is involved in the pathogenesis of cystic fibrosis. Various studies have demonstrated that abnormalities or impaired autophagy are associated with cardiovascular diseases including pulmonary vascular disease. Autophagy plays a key role in maintaining normal vascular biological functions and vascular cell tissue homeostasis, and also plays an important role in the pathogenesis of various vascular diseases. For example, recent studies have found that autophagy participates in the occurrence and development of pulmonary hypertension. In addition, autophagy plays a central role in both innate and adaptive immune responses in immune cells or other cells with immune function. Thus, autophagy is the important cellular biological mechanism which causes cell fighting against pathogenic microorganisms including viruses, bacteria, and parasites. In this chapter, we discuss the work related to autophagy and other lung diseases.

Immune Signaling and Autophagy Regulation.

Autophagy is one of the key degradation systems in organisms. Starvation and nutrient deprivation induce autophagy activation, providing energy and anabolic substances to maintain energy homeostasis. A variety of signals participate in the induction of autophagy, including endoplasmic reticulum stress, oxidative stress, and activation of immune signals. Autophagy is closely related to immunity and inflammation. Autophagy-related gene mutations increase the risk of infectious diseases and malignancies. Autophagy can be regarded as an effector of the immune system to eliminate invading pathogens and is also involved in the immune system recognizing the invasion of pathogens. Autophagy plays important roles in regulating innate immunity and adaptive immunity. In terms of innate immunity, autophagy not only participates in the clearance of pathogens and cell debris after apoptosis but also plays a protective role against toxins, regulates cytokine production, and activates the inflammasome. In the adaptive immune response, autophagy plays an important regulatory role in thymic selection, T cell maturation, T cell polarization, T cell and B cell homeostasis, antigen processing, antigen presentation, and antibody response. On the other hand, autophagy is regulated by immunological and stress signals. The crosstalk between these signaling pathways helps maintain homeostasis and physiological functions. Dysfunction of these regulatory networks is the cause of several kinds of diseases.

Autophagy and the Immune Response.

Innate immunity and adaptive immunity play critical roles in maintaining normal physiological functions and the development of diseases. In innate immune responses, heterogeneous autophagy can directly remove intracellular pathogens while activating PRRs, including TLRs and NLRs, to trigger their signal transduction pathways and promote NKT cell activation, cytokine secretion, and phagocytosis. In adaptive immune responses, the autophagy reaction has an important effect on the homeostasis, function, and differentiation of T lymphocytes, the survival, and development of B lymphocytes and the survival of plasma cells. This review highlights the key role that autophagy plays in the innate immune system and the acquired immune system. Further clarifying the mechanism by which autophagy regulates the immune system is essential for elucidating the precise mechanisms of various diseases and for developing new treatment methods.

Seeking new anti-cancer agents from autophagy-regulating natural products.

Natural products are an important original source of many widely used drugs, including anti-cancer drugs. Early research efforts for seeking anti-cancer therapy from the natural products are mainly focused on the compounds with cytotoxicity capability. The good examples include vinblastine, vincristine, the camptothecin derivatives; topotecan, irinotecan, epipodophyllotoxin derivatives and paclitaxel. In a recent decade, the fundamental progression has been made in the understanding of molecular and cellular mechanisms regarding tumor initiation, metastasis, therapeutic resistance, immune escape, and relapse, which provide a great opportunity for the development of new mechanism-based anticancer drugs, especially drugs against new molecular and cellular targets. Autophagy, a critical cell homeostasis mechanism and promising drug target involved in a verity of human diseases including cancer, can be modulated by many compounds derived from natural products. In this review, we'll give a short introduction of autophagy and discuss the roles of autophagy in the tumorigenesis and progression. And then, we summarize the accumulated evidences to show the anti-tumor effects of several compounds derived from natural products through modulation of autophagy activity.

Autophagy-inducing natural compounds: a treasure resource for developing therapeutics against tissue fibrosis.

Tissue fibrosis is a common pathologic change of many chronic diseases, which is characterized by extracellular matrix accumulation in tissues and dysfunction of the injured organs. Despite there recently gain mechanistic insight into the pathogenesis of tissue fibrosis, therapeutics for tissue fibrosis and thus many chronic diseases remain a significant clinical unmet need. Recent progressions indicate that autophagy, a conserved lysosomal degradation process in eukaryotic cells, not only plays an important regulatory role in maintaining cellular and tissue homeostasis, but also contributes to the development and progression of tissue fibrosis in a diversity of organs. Interestingly, a number of natural compounds derived from plant or Chinese Herb Medicines (CHM), have been identified as modulators of autophagy, and may function as potential therapeutic agents for the treatment of different fibrotic diseases. In this review, we focus on several plant natural compounds that have well-known anti-fibrotic effects through regulating autophagic signal pathways or autophagy activity. These findings should provide important therapeutic clues and strategy for the development of new anti-fibrosis drugs.

Autophagy as a target for development of anti-diabetes drugs derived from natural compounds.

Patients with diabetes have a high level of blood glucose because their body cannot produce enough insulin or properly respond to this hormone. In both situations, it has become evident that persistent high concentrations of glucose, insulin, insulin-like growth factor, and insulin resistance lead to dysfunction and destruction of autophagic activity in the cells of islet and other organs involved in complications of diabetes, including the liver, cardiovascular, and nervous systems. Accumulating evidences have revealed that autophagy is a novel therapeutic target with a wide range of beneficial effects on diabetes and that plenty of drugs and natural products are involved in autophagy modulation, either inducing or inhibiting autophagy, through multiple signaling pathways. In this review, we summarize the roles of several clinical drugs and compounds derived from natural products in diabetes and its complications through regulation of autophagy, expecting to inspire further investigation of the underlying mechanisms of these compounds and to facilitate their better clinical application.

[Morphological analysis of autophagy].

Autophagy is an important homeostatic cellular recycling mechanism responsible for degrading injured or dysfunctional subcellular organelles and proteins in all living cells. The process of autophagy can be divided into three relatively independent steps: the initiation of phagophore, the formation of autophagosome and the maturation/degradation stage. Different morphological characteristics and molecular marker changes can be observed at these stages. Morphological approaches are useful to produce novel knowledge that would not be achieved through other experimental methods. Here we summarize the morphological methods in monitoring autophagy, the principles in data interpretation and the cautions that should be considered in the study of autophagy.

[New methods to detect autophagic flux].

Autophagy is a crucial biological process of eukaryotes, which is involved in cell growth, survival and energy metabolism, while the premise of the autophagy function is activated autophagic flux. It has been confirmed that impaired autophagic flux promotes pathogenesis of many chronic inflammatory diseases, especially cancer, neurodegenerative disease and tissue fibrosis, therefore the analysis of autophagic flux state is important for revealing autophagy function and the mechanism of autophagy related diseases. Given that autophagy is a dynamic process with multiple steps, it is very hard to observe the real state of autophagic flux. Summarized here is the novel concept and current approach to detect autophagic flux. This knowledge is crucial for the researching of the biological function of autophagy, and may provide some strategies for developing autophagy-related drug.

[Progress of autophagy screening systems].

Autophagy is an active research area in the biomedical field as its role has been identified in many physiological and pathological processes. Accordingly, there is a growing demand to identify, quantify and manipulate the process accurately. Meanwhile, there is great interest in identifying compounds that modulate autophagy because they may have applications in the treatment of a variety of autophagy-related diseases. In this review, we summarize the current status of autophagy screening systems to facilitate identification of autophagy modulators.

[Advances in the studies of the link between diabetes and cancer].

Diabetes and cancer are two major chronic diseases with tremendous impact on human health worldwide. Clinical and basic studies demonstrate that diabetes can promote carcinogenesis and tumor progression. High insulin and high insulin-like growth factor are considered to be the major risk factors for cancer. Chronic inflammation and aberrant metabolism also participate in cancer development. It is noteworthy that therapies used for treatment of diabetes may increase or decrease the risk of cancer. Revealing the mechanisms that connect diabetes to cancer will be crucial for prevention and treatment of diabetes-related cancers.

Targeting acute myeloid leukemia with a proapoptotic peptide conjugated to a Toll-like receptor 2-mediated cell-penetrating peptide.

Cell-penetrating peptides provide a unique platform to create a new generation of cancer therapeutics with enhanced efficacy and diminished toxicity. In our study, enhanced expression of toll-like receptor 2 (TLR2) was observed in acute myeloid leukemia (AML) cells. Screening of a phage display peptide library using Biopanning and Rapid Analysis of Selective Interactive Ligands (BRASIL) identified a TLR2-binding peptide motif, Pep2. We show that the TLR2-binding peptide motif targeted and penetrated into leukemia cells in a TLR2-dependent manner. Moreover, a synthetic, chimeric peptide composed of the TLR2-binding motif linked to a programmed cell death-inducing sequence, D(KLAKLAK)2, induced apoptosis in AML cells with high TLR2 expression (TLR2(high)) but not in chronic myeloid leukemia (CML) cells with low TLR2 expression (TLR2(low)). The antileukemia activity of this chimeric peptide was confirmed in leukemia patient samples and an animal model of myeloid leukemia, as the development of leukemia was significantly delayed in mice with TLR2(high) AML compared to TLR2(low) CML NOD/SCID mice. TUNEL assays on bone marrow tissue slices revealed that the chimerical peptide induced leukemia cell apoptosis in a TLR2-dependent manner. Together, our findings indicate that TLR2 is a potential therapeutic target for the prevention and treatment of AML, and the prototype, Pep2-D(KLAKLAK)2, is a promising drug candidate in this setting.

Loss of immunity-supported senescence enhances susceptibility to hepatocellular carcinogenesis and progression in Toll-like receptor 2-deficient mice.

UNLABELLED: Hepatocellular carcinoma (HCC) is a complication at the endstage of chronic inflammatory liver diseases with dismal prognosis. Targeting of Toll-like receptor (TLR) 2 attenuates tumor metastases; we hypothesized that blocking TLR2 might also play a crucial role in reducing hepatocarcinogenesis. Surprisingly, we found that the genetic deletion of TLR2 increased susceptibility to diethylnitrosamine (DEN), a genotoxic carcinogen that can induce HCC. Indeed, TLR2-deficient mice showed a significant increase in carcinogenesis and progression of HCC as indicated by increases in tumor nodule size, tumor volume, and animal death. The enhanced susceptibility to DEN-induced HCC was associated with a broad-spectrum reduction in the immune response to DEN-induced liver injury. We found that TLR2 deficiency caused a decrease in the infiltration of macrophages and an attenuation of apoptosis signal regulating kinase 1 (ASK1) / p38 mitogen-activated protein kinase (p38 MAPK) / nuclear factor kappa B (NF-κB) signaling, which led to a decrease in the expression of interferon-gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukin (IL)-1α/ß, IL-6, and Cxcl-2 as well as suppression of autophagy flux and increases in oxidative stress and p62 aggregation in liver tissue. The defects in immune networks resulted in suppressed p21- and p16/pRb-dependent senescence, which caused an increase in proliferation and a decrease in apoptotic and autophagy-associated cell death in mouse livers. Restoring cellular senescence and autophagy flux by treating TLR2-deficient mice with IFN-γ, a T helper 1 (Th1) cytokine and positive modulator of senescence and autophagy, could attenuate the carcinogenesis and progression of HCC associated with TLR2-deficient animals. CONCLUSION: The loss of immune networks supporting cellular senescence and autophagy flux is attributed to enhanced susceptibility to DEN-induced hepatocellular carcinogenesis and progression in TLR2-deficient mice. These findings may be used to prevent the development of liver cancer.