Autophagy Inhibition-induced Cytosolic DNA Sensing Combined with Differentiation Therapy Induces Irreversible Myeloid Differentiation in Leukemia Cells.

Accumulating evidence indicates that various oncogenic mutations interfere with normal myeloid differentiation of leukemogenic cells during the early process of acute myeloid leukemia (AML) development. Differentiation therapy is a therapeutic strategy capable of terminating leukemic expansion by reactivating the differentiation potential; however, the plasticity and instability of leukemia cells counteract the establishment of treatments aimed at irreversibly inducing and maintaining their differentiation states. On the basis of our previous observation that autophagy inhibitor treatment induces the accumulation of cytosolic DNA and activation of cytosolic DNA-sensor signaling selectively in leukemia cells, we herein examined the synergistic effect of cytosolic DNA-sensor signaling activation with conventional differentiation therapy on AML. The combined treatment succeeded in inducing irreversible differentiation in AML cell lines. Mechanistically, cytosolic DNA was sensed by absent in melanoma 2 (AIM2), a cytosolic DNA sensor. Activation of the AIM2 inflammasome resulted in the accumulation of p21 through the inhibition of its proteasomal degradation, thereby facilitating the myeloid differentiation. Importantly, the combined therapy dramatically reduced the total leukemia cell counts and proportion of blast cells in the spleens of AML mice. Collectively, these findings indicate that the autophagy inhibition-cytosolic DNA-sensor signaling axis can potentiate AML differentiation therapy. SIGNIFICANCE: Clinical effects on AML therapy are closely associated with reactivating the normal myeloid differentiation potential in leukemia cells. This study shows that autophagosome formation inhibitors activate the cytosolic DNA-sensor signaling, thereby augmenting conventional differentiation therapy to induce irreversible differentiation and cell growth arrest in several types of AML cell lines.

Immune Mechanisms of Pulmonary Fibrosis with Bleomycin.

Fibrosis and structural remodeling of the lung tissue can significantly impair lung function, often with fatal consequences. The etiology of pulmonary fibrosis (PF) is diverse and includes different triggers such as allergens, chemicals, radiation, and environmental particles. However, the cause of idiopathic PF (IPF), one of the most common forms of PF, remains unknown. Experimental models have been developed to study the mechanisms of PF, and the murine bleomycin (BLM) model has received the most attention. Epithelial injury, inflammation, epithelial-mesenchymal transition (EMT), myofibroblast activation, and repeated tissue injury are important initiators of fibrosis. In this review, we examined the common mechanisms of lung wound-healing responses after BLM-induced lung injury as well as the pathogenesis of the most common PF. A three-stage model of wound repair involving injury, inflammation, and repair is outlined. Dysregulation of one or more of these three phases has been reported in many cases of PF. We reviewed the literature investigating PF pathogenesis, and the role of cytokines, chemokines, growth factors, and matrix feeding in an animal model of BLM-induced PF.

CX3CL1/CX3CR1 interaction protects against lipotoxicity-induced nonalcoholic steatohepatitis by regulating macrophage migration and M1/M2 status.

BACKGROUND AND OBJECTIVES: Chemokine (C-X3-C motif) ligand 1 (CX3CL1) and its receptor CX3CR1 regulate the migration and activation of immune cells and are involved in the pathogenesis of nonalcoholic steatohepatitis (NASH), but the mechanism remains elusive. Here, the roles of CX3CL1/CX3CR1 in the macrophage migration and polarization in the livers of NASH mice were investigated. METHODS AND RESULTS: The expression of Cx3cl1 and Cx3cr1 was markedly upregulated in the livers of lipotoxicity-induced NASH mice. CX3CR1 was predominantly expressed by F4/80+ macrophages and to a lesser degree by hepatic stellate cells or endothelial cells in the livers of NASH mice. Flow cytometry analysis revealed that, compared with chow-fed mice, NASH mice exhibited a significant increase in CX3CR1+ expression by liver macrophages (LMs), particularly M1 LMs. CX3CR1 deficiency caused a significant increase in inflammatory monocyte/macrophage infiltration and a shift toward M1 dominant macrophages in the liver, thereby exacerbating the progression of NASH. Moreover, transplantation of Cx3cr1-/- bone marrow was sufficient to cause glucose intolerance, inflammation, and fibrosis in the liver. In addition, deletion of CCL2 in Cx3cr1-/- mice alleviated NASH progression by decreasing macrophage infiltration and inducing a shift toward M2 dominant LMs. Importantly, overexpression of CX3CL1 in vivo protected against hepatic fibrosis in NASH. CONCLUSION: Pharmacological therapy targeting liver CX3CL1/CX3CR1 signaling might be a candidate for the treatment of NASH.

CCR2- and CCR5-mediated macrophage infiltration contributes to glomerular endocapillary hypercellularity in antibody-induced lupus nephritis.

OBJECTIVES: LN comprises various glomerular lesions, including endocapillary hypercellularity with macrophage infiltration. In this study, we aimed to clarify the involvement of macrophage-tropic chemokine receptors in the pathogenesis of these glomerular lesions. METHODS: MRL/lpr mouse-derived monoclonal IgG3 antibody-producing hybridomas, 2B11.3 and B1, were injected intraperitoneally into BALB/c mice [wild type (WT)] to induce endocapillary hypercellularity and wire-loop lesions, respectively. The expression of chemokine and chemokine receptors was analysed by quantitative real-time PCR and IF. The roles of chemokine receptors in these lesions were evaluated using chemokine receptor-deficient mice or a selective CCR5 antagonist, maraviroc. RESULTS: 2B11.3 caused glomerular endocapillary hypercellularity with a significant number of glomerular CD68-positive macrophages. Further, enhanced expression of CCL2, CCL3, CCR2, CCR5 and CX3CR1 was observed in the renal cortex, compared with B1 injection, which induced wire-loop lesions. In 2B11.3-induced glomerular lesions, CD68 -positive glomerular macrophages expressed CCL2, CCL3, CCR2, CCR5 and CX3CR1, while glomerular endothelial cells expressed CCL2, CCL3, CX3CL1 and CCR2. When 2B11.3 was injected, CCR2-/- and CCR5-/-, but not CX3CR1-/-, mice exhibited reduced endocapillary hypercellularity, attenuated glomerular macrophage infiltration and improved serum blood urea nitrogen levels. Only CCR2-/- mice developed wire-loop lesions. B1 injection caused wire-loop lesions in these chemokine receptor-deficient mice to a similar extent as WT. Maraviroc treatment reduced 2B11.3-induced endocapillary hypercellularity and improved serum blood urea nitrogen levels. CONCLUSION: CCR2 and CCR5 regulate glomerular macrophage infiltration and contribute to the development of glomerular endocapillary hypercellularity in LN. CCR5 inhibition can be a specific therapy for endocapillary hypercellularity without inducing wire-loop lesions.

Crucial contribution of GPR56/ADGRG1, expressed by breast cancer cells, to bone metastasis formation.

From a mouse triple-negative breast cancer cell line, 4T1, we previously established 4T1.3 clone with a high capacity to metastasize to bone after its orthotopic injection into mammary fat pad of immunocompetent mice. Subsequent analysis demonstrated that the interaction between cancer cells and fibroblasts in a bone cavity was crucial for bone metastasis focus formation arising from orthotopic injection of 4T1.3 cells. Here, we demonstrated that a member of the adhesion G-protein-coupled receptor (ADGR) family, G-protein-coupled receptor 56 (GPR56)/adhesion G-protein-coupled receptor G1 (ADGRG1), was expressed selectively in 4T1.3 grown in a bone cavity but not under in vitro conditions. Moreover, fibroblasts present in bone metastasis sites expressed type III collagen, a ligand for GPR56/ADGRG1. Consistently, GPR56/ADGRG1 proteins were detected in tumor cells in bone metastasis foci of human breast cancer patients. Deletion of GPR56/ADGRG1 from 4T1.3 cells reduced markedly intraosseous tumor formation upon their intraosseous injection. Conversely, intraosseous injection of GPR56/ADGRG1-transduced 4T1, TS/A (mouse breast cancer cell line), or MDA-MB-231 (human breast cancer cell line) exhibited enhanced intraosseous tumor formation. Furthermore, we proved that the cleavage at the extracellular region was indispensable for GPR56/ADGRG1-induced increase in breast cancer cell growth upon its intraosseous injection. Finally, inducible suppression of Gpr56/Adgrg1 gene expression in 4T1.3 cells attenuated bone metastasis formation with few effects on primary tumor formation in the spontaneous breast cancer bone metastasis model. Altogether, GPR56/ADGRG1 can be a novel target molecule to develop a strategy to prevent and/or treat breast cancer metastasis to bone.

CC chemokine ligand 3 deficiency ameliorates diet-induced steatohepatitis by regulating liver macrophage recruitment and M1/M2 status in mice.

BACKGROUND AND AIMS: The global prevalence of nonalcoholic fatty liver disease (NAFLD) is increasing. Chemokines and their receptors have potential as therapeutic targets of NAFLD. We investigated the role of CC chemokine ligand 3 (CCL3) in the development of murine and human NAFLD. METHODS: CCL3-knockout mice (CCL3-/-) and littermate CCL3 wild-type control mice (WT) were fed a high-cholesterol and high-fat (CL) diet for 16 weeks to induce NAFLD. We investigated the impact of CCL3 gene deletion in bone marrow cells and leptin-deficient ob/ob mice on CL diet-induced steatohepatitis. We assayed the serum CCL3 levels in 36 patients with biopsy-proven NAFLD and nine healthy control subjects. RESULTS: Compared with normal chow (NC), the CL diet induced steatohepatitis and hepatic fibrosis and elevated the plasma CCL3 level. In the liver, CCL3 protein colocalized with F4/80+ macrophages, especially CD11c+ M1-like macrophages, rather than other cell types. CCL3-/- attenuated CL diet-induced steatohepatitis and fibrosis associated with M2-dominant liver macrophages compared with the WT. The reconstitution of bone marrow (BM) cells from CCL3-/- attenuated steatohepatitis in WT mice fed a CL diet. Furthermore, crossing CCL3-/- onto the ob/ob background prevented CL diet-induced NAFLD in ob/ob mice, which was associated with a lesser inflammatory phenotype of liver macrophages. Also, the serum and hepatic levels of CCL3 were significantly increased in patients with non-alcoholic steatohepatitis (NASH) compared to those with simple fatty liver (NAFL) and healthy subjects. CONCLUSION: Our data indicate that CCL3 facilitates macrophage infiltration into the liver and M1 polarization in the progression of steatohepatitis and highlight the need for further studies to determine the effect of CCL3-CCR1 and -CCR5 signaling blockade on the treatment of NAFLD.

Crosstalk between Cancer Cells and Fibroblasts for the Production of Monocyte Chemoattractant Protein-1 in the Murine 4T1 Breast Cancer.

The chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2) is shown to promote the progression of breast cancer. We previously identified cancer cell-derived granulocyte-macrophage colony-stimulating factor (GM-CSF) as a potential regulator of MCP-1 production in the murine 4T1 breast cancer, but it played a minimum role in overall MCP-1 production. Here, we evaluated the crosstalk between 4T1 cells and fibroblasts. When fibroblasts were co-cultured with 4T1 cells or stimulated with the culture supernatants of 4T1 cells (4T1-sup), MCP-1 production by fibroblasts markedly increased. 4T1 cells expressed mRNA for platelet-derived growth factor (PDGF)-a, b and c, and the PDGF receptor inhibitor crenolanib almost completely inhibited 4T1-sup-induced MCP-1 production by fibroblasts. However, PDGF receptor antagonists failed to reduce MCP-1 production in tumor-bearing mice. Histologically, 4T1 tumors contained a small number of αSMA-positive fibroblasts, and Mcp-1 mRNA was mainly associated with macrophages, especially those surrounding necrotic lesions on day 14, by in situ hybridization. Thus, although cancer cells have the capacity to crosstalk with fibroblasts via PDGFs, this crosstalk does not play a major role in MCP-1 production or cancer progression in this model. Unraveling complex crosstalk between cancer cells and stromal cells will help us identify new targets to help treat breast cancer patients.

Cytoplasmic DNA accumulation preferentially triggers cell death of myeloid leukemia cells by interacting with intracellular DNA sensing pathway.

Accumulating evidence indicates the presence of cytoplasmic DNAs in various types of malignant cells, and its involvement in anti-cancer drug- or radiotherapy-mediated DNA damage response and replication stress. However, the pathophysiological roles of cytoplasmic DNAs in leukemias remain largely unknown. We observed that during hematopoietic stem cell transplantation (HSCT) in mouse myeloid leukemia models, double-stranded (ds)DNAs were constitutively secreted in the form of extracellular vesicles (EVs) from myeloid leukemia cells and were transferred to the donor cells to dampen their hematopoietic capabilities. Subsequent analysis of cytoplasmic DNA dynamics in leukemia cells revealed that autophagy regulated cytoplasmic dsDNA accumulation and subsequent redistribution into EVs. Moreover, accumulated cytoplasmic dsDNAs activated STING pathway, thereby reducing leukemia cell viability through reactive oxygen species (ROS) generation. Pharmaceutical inhibition of autophagosome formation induced cytoplasmic DNA accumulation, eventually triggering cytoplasmic DNA sensing pathways to exert cytotoxicity, preferentially in leukemia cells. Thus, manipulation of cytoplasmic dsDNA dynamics can be a novel and potent therapeutic strategy for myeloid leukemias.

Cancer non-stem cells as a potent regulator of tumor microenvironment: a lesson from chronic myeloid leukemia.

A limited subset of human leukemia cells has a self-renewal capacity and can propagate leukemia upon their transplantation into animals, and therefore, are named as leukemia stem cells, in the early 1990's. Subsequently, cell subpopulations with similar characteristics were detected in various kinds of solid cancers and were denoted as cancer stem cells. Cancer stem cells are presently presumed to be crucially involved in malignant progression of solid cancer: chemoresitance, radioresistance, immune evasion, and metastasis. On the contrary, less attention has been paid to cancer non-stem cell population, which comprise most cancer cells in cancer tissues, due to the lack of suitable markers to discriminate cancer non-stem cells from cancer stem cells. Chronic myeloid leukemia stem cells generate a larger number of morphologically distinct non-stem cells. Moreover, accumulating evidence indicates that poor prognosis is associated with the increases in these non-stem cells including basophils and megakaryocytes. We will discuss the potential roles of cancer non-stem cells in fostering tumor microenvironment, by illustrating the roles of chronic myeloid leukemia non-stem cells including basophils and megakaryocytes in the pathogenesis of chronic myeloid leukemia, a typical malignant disorder arising from leukemic stem cells.

Expansion of senescent megakaryocyte-lineage cells maintains CML cell leukemogenesis.

BCR-ABL, an oncogenic fusion gene, plays a central role in the pathogenesis of chronic myeloid leukemia (CML). Oncogenic signaling induces oncogene-induced senescence and senescence-associated secretory phenotype (SASP), which is characterized by enhanced production of various cytokines. BCR-ABL gene transduction confers senescent phenotype in vitro; however, the in vivo relevance of senescence has not been explored in this context. Transplantation of BCR-ABL-expressing hematopoietic stem/progenitor cells caused CML in mice with an increase in bone marrow BCR-ABL+CD41+CD150+ leukemic megakaryocyte-lineage (MgkL) cells, which exhibited enhanced senescence-associated ß-galactosidase staining and increased expression of p16 and p21, key molecules that are crucially involved in senescence. Moreover, knockout of p16 and p21 genes reduced both BCR-ABL-induced abnormal megakaryopoiesis and the maintenance of CML cell leukemogenic capacity, as evidenced by attenuated leukemogenic capacity at secondary transplantation. The expression of transforming growth factor-ß1 (TGF-ß1), a representative SASP molecule, was enhanced in the leukemic MgkL cells, and TGF-ß1 inhibition attenuated CML cell leukemogenic capacity both in vitro and in vivo. Furthermore, BCR-ABL-expressing MgkL cells displayed enhanced autophagic activity, and autophagy inhibition reduced bone marrow MgkL cell number and prolonged the survival of CML mice, which had transiently received the tyrosine kinase inhibitor, imatinib, earlier. Thus, BCR-ABL induced the expansion of senescent leukemic MgkL cells, which supported CML leukemogenesis by providing TGF-ß1.

Prevention of CaCl2-induced aortic inflammation and subsequent aneurysm formation by the CCL3-CCR5 axis.

Inflammatory mediators such as cytokines and chemokines are crucially involved in the development of abdominal aortic aneurysm (AAA). Here we report that CaCl2 application into abdominal aorta induces AAA with intra-aortic infiltration of macrophages as well as enhanced expression of chemokine (C-C motif) ligand 3 (CCL3) and MMP-9. Moreover, infiltrating macrophages express C-C chemokine receptor 5 (CCR5, a specific receptor for CCL3) and MMP-9. Both Ccl3-/- mice and Ccr5-/- but not Ccr1-/- mice exhibit exaggerated CaCl2-inducced AAA with augmented macrophage infiltration and MMP-9 expression. Similar observations are also obtained on an angiotensin II-induced AAA model. Immunoneutralization of CCL3 mimics the phenotypes observed in CaCl2-treated Ccl3-/- mice. On the contrary, CCL3 treatment attenuates CaCl2-induced AAA in both wild-type and Ccl3-/- mice. Consistently, we find that the CCL3-CCR5 axis suppresses PMA-induced enhancement of MMP-9 expression in macrophages. Thus, CCL3 can be effective to prevent the development of CaCl2-induced AAA by suppressing MMP-9 expression.

Involvement of a Transcription factor, Nfe2, in Breast Cancer Metastasis to Bone.

Patients with triple negative breast cancer (TNBC) is frequently complicated by bone metastasis, which deteriorates the life expectancy of this patient cohort. In order to develop a novel type of therapy for bone metastasis, we established 4T1.3 clone with a high capacity to metastasize to bone after orthotopic injection, from a murine TNBC cell line, 4T1.0. To elucidate the molecular mechanism underlying a high growth ability of 4T1.3 in a bone cavity, we searched for a novel candidate molecule with a focus on a transcription factor whose expression was selectively enhanced in a bone cavity. Comprehensive gene expression analysis detected enhanced Nfe2 mRNA expression in 4T1.3 grown in a bone cavity, compared with in vitro culture conditions. Moreover, Nfe2 gene transduction into 4T1.0 cells enhanced their capability to form intraosseous tumors. Moreover, Nfe2 shRNA treatment reduced tumor formation arising from intraosseous injection of 4T1.3 clone as well as another mouse TNBC-derived TS/A.3 clone with an augmented intraosseous tumor formation ability. Furthermore, NFE2 expression was associated with in vitro growth advantages of these TNBC cell lines under hypoxic condition, which mimics the bone microenvironment, as well as Wnt pathway activation. These observations suggest that NFE2 can potentially contribute to breast cancer cell survival in the bone microenvironment.

Emergence of Cancer-Associated Fibroblasts as an Indispensable Cellular Player in Bone Metastasis Process.

Bone metastasis is frequently complicated in patients with advanced solid cancers such as breast, prostate and lung cancers, and impairs patients' quality of life and prognosis. At the first step of bone metastasis, cancer cells adhere to the endothelium in bone marrow and survive in a dormant state by utilizing hematopoietic niches present therein. Once a dormant stage is disturbed, cancer cells grow through the interaction with various bone marrow resident cells, particularly osteoclasts and osteoblasts. Consequently, osteoclast activation is a hallmark of bone metastasis. As a consequence, the drugs targeting osteoclast activation are frequently used to treat bone metastasis but are not effective to inhibit cancer cell growth in bone marrow. Thus, additional types of resident cells are presumed to contribute to cancer cell growth in bone metastasis sites. Cancer-associated fibroblasts (CAFs) are fibroblasts that accumulate in cancer tissues and can have diverse roles in cancer progression and metastasis. Given the presence of CAFs in bone metastasis sites, CAFs are emerging as an important cellular player in bone metastasis. Hence, in this review, we will discuss the potential roles of CAFs in tumor progression, particularly bone metastasis.

Two-Faced Roles of Tumor-Associated Neutrophils in Cancer Development and Progression.

Neutrophils are the most abundant circulating leukocytes in humans. Neutrophil infiltration into tumor tissues has long been observed but its roles have been ignored due to the presumed short life cycle and metabolic incompetence of neutrophils. Recent advances in neutrophil biology research have revealed that neutrophils have a longer life cycle with a potential to express various bioactive molecules. Clinical studies have simultaneously unraveled an increase in the neutrophil-lymphocyte ratio (NLR), a ratio of absolute neutrophil to absolute lymphocyte numbers in cancer patient peripheral blood and an association of higher NLR with more advanced or aggressive disease. As a consequence, tumor-associated neutrophils (TANs) have emerged as important players in tumor microenvironment. The elucidation of the roles of TANs, however, has been hampered by their multitude of plasticity in terms of phenotypes and functionality. Difficulties are further enhanced by the presence of a related cell population-polymorphonuclear leukocyte (PMN)-myeloid-derived suppressor cells (MDSCs)-and various dissimilar aspects of neutrophil biology between humans and mice. Here, we discuss TAN biology in various tumorigenesis processes, and particularly focus on the context-dependent functional heterogeneity of TANs.

Pivotal Involvement of the CX3CL1-CX3CR1 Axis for the Recruitment of M2 Tumor-Associated Macrophages in Skin Carcinogenesis.

We previously revealed the crucial roles of a chemokine, CX3CL1, and its receptor, CX3CR1, in skin wound healing. Although repeated wounds frequently develop into skin cancer, the roles of CX3CL1 in skin carcinogenesis remain elusive. Here, we proved that CX3CL1 protein expression and CX3CR1+ macrophages were observed in human skin cancer tissues. Similarly, we observed the enhancement of CX3CL1 expression and the abundant accumulation of CX3CR1+ tumor-associated macrophages with M2-like phenotypes in the skin carcinogenesis process induced by the combined treatment with 7,12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol-13-acetate. In this mouse skin carcinogenesis process, CX3CR1+ tumor-associated macrophages exhibited M2-like phenotypes with the expression of Wnt3a and angiogenic molecules including VEGF and matrix metalloproteinase 9. Compared with wild-type mice, CX3CR1-deficient mice showed fewer numbers of skin tumors with a lower incidence. Concomitantly, M2-macrophage numbers and neovascularization were reduced with the depressed expression of angiogenic factors and Wnt3a. Thus, the CX3CL1-CX3CR1 axis can crucially contribute to skin carcinogenesis by regulating the accumulation and functions of tumor-associated macrophages. Thus, this axis can be a good target for preventing and/or treating skin cancers.

CCL4 Signaling in the Tumor Microenvironment.

CCL4, a CC chemokine, previously known as macrophage inflammatory protein (MIP)-1ß, has diverse effects on various types of immune and nonimmune cells by the virtue of its interaction with its specific receptor, CCR5, in collaboration with related but distinct CC chemokines such as CCL3 and CCL5, which can also bind CCR5. Several lines of evidence indicate that CCL4 can promote tumor development and progression by recruiting regulatory T cells and pro-tumorigenic macrophages, and acting on other resident cells present in the tumor microenvironment, such as fibroblasts and endothelial cells, to facilitate their pro-tumorigenic capacities. These observations suggest the potential efficacy of CCR5 antagonists for cancer treatment. On the contrary, under some situations, CCL4 can enhance tumor immunity by recruiting cytolytic lymphocytes and macrophages with phagocytic ability. Thus, presently, the clinical application of CCR5 antagonists warrants more detailed analysis of the role of CCL4 and other CCR5-binding chemokines in the tumor microenvironment.

Regulation of stimulus-induced interleukin-8 gene transcription in human adrenocortical carcinoma cells - Role of AP-1 and NF-κB.

Stimulation of H295R adrenocortical carcinoma cells with angiotensin II or cytokines induces the secretion of the chemokine interleukin-8 (IL-8). Here, we have analyzed the molecular mechanism of stimulus-induced IL-8 expression. IL-8 expression and IL-8 promoter activity increased in H295R cells expressing an activated Gαq-coupled designer receptor. H295R cells stimulated with either interleukin-1ß (IL-1ß) or phorbol ester also showed elevated IL-8 mRNA levels and higher IL-8 promoter activities. Deletion and point mutations of the IL-8 promoter revealed that the AP-1 binding site within the IL-8 promoter is essential to connect designer receptor stimulation with the transcriptional activation of the IL-8 gene. Expression of a constitutively active mutant of c-Jun, or expression of constitutively active mutants of the protein kinases MEKK1 and MKK6 confirmed that the IL-8 gene is a bona fide target of AP-1 in adrenocortical carcinoma cells. Upregulation of IL-8 expression in IL-1ß-treated H295R cells required NF-κB while the phorbol ester TPA used both the AP-1 and NF-κB sites of the IL-8 gene to stimulate IL-8 expression. These data were corroborated in experiments with chromatin-embedded AP-1 or NF-κB-responsive reporter genes. While stimulation of Gαq-coupled designer receptors increased the AP-1 activity in the cells, IL-1ß specifically stimulated NF-κB-regulated transcription. Stimulation of the cells with TPA increased both AP-1 and NF-κB activities. We conclude that stimulation of Gαq-coupled designer receptors or IL-1 receptors triggers distinct signaling pathways in H295R cells leading to the activation of either AP-1 or NF-κB. Nevertheless, both signaling cascades converge to the IL-8 gene, inducing IL-8 gene transcription.

Type I interferon-driven susceptibility to Mycobacterium tuberculosis is mediated by IL-1Ra.

The bacterium Mycobacterium tuberculosis (Mtb) causes tuberculosis and is responsible for more human mortality than any other single pathogen1. Progression to active disease occurs in only a fraction of infected individuals and is predicted by an elevated type I interferon (IFN) response2-7. Whether or how IFNs mediate susceptibility to Mtb has been difficult to study due to a lack of suitable mouse models6-11. Here, we examined B6.Sst1S congenic mice that carry the 'susceptible' allele of the Sst1 locus that results in exacerbated Mtb disease12-14. We found that enhanced production of type I IFNs was responsible for the susceptibility of B6.Sst1S mice to Mtb. Type I IFNs affect the expression of hundreds of genes, several of which have previously been implicated in susceptibility to bacterial infections6,7,15-18. Nevertheless, we found that heterozygous deficiency in just a single IFN target gene, Il1rn, which encodes interleukin-1 receptor antagonist (IL-1Ra), is sufficient to reverse IFN-driven susceptibility to Mtb in B6.Sst1S mice. In addition, antibody-mediated neutralization of IL-1Ra provided therapeutic benefit to Mtb-infected B6.Sst1S mice. Our results illustrate the value of the B6.Sst1S mouse to model IFN-driven susceptibility to Mtb, and demonstrate that IL-1Ra is an important mediator of type I IFN-driven susceptibility to Mtb infections in vivo.

IL17RB expression might predict prognosis and benefit from gemcitabine in patients with resectable pancreatic cancer.

BACKGROUND: (Interleukin 17 Receptor Beta) IL17RB has been implicated in several malignancies. However, its role in the progression of and chemosensitivity in pancreatic cancer remains unknown. We aimed to determine the clinical significance of IL17RB expression in the prognosis of resectable pancreatic cancer and in the benefits from gemcitabine treatment. MATERIALS AND METHODS: We used microarray and immunohistochemical staining techniques to evaluate IL17RB expression in 91 resectable pancreatic cancer tissues and their respective matched adjacent non-cancerous tissues. Quantitative real-time PCR and Western blotting were used to evaluate IL17RB in human pancreatic cancer cell lines after gemcitabine treatment. The correlation between IL17RB expression and overall survival and disease-free survival times were analyzed. RESULTS: IL17RB expression correlated with lymph node metastasis and (Vascular endothelial growth factor) VEGF expression. Cox proportional model showed that high IL17RB expression is a significant negative prognostic factor for both (overall survival) OS and (disease-free survival) DFS. Kaplan-Meier survival curves confirmed significantly reduced median overall and DFS time in high IL17RB patients as compared with low IL17RB patients. Furthermore, Cox proportional model confirmed a correlation between adjuvant treatment with gemcitabine-based chemotherapy and IL17RB expression. Kaplan-Meier survival curves showed that low IL17RB expression was associated with longer OS and DFS in patients than high IL17RB expression and gemcitabine-based adjuvant chemotherapy. In human pancreatic cancer cell lines, the messenger RNA and protein levels of IL17RB were significantly enhanced after gemcitabine treatment. CONCLUSIONS: IL17RB predicts the prognosis and benefit from gemcitabine in patients with resectable pancreatic cancer.

CCL2-Mediated Reversal of Impaired Skin Wound Healing in Diabetic Mice by Normalization of Neovascularization and Collagen Accumulation.

Patients with diabetes frequently present with complications such as impaired skin wound healing. Skin wound sites display a markedly enhanced expression of CCL2, a potent macrophage chemoattractant, together with macrophage infiltration during the early inflammatory phase in skin wound healing of healthy individuals, but the association of CCL2 with delayed skin wound healing in patients with diabetes remains elusive. In this study, we showed that, compared with control mice, mice with streptozotocin-induced diabetes displayed impaired healing after excisional skin injury, with decreased neovascularization, CCL2 expression, and macrophage infiltration. Compromised skin wound healing in mice with diabetes was reversed by the administration of topical CCL2 immediately after the injury, as evidenced by normalization of wound closure rates, neovascularization, collagen accumulation, and infiltration of macrophages expressing vascular endothelial growth factor, a potent angiogenic factor, and transforming growth factor-ß. CCL2 treatment further increased the accumulation of endothelial progenitor cells at the wound sites of mice with diabetes and eventually accelerated neovascularization. Thus, the topical application of CCL2 can be an effective therapeutic option for the treatment of patients with diabetes with defective wound repair, promoting neovascularization and collagen accumulation at skin wound sites.