Cytokine CCL9 Mediates Oncogenic KRAS-Induced Pancreatic Acinar-to-Ductal Metaplasia by Promoting Reactive Oxygen Species and Metalloproteinases.

Pancreatic ductal adenocarcinoma (PDAC) can originate from acinar-to-ductal metaplasia (ADM). Pancreatic acini harboring oncogenic Kras mutations are transdifferentiated to a duct-like phenotype that further progresses to become pancreatic intraepithelial neoplasia (PanIN) lesions, giving rise to PDAC. Although ADM formation is frequently observed in KrasG12D transgenic mouse models of PDAC, the exact mechanisms of how oncogenic KrasG12D regulates this process remain an enigma. Herein, we revealed a new downstream target of oncogenic Kras, cytokine CCL9, during ADM formation. Higher levels of CCL9 and its receptors, CCR1 and CCR3, were detected in ADM regions of the pancreas in p48cre:KrasG12D mice and human PDAC patients. Knockdown of CCL9 in KrasG12D-expressed pancreatic acini reduced KrasG12D-induced ADM in a 3D organoid culture system. Moreover, exogenously added recombinant CCL9 and overexpression of CCL9 in primary pancreatic acini induced pancreatic ADM. We also showed that, functioning as a downstream target of KrasG12D, CCL9 promoted pancreatic ADM through upregulation of the intracellular levels of reactive oxygen species (ROS) and metalloproteinases (MMPs), including MMP14, MMP3 and MMP2. Blockade of MMPs via its generic inhibitor GM6001 or knockdown of specific MMP such as MMP14 and MMP3 decreased CCL9-induced pancreatic ADM. In p48cre:KrasG12D transgenic mice, blockade of CCL9 through its specific neutralizing antibody attenuated pancreatic ADM structures and PanIN lesion formation. Furthermore, it also diminished infiltrating macrophages and expression of MMP14, MMP3 and MMP2 in the ADM areas. Altogether, our results provide novel mechanistic insight into how oncogenic Kras enhances pancreatic ADM through its new downstream target molecule, CCL9, to initiate PDAC.

CTHRC1 promotes colorectal cancer progression by recruiting tumor-associated macrophages via up-regulation of CCL15.

Tumor-associated macrophages (TAMs) represent a key factor in the tumor immune microenvironment (TME), exerting significant influence over tumor migration, invasion, immunosuppressive features, and drug resistance. Collagen triple helix repeat containing 1 (CTHRC1), a 30 KDa protein which was secreted during the tissue-repair process, is highly expressed in several malignant tumors, including colorectal cancer (CRC). Previous studies demonstrated that CTHRC1 expression in TAMs was positively correlated to M2 macrophage polarization and liver metastasis, while our discovery suggesting a novel mechanism that CTHRC1 secreted from cancer cell could indirectly interplay with TAMs. In this study, the high expression level of CTHRC1 was evaluated in CRC based on GEO and TCGA databases. Further, CTHRC1 was detected high in all stages of CRC patients by ELISA and was correlated to poor prognosis. Multispectral imaging of IHC demonstrated that M2 macrophage infiltration was increased accompanied with CTHRC1 enrichment, suggesting that CTHRC1 may have chemotactic effect on macrophages. In vitro, CTHRC1 could have chemotactic ability of macrophage in the presence of HT-29 cell line. Cytokine microarray revealed that CTHRC1 could up-regulate the CCL15 level of HT-29, pathway analysis demonstrated that CTHRC1 could regulate CCL15 by controlling the TGFß activation and Smad phosphorylation level. In vivo, knocking down of CTHRC1 from CT-26 also inhibits tumor formation. In conclusion, CTHRC1 could promote the chemotactic ability of macrophages by up-regulating CCL15 via TGFß/Smad pathway; additionally, a high level of CTHRC1 could promote macrophage's M2 polarization. This discovery may be related to tumor immune tolerance and tumor immunotherapy resistance in CRC. KEY MESSAGES: CTHRC1 promotes CRC progression by up-regulating CCL15 via TGF-ß/Smad pathways to further recruit tumor-associated macrophages. By the means of autocrine or paracrine, CTHRC1 can indeed promote macrophage chemotaxis and enhance the infiltration of macrophages in tumor tissues but in the presence of tumor cells. CAFs were another source of CTHRC1, indicating CTHRC1 can infiltrate tumor islet as well as the stomal and be secreted from both tumor cells and CAFs. This study validated CTHRC1 as a potential immune therapy target CRC.

Development of a Novel Anti-Mouse CCR6 Monoclonal Antibody (C6Mab-13) by N-Terminal Peptide Immunization.

The CC chemokine receptor 6 (CCR6) is a G protein-coupled receptor family member that is highly expressed in B lymphocytes, certain subsets of effector and memory T cells, and immature dendritic cells. CCR6 has only one chemokine ligand, CCL20. The CCL20-CCR6 axis has been recognized as a therapeutic target for autoimmune diseases and tumor. This study developed specific monoclonal antibodies (mAbs) against mouse CCR6 (mCCR6) using the peptide immunization method. The established anti-mCCR6 mAb, C6Mab-13 (rat IgG1, kappa), reacted with mCCR6-overexpressed Chinese hamster ovary-K1 (CHO/mCCR6), and mCCR6-endogenously expressed P388 (mouse lymphoid neoplasma) and J774-1 (mouse macrophage-like) cells in flow cytometry. The dissociation constant (KD) of C6Mab-13 for CHO/mCCR6 cells was determined to be 2.8 × 10-9 M, indicating that C6Mab-13 binds to mCCR6 with high affinity. In summary, C6Mab-13 is useful for detecting mCCR6-expressing cells through flow cytometry.

Colivelin, a synthetic derivative of humanin, ameliorates endothelial injury and glycocalyx shedding after sepsis in mice.

Endothelial dysfunction plays a central role in the pathogenesis of sepsis-mediated multiple organ failure. Several clinical and experimental studies have suggested that the glycocalyx is an early target of endothelial injury during an infection. Colivelin, a synthetic derivative of the mitochondrial peptide humanin, has displayed cytoprotective effects in oxidative conditions. In the current study, we aimed to determine the potential therapeutic effects of colivelin in endothelial dysfunction and outcomes of sepsis in vivo. Male C57BL/6 mice were subjected to a clinically relevant model of polymicrobial sepsis by cecal ligation and puncture (CLP) and were treated with vehicle or colivelin (100-200 µg/kg) intraperitoneally at 1 h after CLP. We observed that vehicle-treated mice had early elevation of plasma levels of the adhesion molecules ICAM-1 and P-selectin, the angiogenetic factor endoglin and the glycocalyx syndecan-1 at 6 h after CLP when compared to control mice, while levels of angiopoietin-2, a mediator of microvascular disintegration, and the proprotein convertase subtilisin/kexin type 9, an enzyme implicated in clearance of endotoxins, raised at 18 h after CLP. The early elevation of these endothelial and glycocalyx damage biomarkers coincided with lung histological injury and neutrophil inflammation in lung, liver, and kidneys. At transmission electron microscopy analysis, thoracic aortas of septic mice showed increased glycocalyx breakdown and shedding, and damaged mitochondria in endothelial and smooth muscle cells. Treatment with colivelin ameliorated lung architecture, reduced organ neutrophil infiltration, and attenuated plasma levels of syndecan-1, tumor necrosis factor-α, macrophage inflammatory protein-1α and interleukin-10. These therapeutic effects of colivelin were associated with amelioration of glycocalyx density and mitochondrial structure in the aorta. At molecular analysis, colivelin treatment was associated with inhibition of the signal transducer and activator of transcription 3 and activation of the AMP-activated protein kinase in the aorta and lung. In long-term outcomes studies up to 7 days, co-treatment of colivelin with antimicrobial agents significantly reduced the disease severity score when compared to treatment with antibiotics alone. In conclusion, our data support that damage of the glycocalyx is an early pathogenetic event during sepsis and that colivelin may have therapeutic potential for the treatment of sepsis-associated endothelial dysfunction.

Potential neurotoxic activity of diverse molecules released by astrocytes.

Astrocytes are the main support cells of the central nervous system. They also participate in neuroimmune reactions. In response to pathological and immune stimuli, astrocytes transform to reactive states characterized by increased release of inflammatory mediators. Some of these molecules are neuroprotective and inflammation resolving while others, including reactive oxygen species (ROS), nitric oxide (NO), matrix metalloproteinase (MMP)- 9, L-glutamate, and tumor necrosis factor α (TNF), are well-established toxins known to cause damage to surrounding cells and tissues. We hypothesized that similar to microglia, the brain immune cells, reactive astrocytes can release a broader set of diverse molecules that are potentially neurotoxic. A literature search was conducted to identify such molecules using the following two criteria: 1) evidence of their expression and secretion by astrocytes and 2) direct neurotoxic action. This review describes 14 structurally diverse molecules as less-established astrocyte neurotoxins, including C-X-C motif chemokine ligand (CXCL)10, CXCL12/CXCL12(5-67), FS-7-associated surface antigen ligand (FasL), macrophage inflammatory protein (MIP)- 2α, TNF-related apoptosis inducing ligand (TRAIL), pro-nerve growth factor (proNGF), pro-brain-derived neurotrophic factor (proBDNF), chondroitin sulfate proteoglycans (CSPGs), cathepsin (Cat)B, group IIA secretory phospholipase A2 (sPLA2-IIA), amyloid beta peptides (Aß), high mobility group box (HMGB)1, ceramides, and lipocalin (LCN)2. For some of these molecules, further studies are required to establish either their direct neurotoxic effects or the full spectrum of stimuli that induce their release by astrocytes. Only limited studies with human-derived astrocytes and neurons are available for most of these potential neurotoxins, which is a knowledge gap that should be addressed in the future. We also summarize available evidence of the role these molecules play in select neuropathologies where reactive astrocytes are a key feature. A comprehensive understanding of the full spectrum of neurotoxins released by reactive astrocytes is key to understanding neuroinflammatory diseases characterized by the adverse activation of these cells and may guide the development of novel treatment strategies.

Hepatitis B Virus X Protein Modulates Chemokine CCL15 Upregulation in Hepatocellular Carcinoma.

BACKGROUND: Hepatitis B virus X protein (HBx) is an indispensable progression factor in Hepatocellular Carcinoma (HCC). CCL15 could be a peculiar proteomic biomarker of HCC with tumorigenesis and tumor invasion. OBJECTIVE: The aim of the study was to explore the relationship between HBx and CCL15 expression in HCC. METHODS: HBV-positive HCC pathological tissue samples and corresponding adjacent non-tumor liver tissues were collected. The expression of HBx and CCL15 was analyzed by immunohistochemistry, real-time Polymerase Chain Reaction (PCR), and western blot analysis in tissues or in vitro. RESULTS: The levels of CCL15 mRNA and protein expression in HCC samples were observably higher than those of adjacent non-tumor liver tissues. The CCL15 was significantly associated with the expression of HBx in HBV-positive HCC samples. The up-regulation of HBx induced CCL15 expression in vitro. The high expression score of CCL15 was significant associated with the poor prognosis of HCC patients. CONCLUSION: The CCL15 expression was observably associated with HBx in HCC patients. The CCL15 may be considered as an indicator in the clinical management of HBV-associated HCC.

Endothelial cell­derived CCL15 mediates the transmigration of fibrocytes through the CCL15­CCR1 axis in vitro.

Wound healing is a complex physiological process in which fibrocytes serve a vital role. However, the mechanism underlying the recruitment of fibrocytes during wound healing remains largely unknown. The present study aimed to investigate whether endothelial cells are involved in the recruitment of fibrocytes in wound healing. To mimic the in vivo angiogenic process, a co­culture system consisting of endothelial cells and fibrocytes was achieved using a permeable Transwell co­culture system. The expression of chemokines produced by endothelial cells with or without co­culture was then measured using a gene chip. Based on the dataset from chip analysis, chemokine ligand 15 (CCL15) produced by endothelial cells was identified, which likely serves a regulatory role in mediating the transmigration of fibrocytes. Overexpression of CCL15 in endothelial cells or chemokine receptor 1 (CCR1) in fibrocytes promoted the transmigration of fibrocytes, whilst silencing the expression of CCL15 in endothelial cells or that of CCR1 in fibrocytes attenuated the transmigration of fibrocytes. Results from the present study suggested that the CCL15­CCR1 axis between endothelial cells and fibrocytes serves a vital role in mediating the recruitment of fibrocytes during wound healing.

Reduced Mrp2 surface availability as PI3Kγ-mediated hepatocytic dysfunction reflecting a hallmark of cholestasis in sepsis.

Sepsis-associated liver dysfunction manifesting as cholestasis is common during multiple organ failure. Three hepatocytic dysfunctions are considered as major hallmarks of cholestasis in sepsis: impairments of microvilli covering canalicular membranes, disruptions of tight junctions sealing bile-collecting canaliculae and disruptions of Mrp2-mediated hepatobiliary transport. PI3Kγ loss-of-function was suggested as beneficial in early sepsis. Yet, the PI3Kγ-regulated cellular processes in hepatocytes remained largely unclear. We analysed all three sepsis hallmarks for responsiveness to massive PI3K/Akt signalling and PI3Kγ loss-of-function, respectively. Surprisingly, neither microvilli nor tight junctions were strongly modulated, as shown by electron microscopical studies of mouse liver samples. Instead, quantitative electron microscopy proved that solely Mrp2 surface availability, i.e. the third hallmark, responded strongly to PI3K/Akt signalling. Mrp2 plasma membrane levels were massively reduced upon PI3K/Akt signalling. Importantly, Mrp2 levels at the plasma membrane of PI3Kγ KO hepatocytes remained unaffected upon PI3K/Akt signalling stimulation. The effect explicitly relied on PI3Kγ's enzymatic ability, as shown by PI3Kγ kinase-dead mice. Keeping the surface availability of the biliary transporter Mrp2 therefore is a cell biological process that may underlie the observation that PI3Kγ loss-of-function protects from hepatic excretory dysfunction during early sepsis and Mrp2 should thus take center stage in pharmacological interventions.

Chinese herbal medicine Baoyuan Jiedu decoction inhibits the accumulation of myeloid derived suppressor cells in pre-metastatic niche of lung via TGF-ß/CCL9 pathway.

Baoyuan Jiedu (BYJD for short) decoction, a traditional Chinese medicine formula, is composed of Astragalus, Ginseng, Aconite root, Honeysuckle, Angelica, Licorice, which has the functions of nourishing qi and blood, enhancing immune function, improving quality of life and prolonging survival time of tumor patients. The present study aimed to investigate the effect and mechanism of BYJD decoction on reversing the pre-metastatic niche. We showed that BYJD decoction could prolong the survival time of 4T1 tumor-bearing mice. Moreover, we found that the BYJD decoction inhibited the formation of lung pre-metastatic niche and inhibited recruitment of myeloid derived suppressor cells (MDSCs) in the lung. Mechanistically, we showed that the proteins and genes expression of TGF-ß, Smad2, Smad3, p-Smad2/3, Smad4, CCL9 in the TGF-ß/CCL9 signaling pathway were suppressed by BYJD decoction. In line with the above findings, our results confirm that BYJD decoction inhibits the accumulation of MDSC in pre-metastatic niche of lung via TGF-ß/CCL9 pathway.

Blockade of DC-SIGN+ Tumor-Associated Macrophages Reactivates Antitumor Immunity and Improves Immunotherapy in Muscle-Invasive Bladder Cancer.

Tumor-associated macrophages (TAM) play an indispensable role in the modulation of the cancer immune microenvironment. Despite the fact that TAMs may exert both antitumor and protumor activities, the molecular mechanisms involved remain poorly understood. Here, we characterized a subpopulation of TAMs expressing dendritic cell-specific C-type lectin (DC-SIGN) and investigated its relevance to the prognosis and immune microenvironment of muscle-invasive bladder cancer (MIBC). DC-SIGN+ TAMs were abundant in a significant proportion of human MIBC specimens. High levels of DC-SIGN+ TAMs were associated with dismal prognosis and unresponsiveness to adjuvant chemotherapy in MIBC. Notably, multiple anti-inflammatory cytokines were enriched in DC-SIGN+ TAMs. RNA-seq analysis revealed that multiple M2-like signaling pathways were significantly upregulated in DC-SIGN+ TAMs. High infiltration of DC-SIGN+ TAMs was associated with CD8+ T-cell tolerance in MIBC. Moreover, abrogating DC-SIGN function using a neutralizing antibody led to impaired expression of anti-inflammatory cytokines and augmented PD-1 inhibitor pembrolizumab-mediated cytotoxic effects of CD8+T cells toward MIBC cells. In summary, these results suggest that DC-SIGN+ TAM infiltration is closely linked to a protumor immune microenvironment and may serve as a promising therapeutic target in the immunotherapy of MIBC. SIGNIFICANCE: DC-SIGN+ TAMs have an immunosuppressive and tumor-promoting function and may serve as a prognostic indicator and therapeutic target in MIBC.

Circadian Clock Gene Bmal1 Regulates Bilirubin Detoxification: A Potential Mechanism of Feedback Control of Hyperbilirubinemia.

Controlling bilirubin to a low level is necessary in physiology because of its severe neurotoxicity. Therefore, it is of great interest to understand the regulatory mechanisms for bilirubin homeostasis. In this study, we uncover a critical role for circadian clock in regulation of bilirubin detoxification and homeostasis. Methods: The mRNA and protein levels of Bmal1 (a core clock gene), metabolic enzymes and transporters were measured by qPCR and Western blotting, respectively. Luciferase reporter, mobility shift and chromatin immunoprecipitation were used to investigate transcriptional gene regulation. Experimental hyperbilirubinemia was induced by injection of bilirubin or phenylhydrazine. Unconjugated bilirubin (UCB) and conjugated bilirubin were assessed by ELISA. Results: We first demonstrated diurnal variations in plasma UCB levels and in main bilirubin-detoxifying genes Ugt1a1 and Mrp2. Of note, the circadian UCB levels were antiphase to the circadian expressions of Ugt1a1 and Mrp2. Bmal1 ablation abrogated the circadian rhythms of UCB and bilirubin-induced hepatotoxicity in mice. Bmal1 ablation also decreased mRNA and protein expressions of both Ugt1a1 and Mrp2 in mouse livers, and blunted their circadian rhythms. A combination of luciferase reporter, mobility shift, and chromatin immunoprecipitation assays revealed that Bmal1 trans-activated Ugt1a1 and Mrp2 through specific binding to the E-boxes in the promoter region. Further, Bmal1 ablation caused a loss of circadian time-dependency in bilirubin clearance and sensitized mice to chemical induced-hyperbilirubinemia. Moreover, bilirubin stimulated Bmal1 expression through antagonism of Rev-erbα, constituting a feedback mechanism in bilirubin detoxification. Conclusion: These data supported a dual role for circadian clock in regulation of bilirubin detoxification, generating circadian variations in bilirubin level via direct transactivation of detoxifying genes Ugt1a1 and Mrp2, and defending the body against hyperbilirubinemia via Rev-erbα antagonism. Thereby, our study provided a potential mechanism for management of bilirubin related diseases.

C-C Motif Chemokine Ligand 15 May Be a Useful Biomarker for Predicting the Prognosis of Patients with Chronic Hypersensitivity Pneumonitis.

BACKGROUND: Chronic hypersensitivity pneumonitis (CHP) is characterized by lymphocytic inflammation and progressive fibrosis of the lung caused by a variety of inhaled antigens. Due to the difficulty of accurately diagnosing CHP, and the poor prognosis associated with the condition, a novel clinical biomarker is urgently needed. OBJECTIVE: To investigate the usefulness of C-C motif chemokine ligand 15 (CCL15), which had been demonstrated to highly express in the lungs of CHP patients, as a clinical biomarker for CHP. METHOD: Immunohistochemical investigations were performed on lung tissue from CHP patients, and CCL15 levels in serum and bronchoalveolar lavage fluid (BALF) were measured via the enzyme-linked immunosorbent assay. RESULTS: Immunohistochemistry investigations revealed high CCL15 expression in the lungs of CHP patients. Serum CCL15 levels in CHP patients (29.1 ± 2.1 µg/mL) were significantly higher than those of idiopathic pulmonary fibrosis patients (19.7 ± 1.3 µg/mL, p = 0.01) and healthy subjects (19.5 ± 1.7 µg/mL, p = 0.003). When BALF CCL15 level was divided by BALF albumin (Alb) level (BALF CCL15/Alb), it was significantly inversely correlated with forced vital capacity (ß = -0.47, p = 0.0006), percentage of predicted carbon monoxide diffusion capacity of the lung (ß = -0.41, p = 0.0048), and BALF lymphocyte count (ß = -0.34, p = 0.01) in CHP patients. Multivariate Cox proportional hazards analysis revealed that high BALF CCL15/Alb and poor prognosis were statistically significantly independently correlated in CHP patients (HR 1.1, 95% CI 1.03-1.18, p = 0.004). CONCLUSION: The results of the current study suggest that CCL15 may be a useful prognostic biomarker for CHP. CCL15 was highly expressed in the lung tissue of CHP patients, and BALF CCL15/Alb was significantly associated with CHP prognosis.

OMICs approaches-assisted identification of macrophages-derived MIP-1γ as the therapeutic target of botanical products TNTL in diabetic retinopathy.

BACKGROUND: Inflammatory reaction in the dysfunction of retinal endotheliocytes has been considered to play a vital role in diabetic retinopathy (DR). Anti-inflammatory therapy so far gains poor outcome as DR treatment. This study aims to identify a novel therapeutic target of DR from the OMICs studies of a traditional anti-DR botanical products TNTL. METHODS: Hyperglycemic mice were treated with TNTL. The anti-hyperglycemic effect of TNTL was validated to confirm the biological consistency of the herbal products from batches. Improvement of DR by TNTL was examined by various assays on the retina. Next-generation transcriptome sequencing and cytokine array was used to identify the therapeutic targets. In vitro study was performed to validate the target. RESULTS: We observed that TNTL at its high doses possessed anti-hyperglycemic effect in murine type I diabetic model, while at its doses without reducing blood glucose, it suppressed DR incidence. TNTL restored the blood-retina barrier integrity, suppressed retinal neovascularization, and attenuated the retinal ganglion cell degeneration. Transcriptomic analysis on the retina tissue of hyperglycemic mice with or without TNTL revealed that the inflammatory retina microenvironment was significantly repressed. TNTL treatment suppressed pro-inflammatory macrophages in the retina, which resulted in the inactivation of endothelial cell migration, restoration of endothelial cell monolayer integrity, and prevention of leakage. Cytokine array analysis suggested that TNTL could significantly inhibit the secretion of MIP1γ from pro-inflammatory macrophages. Prevention of endothelial dysfunction by TNTL may be mediated by the inhibition of MIP1γ/CCR1 axis. More specifically, TNTL suppressed MIP1γ release from pro-inflammatory macrophages, which in turn inhibited the activation of CCR1-associated signaling pathways in endothelial cells. CONCLUSION: Our findings demonstrated that TNTL might be an alternative treatment to DR, and the primary source of potential drug candidates against DR targeting MIP1γ/CCR1 axis in the retinal microenvironment.

Metformin enhances gefitinib efficacy by interfering with interactions between tumor-associated macrophages and head and neck squamous cell carcinoma cells.

BACKGROUND: Tumor-associated macrophages (TAMs) play an important role in drug resistance in many tumors, including head and neck squamous cell carcinoma (HNSCC). However, how TAMs interact with HNSCC cells to induce drug resistance, especially under hypoxic conditions, is unclear. In this study, we investigated the mechanism of TAM-induced gefitinib resistance in HNSCC cells and sought for novel therapeutic strategies. METHODS: The effects of hypoxia-treated HNSCC cells on the migration and polarization of macrophages were analyzed. Recombinant cytokine proteins and neutralizing antibodies were used as controls. In addition, we assessed the cytotoxic effects of gefitinib on HNSCC cells treated with M2-type macrophage conditioned medium, and carried out a cytokine antibody array analysis, thereby revealing the key factor CCL15. The relationship between serum CCL15 expression levels and prognosis in HNSCC patients was analyzed. In addition, we performed bioinformatic analyses to pursue the mechanisms of CCL15-induced gefitinib resistance. Finally, metformin was used to evaluate the sensitizing effects of gefitinib treatment on HNSCC cells in vitro and in vivo. RESULTS: We found that HNSCC cells recruited macrophages by secreting VEGF and polarized the macrophages to the M2 phenotype through IL-6. Conversely, we found that M2-type TAMs promoted HNSCC cell resistance to gefitinib through paracrine CCL15 signaling. The serum CCL15 levels in HNSCC patients showed a significant correlation with patient prognosis. Furthermore, we found that M2-type TAMs could suppress the sensitivity of HNSCC cells to gefitinib through the CCL15-CCR1-NF-κB pathway. In addition, we found that metformin not only inhibited CCL15 expression in M2-type TAMs enhanced by hypoxia, but also suppressed CCR1 surface expression in HNSCC cells. Encouragingly, we found that metformin sensitized HNSCC cells to gefitinib treatment in vitro and in vivo. CONCLUSIONS: Based on our data we conclude that we have identified a novel interaction between M2-type TAMs and HNSCC cells that contributes to gefitinib resistance. We also found that metformin inhibited the cross-talk between macrophages and tumor cells, thereby eliciting therapeutic effects both in vitro and in vivo.

The prominent impairment of liver/intestinal cytochrome P450 and intestinal drug transporters in sepsis-induced acute kidney injury over acute and chronic renal ischemia, a mouse model comparison.

Drug dosing adjustment in sepsis-induced acute kidney injury (sepsis-AKI) is currently adjusted based on renal function. Sepsis is a multiorgan injury, and thus, drug metabolism in sepsis-AKI might be interfered by non-renal factors such as changes in functions of drug-metabolizing enzymes in the liver and functions of intestinal drug transporters. We compared the defect on mouse CYP3A11 (human CYP3A4 representative) in liver and intestine along with several intestinal drug transporters (MDR1a, MRP2, and OATP3) in three mouse models; chronic ischemic reperfusion injury (Chr I/R; 4-week), acute ischemic reperfusion injury (Acute I/R; 24-h), and cecal ligation and puncture (CLP; 24-h) as representative of sepsis-AKI. Decreased expression of CYP3A11 and drug transporters was demonstrated in all models. Among these models, sepsis-AKI had the least severe renal injury (increased BUN and Scr) with the most severe liver injury (increased ALT and changes in liver histopathology), the most severe intestinal leakage (increased serum (1→3)-ß-D-glucan) and the highest increase in serum IL-6. A reduced expression and activity of liver and intestinal CYP3A11 along with intestinal efflux-drug transporter expressions (MDR1a and MRP2), but not drug uptake transporter (OATP3), was predominant in sepsis-AKI compared with acute I/R. Additionally, a reduction of CYP3A4 expression with IL-6 was demonstrated on HepG2 cells implying a direct injury of IL-6 on human liver cells. Differences in drug metabolism were reported between sepsis-AKI and ischemic-AKI confirming that drug dosing adjustment in sepsis-AKI depends not just only on renal function but also on several non-renal factors. Further studies are warranted.

CCL9/CCR1 induces myeloid­derived suppressor cell recruitment to the spleen in a murine H22 orthotopic hepatoma model.

Myeloid­derived suppressor cells (MDSCs) are the major negative regulators of immune responses and expand in numerous tumor models. They contribute to tumor progression and metastasis, and are involved in limiting the effects of cancer immunotherapy. To selectively target MDSCs, it is required to understand the molecular mechanisms that drive MDSC expansion. The mechanisms of their accumulation in tumor tissue have been extensively studied, while the mechanisms of their expansion in lymphoid organs have been rarely explored. The spleen is the largest lymphoid organ in the human body. A previous study by our group reported that a negative immune status in the spleen facilitated tumor growth, with MDSCs being the major immunosuppressive cells. In the present study, a murine H22 orthotopic hepatoma model was established and the mechanisms of splenic MDSC accumulation were studied, including MDSC proliferation, apoptosis and chemotaxis. The proliferation and apoptosis of splenic MDSCs did not differ between normal and tumor­bearing (TB) mice. Cytokine array and ELISA of splenic tissues indicated elevated chemokine (C­C motif) ligand 9 (CCL9) levels in TB mice. Furthermore, splenic macrophages were able to secrete CCL9. Flow cytometric analysis revealed that splenic MDSCs from TB mice also overexpressed C­C motif chemokine receptor 1 (CCR1), the receptor for CCL9. Taken together, the present results indicate that CCL9 secreted by splenic macrophages induces a CCR1­dependent accumulation of MDSCs in the spleen in a murine H22 hepatoma model.

Markers Involved in Innate Immunity and Neutrophil Activation are Elevated during Acute Human Anaphylaxis: Validation of a Microarray Study.

BACKGROUND: We have previously identified the upregulation of the innate immune response, neutrophil activation, and apoptosis during anaphylaxis using a microarray approach. This study aimed to validate the differential gene expression and investigate protein concentrations of "hub genes" and upstream regulators during anaphylaxis. METHODS: Samples were collected from patients with anaphylaxis on their arrival at the emergency department, and after 1 and 3 h. mRNA levels of 11 genes (interleukin-6 [IL-6], IL-10, oncostatin M [OSM], S100A8, S100A9, matrix metalloproteinase 9 [MMP9], FASL, toll-like receptor 4 [TLR4], MYD88, triggering receptor expressed on myeloid cells 1 [TREM1], and cluster of differentiation 64 [CD64]) were measured in peripheral blood leucocytes using qPCR. Serum protein concentrations were measured by ELISA or cytometric bead array for 6 of these candidates. RESULTS: Of 69 anaphylaxis patients enrolled, 36 (52%) had severe reactions, and 38 (55%) were female. Increases in both mRNA and protein of IL-10, S100A9, MMP9, and TREM1 were observed. OSM, S100A8, TLR4, and CD64 were upregulated and IL-6 protein concentrations were increased during anaphylaxis. Both FASL and soluble Fas ligand decreased during anaphylaxis. CONCLUSION: These results provide evidence for the involvement of innate immune pathways and myeloid cells during human anaphylaxis, validating previous microarray findings. Elevated S100A8, S100A9, TLR4, and TREM1 expression, and increased S100A9 and soluble TREM1 protein concentrations strongly suggest that neutrophils are activated during acute anaphylaxis.

Kallikrein-related peptidases are activators of the CC chemokine CCL14.

Chemokine CCL14 is inactive in its proform. Here, we show that inflammation- and cancer-associated kallikrein-related peptidases KLK5 and KLK8 remove the N-terminal eight amino acids from the proform thereby converting CCL14 to its active state. Activity of the chemokine is demonstrated by migration of myeloid cells expressing relevant receptors.

Involvement of Macrophage Inflammatory Protein-1 Family Members in the Development of Diabetic Neuropathy and Their Contribution to Effectiveness of Morphine.

Current investigations underline the important roles of C-C motif ligands in the development of neuropathic pain; however, their participation in diabetic neuropathy is still undefined. Therefore, the goal of our study was to evaluate the participation of macrophage inflammatory protein-1 (MIP-1) family members (CCL3, CCL4, CCL9) in a streptozotocin (STZ)-induced mouse model of diabetic neuropathic pain. Single intrathecal administration of each MIP-1 member (10, 100, or 500 ng/5 µl) in naïve mice evoked hypersensitivity to mechanical (von Frey test) and thermal (cold plate test) stimuli. Concomitantly, protein analysis has shown that, 7 days following STZ injection, the levels of CCL3 and CCL9 (but not CCL4) are increased in the lumbar spinal cord. Performed additionally, immunofluorescence staining undoubtedly revealed that CCL3, CCL9, and their receptors (CCR1 and CCR5) are expressed predominantly by neurons. In vitro studies provided evidence that the observed expression of CCL3 and CCL9 may be partially of glial origin; however, this observation was only partially possible to confirm by immunohistochemical study. Single intrathecal administration of CCL3 or CCL9 neutralizing antibody (2 and 4 µg/5 µl) delayed neuropathic pain symptoms as measured at day 7 following STZ administration. Single intrathecal injection of a CCR1 antagonist (J113863; 15 and 20 µg/5 µl) also attenuated pain-related behavior as evaluated at day 7 after STZ. Both neutralizing antibodies, as well as the CCR1 antagonist, enhanced the effectiveness of morphine in STZ-induced diabetic neuropathy. These findings highlight the important roles of CCL3 and CCL9 in the pathology of diabetic neuropathic pain and suggest that they play pivotal roles in opioid analgesia.

Inflammatory Molecule, PSGL-1, Deficiency Activates Macrophages to Promote Colorectal Cancer Growth through NFκB Signaling.

P-selectin glycoprotein ligand 1 (SELPLG/PSGL-1) is an inflammatory molecule that is functionally related to immune cell differentiation and leukocyte mobilization. However, the role of PSGL-1 in tumor development remains unknown. Therefore, this study investigates the mechanistic role of PSGL-1 in the development of intestinal tumors in colorectal cancer. ApcMin/+ mice are highly susceptible to spontaneous intestinal adenoma formation, and were crossbred with PSGL1-null mice to generate compound transgenic mice with a ApcMin/+;PSGL-1-/- genotype. The incidence and pathologic features of the intestinal tumors were compared between the ApcMin/+ mice and ApcMin/+;PSGL-1-/- mice. Importantly, PSGL-1-deficient mice showed increased susceptibility to develop intestinal tumors and accelerated tumor growth. Mechanistically, increased production of the mouse chemokine ligand 9 (CCL9/MIP-1γ) was found in the PSGL-1-deficient mice, and the macrophages are likely the major source of macrophage inflammatory protein-1 gamma (MIP-1γ). Studies in vitro demonstrated that macrophage-derived MIP-1γ promoted colorectal cancer tumor cell growth through activating NFκB signaling. Conversely, restoration of the PSGL-1 signaling via bone marrow transplantation reduced MIP-1γ production and attenuated the ability of ApcMin/+;PSGL-1-/- mice to generate intestinal tumors. In human colorectal cancer clinical specimens, the presence of PSGL-1-positive cells was associated with a favorable tumor-node-metastasis staging and decreased lymph node metastasis.Implications:PSGL-1 deficiency and inflammation render intestinal tissue more vulnerable to develop colorectal tumors through a MIP-1γ/NFκB signaling axis. Mol Cancer Res; 15(4); 467-77. ©2017 AACR.