KC Protein Levels Research Articles

Dysregulation of microRNA biogenesis in the small intestine after ethanol and burn injury

Ethanol exposure at the time of burn injury is a major contributor to post-burn pathogenesis. Many of the adverse effects associated with ethanol and burn injury are linked to an impaired intestinal barrier. The combined insult causes intestinal inflammation, resulting in tissue damage, altered tight junction expression, and increased intestinal permeability. MicroRNAs play a critical role in maintaining intestinal homeostasis including intestinal inflammation and barrier function. Specifically, miR-150 regulates inflammatory mediators which can contribute to gut barrier disruption. The present study examined whether ethanol and burn injury alter expression of microRNA processing enzymes (Drosha, Dicer, and Argonaute-2) and miR-150 in the small intestine. Male mice were gavaged with ethanol (~2.9g/kg) 4h prior to receiving a ~12.5% total body surface area full thickness burn. One or three days after injury, mice were euthanized and small intestinal epithelial cells (IECs) were isolated and analyzed for expression of microRNA biogenesis components and miR-150. Dicer mRNA and protein levels were not changed following the combined insult. Drosha and Argonaute-2 mRNA and protein levels were significantly reduced in IECs one day after injury; which accompanied reduced miR-150 expression. To further determine the role of miR-150 in intestinal inflammation, young adult mouse colonocytes were transfected with a miR-150 plasmid and stimulated with LPS (100ng/ml). miR-150 overexpression significantly reduced IL-6 and KC protein levels compared to vector control cells challenged with LPS. These results suggest that altered microRNA biogenesis and associated decrease in miR-150 likely contribute to increased intestinal inflammation following ethanol and burn injury.

Local application of adipose-derived mesenchymal stromal cells in experimental inflammatory OA results in interleukin-1β-mediated attraction of PMNs and reduced S100A8/A9 release

Purpose: Recent studies have demonstrated that mild synovitis in early phases of osteoarthritis (OA) is conducive to development of joint damage. OA synovitis is characterized by elevated levels of pro-inflammatory factors like S100A8, S100A9, interleukin-6 (IL-6), and interleukin-1 beta (IL-1β). S100A8/A9 was found to be crucial in mediating joint destruction in inflammatory experimental OA. Previously we found that adipose-derived mesenchymal stromal cells (ASCs) exhibit immunosuppressive characteristics and reduce joint pathology after local application into mouse knee joints with experimental inflammatory OA. This protective effect is only perceived after intra-articular injection in early but not late stage OA, suggesting that the effect may be mediated by an inflammatory environment. Our objective was to examine the working mechanism of ASCs after early injection in experimental OA. Methods: Experimental OA was induced by injection of collagenase into murine knee joints (CiOA). Total knee joints were stained with haematoxylin/eosin and the PMN-specific antibody NIMP-R14. ASCs were isolated from murine adipose tissue and stimulated for 24 hours with IL-1β or S100A8/A9. Gene expression in stimulated cells was analyzed using qPCR. Protein levels of chemokines and cytokines were measured in culture supernatant using Luminex. Migration of MACS isolated bone marrow (BM-) PMNs towards ASC-conditioned medium (CM) was examined using Transwell membranes. ASCs were co-cultured with BM-PMNs and analyzed using histology and Luminex. Results: Injection of ASCs into day 7 CiOA knee joints (when synovitis and IL-1β and S100A8/A9 levels are highest) caused a strong attraction of particularly PMN-like cells and their clustering around ASCs in the synovium shortly after injection (6 hours), which was confirmed by immunohistochemistry. IL-1β stimulation of ASCs in vitro strongly increased gene expression of PMN-attracting chemokines KC, CXCL5, and CXCL7 as well as protein levels of KC, whereas S100A8/A9 did not. The migration of BM-PMNs through Transwell membranes towards CM of IL-1β-stimulated ASCs was significantly increased (from 5% to 10%) when compared to CM of non-stimulated ASCs. Next, ASCs were co-cultured with BM-PMNs in the presence or absence of IL-1β. After 6 hours, a clear clustering of neutrophils around ASCs was observed, with a significant increase in the number of ASCs clustering with PMNs, as well as a significantly elevated number of clustering PMNs per ASC after IL-1β stimulation. Interestingly, association of PMNs with ASCs lead to a significantly diminished release of KC protein by ASCs (69% and 76% lower after 24 h and 48 h respectively), as well as a significantly reduced release of S100A8/A9 protein by the PMNs. This coincided with lowering of S100A8/A9 levels in washouts of inflamed synovium 6 h and 48 h after injection of ASCs in day 7 CiOA knee joints. Conclusions: Local application of ASCs in inflamed CiOA knee joints results in attraction and clustering of PMNs with ASCs in the synovium. This presumably runs via IL-1β-mediated up-regulation of chemokine release by ASCs, and ultimately results in significantly lowered levels of S100A8/A9. This research was supported by the Dutch Arthritis Association.

Pemetrexed + Sorafenib] lethality is increased by inhibition of ERBB1/2/3-PI3K-NFκB compensatory survival signaling.

In the completed phase I trial NCT01450384 combining the anti-folate pemetrexed and the multi-kinase inhibitor sorafenib it was observed that 20 of 33 patients had prolonged stable disease or tumor regression, with one complete response and multiple partial responses. The pre-clinical studies in this manuscript were designed to determine whether [pemetrexed + sorafenib] –induced cell killing could be rationally enhanced by additional signaling modulators. Multiplex assays performed on tumor material that survived and re-grew after [pemetrexed + sorafenib] exposure showed increased phosphorylation of ERBB1 and of NFκB and IκB; with reduced IκB and elevated G-CSF and KC protein levels. Inhibition of JAK1/2 downstream of the G-CSF/KC receptors did not enhance [pemetrexed + sorafenib] lethality whereas inhibition of ERBB1/2/4 using kinase inhibitory agents or siRNA knock down of ERBB1/2/3 strongly promoted killing. Inhibition of ERBB1/2/4 blocked [pemetrexed + sorafenib] stimulated NFκB activation and SOD2 expression; and expression of IκB S32A S36A significantly enhanced [pemetrexed + sorafenib] lethality. Sorafenib inhibited HSP90 and HSP70 chaperone ATPase activities and reduced the interactions of chaperones with clients including c-MYC, CDC37 and MCL-1. In vivo, a 5 day transient exposure of established mammary tumors to lapatinib or vandetanib significantly enhanced the anti-tumor effect of [pemetrexed + sorafenib], without any apparent normal tissue toxicities. Identical data to that in breast cancer were obtained in NSCLC tumors using the ERBB1/2/4 inhibitor afatinib. Our data argue that the combination of pemetrexed, sorafenib and an ERBB1/2/4 inhibitor should be explored in a new phase I trial in solid tumor patients.

Abstract B43: Targeting tumor microenvironment for treatment of K-ras mutant lung cancer

Abstract Activating mutations of K-ras are one of the most common molecular alterations associated with lung cancer development. Several attempts to develop therapies directly targeting K-ras have been failed thus far, clearly state the need for new strategies to bring clinical benefits to patients displaying such a molecular profile. Using a conditional K-ras mutant lung cancer mouse model (CC-LR) we previously showed that K-ras mutated lung tumors have intrinsic inflammatory characteristics associated with NF-κB pathway activation. Therefore, CC-LR mice were crossed with IKK-β f/f mice to develop a K-ras mutant mouse with lack of NF-κB activity in airway epithelium. This changed the bronchoalveolar lavage fluid (BALF) cellular component of the CC-LR mice (mostly by reduction of macrophages), and resulted in a ∼70% (3.4-fold) reduction in lung surface tumor number. Lung of CC-LR mice also shows high protein level of IL-6 (downstream to NF-κB activation) and increased STAT3 gene expression (downstream signal to IL-6) plus infiltration of myeloid and Th17 cells. Accordingly, we genetically ablated IL-6 in the CC-LR mice. This significantly inhibited lung cancer development (1.7-fold, 41%) suggesting a role for IL-6 mediated signaling in tumor promotion probably through STAT3 pathway activation. IL-6 and STAT3 are required for differentiation of Th17 cells from naïve T cells. Th17 cells mostly produce IL-17A that binds to the IL-17 receptor (IL-17R). IL-17R signaling is required for lung CXC chemokine expression and myeloid cell recruitment. Furthermore, IL-17A induces more production of IL-6 by epithelial cells. So, CC-LR mice were crossed with IL-17A KO mice. Lack of IL-17A in CC-LR mice resulted in a ∼70% (3.4-fold) tumor reduction. It also decreased the expression of pro-inflammatory mediators and reduced recruitment of myeloid cells. These reults suggest that K-ras initiated airway inflammation through NF-κB mediated activation of the IL-6/STAT3/Th17 signaling promotes lung tumorigenesis, and introduce IL-6 as a potential druggable target for lung cancer patients who harbor K-ras mutations. This prompted us to block IL-6 pathway using a monoclonal anti-IL-6 immunotherapy in our K-ras mutant model. Six week old CC-LR mice were injected intraperitoneally with 20 mg/kg dose of an anti-IL-6 monoclonal IgG1 antibody, twice a week, for a period of 8 weeks. Flowcytometry and qPCR analysis of total lung in non-treated CC-LR mice showed 2-fold increase in the population of tumor associated macrophage (TAM) and Arginase-1 mRNA expression (3 fold) during tumor progression from age 6 weeks to 14 weeks suggesting that epithelial signal initiated by K-ras mutation dictates an inflammatory type 2 pro-tumor fate for lung microenvironment. We further found that anti-IL-6 treatment suppressed tumor development by ∼78% (4.6-fold) in CC-LR mice with a significant reduction in the expression levels of Ki-67, VEGF, MMP9, CD31 and phospho-STAT3 in lung tissue. It also reduced TGFβ and KC protein levels in BALF with a significant decrease in Arginase1 mRNA expression (3-fold) and TAM population (5-fold) in total lung homogenate. We conclude that K-ras mutation drives an immunosuppressive pro-tumor response and blocking IL-6 shifts this to an anti-tumor lung microenvironment. Therefore we propose pharmacological targeting of IL-6 as a potential therapeutic strategy alone or in combination with conventional cytotoxic therapy for lung cancer patients with K-ras mutation. Funded by: American Cancer Society, RSG-11-115-01-CNE, and American Lung Association/LUNGevity Foundation, LCD-114696-N Citation Format: Mauricio da Silva Caetano, Seon Hee Chang, Amber M. Cumpian, Soudabeh Daliri, Maria Miguelina De La Garza, Cesar E. Ochoa, Chen Dong, Seyed Javad Moghaddam. Targeting tumor microenvironment for treatment of K-ras mutant lung cancer. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B43. doi: 10.1158/1557-3125.RASONC14-B43

Abstract A13: Pharmacologic targeting of IL-6 suppresses tumor progression in a non-small cell lung cancer mouse model with K-ras mutation through re-educating the tumor microenvironment.

Abstract Activating mutations of K-ras are one of the most common molecular alterations associated with lung cancer development. Several attempts to develop therapies directly targeting K-ras have failed thus far, clearly state the need for new strategies to bring clinical benefits to patients displaying such molecular profile. Using a conditional K-ras mutated lung cancer mouse model (CC-LR) we previously showed that K-ras mutated lung tumors have intrinsic inflammatory characteristics and we also demonstrated that chronic obstructive pulmonary disease (COPD)-like airway inflammation, an independent risk factor for lung cancer, promotes lung tumor in this model. CC-LR mice shows high protein level of interleukin 6 (IL-6) and increased STAT3 (downstream signal to IL6) gene expression, and we have further shown that genetic ablation of IL-6 in CC-LR mice results in significant tumor reduction. High levels of IL-6 and up-regulation of STAT3 and its target genes have been observed in the lungs of smokers with COPD, and in 50% of human lung adenocarcinomas. These indicate an essential role for IL-6 in lung cancer promotion, and introduce IL-6 as a potential druggable target for lung cancer patients who harbor K-ras mutations. This prompted us to block IL-6 pathway using a monoclonal anti-IL-6 immunotherapy in our K-ras mutated lung cancer mouse model. Six week old CC-LR mice were injected intraperitoneally (I.P.) with 20 mg/kg dose of an anti-IL-6 monoclonal IgG1 antibody, twice a week, for a period of eight weeks. Flowcytometry analysis of total lung inflammatory cell population in non-treated CC-LR mice showed 2-fold increase in the population of tumor associated macrophage (TAM) (CD45+/CD11b+/GR1+/F4/80+/MCSFR+) during tumor progression from age 6 weeks to 14 weeks. qPCR analysis of total lung RNA from non-treated CC-LR mice showed that there is an increase in the level of Arginase-1 (3 fold) with no change in the level of inducible Nitric Oxide Synthetize (NOS2). This suggests that epithelial signal initiated by K-ras mutation may dictate the fate of lung microenvironment by creating an inflammatory pro-tumor type 2 response. Then we found that anti-IL-6 treatment suppresses tumor progression by ~78% (4.6-fold) in CC-LR mice which is associated with a significant reduction in the quantity of inflammatory cells in bronchoalveolar lavage fluid (BALF). It also decreased the expression of proliferation marker: Ki-67, and pro angiogenic factors: VEGF, MMP9 and CD31 and phospho-STAT3 in lung tissue. ELISA analysis of BALF from CC-LR mice also showed that anti-IL-6 treatment reduces TGFβ and KC protein levels with no change in level of IL-17. qPCR and flowcytometry analysis of total lung homogenate of CC-LR mice further showed that IL-6 blockade induces a significant decrease in Arginase1 expression (3-fold) and TAM population (5-fold). Taking together here we showed that K-ras mutated lung tumor drives an immunosuppressive pro-tumor microenvironment, with an M2 TAM polarization and blocking the suppressive actions of IL-6 shifts this to an anti-tumor lung microenvironment. Therefore we propose pharmacological targeting of IL-6 as a potential therapeutic strategy alone or in combination with conventional cytotoxic therapy for lung cancer patients with K-ras mutation. Funded by: American Cancer Society, RSG-11-115-01-CNE, and American Lung Association/LUNGevity Foundation, LCD-114696-N Citation Format: Mauricio S. Caetano, Amber M. Cumpian, Lei Gong, Cesar E. Ochoa, Seon H. Chang, Huiyuan Zhang, Stephanie Watowich, Cinthya Sternberg, Carlos G. Ferreira, Seyed J. Moghaddam. Pharmacologic targeting of IL-6 suppresses tumor progression in a non-small cell lung cancer mouse model with K-ras mutation through re-educating the tumor microenvironment. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr A13.

Protective effect of Korean Red Ginseng extract against Helicobacter pylori-induced gastric inflammation in Mongolian gerbils

Helicobacter pylori-induced gastric inflammation includes induction of inflammatory mediators interleukin (IL)-8 and inducible nitric oxide synthase (iNOS), which are mediated by oxidant-sensitive transcription factor NF-κB. High levels of lipid peroxide (LPO) and increased activity of myeloperoxidase (MPO), a biomarker of neutrophil infiltration, are observed in H. pylori-infected gastric mucosa. Panax ginseng Meyer, a Korean herb medicine, is widely used in Asian countries for its biological activities including anti-inflammatory efficacy. The present study aims to investigate whether Korean Red Ginseng extract (RGE) inhibits H. pylori-induced gastric inflammation in Mongolian gerbils. One wk after intragastric inoculation with H. pylori, Mongolian gerbils were fed with either the control diet or the diet containing RGE (200 mg RGE/gerbil) for 6 wk. The following were determined in gastric mucosa: the number of viable H. pylori in stomach; MPO activity; LPO level; mRNA and protein levels of keratinocyte chemoattractant factor (KC, a rodent IL-8 homolog), IL-1β, and iNOS; protein level of phospho-IκBα (which reflects the activation of NF-κB); and histology. As a result, RGE suppressed H. pylori-induced mRNA and protein levels of KC, IL-1β, and iNOS in gastric mucosa. RGE also inhibited H. pylori-induced phosphorylation of IκBα and increases in LPO level and MPO activity of gastric mucosa. RGE did not affect viable H. pylori colonization in the stomach, but improved the histological grade of infiltration of polymorphonuclear neutrophils, intestinal metaplasia, and hyperplasia. In conclusion, RGE inhibits H. pylori-induced gastric inflammation by suppressing induction of inflammatory mediators (KC, IL-1β, iNOS), MPO activity, and LPO level in H. pylori-infected gastric mucosa.

IL-1β Augments TNF-α–Mediated Inflammatory Responses from Lung Epithelial Cells

Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) mediate the development of numerous inflammatory lung diseases. Since IL-1beta is typically activated in situations where TNF-alpha is produced, it was hypothesized that IL-1beta alters TNF-alpha-induced proinflammatory epithelial cell function by altering TNF receptor shedding and surface abundance. In this study, the impact of IL-1beta on TNF-alpha-mediated chemokine production as well as TNF receptor surface expression and shedding were investigated from mouse pulmonary epithelial cells (MLE-15). Interleukin-1beta rapidly and persistently enhanced soluble and surface TNFR2. These effects were dependent on TNFR1 expression. TNFR2 small-interfering RNA (siRNA) shifted IL-1beta responses, significantly increasing surface and shed TNFR1 implying IL-1beta selectively modifies TNF receptors depending on cellular receptor composition. mRNA expression of both receptors was unaltered by IL-1beta up to 24 h or in combination with TNF-alpha indicating effects were post-transcriptional. Interleukin-1beta pretreatment enhanced TNF-alpha-induced macrophage inflammatory protein (MIP)-2 and KC mRNA expression as well as MIP-2 and KC protein levels at the same time point analyzed. Experiments utilizing siRNA against the TNF receptors and a TNFR1 neutralizing antibody demonstrated TNF-alpha induced MIP-2 through TNFR1, whereas both receptors may have contributed to KC production. These data suggest IL-1beta modulates TNF-alpha-mediated inflammatory lung diseases by enhancing epithelial cell TNF receptor surface expression.

TACI‐Ig prevents the development of airway hyperresponsiveness in a murine model of asthma

Increased levels of serum IgE are associated with greater asthma prevalence and disease severity. IgE depletion using an anti-IgE monoclonal antibody has met with success in the treatment of moderate-to-severe and severe persistent allergic asthma. To test whether B cell-targeted therapy is a more effective treatment for airway hyperresponsiveness (AHR) in a murine model compared with IgE-depletion. We delivered soluble mTACI-Ig, a receptor for the B cell survival factors BLyS (B Lymphocyte Stimulator) and APRIL (A PRoliferation-Inducing Ligand), or anti-IgE to allergen-sensitized mice before airway challenge with allergen. mTACI-Ig treatment reduced circulating mature B cell levels in the blood, while anti-IgE treatment had no effect on B cell counts. Both mTACI-Ig and anti-IgE decreased the levels of total and allergen-specific IgE in the serum. Histopathologic analysis of lungs showed a reduction in disease severity scores for both treatment groups, but results were more pronounced in mTACI-Ig-treated mice. Neutrophil and eosinophil numbers in the bronchoalveolar lavage (BAL) were significantly reduced following mTACI-Ig treatment, but not after anti-IgE delivery. BLyS and APRIL blockade also resulted in a significant decrease in IL-4 and eotaxin mRNA and IL-4 and KC protein levels in total lung homogenates and BAL fluid, respectively. Finally, mTACI-Ig treatment was more effective than anti-IgE treatment in reducing AHR to inhaled antigen. Our data demonstrate that delivery of mTACI-Ig is a more effective treatment than anti-IgE mAb in a murine model of AHR.

The chemokine KC regulates HGF-stimulated epithelial cell morphogenesis.

Hepatocyte growth factor (HGF) induces migration, proliferation, and branching in renal epithelial cells from the inner medullary collecting duct (mIMCD-3 cells). Microarray analysis after HGF stimulation of these cells revealed upregulation of the chemokine KC. We found that both the message and protein levels of KC are increased after HGF treatment and that mIMCD-3 cells express the KC receptor CXCR2. Treatment with KC results in stimulation of mIMCD-3 cell proliferation but has no effect on basal rates of cell migration or branching morphogenesis. In contrast to its known stimulatory effect on neutrophil migration, KC markedly inhibits HGF-mediated cell migration and branching morphogenesis, resulting in shorter tubules with fewer branch points. Examination of the mechanism of this effect reveals that KC does not alter phosphorylation of the c-met receptor or the initial activation of the MAPK or phosphoinositide 3-kinase (PI 3-K) signaling pathways. However, sustained activation of the PI 3-K pathway by HGF was inhibited by treatment with KC, and mimicking this effect by treatment with LY-294002 2 h after HGF stimulation reproduced the inhibition of HGF-stimulated branching morphogenesis. These data demonstrate that HGF-mediated KC production can act in an autocrine fashion to downregulate excessive branching and migration of renal epithelial cells in response to HGF, while still supporting cell proliferation. These characteristics may play a role in modulating the response to HGF during developmental tubule formation and/or during the repair of the tubular architecture following injury.

CXC chemokine receptor-2 ligands are required for neutrophil-mediated host defense in experimental brain abscesses.

We have developed a mouse brain abscess model by using Staphylococcus aureus, one of the main etiologic agents of brain abscesses in humans. Direct damage to the blood-brain barrier was observed from 24 h to 7 days after S. aureus exposure as demonstrated by the accumulation of serum IgG in the brain parenchyma. Evaluation of brain abscesses by immunohistochemistry and flow cytometry revealed a prominent neutrophil infiltrate. To address the importance of neutrophils in the early containment of S. aureus infection in the brain, mice were transiently depleted of neutrophils before implantation of bacteria-laden beads. Neutrophil-depleted animals consistently demonstrated more severe brain abscesses and higher CNS bacterial burdens compared with control animals. S. aureus led to the induction of numerous chemokines in the brain, including macrophage-inflammatory protein (MIP)-1alpha/CCL3, MIP-1beta/CCL4, MIP-2/CXCL1, monocyte chemoattractant protein-1/CCL2, and TCA-3/CCL1, within 6 h after bacterial exposure. These chemokines also were expressed by both primary cultures of neonatal mouse microglia and astrocytes exposed to heat-inactivated S. aureus in vitro. Because neutrophils constitute the majority of the cellular infiltrate in early brain abscess development, subsequent analysis focused on MIP-2 and KC/CXCL1, two neutrophil-attracting CXC chemokines. Both MIP-2 and KC protein levels were significantly elevated in the brain after S. aureus exposure. Neutrophil extravasation into the brain parenchyma was impaired in CXCR2 knockout mice and was associated with increased bacterial burdens. These studies demonstrate the importance of the CXCR2 ligands MIP-2 and KC and neutrophils in the acute host response to S. aureus in the brain.