Investigation of the effects of isoeugenol-based phenolic compounds on migration and proliferation of HT29 colon cancer cells at cellular and molecular level.

Colorectal cancer is a type of cancer encountered worldwide and ranks third among all cancer types in terms of incidence. Polyphenols have been shown to have a wide range of biological functions, including a significant impact on cancer start, development, and promotion through regulating many signaling pathways. The aim of this study was to investigate the anticancer effects of isoeugenol based compounds 1, 2 on HT29 colorectal cancer cell line in vitro. MTT test and scratch assay were carried out to determine the effect of these compounds on HT29 cell proliferation and migration respectively. In addition, mRNA expression levels of apoptosis and metastasis-related genes (p53, Bcl2, Bax, Caspase 3, Caspase7, Caspase8, Caspase9, HIF1-α, VEGF, MMP-2, MMP-9) were examined by quantitative real-time PCR. The results indicated that 1 and 2 inhibited HT29 cell proliferation and induced apoptosis by increasing the Bax/Bcl2 ratio and Caspase-9 and Caspase-3 mRNA expression. In conclusion, the results of this study showed that the treatment of these compounds significantly suppressed the mRNA expressions of metastasis-related genes such as Matrix Metalloproteinase-2, Matrix Metalloproteinase-9, Vascular Endothelial Growth Factor and Hypoxia­Inducible Factor 1α.

A New Monoterpene Alkaloid from Incarvillea sinensis with Migration Inhibitory Activity on Cancer Cell.

A novel monoterpene alkaloid, named incarvine G, was isolated from the Incarvillea sinensis Lam. Its chemical structure was elucidated using comprehensive spectroscopic methods. Incarvine G is an ester compound comprised of a monoterpene alkaloid and glucose. This compound showed evident inhibition on cell migration, invasion, and cytoskeleton formation of human MDA-MB-231 with low cytotoxicity.

Dexmedetomidine, an Alpha 2a Adrenergic Receptor Agonist, Mitigates Experimental Autoimmune Encephalomyelitis by Desensitization of CXCR7 in Microglia.

The autoimmune disease multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), is characterized by an ascending paralysis that is characterized by extensive infiltration of the central nervous system by inflammatory cells. Although several studies to some extent uncover the cellular mechanisms of microglia that govern EAE pathogenesis, the molecular mechanisms that orchestrate the movement of microglia remain unknown, and potential novel therapeutic strategies are still required. In this study, we report that dexmedetomidine, an alpha 2a adrenergic receptor agonist, attenuates the clinical severity of EAE with less infiltration of microglia. During EAE, dexmedetomidine inhibits SDF-1- and I-TAC-induced chemotaxis of microglia mediated by CXCR7 but not CXCR4 or CXCR3. Most importantly, the alpha 2a adrenergic receptor is essential in dexmedetomidine-induced CXCR7 desensitization in microglia. Further experiments confirmed that CXCR7 desensitization required atypical protein kinase C ζ activation, while conventional and novel protein kinase C isoforms were not involved. Altogether, our data elucidate the mechanism of dexmedetomidine-induced CXCR7 desensitization in microglia and amelioration in EAE, which might lead to a better understanding of the therapeutic effects of dexmedetomidine as well as its implications for CXCR7 desensitization in autoimmune disease.

Anticancer effect of (S)-crizotinib on osteosarcoma cells by targeting MTH1 and activating reactive oxygen species.

MTH1 has become a new rising star in the field of 'cancer phenotypic lethality' and can be targeted in many kinds of tumors. This study aimed to explore the anticancer effect of MTH1-targeted drug (S)-crizotinib on osteosarcoma (OS) cells. We detected MTH1 expression in OS tissues and cells using immunohistochemistry and western blot. The effects of MTH1 on OS cell viability were explored using the siRNA technique and CCK8. The anticancer effects of the MTH1-targeted drug (S)-crizotinib on OS cells were explored by in-vitro assays. The intracellular 8-oxo-dGTP level and oxygen reactive species (ROS) of OS cells were detected by Cy3-conjugated avidin staining and dichlorofluorescein diacetate staining, respectively. The expression of MTH1 was significantly higher in OS tissues and cell lines than that in the corresponding adjacent tissues and osteoblastic cell line. The proliferation of OS cells was significantly inhibited through knockdown of MTH1 by siRNA technology. (S)-Crizotinib could inhibit the proliferation of OS cells with an increase in the apoptosis levels and causing G0/G1 arrest by targeting MTH1 and activating ROS. In addition, (S)-crizotinib could inhibit the migration of OS cells. (S)-Crizotinib could suppress the proliferation and migration, cause G0/G1 arrest, and increase the apoptosis level of OS cells by targeting MTH1 and activating ROS. This study will provide a promising therapeutic target and the theoretical basis for the clinical application of (S)-crizotinib in OS.

Resolvin D1 Polarizes Primary Human Macrophages toward a Proresolution Phenotype through GPR32.

Resolvin D1 (RvD1) was shown to be a potent anti-inflammatory and proresolution lipid mediator in several animal models of inflammation, but its mechanism of action in humans is not clear. We show that the RvD1 receptor GPR32 is present on resting, proinflammatory M(LPS) and alternatively activated primary human M(IL-4) macrophages, whereas TGF-ß and IL-6 reduce its membrane expression. Accordingly, stimulation of resting primary human macrophages with 10 nM RvD1 for 48 h maximally reduced the secretion of the proinflammatory cytokines IL-1ß and IL-8; abolished chemotaxis to several chemoattractants like chemerin, fMLF, and MCP-1; and doubled the phagocytic activity of these macrophages toward microbial particles. In contrast, these functional changes were not accompanied by surface expression of markers specific for alternatively activated M(IL-4) macrophages. Similar proresolution effects of RvD1 were observed when proinflammatory M(LPS) macrophages were treated with RvD1. In addition, we show that these RvD1-mediated effects are GPR32 dependent because reduction of GPR32 expression by small interfering RNA, TGF-ß, and IL-6 treatment ablated these proresolution effects in primary human macrophages. Taken together, our results indicate that in humans RvD1 triggers GPR32 to polarize and repolarize macrophages toward a proresolution phenotype, supporting the role of this mediator in the resolution of inflammation in humans.

Tyrosine kinase inhibitor TKI-258 inhibits cell motility in oral squamous cell carcinoma in vitro.

BACKGROUND: Oral squamous cell carcinoma is extremely invasive, and this behavior is regulated by binding of extracellular molecules to the cell membrane receptors. The TKI-258 inhibits phosphorylation of FGFRs VEGFRs and PDGFRs. Our aim was to analyze the effect of TKI-258 treatment in cell movement using SCC-4 cell line from human oral squamous cell carcinoma. METHODS: F-actin was stained with rhodamine phalloidin, and confocal analysis was performed. The migration and invasion (membrane covered with Matrigel™ ) three-dimensional assays were performed, and control and cells treated with TKI-258 that migrated through the membrane were counted after 24 h. RESULTS: Control cells presented abundant cytoplasm with F-actin wide distributed and evident cell cortex; however, treated (1, 5 and 10 µM TKI-258) cells showed round morphology, scanty cytoplasm, F-actin disorganized and preserved cell cortex. TKI-258 (1, 5, and 10 µM) treatment inhibits migrating cells (ANOVA, F = 97.749, d.f. = 3, 10; P < 0.0001), and it was concentration dependent. Invading cell treated with 5 µM TKI-258 was significantly lower (t = 6.708, d.f. = 5, P < 0.001). CONCLUSIONS: These results suggest that the tyrosine kinase inhibitor TKI-258 has an inhibitory effect on cell motility, affecting F-actin, cell migration, and cell invasion, and probably, these processes are regulated by signaling pathways FGFRs and/or PDGFRs and/or VEGFRs.

The Positively Charged COOH-terminal Glycosaminoglycan-binding CXCL9(74-103) Peptide Inhibits CXCL8-induced Neutrophil Extravasation and Monosodium Urate Crystal-induced Gout in Mice.

The ELR(-)CXC chemokine CXCL9 is characterized by a long, highly positively charged COOH-terminal region, absent in most other chemokines. Several natural leukocyte- and fibroblast-derived COOH-terminally truncated CXCL9 forms missing up to 30 amino acids were identified. To investigate the role of the COOH-terminal region of CXCL9, several COOH-terminal peptides were chemically synthesized. These peptides display high affinity for glycosaminoglycans (GAGs) and compete with functional intact chemokines for GAG binding, the longest peptide (CXCL9(74-103)) being the most potent. The COOH-terminal peptide CXCL9(74-103) does not signal through or act as an antagonist for CXCR3, the G protein-coupled CXCL9 receptor, and does not influence neutrophil chemotactic activity of CXCL8 in vitro. Based on the GAG binding data, an anti-inflammatory role for CXCL9(74-103) was further evidenced in vivo. Simultaneous intravenous injection of CXCL9(74-103) with CXCL8 injection in the joint diminished CXCL8-induced neutrophil extravasation. Analogously, monosodium urate crystal-induced neutrophil migration to the tibiofemural articulation, a murine model of gout, is highly reduced by intravenous injection of CXCL9(74-103). These data show that chemokine-derived peptides with high affinity for GAGs may be used as anti-inflammatory peptides; by competing with active chemokines for binding and immobilization on GAGs, these peptides may lower chemokine presentation on the endothelium and disrupt the generation of a chemokine gradient, thereby preventing a chemokine from properly performing its chemotactic function. The CXCL9 peptide may serve as a lead molecule for further development of inhibitors of inflammation based on interference with chemokine-GAG interactions.

Inhibition of CXCL12-mediated chemotaxis of Jurkat cells by direct immunotoxicants.

Directional migration of cells to specific locations is required in tissue development, wound healing, and immune responses. Immune cell migration plays a crucial role in both innate and adaptive immunity. Chemokines are small pro-inflammatory chemoattractants that control the migration of leukocytes. In addition, they are also involved in other immune processes such as lymphocyte development and immune pathology. In a previous toxicogenomics study using the Jurkat T cell line, we have shown that the model immunotoxicant TBTO inhibited chemotaxis toward the chemokine CXCL12. In the present work, we aimed at assessing a novel approach to detecting chemicals that affect the process of cell migration. For this, we first evaluated the effects of 31 chemicals on mRNA expression of genes that are known to be related to cell migration. With this analysis, seven immunotoxicants were identified as potential chemotaxis modulators, of which five (CoCl2 80 µM, MeHg 1 µM, ochratoxin A 10 µM, S9-treated ochratoxin A 10 µM, and TBTO 100 nM) were confirmed as chemotaxis inhibitor in an in vitro trans-well chemotaxis assay using the chemokine CXCL12. The transcriptome data of the five compounds together with previously obtained protein phosphorylation profiles for two out of five compounds (i.e., ochratoxin A and TBTO) revealed that the mechanisms behind the chemotaxis inhibition are different for these immunotoxicants. Moreover, the mTOR inhibitor rapamycin had no effect on the chemotaxis of Jurkat cells, indicating that the mTOR pathway is not involved in CXCL12-mediated chemotaxis of Jurkat cells, which is opposite to the findings on human primary T cells (Munk et al. in PLoS One 6(9):e24667, 2011). Thus, the results obtained from the chemotaxis assay conducted with Jurkat cells might not fully represent the results obtained with human primary T cells. Despite this difference, the present study indicated that some compounds may exert their immunotoxic effects through inhibition of CXCL12-mediated chemotaxis.

Sensitization of breast cancer cells to doxorubicin via stable cell line generation and overexpression of DFF40.

There are a number of reports demonstrating a relationship between the alterations in DFF40 expression and development of some cancers. Here, increased DFF40 expression in T-47D cells in the presence of doxorubicin was envisaged for therapeutic usage. The T-47D cells were transfected with an eukaryotic expression vector encoding the DFF40 cDNA. Following incubation with doxorubicin, propidium iodide (PI) staining was used for cell cycle distribution analysis. The rates of apoptosis were determined by annexin V/PI staining. Apoptosis was also evaluated using the DNA laddering analysis. The viability of DFF40-transfected cells incubated with doxorubicin was significantly decreased compared with control cells. However, there were no substantial changes in the cell cycle distribution of pIRES2-DFF40 cells incubated with doxorubicin compared to control cells. The expression of DFF40, without doxorubicin incubation, had also no significant effect on the cell cycle distribution. There was no DNA laddering in cells transfected with the empty pIRES2 vector when incubated with doxorubicin. In contrast, DNA laddering was observed in DFF40 transfected cells in the presence of doxorubicin after 48 h. Also, the expression of DFF40 and DFF45 was increased in DFF40 transfected cells in the presence of doxorubicin enhancing cell death. Collectively our results indicated that co-treatment of DFF40-transfected cells with doxorubicin can enhance the killing of these tumor cells via apoptosis. Thus, modulation of DFF40 level may be a beneficial strategy for treatment of chemo-resistant cancers.

Targeting cell migration in rheumatoid arthritis.

PURPOSE OF REVIEW: To provide an update of past failures, future prospects and key challenges facing the therapeutic targeting of chemokines and their receptors in rheumatoid arthritis. RECENT FINDINGS: Clinical trials in rheumatoid arthritis have been undertaken with small molecule antagonists or neutralizing antibodies targeting CCR1, CCR5 and CXCL10. Some encouraging results have emerged. Laboratory and clinical research has identified CCL19, CXCL13 and CXCL12, and their receptors, as potential future targets. Developments in our appreciation of posttranslational chemokine modification highlight the complexity of chemokine networks operating in inflamed tissues, and the substantial gaps in existing knowledge. SUMMARY: Despite previous disappointments, there are still reasons to be optimistic that drugs targeting chemokines and their receptors could be developed for the treatment of rheumatoid arthritis. However, a deeper understanding of the chemokine networks at work in inflamed joints is a necessary prerequisite.

Glucocorticoids induce effector T cell depolarization via ERM proteins, thereby impeding migration and APC conjugation.

Glucocorticoids (GCs) repress lymphocyte function by controlling gene expression. In this study, we investigated Ag-specific effector T cells and provide evidence that GCs also modulate these cells' cytoskeletal architecture by nongenomic mechanisms. Following GC treatment, effector T cells rapidly lose their polarized morphology, which impedes both their migratory capacity and their interaction with APCs. The cytoskeleton rearrangements are preceded by an activation of ezrin-radixin-moesin proteins, which transiently increases the cellular rigidity but seems to occur independently of altered tyrosine phosphorylation. Phospholipase C activity is critically involved in mediating these nongenomic effects, because its inhibition prevents both T cell depolarization and ezrin-radixin-moesin phosphorylation after GC exposure. GC administration in vivo induced similar morphological changes in effector T cells as observed in vitro, suggesting that the above process plays a role in modulating inflammatory diseases. Taken together, our findings identify a novel mechanism through which GCs rapidly repress T cell function independently of gene transcription.

Macrolactone Nuiapolide, Isolated from a Hawaiian Marine Cyanobacterium, Exhibits Anti-Chemotactic Activity.

A new bioactive macrolactone, nuiapolide (1) was identified from a marine cyanobacterium collected off the coast of Niihau, near Lehua Rock. The natural product exhibits anti-chemotactic activity at concentrations as low as 1.3 µM against Jurkat cells, cancerous T lymphocytes, and induces a G2/M phase cell cycle shift. Structural characterization of the natural product revealed the compound to be a 40-membered macrolactone with nine hydroxyl functional groups and a rare tert-butyl carbinol residue.

Disease-modifying therapy in multiple sclerosis and chronic inflammatory demyelinating polyradiculoneuropathy: common and divergent current and future strategies.

Multiple sclerosis (MS) and chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) represent chronic, autoimmune demyelinating disorders of the central and peripheral nervous system. Although both disorders share some fundamental pathogenic elements, treatments do not provide uniform effects across both disorders. We aim at providing an overview of current and future disease-modifying strategies in these disorders to demonstrate communalities and distinctions. Intravenous immunoglobulins (IVIG) have demonstrated short- and long-term beneficial effects in CIDP but are not effective in MS. Dimethyl fumarate (BG-12), teriflunomide and laquinimod are orally administered immunomodulatory drugs that are already approved or likely to be approved in the near future for the basic therapy of patients with relapsing-remitting MS (RRMS) due to positive results in Phase III clinical trials. However, clinical trials with these drugs in CIDP have not (yet) been initiated. Natalizumab and fingolimod are approved for the treatment of RRMS, and trials to evaluate their safety and efficacy in CIDP are now planned. Alemtuzumab, ocrelizumab and daclizumab respresent monoclonal antibodies in advanced stages of clinical development for their use in RRMS patients. Attempts to study the safety and efficacy of alemtuzumab and B cell-depleting anti-CD20 antibodies, i.e. rituximab, ocrelizumab or ofatumumab, in CIDP patients are currently under way. We provide an overview of the mechanism of action and clinical data available on disease-modifying immunotherapy options for MS and CIDP. Enhanced understanding of the relative effects of therapies in these two disorders may aid rational treatment selection and the development of innovative treatment approaches in the future.

Retinoid X receptor agonists impair arterial mononuclear cell recruitment through peroxisome proliferator-activated receptor-γ activation.

Mononuclear cell migration into the vascular subendothelium constitutes an early event of the atherogenic process. Because the effect of retinoid X receptor (RXR)α on arterial mononuclear leukocyte recruitment is poorly understood, this study investigated whether RXR agonists can affect this response and the underlying mechanisms involved. Decreased RXRα expression was detected after 4 h stimulation of human umbilical arterial endothelial cells with TNF-α. Interestingly, under physiological flow conditions, TNF-α-induced endothelial adhesion of human mononuclear cells was concentration-dependently inhibited by preincubation of the human umbilical arterial endothelial cells with RXR agonists such as bexarotene or 9-cis-retinoid acid. RXR agonists also prevented TNF-α-induced VCAM-1 and ICAM-1 expression, as well as endothelial growth-related oncogene-α and MCP-1 release. Suppression of RXRα expression with a small interfering RNA abrogated these responses. Furthermore, inhibition of MAPKs and NF-κB pathways were involved in these events. RXR agonist-induced antileukocyte adhesive effects seemed to be mediated via RXRα/peroxisome proliferator-activated receptor (PPAR)γ interaction, since endothelial PPARγ silencing abolished their inhibitory responses. Furthermore, RXR agonists increased RXR/PPARγ interaction, and combinations of suboptimal concentrations of both nuclear receptor ligands inhibited TNF-α-induced mononuclear leukocyte arrest by 60-65%. In vivo, bexarotene dose-dependently inhibited TNF-α-induced leukocyte adhesion to the murine cremasteric arterioles and decreased VCAM-1 and ICAM-1 expression. Therefore, these results reveal that RXR agonists can inhibit the initial inflammatory response that precedes the atherogenic process by targeting different steps of the mononuclear recruitment cascade. Thus, RXR agonists may constitute a new therapeutic tool in the control of the inflammatory process associated with cardiovascular disease.

Inhibition of CXCR4-CXCL12 chemotaxis in melanoma by AMD11070.

BACKGROUND: Despite intensive research and novel adjuvant therapies, there is currently no cure for metastatic melanoma. The chemokine receptor CXCR4 controls metastasis to sites such as the liver; however, the therapeutic blockade with the existing agents has proven difficult. METHODS: AMD11070, a novel orally bioavailable inhibitor of CXCR4, was tested for its ability to inhibit the migration of melanoma cells compared with the commonly described antagonist AMD3100. RESULTS: AMD11070 abrogated melanoma cell migration and was significantly more effective than AMD3100. Importantly for the clinical context, the expression of B-RAF-V600E did not the affect the sensitivity of AMD11070. CONCLUSION: Liver-resident myofibroblasts excrete CXCL12, which is able to promote the migration of CXCR4-expressing tumour cells from the blood into the liver. Blockade of this axis by AMD11070 thus represents a novel therapeutic strategy for both B-RAF wild-type and mutated melanomas.

Anti-adhesion therapies and the rule of 3 for rare events.

During the open-label trial of natalizumab for Crohn's disease, an isolated case of progressive multifocal leukoencephalopathy (PML) was found. This prompted a more careful review by regulators, physicians, and the pharmaceutical industry. A new gut-specific monoclonal antibody, vedolizumab, has been shown to be effective in inflammatory bowel disease, and in continued trials no patients have developed PML. Given the mortality of PML and lack of effective treatments, patients may remain concerned that PML is a possible risk factor. So, going forward, how do we quantify the risk of this serious adverse event? This review details how we define the maximum risk when no (or very few) events have occurred with an easy-to-use equation.

Critical role for reactive oxygen species in apoptosis induction and cell migration inhibition by diallyl trisulfide, a cancer chemopreventive component of garlic.

Diallyl trisulfide (DATS) is a structurally simple but biologically active constituent of processed garlic with in vivo activity against chemically induced as well as oncogene-driven cancer in experimental rodents. This study offers novel insights into the mechanisms underlying anticancer effects of DATS using human breast cancer cells as a model. Exposure of human breast cancer cells (MCF-7 and MDA-MB-231) and a cell line derived from spontaneously developing mammary tumor of a transgenic mouse (BRI-JM04) to DATS resulted in a dose-dependent inhibition of cell viability that was accompanied by apoptosis induction. A non-tumorigenic normal human mammary cell line (MCF-10A) was resistant to growth inhibition and apoptosis induction by DATS. The DATS-induced apoptosis in MDA-MB-231, MCF-7, and BRI-JM04 cells was associated with reactive oxygen species (ROS) production as evidenced by fluorescence microscopy and flow cytometry using a chemical probe (MitoSOX Red). Overexpression of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) as well as Mn-SOD conferred significant protection against DATS-induced ROS production and apoptotic cell death in MDA-MB-231 and MCF-7 cells. Activation of Bak, but not Bax, resulting from DATS treatment was markedly suppressed by overexpression of Mn-SOD. The DATS treatment caused ROS generation, but not activation of Bax or Bak, in MCF-10A cells. Furthermore, the DATS-mediated inhibition of cell migration was partially but significantly attenuated by Cu,Zn-SOD and Mn-SOD overexpression in association with changes in levels of proteins involved in epithelial-mesenchymal transition. The DATS-mediated induction of heme oxygenase-1 was partially attenuated by overexpression of Mn-SOD. These results provide novel mechanistic insights indicating a critical role for ROS in anticancer effects of DATS.

A small-molecule macrophage migration inhibitory factor antagonist protects against glomerulonephritis in lupus-prone NZB/NZW F1 and MRL/lpr mice.

Autoimmunity leads to the activation of innate effector pathways, proinflammatory cytokine production, and end-organ injury. Macrophage migration inhibitory factor (MIF) is an upstream activator of the innate response that mediates the recruitment and retention of monocytes via CD74 and associated chemokine receptors, and it has a role in the maintenance of B lymphocytes. High-expression MIF alleles also are associated with end-organ damage in different autoimmune diseases. We assessed the therapeutic efficacy of (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1), an orally bioavailable MIF antagonist, in two distinct models of systemic lupus erythematosus: the NZB/NZW F1 and the MRL/lpr mouse strains. ISO-1, like anti-MIF, inhibited the interaction between MIF and its receptor, CD74, and in each model of disease, it reduced functional and histological indices of glomerulonephritis, CD74(+) and CXCR4(+) leukocyte recruitment, and proinflammatory cytokine and chemokine expression. Neither autoantibody production nor T and B cell activation were significantly affected, pointing to the specificity of MIF antagonism in reducing excessive proinflammatory responses. These data highlight the feasibility of targeting the MIF-MIF receptor interaction by small-molecule antagonism and support the therapeutic value of downregulating MIF-dependent pathways of tissue damage in systemic lupus erythematosus.

CCR2-antagonist prophylaxis reduces pulmonary immune pathology and markedly improves survival during influenza infection.

Infection with influenza virus induces severe pulmonary immune pathology that leads to substantial human mortality. Although antiviral therapy is effective in preventing infection, no current therapy can prevent or treat influenza-induced lung injury. Previously, we reported that influenza-induced pulmonary immune pathology is mediated by inflammatory monocytes trafficking to virus-infected lungs via CCR2 and that influenza-induced morbidity and mortality are reduced in CCR2-deficient mice. In this study, we evaluated the effect of pharmacologically blocking CCR2 with a small molecule inhibitor (PF-04178903) on the entry of monocytes into lungs and subsequent morbidity and mortality in influenza-infected mice. Subcutaneous injection of mice with PF-04178903 was initiated 1 d prior to infection with influenza strain H1N1A/Puerto Rico/8/34. Compared with vehicle controls, PF-04178903-treated mice demonstrated a marked reduction in mortality (75 versus 0%) and had significant reductions in weight loss and hypothermia during subsequent influenza infection. Drug-treated mice also displayed significant reductions in bronchoalveolar lavage fluid total protein, albumin, and lactose dehydrogenase activity. Administration of PF-04178903 did not alter viral titers, severity of secondary bacteria infections (Streptococcus pneumoniae), or levels of anti-influenza-neutralizing Abs. Drug-treated mice displayed an increase in influenza nucleoprotein-specific cytotoxic T cell activity. Our results suggest that CCR2 antagonists may represent an effective prophylaxis against influenza-induced pulmonary immune pathology.

Cathelicidin LL-37 increases lung epithelial cell stiffness, decreases transepithelial permeability, and prevents epithelial invasion by Pseudomonas aeruginosa.

In addition to its antibacterial activity, the cathelicidin-derived LL-37 peptide induces multiple immunomodulatory effects on host cells. Atomic force microscopy, F-actin staining with phalloidin, passage of FITC-conjugated dextran through a monolayer of lung epithelial cells, and assessment of bacterial outgrowth from cells subjected to Pseudomonas aeruginosa infection were used to determine LL-37's effect on epithelial cell mechanical properties, permeability, and bacteria uptake. A concentration-dependent increase in stiffness and F-actin content in the cortical region of A549 cells and primary human lung epithelial cells was observed after treatment with LL-37 (0.5-5 µM), sphingosine 1-phosphate (1 µM), or LPS (1 µg/ml) or infection with PAO1 bacteria. Other cationic peptides, such as RK-31, KR-20, or WLBU2, and the antibacterial cationic steroid CSA-13 did not reproduce the effect of LL-37. A549 cell pretreatment with WRW4, an antagonist of the transmembrane formyl peptide receptor-like 1 protein attenuated LL-37's ability to increase cell stiffness. The LL-37-mediated increase in cell stiffness was accompanied by a decrease in permeability and P. aeruginosa uptake by a confluent monolayer of polarized normal human bronchial epithelial cells. These results suggested that the antibacterial effect of LL-37 involves an LL-37-dependent increase in cell stiffness that prevents epithelial invasion by bacteria.