A novel inhibitor of the alternative pathway of complement reverses inflammation and bone destruction in experimental arthritis

Complement is an important component of the innate and adaptive immune response, yet complement split products generated through activation of each of the three complement pathways (classical, alternative, and lectin) can cause inflammation and tissue destruction. Previous studies have shown that complement activation through the alternative, but not classical, pathway is required to initiate antibody-induced arthritis in mice, but it is unclear if the alternative pathway (AP) plays a role in established disease. Previously, we have shown that human complement receptor of the immunoglobulin superfamily (CRIg) is a selective inhibitor of the AP of complement. Here, we present the crystal structure of murine CRIg and, using mutants, provide evidence that the structural requirements for inhibition of the AP are conserved in human and mouse. A soluble form of CRIg reversed inflammation and bone loss in two experimental models of arthritis by inhibiting the AP of complement in the joint. Our data indicate that the AP of complement is not only required for disease induction, but also disease progression. The extracellular domain of CRIg thus provides a novel tool to study the effects of inhibiting the AP of complement in established disease and constitutes a promising therapeutic with selectivity for a single complement pathway

Host-Detrimental Role of Esx-1-Mediated Inflammasome Activation in Mycobacterial Infection

The Esx-1 (type VII) secretion system is a major virulence determinant of pathogenic mycobacteria, including Mycobacterium marinum. However, the molecular events and host-pathogen interactions underlying Esx-1-mediated virulence in vivo remain unclear. Here we address this problem in a non-lethal mouse model of M. marinum infection that allows detailed quantitative analysis of disease progression. M. marinum established local infection in mouse tails, with Esx-1-dependent formation of caseating granulomas similar to those formed in human tuberculosis, and bone deterioration reminiscent of skeletal tuberculosis. Analysis of tails infected with wild type or Esx-1-deficient bacteria showed that Esx-1 enhanced generation of proinflammatory cytokines, including the secreted form of IL-1β, suggesting that Esx-1 promotes inflammasome activation in vivo. In vitro experiments indicated that Esx-1-dependent inflammasome activation required the host NLRP3 and ASC proteins. Infection of wild type and ASC-deficient mice demonstrated that Esx-1-dependent inflammasome activation exacerbated disease without restricting bacterial growth, indicating a host-detrimental role of this inflammatory pathway in mycobacterial infection. These findings define an immunoregulatory role for Esx-1 in a specific host-pathogen interaction in vivo, and indicate that the Esx-1 secretion system promotes disease and inflammation through its ability to activate the inflammasome

Quantification of Tumor Burden in a Genetically Engineered Mouse Model of Lung Cancer by Micro-CT and Automated Analysis

AbstractGenetically engineered mouse models (GEMMs) of lung cancer closely recapitulate the human disease but suffer from the difficulty of evaluating tumor growth by conventional methods. Herein, a novel automated image analysis method for estimating the lung tumor burden from in vivo micro-computed tomography (micro-CT) data is described. The proposed tumor burden metric is the segmented soft tissue volume contained within a chest space region of interest, excluding an estimate of the heart volume. The method was validated by comparison with previously published manual analysis methods and applied in two therapeutic studies in a mutant K-ras GEMM of non–small cell lung carcinoma. Mice were imaged by micro-CT pre-treatment and stratified into four treatment groups: an antibody inhibiting vascular endothelial growth factor (anti-VEGF), chemotherapy, combination of anti-VEGF and chemotherapy, or control antibody. In the first study, post-treatment imaging was performed 4 weeks later. In the second study, mice were scanned serially on a high-throughput scanner every 2 weeks for 8 weeks during treatment. In both studies, the automated tumor burden estimates were well correlated with manual metrics (r value range: 0.83-0.93, P < .0001) and showed a similar, significant reduction in tumor growth in mice treated with anti-VEGF alone or in combination with chemotherapy. Given the fully automated nature of this technique, the proposed analysis method can provide a valuable tool in preclinical drug research for screening and randomizing animals into treatment groups and evaluating treatment efficacy in mouse models of lung cancer in a highly robust and efficient manner

A Rare Population of CD24+ITGB4+Notchhi Cells Drives Tumor Propagation in NSCLC and Requires Notch3 for Self-Renewal

Sustained tumor progression has been attributed to a distinct population of tumor-propagating cells (TPCs). To identify TPCs relevant to lung cancer pathogenesis, we investigated functional heterogeneity in tumor cells isolated from Kras-driven mouse models of non-small-cell lung cancer (NSCLC). CD24(+)ITGB4(+)Notch(hi) cells are capable of propagating tumor growth in both a clonogenic and an orthotopic serial transplantation assay. While all four Notch receptors mark TPCs, Notch3 plays a nonredundant role in tumor cell propagation in two mouse models and in human NSCLC. The TPC population is enriched after chemotherapy, and the gene signature of mouse TPCs correlates with poor prognosis in human NSCLC. The role of Notch3 in tumor propagation may provide a therapeutic target for NSCLC

Anti-VEGF antibody therapy does not promote metastasis in genetically engineered mouse tumour models.

Resistance to anti-angiogenic therapy can occur via several potential mechanisms. Unexpectedly, recent studies showed that short-term inhibition of either VEGF or VEGFR enhanced tumour invasiveness and metastatic spread in preclinical models. In an effort to evaluate the translational relevance of these findings, we examined the consequences of long-term anti-VEGF monoclonal antibody therapy in several well-validated genetically engineered mouse tumour models of either neuroendocrine or epithelial origin. Anti-VEGF therapy decreased tumour burden and increased overall survival, either as a single agent or in combination with chemotherapy, in all four models examined. Importantly, neither short- nor long-term exposure to anti-VEGF therapy altered the incidence of metastasis in any of these autochthonous models, consistent with retrospective analyses of clinical trials. In contrast, we observed that sunitinib treatment recapitulated previously reported effects on tumour invasiveness and metastasis in a pancreatic neuroendocrine tumour (PNET) model. Consistent with these results, sunitinib treatment resulted in an up-regulation of the hypoxia marker GLUT1 in PNETs, whereas anti-VEGF did not. These results indicate that anti-VEGF mediates anti-tumour effects and therapeutic benefits without a paradoxical increase in metastasis. Moreover, these data underscore the concept that drugs targeting VEGF ligands and receptors may affect tumour metastasis in a context-dependent manner and are mechanistically distinct from one another. J Pathol 2012 Aug; 227(4):417-30

Complement C3 Is Activated in Human AD Brain and Is Required for Neurodegeneration in Mouse Models of Amyloidosis and Tauopathy

Complement pathway overactivation can lead to neuronal damage in various neurological diseases. Although Alzheimer's disease (AD) is characterized by β-amyloid plaques and tau tangles, previous work examining complement has largely focused on amyloidosis models. We find that glial cells show increased expression of classical complement components and the central component C3 in mouse models of amyloidosis (PS2APP) and more extensively tauopathy (TauP301S). Blocking complement function by deleting C3 rescues plaque-associated synapse loss in PS2APP mice and ameliorates neuron loss and brain atrophy in TauP301S mice, improving neurophysiological and behavioral measurements. In addition, C3 protein is elevated in AD patient brains, including at synapses, and levels and processing of C3 are increased in AD patient CSF and correlate with tau. These results demonstrate that complement activation contributes to neurodegeneration caused by tau pathology and suggest that blocking C3 function might be protective in AD and other tauopathies

Multimodal Microvascular Imaging Reveals that Selective Inhibition of Class I PI3K Is Sufficient to Induce an Antivascular Response

The phosphatidylinositol 3-kinase (PI3K) pathway is a central mediator of vascular endothelial growth factor (VEGF)-driven angiogenesis. The discovery of small molecule inhibitors that selectively target PI3K or PI3K and mammalian target of rapamycin (mTOR) provides an opportunity to pharmacologically determine the contribution of these key signaling nodes in VEGF-A-driven tumor angiogenesis in vivo. This study used an array of microvascular imaging techniques to monitor the antivascular effects of selective class I PI3K, mTOR, or dual PI3K/ mTOR inhibitors in colorectal and prostate cancer xenograft models. Micro-computed tomography (micro-CT) angiography, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), vessel size index (VSI) MRI, and DCE ultrasound (DCE-U/S) were employed to quantitatively evaluate the vascular (structural and physiological) response to these inhibitors. GDC-0980, a dual PI3K/mTOR inhibitor, was found to reduce micro-CT angiography vascular density, while VSI MRI demonstrated a significant reduction in vessel density and an increase in mean vessel size, consistent with a loss of small functional vessels and a substantial antivascular response. DCE-MRI showed that GDC-0980 produces a strong functional response by decreasing the vascular permeability/perfusion-related parameter, Ktrans. Interestingly, comparable antivascular effects were observed for both GDC-980 and GNE-490 (a selective class I PI3K inhibitor). In addition, mTOR-selective inhibitors did not affect vascular density, suggesting that PI3K inhibition is sufficient to generate structural changes, characteristic of a robust antivascular response. This study supports the use of noninvasive microvascular imaging techniques (DCE-MRI, VSI MRI, DCE-U/S) as pharmacodynamic assays to quantitatively measure the activity of PI3K and dual PI3K/mTOR inhibitors in vivo