Exacerbation of lung radiation injury by viral infection: the role of Clara cells and Clara cell secretory protein.

Viral infections have been associated with exacerbation of disease in human cases of idiopathic pulmonary fibrosis. Since pulmonary fibrosis is a common outcome after irradiation to the lung, we hypothesized that viral infection after radiation exposure would exacerbate radiation-induced lung injury. Epithelial injury, a frequent outcome after infection, has been hypothesized to contribute to the pathogenesis of pulmonary fibrosis and bronchiolar epithelial Clara cells participate in epithelial repair. Therefore, it was further hypothesized that altered responses after irradiation involve the bronchiolar epithelial Clara cells. C57BL/6J or CCSP(-/-) mice were irradiated with 0 (sham), 5, 10 or 15 Gy to the whole thorax. At ten weeks post-irradiation, animals were mock infected or infected with influenza A virus and body weight and survival were monitored. Pulmonary function was assessed by whole-body plethysmography. The Clara cell markers, CCSP and Cyp2f2, were measured in the lung by qRT-PCR, and protein expression was visualized in the lung by immunofluorescence. Following pulmonary function tests, mice were sacrificed and tissues were collected for pathological analysis. In 15 Gy irradiated animals infected with influenza A virus, accelerated respiratory rates, reduced pulmonary function, and exacerbated lung pathology occurred earlier post-irradiation than previously observed after irradiation alone, suggesting infection accelerates the development of radiation injury. After irradiation alone, CCSP and Cyp2f2 mRNA levels were reduced, correlating with reductions in the number of Clara cells lining the airways. When combined with infection, these markers further declined and an apparent delay in recovery of mRNA expression was observed, suggesting that radiation injury leads to a chronic reduction in the number of Clara cells that may potentiate the epithelial injury observed after influenza A virus infection. This novel finding may have considerable therapeutic implications with respect to both thoracic tumor patients and recipients of bone marrow transplants.

Conditional expression of human ß-hexosaminidase in the neurons of Sandhoff disease rescues mice from neurodegeneration but not neuroinflammation.

This study evaluated whether GM(2) ganglioside storage is necessary for neurodegeneration and neuroinflammation by performing ß-hexosaminidase rescue experiments in neurons of HexB(-/-) mice. We developed a novel mouse model, whereby the expression of the human HEXB gene was targeted to neurons of HexB(-/-) mice by the Thy1 promoter. Despite ß-hexosaminidase restoration in neurons was sufficient in rescuing HexB(-/-) mice from GM(2) neuronal storage and neurodegeneration, brain inflammation persisted, including the presence of large numbers of reactive microglia/macrophages due to persisting GM(2) presence in this cell type. In conclusion, our results suggest that neuroinflammation is not sufficient to elicit neurodegeneration as long as neuronal function is restored.

Osteoarthritis accelerates and exacerbates Alzheimer's disease pathology in mice.

BACKGROUND: The purpose of this study was to investigate whether localized peripheral inflammation, such as osteoarthritis, contributes to neuroinflammation and neurodegenerative disease in vivo. METHODS: We employed the inducible Col1-IL1ßXAT mouse model of osteoarthritis, in which induction of osteoarthritis in the knees and temporomandibular joints resulted in astrocyte and microglial activation in the brain, accompanied by upregulation of inflammation-related gene expression. The biological significance of the link between peripheral and brain inflammation was explored in the APP/PS1 mouse model of Alzheimer's disease (AD) whereby osteoarthritis resulted in neuroinflammation as well as exacerbation and acceleration of AD pathology. RESULTS: Induction of osteoarthritis exacerbated and accelerated the development of neuroinflammation, as assessed by glial cell activation and quantification of inflammation-related mRNAs, as well as Aß pathology, assessed by the number and size of amyloid plaques, in the APP/PS1; Col1-IL1ßXAT compound transgenic mouse. CONCLUSION: This work supports a model by which peripheral inflammation triggers the development of neuroinflammation and subsequently the induction of AD pathology. Better understanding of the link between peripheral localized inflammation, whether in the form of osteoarthritis, atherosclerosis or other conditions, and brain inflammation, may prove critical to our understanding of the pathophysiology of disorders such as Alzheimer's, Parkinson's and other neurodegenerative diseases.

The trigeminal retrograde transfer pathway in the treatment of neurodegeneration.

The trigeminal sensory system was evaluated for the retrograde transfer of gene therapy vectors into the CNS. The feline immunodeficiency viral vector, FIV(HEXB), encoding for the human HEXB gene, was injected intra-articularly in the temporomandibular joint of 12 week-old HexB(-/-) mice displaying clinical and histopathological signs of Sandhoff disease. This treatment regiment reduced GM(2) storage and ameliorated neuroinflammation in the brain of HexB(-/-) mice, as well as attenuated behavioral deficits. In conclusion, retrograde transfer along trigeminal sensory nerves may prove to be a valuable route of gene therapy administration for the treatment of lysosomal storage disorders and other neurodegenerative diseases.

Spinal interleukin-1beta in a mouse model of arthritis and joint pain.

OBJECTIVE: Pain from arthritis has been associated with peripheral sensitization of primary sensory afferents and the development of inflammation at the dorsal horns. This study was undertaken to determine whether the role of spinal interleukin-1beta (IL-1beta) in central processing of pain is important in the development of arthritis. METHODS: Col1-IL-1betaXAT mice and GFAP-IL-1betaXAT mice were injected with the feline immunodeficiency virus (FIV) (Cre) vector in the right and left temporomandibular joints (TMJs), or in the cisterna magna, respectively, to induce IL-1beta expression in the dorsal horns of the spinal horn. To inhibit intrathecal IL-1 receptor type I (IL-1RI) signaling, FIV(IL-1Ra) vector was injected into the cisterna magna of Col1-IL-1betaXAT mice. The effects of IL-1RI receptor inhibition in GFAP-IL-1betaXAT mice were studied in the GFAP-IL-1betaXAT-IL-1RI(-/-) compound mouse model. Neuroinflammatory, sensory, and behavioral changes were evaluated in conjunction with arthritic changes in the TMJ, assessed by histopathologic and immunohistochemical analyses. RESULTS: Induction of an osteoarthritis-like condition in the TMJ in the Col1-IL-1betaXAT mouse model resulted in up-regulation of murine IL-1beta at the dorsal horns. Moreover, intrathecal inhibition of IL-1RI in Col1-IL-1betaXAT mice with arthritis led to amelioration of joint pathology and attenuation of the attendant joint pain. Overexpression of spinal IL-1beta in the recently developed GFAP-IL-1betaXAT somatic mosaic model of neuroinflammation led to development of arthritis-like pathology accompanied by increased pain-like behavior. CONCLUSION: Our results indicate that joint pathology and pain are dependent on spinal IL-1beta, and suggest the presence of a bidirectional central nervous system-peripheral joints crosstalk that may contribute to the development, expansion, and exacerbation of arthritis.

Peripheral blood mononuclear cell infiltration and neuroinflammation in the HexB-/- mouse model of neurodegeneration.

Myeloid-derived immune cells, including microglia, macrophages and monocytes, have been previously implicated in neurodegeneration. We investigated the role of infiltrating peripheral blood mononuclear cells (PBMC) in neuroinflammation and neurodegeneration in the HexB-/- mouse model of Sandhoff disease. Ablation of the chemokine receptor CCR2 in the HexB-/- mouse resulted in significant inhibition of PBMC infiltration into the brain, decrease in TNFalpha and MHC-II mRNA abundance and retardation in clinical disease development. There was no change in the level of GM2 storage and pro-apoptotic activity or astrocyte activation in HexB-/-; Ccr2-/- double knockout mice, which eventually succumbed secondary to GM2 gangliosidosis.

Sequential down-regulation of E-cadherin with squamous cell carcinoma progression: loss of E-cadherin via a prostaglandin E2-EP2 dependent posttranslational mechanism.

The incidence of skin cancer is on the rise, with over 1 million new cases yearly. Although it is known that squamous cell cancers (SCC) are caused by UV light, the mechanism(s) involved remains poorly understood. In vitro studies with epithelial cells or reports examining malignant skin lesions suggest that loss of E-cadherin-mediated cell-cell contacts may contribute to SCCs. Other studies show a pivotal role for cyclooxygenase-dependent prostaglandin E2 (PGE2) synthesis in this process. Using chronically UV-irradiated SKH-1 mice, we show a sequential loss of E-cadherin-mediated cell-cell contacts as lesions progress from dysplasia to SCCs. This E-cadherin down-regulation was also evident after acute UV exposure in vivo. In both chronic and acute UV injury, E-cadherin levels declined at a time when epidermal PGE2 synthesis was enhanced. Inhibition of PGE2 synthesis by indomethacin in vitro, targeted deletion of EP2 in primary mouse keratinocyte (PMK) cultures or deletion of the EP2 receptor in vivo abrogated this UV-induced E-cadherin down-regulation. In contrast, addition of PGE2 or the EP2 receptor agonist butaprost to PMK produced a dose- and time-dependent decrease in E-cadherin. We also show that UV irradiation, via the PGE2-EP2 signaling pathway, may initiate tumorigenesis in keratinocytes by down-regulating E-cadherin-mediated cell-cell contacts through its mobilization away from the cell membrane, internalization into the cytoplasm, and shuttling through the lysosome and proteasome degradation pathways. Further understanding of how UV-PGE2-EP2 down-regulates E-cadherin may lead to novel chemopreventative strategies for the treatment of skin and other epithelial cancers.

Intraperitoneal inoculation of Sandhoff mouse neonates with an HIV-1 based lentiviral vector exacerbates the attendant neuroinflammation and disease phenotype.

We aimed to evaluate the efficacy of VSV-G pseudotyped, defective HIV-1 based lentiviral vectors for the neonatal transfer of therapeutic genes following systemic administration in Sandhoff mouse pups. Despite transgene expression in mouse brains, these animals presented with significant exacerbation and acceleration of the disease neurological phenotype. We observed an increase and acceleration in the presence of MHC-II and CD45+ cells in their brains, along with neuroinflammation, but not in control heterozygous or wild type littermates that also received the same treatment.

Sustained hippocampal IL-1 beta overexpression mediates chronic neuroinflammation and ameliorates Alzheimer plaque pathology.

Neuroinflammation is a conspicuous feature of Alzheimer disease (AD) pathology and is thought to contribute to the ultimate neurodegeneration that ensues. IL-1 beta has emerged as a prime candidate underlying this response. Here we describe a transgenic mouse model of sustained IL-1 beta overexpression that was capable of driving robust neuroinflammation lasting months after transgene activation. This response was characterized by astrocytic and microglial activation in addition to induction of proinflammatory cytokines. Surprisingly, when triggered in the hippocampus of the APPswe/PS1dE9 mouse model of AD, 4 weeks of IL-1 beta overexpression led to a reduction in amyloid pathology. Congophilic plaque area fraction and frequency as well as insoluble amyloid beta 40 (A beta 40) and A beta 42 decreased significantly. These results demonstrate a possible adaptive role for IL-1 beta-driven neuroinflammation in AD and may help explain recent failures of antiinflammatory therapeutics for this disease.

Amelioration of pain and histopathologic joint abnormalities in the Col1-IL-1beta(XAT) mouse model of arthritis by intraarticular induction of mu-opioid receptor into the temporomandibular joint.

OBJECTIVE: To evaluate opioid receptor function as a basis for novel antinociceptive therapy in arthritis. METHODS: We induced human mu-opioid receptor (HuMOR) expression in arthritic joints of mice, using the feline immunodeficiency virus (FIV) vector, which is capable of stably transducing dividing, growth-arrested, and terminally differentiated cells. Male and female Col1-IL-1beta(XAT)-transgenic mice developed on a C57BL/6J background and wild-type littermates were studied. RESULTS: A single injection of FIV(HuMOR) into the temporomandibular joints of Col1-IL-1beta(XAT)-transgenic mice 1 week prior to induction of arthritis prevented the development of orofacial pain and joint dysfunction, and reduced the degree of histopathologic abnormality in the joint. In addition, FIV(HuMOR) prevented the attendant sensitization of trigeminal sensory neurons and activation of astroglia in brainstem trigeminal sensory nuclei. These effects were mediated by the transduction of primary sensory neurons via transport of FIV vectors from peripheral nerve endings to sensory ganglia, as evidenced by HuMOR expression in neuronal cell bodies located in the trigeminal ganglia, as well as in their proximal and distal nerve branches located in the main sensory and subnucleus caudalis of the brainstem and joints, respectively. The presence of MOR ligands predominantly in the descending trigeminal nucleus suggested that the observed antinociception occurred at the subnucleus caudalis. Articular chondrocytes and meniscal tissue were also infected by FIV(HuMOR), which presumably exerted an antiinflammatory effect on cartilage. CONCLUSION: Our results indicate that prophylactic therapy with MOR overexpression in joints can successfully prevent the development of pain, dysfunction, and histopathologic abnormalities in the joints in arthritis. These findings may provide a basis for the future development of spatiotemporally controlled antinociceptive and antiinflammatory therapy for arthritis.

Intraarticular induction of interleukin-1beta expression in the adult mouse, with resultant temporomandibular joint pathologic changes, dysfunction, and pain.

OBJECTIVE: To examine the effects of intraarticular induction of interleukin-1beta (IL-1beta) expression in adult mice. METHODS: We used somatic mosaic analysis in a novel transgenic mouse with an inducible IL-1beta transcription unit. Transgene activation was induced by Cre recombinase in the temporomandibular joints (TMJs) of adult transgenic mice (conditional knockin model). The effects of intraarticular IL-1beta induction were subsequently evaluated at the cellular, histopathologic, and behavioral levels. RESULTS: We developed transgenic mice capable of germline transmission of a dormant transcription unit consisting of the mature form of human IL-1beta as well as the reporter gene beta-galactosidase driven by the rat procollagen 1A1 promoter. Transgene activation by a feline immunodeficiency virus Cre vector resulted in histopathologic changes, including articular surface fibrillations, cartilage remodeling, and chondrocyte cloning. We also demonstrated up-regulation of genes implicated in arthritis (cyclooxygenase 2, IL-6, matrix metalloproteinase 9). There was a lack of inflammatory cells in these joints. Behavioral changes, including increased orofacial grooming and decreased resistance to mouth opening, were used as measures of nociception and joint dysfunction, respectively. The significant increase in expression of the pain-related neurotransmitter calcitonin gene-related peptide (CGRP) in the sensory ganglia as well as the auxiliary protein CGRP receptor component protein of the calcitonin-like receptor in the brainstem further substantiated the induction of pain. CONCLUSION: Induction of IL-1beta expression in the TMJs of adult mice led to pathologic development, dysfunction, and related pain in the joints. The somatic mosaic model presented herein may prove useful in the preclinical evaluation of existing and new treatments for the management of joint pathologic changes and pain, such as in osteoarthritis.

Transcriptional and posttranslational regulation of Cre recombinase by RU486 as the basis for an enhanced inducible expression system.

Genetic studies often require the employment of an inducible expression system, whereby the expression of a particular gene can be regulated by the exogenous administration of an inert ligand. Cre/loxP-based systems have been previously described as the basis for inducible expression systems by exerting site-specific DNA recombination. In our effort to enhance the properties of the RU486-responsive CrePr1 construct, we have developed the dual GLVP/CrePr system, in which RU486 confers activity control at both the transcriptional and the posttranslational level of CrePr1. This was achieved by placing CrePr1 transcriptional regulation under the control of the RU486-sensitive chimeric regulator GLVP. Stable cell lines harboring the dual GLVP/CrePr as well as the single CrePr1 system were developed. Our results indicate that the dually regulated system is highly inducible by RU486 while maintaining minimal basal activity ("leakage"), characteristics that can be employed in the development of transgenic mice in which genetic pathways can be turned on or turned off after exogenous administration of RU486 at physiologically inert doses.