Targeted drug delivery via caveolae-associated protein PV1 improves lung fibrosis.

Systemic administration of bio-therapeutics can result in only a fraction of drug reaching targeted tissues, with the majority of drug being distributed to tissues irrelevant to the drug's site of action. Targeted delivery to specific organs may allow for greater accumulation, better efficacy, and improved safety. We investigated how targeting plasmalemma vesicle-associated protein (PV1), a protein found in the endothelial caveolae of lungs and kidneys, can promote accumulation in these organs. Using ex vivo fluorescence imaging, we show that intravenously administered αPV1 antibodies localize to mouse lungs and kidneys. In a bleomycin-induced idiopathic pulmonary fibrosis (IPF) mouse model, αPV1 conjugated to Prostaglandin E2 (PGE2), a known anti-fibrotic agent, significantly reduced collagen content and fibrosis whereas a non-targeted PGE2 antibody conjugate failed to slow fibrosis progression. Our results demonstrate that PV1 targeting can be utilized to deliver therapeutics to lungs and this approach is potentially applicable for various lung diseases.

T follicular helper-like cells contribute to skin fibrosis.

Systemic sclerosis (SSc) is a debilitating inflammatory and fibrotic disease that affects the skin and internal organs. Although the pathophysiology of SSc remains poorly characterized, mononuclear cells, mainly macrophages and T cells, have been implicated in inflammation and fibrosis. Inducible costimulator (ICOS), which is expressed on a subset of memory T helper (TH) and T follicular helper (TFH) cells, has been shown to be increased in SSc and associated with disease pathology. However, the identity of the relevant ICOS+ T cells and their contribution to inflammation and fibrosis in SSc are still unknown. We show that CD4+ ICOS-expressing T cells with a TFH-like phenotype infiltrate the skin of patients with SSc and are correlated with dermal fibrosis and clinical disease status. ICOS+ TFH-like cells were found to be increased in the skin of graft-versus-host disease (GVHD)-SSc mice and contributed to dermal fibrosis via an interleukin-21- and matrix metalloproteinase 12-dependent mechanism. Administration of an anti-ICOS antibody to GVHD-SSc mice prevented the expansion of ICOS+ TFH-like cells and inhibited inflammation and dermal fibrosis. Interleukin-21 neutralization in GVHD-SSc mice blocked disease pathogenesis by reducing skin fibrosis. These results identify ICOS+ TFH-like profibrotic cells as key drivers of fibrosis in a GVHD-SSc model and suggest that inhibition of these cells could offer therapeutic benefit for SSc.

Divergent roles for Clusterin in Lung Injury and Repair.

Lung fibrosis is an unabated wound healing response characterized by the loss and aberrant function of lung epithelial cells. Herein, we report that extracellular Clusterin promoted epithelial cell apoptosis whereas intracellular Clusterin maintained epithelium viability during lung repair. Unlike normal and COPD lungs, IPF lungs were characterized by significantly increased extracellular Clusterin whereas the inverse was evident for intracellular Clusterin. In vitro and in vivo studies demonstrated that extracellular Clusterin promoted epithelial cell apoptosis while intercellular Clusterin modulated the expression of the DNA repair proteins, MSH2, MSH6, OGG1 and BRCA1. The fibrotic response in Clusterin deficient (CLU-/-) mice persisted after bleomycin and it was associated with increased DNA damage, reduced DNA repair responses, and elevated cellular senescence. Remarkably, this pattern mirrored that observed in IPF lung tissues. Together, our results show that cellular localization of Clusterin leads to divergent effects on epithelial cell regeneration and lung repair during fibrosis.

In Situ Imaging of Tissue Remodeling with Collagen Hybridizing Peptides.

Collagen, the major structural component of nearly all mammalian tissues, undergoes extensive proteolytic remodeling during developmental states and a variety of life-threatening diseases such as cancer, myocardial infarction, and fibrosis. While degraded collagen could be an important marker of tissue damage, it is difficult to detect and target using conventional tools. Here, we show that a designed peptide (collagen hybridizing peptide: CHP), which specifically hybridizes to the degraded, unfolded collagen chains, can be used to image degraded collagen and inform tissue remodeling activity in various tissues: labeled with 5-carboxyfluorescein and biotin, CHPs enabled direct localization and quantification of collagen degradation in isolated tissues within pathologic states ranging from osteoarthritis and myocardial infarction to glomerulonephritis and pulmonary fibrosis, as well as in normal tissues during developmental programs associated with embryonic bone formation and skin aging. The results indicate the general correlation between the level of collagen remodeling and the amount of denatured collagen in tissue and show that the CHP probes can be used across species and collagen types, providing a versatile tool for not only pathology and developmental biology research but also histology-based disease diagnosis, staging, and therapeutic screening. This study lays the foundation for further testing CHP as a targeting moiety for theranostic delivery in various animal models.

Resolution of Skin Fibrosis by Neutralization of the Antifibrinolytic Function of Plasminogen Activator Inhibitor 1.

OBJECTIVE: Systemic sclerosis (SSc) is a fibrotic disease characterized by an obliterative vasculopathy with thrombosis and impairment of the coagulation-fibrinolysis balance. Plasminogen activator inhibitor 1 (PAI-1) is the major inhibitor of profibrinolytic plasminogen activators (PAs). This study was undertaken to evaluate the contribution of PAI-1 to SSc pathology in the skin. METHODS: PAI-1 was evaluated in skin from patients with diffuse SSc (dSSc) and those with limited SSc (lSSc) by immunohistochemistry. The contribution of PAI-1 to SSc pathology was tested in vivo in murine graft-versus-host disease (GVHD) and bleomycin models of progressive skin fibrosis and in vitro in dermal human microvascular endothelial cells (HMVECs) using a monoclonal antibody that selectively prevents the binding of PAI-1 to PA. RESULTS: Skin from patients with dSSc and those with lSSc showed increased PAI-1 levels in the epidermis and microvessel endothelium. PAI-1 neutralization in the GVHD model led to a dramatic, dose-dependent improvement in clinical skin score, concomitant with vasculopathy resolution, including a reduction in fibrinolysis regulators and vascular injury markers, as well as reduced inflammation. Resolution of vasculopathy and inflammation was associated with resolution of skin fibrosis, as assessed by reduction in collagen content and expression of key profibrotic mediators, including transforming growth factor ß1 and tissue inhibitor of metalloproteinases 1. Similar to the GVHD model, PAI-1 neutralization reduced dermal inflammation and fibrosis in the bleomycin model. PAI-1 neutralization stimulated plasmin-mediated metalloproteinase 1 activation in dermal HMVECs. CONCLUSION: Our findings indicate that neutralization of the antifibrinolytic function of PAI-1 resolves skin fibrosis by limiting the extent of initial vascular injury and connective tissue inflammation. These data suggest that PAI-1 represents an important checkpoint in disease pathology in human SSc.

IL-9 governs allergen-induced mast cell numbers in the lung and chronic remodeling of the airways.

RATIONALE: IL-9 is a pleiotropic cytokine that has multiple effects on structural as well as numerous hematopoietic cells, which are central to the pathogenesis of asthma. OBJECTIVES: The contribution of IL-9 to asthma pathogenesis has thus far been unclear, due to conflicting reports in the literature. These earlier studies focused on the role of IL-9 in acute inflammatory models; here we have investigated the effects of IL-9 blockade during chronic allergic inflammation. METHODS: Mice were exposed to either prolonged ovalbumin or house dust mite allergen challenge to induce chronic inflammation and airway remodeling. MEASUREMENTS AND MAIN RESULTS: We found that IL-9 governs allergen-induced mast cell (MC) numbers in the lung and has pronounced effects on chronic allergic inflammation. Anti-IL-9 antibody-treated mice were protected from airway remodeling with a concomitant reduction in mature MC numbers and activation, in addition to decreased expression of the profibrotic mediators transforming growth factor-ß1, vascular endothelial growth factor, and fibroblast growth factor-2 in the lung. Airway remodeling was associated with impaired lung function in the peripheral airways and this was reversed by IL-9 neutralization. In human asthmatic lung tissue, we identified MCs as the main IL-9 receptor expressing population and found them to be sources of vascular endothelial growth factor and fibroblast growth factor-2. CONCLUSIONS: Our data suggest an important role for an IL-9-MC axis in the pathology associated with chronic asthma and demonstrate that an impact on this axis could lead to a reduction in chronic inflammation and improved lung function in patients with asthma.

Antigen-induced increases in pulmonary mast cell progenitor numbers depend on IL-9 and CD1d-restricted NKT cells.

Pulmonary mast cell progenitor (MCp) numbers increase dramatically in sensitized and aerosolized Ag-challenged mice. This increase depends on CD4(+) T cells, as no MCp increase occurs in the lungs of sensitized wild-type (WT) mice after mAb depletion of CD4(+) but not CD8(+) cells before aerosol Ag challenge. Neither the genetic absence of IL-4, IL-4Ralpha chain, STAT-6, IFN-gamma, or IL-12p40 nor mAb blockade of IFN-gamma, IL-3, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17A, IL-12p40, or IL-12p40Rbeta1 before Ag challenge in WT mice reduces the pulmonary MCp increase. However, sensitized and Ag-challenged IL-9-deficient mice and sensitized WT mice given mAb to IL-9 just before Ag challenge show significant reductions in elicited lung MCp/10(6) mononuclear cells of 47 and 66%, respectively. CD1d-deficient mice and WT mice receiving anti-CD1d before Ag challenge also show significant reductions of 65 and 59%, respectively, in elicited lung MCp/10(6) mononuclear cells, revealing an additional requirement for MCp recruitment. However, in Jalpha18-deficient mice, which lack only type 1 or invariant NKT cells, the increase in the numbers of lung MCp with Ag challenge was intact, indicating that their recruitment must be mediated by type 2 NKT cells. Furthermore, anti-CD1d treatment of IL-9-deficient mice or anti-IL-9 treatment of CD1d-deficient mice does not further reduce the significant partial impairment of MCp recruitment occurring with a single deficiency. These findings implicate type 2 NKT cells and IL-9 as central regulators that function in the same pathway mediating the Ag-induced increase in numbers of pulmonary MCp.

Macrophage impairment underlies airway occlusion in primary respiratory syncytial virus bronchiolitis.

Although respiratory syncytial virus (RSV) infection is the most important cause of bronchiolitis in infants, the pathogenesis of RSV disease is poorly described. We studied histopathologic changes in a panel of lung tissue specimens obtained from infants with fatal cases of primary RSV infection. In these tissues, airway occlusion with accumulations of infected, apoptotic cellular debris and serum protein was consistently observed. Similar observations were found after RSV infection in New Zealand black (NZB) mice, which have constitutive deficiencies in macrophage function, but not in BALB/c mice. A deficiency in the number of alveolar macrophages in NZB mice appears to be central to enhanced disease, because depletion of alveolar macrophages in BALB/c mice before RSV exposure resulted in airway occlusion. In mice with insufficient numbers of macrophages, RSV infection yielded an increased viral load and enhanced expression of type I interferon-associated genes at the height of disease. Together, our data suggest that innate, rather than adaptive, immune responses are critical determinants of the severity of RSV bronchiolitis.

Sensing intracellular pathogens-NOD-like receptors.

The innate immune system uses different molecules that sense pathogen associated molecular patterns. These include Toll-like receptors (TLRs), RIG-1-like receptors (RLRs) and the NOD-like receptors (NLRs). The NLRs, consisting of more than 20 related family members, are present in the cytosol and recognize intracellular ligands. Members of the NLR can be grouped into molecules that contain either a CARD or a Pyrin motif. The NOD proteins mediate NF-kappaB activation, whereas Pyrin molecules such as NALP3 regulate IL-1beta and IL-18 production. In this review, we will discuss the role of NLRs in pattern recognition of microbial components and their role in health and disease.

Tumor necrosis factor-alpha triggers mucus production in airway epithelium through an IkappaB kinase beta-dependent mechanism.

Excessive mucus production by airway epithelium is a major characteristic of a number of respiratory diseases, including asthma, chronic bronchitis, and cystic fibrosis. However, the signal transduction pathways leading to mucus production are poorly understood. Here we examined the potential role of IkappaB kinase beta (IKKbeta) in mucus synthesis in vitro and in vivo. Tumor necrosis factor-alpha (TNF-alpha) or transforming growth factor-alpha stimulation of human epithelial cells resulted in mucus secretion as measured by MUC5AC mRNA and protein. TNF-alpha stimulation induced IKKbeta-dependent p65 nuclear translocation, mucus synthesis, and production of cytokines from epithelial cells. TNF-alpha, but not transforming growth factor-alpha, induced mucus production dependent on IKKbeta-mediated NF-kappaB activation. In vivo, TNF-alpha induced NF-kappaB as determined by whole mouse body bioluminescence. This activation was localized to the epithelium as revealed by LacZ staining in NF-kappaB-LacZ transgenic mice. TNF-alpha-induced mucus production in vivo could also be inhibited by administration into the epithelium of an IKKbeta dominant negative adenovirus. Taken together, our results demonstrated the important role of IKKbeta in TNF-alpha-mediated mucus production in airway epithelium in vitro and in vivo.

Persistent expression of chemokine and chemokine receptor RNAs at primary and latent sites of herpes simplex virus 1 infection.

Inflammatory cytokines and infiltrating T cells are readily detected in herpes simplex virus (HSV) infected mouse cornea and trigeminal ganglia (TG) during the acute phase of infection, and certain cytokines continue to be expressed at lower levels in infected TG during the subsequent latent phase. Recent results have shown that HSV infection activates Toll-like receptor signaling. Thus, we hypothesized that chemokines may be broadly expressed at both primary sites and latent sites of HSV infection for prolonged periods of time. Real-time reverse transcriptase-polymrease chain reaction (RT-PCR) to quantify expression levels of transcripts encoding chemokines and their receptors in cornea and TG following corneal infection. RNAs encoding the inflammatory-type chemokine receptors CCR1, CCR2, CCR5, and CXCR3, which are highly expressed on activated T cells, macrophages and most immature dendritic cells (DC), and the more broadly expressed CCR7, were highly expressed and strongly induced in infected cornea and TG at 3 and 10 days postinfection (dpi). Elevated levels of these RNAs persisted in both cornea and TG during the latent phase at 30 dpi. RNAs for the broadly expressed CXCR4 receptor was induced at 30 dpi but less so at 3 and 10 dpi in both cornea and TG. Transcripts for CCR3 and CCR6, receptors that are not highly expressed on activated T cells or macrophages, also appeared to be induced during acute and latent phases; however, their very low expression levels were near the limit of our detection. RNAs encoding the CCR1 and CCR5 chemokine ligands MIP-1alpha, MIP-1beta and RANTES, and the CCR2 ligand MCP-1 were also strongly induced and persisted in cornea and TG during the latent phase. These and other recent results argue that HSV antigens or DNA can stimulate expression of chemokines, perhaps through activation of Toll-like receptors, for long periods of time at both primary and latent sites of HSV infection. These chemokines recruit activated T cells and other immune cells, including DC, that express chemokine receptors to primary and secondary sites of infection. Prolonged activation of chemokine expression could provide mechanistic explanations for certain aspects of HSV biology and pathogenesis.

Essential role for the C5a receptor in regulating the effector phase of synovial infiltration and joint destruction in experimental arthritis.

A characteristic feature of rheumatoid arthritis is the abundance of inflammatory cells in the diseased joint. Two major components of this infiltrate are neutrophils in the synovial fluid and macrophages in the synovial tissue. These cells produce cytokines including tumor necrosis factor alpha and other proinflammatory mediators that likely drive the disease through its effector phases. To investigate what mechanisms underlie the recruitment of these cells into the synovial fluid and tissue, we performed expression analyses of chemoattractant receptors in a related family that includes the anaphylatoxin receptors and the formyl-MetLeuPhe receptor. We then examined the effect of targeted disruption of two abundantly expressed chemoattractant receptors, the receptors for C3a and C5a, on arthritogenesis in a mouse model of disease. We report that genetic ablation of C5a receptor expression completely protects mice from arthritis.