Obliterative bronchiolitis (OB) is the key impediment to the long-term survival of lung transplant recipients and the lack of a robust preclinical model precludes examining OB immunopathogenesis. In the current study, lungs from C57BL/10 H-2 b mice that are MHC compatible, but minor histocompatability antigen incompatible, were transplanted into C57BL/6 mice. Histological features and cytokine profiles of OB were assessed. Moderate rejection (grade A3) developed by day 14, with evidence of OB at that time point. At 21 days, OB was present in 55% of grafts and moderate to severe rejection (grade A3-A4) was present in all mice. At 28 days, OB was present in 44% of mice and severe rejection (grade A4) was present in all. IL-17A, but not IL-17F, splenic mRNA transcripts and serum protein levels were increased only in mice that developed OB, whereas IL-10 transcripts and protein were increased only in non-OB mice. Neutralizing IL-17 prevented OB, down regulated acute rejection, and upregulated systemic IL-10. Collectively, these data show that transplantation of minor histoincompatible lungs from C57BL/10 mice into C57BL/6 mice results in a highly reproducible preclinical model of OB. In addition, these data indicate that neutralizing IL-17A or augmenting IL-10 could be therapeutic interventions to prevent OB.

Pulmonary arterial hypertension (PAH) is characterized by increased pulmonary artery pressure and vascular resistance, typically leading to right heart failure and death. Current therapies improve quality of life of the patients but have a modest effect on long-term survival. A detailed transcriptomics and systems biology view of the PAH lung is expected to provide new testable hypotheses for exploring novel treatments. We completed transcriptomics analysis of PAH and control lung tissue to develop disease-specific and clinical data/tissue pathology gene expression classifiers from expression datasets. Gene expression data were integrated into pathway analyses. Gene expression microarray data were collected from 58 PAH and 25 control lung tissues. The strength of the dataset and its derived disease classifier was validated using multiple approaches. Pathways and upstream regulators analyses was completed with standard and novel graphical approaches. The PAH lung dataset identified expression patterns specific to PAH subtypes, clinical parameters, and lung pathology variables. Pathway analyses indicate the important global role of TNF and transforming growth factor signaling pathways. In addition, novel upstream regulators and insight into the cellular and innate immune responses driving PAH were identified. Finally, WNT-signaling pathways may be a major determinant underlying the observed sex differences in PAH. This study provides a transcriptional framework for the PAH-diseased lung, supported by previously reported findings, and will be a valuable resource to the PAH research community. Our investigation revealed novel potential targets and pathways amenable to further study in a variety of experimental systems.

Primary graft dysfunction (PGD) is a major complication following lung transplantation. We reported that anti-type V collagen (col(V)) T cell immunity was strongly associated with PGD. However, the role of preformed anti-col(V) Abs and their potential target in PGD are unknown. Col(V) immune serum, purified IgG or B cells from col(V) immune rats were transferred to WKY rat lung isograft recipients followed by assessments of lung pathology, cytokines, and PaO2/FiO2, an index of lung dysfunction in PGD. Immune serum, purified IgG, and B cells all induced pathology consistent with PGD within 4 days posttransfer; up-regulated IFN-γ, TNF-α, and IL-1β locally; and induced significant reductions in PaO2/FiO2. Depleting anti-col(V) Abs before transfer demonstrated that IgG2c was a major subtype mediating injury. Confocal microscopy revealed strong apical col(V) expression on lung epithelial, but not endothelial cells; which was consistent with the ability of col(V) immune serum to induce complement-dependent cytotoxicity only in the epithelial cells. Examination of plasma from patients with or without PGD revealed that higher levels of preformed anti-col(V) Abs were strongly associated with PGD development. This study demonstrates a major role for anti-col(V) humoral immunity in PGD, and identifies the airway epithelium as a target in PGD.

Objective. To investigate the inhibition of matrix metalloproteinase 1 (MMP-1), MMP-8, and MMP-13 by doxycycline, and to determine whether the variable hemopexin-like domain of each MMP was responsible for the differences in susceptibility to doxycycline inhibition among these collagenases.Methods. Recombinant human MMP-1 (collagenase 1), MMP-8 (collagenase 2), and MMP-13 (collagenase 3), truncated forms of MMP-8 and MMP-13 lacking the hemopexin-like domain, and a mutant form of truncated MMP-13 were used in these studies. The activity of the full-length MMP in the presence of doxycycline was tested against type II collagen, a natural substrate for the enzymes. A small peptolide substrate was used to determine which structural features of the MMPs were related to sensitivity to doxycycline inhibition.Results The matrix metalloproteinases (MMPs) represent a family of structurally related enzyme proteins that share many structural and functional characteristics (1,2), but differ in substrate specificity and cellular origin. Each of the known MMPs has at least 3 structural domains: the propeptide domain, the catalytic domain, and the hemopexin-like domain. A proline-rich hinge region of variable length is present between the catalytic domain and the hemopexin-like domain. The catalytic domain and the propeptide domain are highly conserved, while the hemopexin-like domain shows significant variability. In the gelatinases, an additional gelatinbinding domain is inserted into the catalytic domain.Three collagenases share the unique ability to cleave the triple helical region of interstitial collagens. MMP-1 (collagenase 1) has been purified from fibroblasts of humans and other animals and is expressed by a variety of connective tissues and epithelia. MMP-8 (collagenase 2) was initially purified from neutrophils, but is also expressed in cartilage (3,4). MMP-13 (collagenase 3) was originally described as rat uterine collagenase (5), but has now been shown to also be produced by human breast carcinomas and to be a major product

erative bronchiolitis (OB), a fibrotic airway lesion, is the leading cause of death after lung transplantation. Type V collagen [col(V)] overexpression and IL-17-mediated anti-col(V) immunity are key contributors to OB pathogenesis. Here, we report a previously undefined role of IL-17 in inducing col(V) overexpression, leading to epithelial mesenchymal transition (EMT) and subsequent OB. We observed IL-17-mediated induction of col(V) ␣1 chains [␣1 (V)] in normal airway epithelial cells in vitro and detected ␣1 (V)-specific antibodies in bronchoalveolar lavage fluid of lung transplant patients. Overexpression of IL-17 and col(V) was detected in OB lesions in patient lung biopsies and in a murine OB model. IL-17 is shown to induce EMT, TGF-␤ mRNA expression, and SMAD3 activation, whereas downregulating SMAD7 expression in vitro. Pharmacological inhibition of TGF-␤RI tyrosine kinase, p38 MAPK, or focal adhesion kinase prevented col(V) overexpression and EMT. In murine orthotopic lung transplants, neutralizing IL-17 significantly decreased TGF-␤ mRNA and protein expression and prevented epithelial repair/ OB. Our findings highlight a feed-forward loop between IL-17 and TGF-␤, leading to induction of col(V) and associated epithelial repair, thus providing one possible link between autoimmunity and OB after lung transplantation. autoimmunity; p38 MAPK; focal adhesion kinase; small-airway epithelial cells; RLE-6TN; mouse transplant model; epithelial-mesenchymal transition OBLITERATIVE BRONCHIOLITIS (OB) is characterized by extensive peribronchiolar fibrosis with plugs of granulation tissues (fibroblasts and collagen) that occlude small airways. OB is the key reason that the 5-yr survival of lung transplant recipients is only 50%, the worst of all major solid organ transplants (42,48).Aberrant epithelial repair is a key event in the transplanted lung (1, 9) in which bronchioles lose resident epithelial cells and become occluded by granulation tissue. Abnormal epithelial repair eventually causes an epithelial-to-mesenchymal transition (EMT), a functional and phenotypic change of epithelial cells into spindle-shaped, migratory (43) and matrix-component-secreting mesenchymal cells (10, 41), and a process associated with lung fibrosis (15,27). However, the direct connection between EMT and the in vivo phenomena of fibrosis and fibro-obliterative disease remains controversial.We and others previously reported that OB is associated with dysregulation of two types of collagen: 1) marked increase in type V collagen [col(V)], a quantitatively minor lung collagen (8,14,40), and 2) a decrease in the major lung collagen type I [col(I)] (2, 53). We have shown that prospective monitoring of patients with human lung transplant revealed a critical role of col(V)-specific cellular immunity in OB pathogenesis (14,40). Although overexpression of col(V), an otherwise quantitatively minor collagen, is involved in OB pathology, mechanisms leading to col(V) overexpression are unknown. Thus a mechanistic understanding of the triggers of col(V)...