Heather L. Benson

Neutralizing IL-17 prevents obliterative bronchiolitis in murine orthotopic lung transplantation.

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‐2b 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.

Type V Collagen-Induced Oral Tolerance Plus Low-Dose Cyclosporine Prevents Rejection of MHC Class I and II Incompatible Lung Allografts

Autoimmunity to type V collagen (col(V)) is a major risk factor for lung allograft rejection. Although col(V)-induced oral tolerance abrogates rejection of minor histoincompatible lung transplants, its ability to prevent rejection of fully MHC incompatible lung allografts is unknown. Rat lung allografts fully incompatible at MHC class I and II loci (Brown Norway (RT1n)) were transplanted into untreated Wistar Kyoto rat recipients (WKY, RT1l), or WKY rats were fed col(V) pretransplantation. To determine whether col(V) enhanced cyclosporine (CsA)-mediated immune suppression, WKY rats were treated with low-dose CsA (5 mg/kg), posttransplant, or oral col(V) plus CsA. The data showed that in contrast to col(V) or CsA, col(V) plus low-dose CsA significantly prevented rejection pathology, down-regulated alloantigen-induced production of IFN-γ and IL-17A, and suppressed chemotaxis for lung macrophages in allograft bronchoalveolar lavage fluid that was associated with lower local levels of MCP-1 (CCL2). Col(V) plus CsA was associated with alloantigen-induced expression of IL-10 in mediastinal lymph node or splenic T cells, intragraft expression of IL-10 and Foxp3 in perivascular and peribronchiolar mononuclear cells, and constitutive production of IL-10 from allograft alveolar macrophages. These data demonstrate that col(V) enhances low-dose CsA-mediated immune suppression, and suggest a role for oral col(V) in immune modulation in lung transplantation.

Role of Complement Activation in Obliterative Bronchiolitis Post–Lung Transplantation

Obliterative bronchiolitis (OB) post-lung transplantation involves IL-17–regulated autoimmunity to type V collagen and alloimmunity, which could be enhanced by complement activation. However, the specific role of complement activation in lung allograft pathology, IL-17 production, and OB is unknown. The current study examines the role of complement activation in OB. Complement-regulatory protein (CRP) (CD55, CD46, complement receptor 1–related protein y/CD46) expression was downregulated in human and murine OB; and C3a, a marker of complement activation, was upregulated locally. IL-17 differentially suppressed complement receptor 1–related protein y expression in airway epithelial cells in vitro. Neutralizing IL-17 recovered CRP expression in murine lung allografts and decreased local C3a production. Exogenous C3a enhanced IL-17 production from alloantigen- or autoantigen (type V collagen)-reactive lymphocytes. Systemically neutralizing C5 abrogated the development of OB, reduced acute rejection severity, lowered systemic and local levels of C3a and C5a, recovered CRP expression, and diminished systemic IL-17 and IL-6 levels. These data indicated that OB induction is in part complement dependent due to IL-17–mediated downregulation of CRPs on airway epithelium. C3a and IL-17 are part of a feed-forward loop that may enhance CRP downregulation, suggesting that complement blockade could be a therapeutic strategy for OB.

IL-17 Induces Type V Collagen Overexpression and EMT via TGF-β dependent Pathways in Obliterative Bronchiolitis

Obliterative 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) α1 chains [α1 (V)] in normal airway epithelial cells in vitro and detected α1 (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-β mRNA expression, and SMAD3 activation, whereas downregulating SMAD7 expression in vitro. Pharmacological inhibition of TGF-βRI 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-β mRNA and protein expression and prevented epithelial repair/OB. Our findings highlight a feed-forward loop between IL-17 and TGF-β, leading to induction of col(V) and associated epithelial repair, thus providing one possible link between autoimmunity and OB after lung transplantation.

Endogenous Matrix Metalloproteinases 2 and 9 Regulate Activation of CD4+ and CD8+ T cells

We reported that inhibiting matrix metalloproteinases (MMP), known to remodel the extracellular matrix, also down-regulated antigen-specific T-cell responses. However, the direct role of MMP2 and MMP9 in regulating intracellular function in T cells is unknown. Markers of cellular activation and cytokine profiles were examined in anti-CD3-stimulated wild-type C57BL/6 mouse-derived CD4(+) or CD8(+) T cells, or MMP2- or MMP9-deficient (-/-) mice. MMP-sufficient T cells were also treated with SB-3CT, a highly selective inhibitor of MMP2 and MMP9. The effect of MMP-specific inhibition on T cell-dependent, antigen-specific murine lung injury was examined in vivo. SB-3CT induced dose-dependent reductions in anti-CD3-stimulated T-cell proliferation. Although MMP2(-/-) cells were reduced 20%, anti-CD3-induced proliferation was down-regulated 80-85% in MMP9(-/-) or in SB-3CT-treated wild-type CD4(+) and CD8(+) T cells. Intracellular calcium flux was augmented in response to MMP inhibition or deficiency in the same cells, and IL-2 production was reduced in CD4(+) and CD8(+) MMP9(-/-) T cells. SB-3CT-mediated MMP2 and MMP9 inhibition abrogated antigen-specific CD8(+) T cell-mediated lung injury in vivo. MMPs, particularly MMP9, may function intracellularly to regulate T-cell activation. T cell-targeted MMP inhibition may provide a novel approach of immune regulation in the treatment of T cell-mediated diseases.

Donor lung derived myeloid and plasmacytoid dendritic cells differentially regulate T cell proliferation and cytokine production

BackgroundDirect allorecognition, i.e., donor lung-derived dendritic cells (DCs) stimulating recipient-derived T lymphocytes, is believed to be the key mechanism of lung allograft rejection. Myeloid (cDCs) and plasmacytoid (pDCs) are believed to have differential effects on T cell activation. However, the roles of each DC type on T cell activation and rejection pathology post lung transplantation are unknown.MethodsUsing transgenic mice and antibody depletion techniques, either or both cell types were depleted in lungs of donor BALB/c mice (H-2d) prior to transplanting into C57BL/6 mice (H-2b), followed by an assessment of rejection pathology, and pDC or cDC-induced proliferation and cytokine production in C57BL/6-derived mediastinal lymph node T cells (CD3+).ResultsDepleting either DC type had modest effect on rejection pathology and T cell proliferation. In contrast, T cells from mice that received grafts depleted of both DCs did not proliferate and this was associated with significantly reduced acute rejection scores compared to all other groups. cDCs were potent inducers of IFNγ, whereas both cDCs and pDCs induced IL-10. Both cell types had variable effects on IL-17A production.ConclusionCollectively, the data show that direct allorecognition by donor lung pDCs and cDCs have differential effects on T cell proliferation and cytokine production. Depletion of both donor lung cDC and pDC could prevent the severity of acute rejection episodes.

Allergic Airway Disease in Mice Alters T and B Cell Responses during an Acute Respiratory Poxvirus Infection

Pulmonary viral infections can exacerbate or trigger the development of allergic airway diseases via multiple mechanisms depending upon the infectious agent. Respiratory vaccinia virus transmission is well established, yet the effects of allergic airway disease on the host response to intra-pulmonary vaccinia virus infection remain poorly defined. As shown here BALB/c mice with preexisting airway disease infected with vaccinia virus developed more severe pulmonary inflammation, higher lung virus titers and greater weight loss compared with mice inoculated with virus alone. This enhanced viremia was observed despite increased pulmonary recruitment of CD8+ T effectors, greater IFNγ production in the lung, and high serum levels of anti-viral antibodies. Notably, flow cytometric analyses of lung CD8+ T cells revealed a shift in the hierarchy of immunodominant viral epitopes in virus inoculated mice with allergic airway disease compared to mice treated with virus only. Pulmonary IL-10 production by T cells and antigen presenting cells was detected following virus inoculation of animals and increased dramatically in allergic mice exposed to virus. IL-10 modulation of host responses to this respiratory virus infection was greatly influenced by the localized pulmonary microenvironment. Thus, blocking IL-10 signaling in virus-infected mice with allergic airway disease enhanced pulmonary CD4+ T cell production of IFNγ and increased serum anti-viral IgG1 levels. In contrast, pulmonary IFNγ and virus-specific IgG1 levels were reduced in vaccinia virus-treated mice with IL-10 receptor blockade. These observations demonstrate that pre-existing allergic lung disease alters the quality and magnitude of immune responses to respiratory poxviruses through an IL-10-dependent mechanism.

Matrix metalloproteinases in T cell mediated pulmonary diseases.

Lung transplantation is the only conclusive treatment for many patients suffering from end-stage pulmonary disease. Unfortunately, the leading cause of death in lung transplant recipients is the development of chronic rejection known as obliterative bronchiolitis, characterized by extensive remodeling. Matrix metalloproteinases (MMPs) are endopeptidases known for their role in matrix remodeling and their involvement in many biological processes including end-stage pulmonary disease and transplant rejection. Our understanding of MMPs involvement in pulmonary immunity is rapidly expanding. As a result there has been some focus on MMPs role in T cell-associated pulmonary diseases, such as pulmonary fibrosis, emphysema, asthma and bronchiolitis obliterans syndrome. However, not much is known about the role of MMPs in regulating immune cell function. It is now commonly known that MMP inhibition via, broad spectrum or specific synthetic or naturally occurring inhibitors (TIMPs) can down regulated many pulmonary disease states. In this review, we explore the idea that T cell targeted MMP inhibition may provide a novel approach of immune regulation in the treatment of T cell-mediated diseases.