Biomedical Sciences ETDs

Publication Date

Winter 11-6-2019

Abstract

Inflammation is a prominent pathologic feature in pulmonary arterial hypertension as demonstrated by pulmonary vascular infiltration of inflammatory cells, including T and B lymphocytes. However, the contribution of the adaptive immune system is not well characterized in pulmonary hypertension (PH) caused by chronic hypoxia (CH). Inflammatory cells, through various mechanisms, have the capability to increase pulmonary vascular resistance in various disease states. CD4+T cells have the potential to play a significant role in the disease process initiated by hypoxia. CD4+T cells are largely responsible for orchestrating downstream immune processes and are critical in focusing and amplifying inflammatory responses by other immune effector cells. Actions of CD4+T cells are essential for the full development of adaptive immune cytotoxicity and antibody production. CD4+T cells are required for initiating and maintaining inflammation, suggesting these cells could play an important role in the pathogenesis of hypoxic PH. Therefore, we tested the hypothesis that CD4+T cells, specifically the CD4+, IL-17a+TH17 cell subset, contribute to CH-induced PH.

We compared indices of PH resulting from CH (PB=380 mmHg; 3 wk) in wild-type (WT) mice and RAG1 knock-out (KO) mice (lack mature T and B cells). Separate sets of mice were adoptively transferred with CD4+or CD8+T cells, or ex vivopolarized TH17 cells prior to normoxic or CH exposure to evaluate the involvement of specific T cell subsets. Sham-treated RAG1 KO mice were protected from PH as indicated by diminished right ventricular systolic pressure (RVSP) and arterial remodeling compared to CH WT mice. Adoptive transfer of CD4+, but not CD8+T cells, restored the PH phenotype in RAG1 KO mice. Interestingly, RAG1 KO mice receiving TH17 cells displayed evidence of PH independent of CH exposure. Further supporting our hypothesis, depletion of CD4+T cells or treatment with SR1001, an inhibitor of TH17 cell development, prevented increased RVSP and arterial remodeling responses to CH. Therefore, our data indicate that CD4+T cells, specifically TH17 cells, contribute to CH-induced PH.

Interleukin-6 (IL-6) is a pleotropic cytokine that signals through the membrane-bound IL-6 receptor (mIL-6R) to induce anti-inflammatory (“classic-signaling”) responses. This cytokine also binds to the soluble IL-6R (sIL-6R) to promote inflammation (“trans-signaling”). mIL-6R expression is restricted to hepatocytes and immune cells. However, activated T cells release sIL-6R into adjacent tissues to induce trans-signaling. These cellular actions require the ubiquitously expressed membrane receptor gp130. IL-6 is produced by pulmonary arterial smooth muscle cells (PASMCs) exposed to CH and IL-6 knockout mice are protected from CH-induced PH. IL-6, through the activation of STAT1 and STAT3 has the potential to contribute to a broad array of downstream effects, such as cell growth and migration. CH-induced PH is associated with increased proliferation and migration of PASMCs to previously non-muscularized vessels of the lung. Therefore, we tested the hypothesis that IL-6 trans-signaling contributes to CH-induced PH and arterial remodeling.

Normoxic and CH (3 wk) mice were given the IL-6 trans-signaling-specific inhibitor sgp130Fc, and indices of PH were measured. Plasma sIL-6R and sgp130 levels were also measured in mice exposed to either normoxia or CH for 5 days. Additionally, PASMC migration was evaluated using a scratch-wound healing assay and proliferation assessed by measuring culture confluency over time and DNA incorporation of the thymidine analog 5-ethynyl-2´-deoxyuridine (EdU). Plasma levels of sgp130 were significantly decreased in mice exposed to CH for 5 days as compared to normoxic mice, while sIL-6R levels were unchanged. Consistent with our hypothesis, sgp130Fc treatment attenuated CH-induced increases in RVSP and pulmonary arterial remodeling as compared to vehicle (saline)-treated mice. In addition, PASMCs cultured in the presence of IL-6 and sIL-6R showed enhanced migration but not proliferation compared to those treated with IL-6 or sIL-6R alone or in the presence of sgp130Fc. These results indicate that IL-6 trans-signaling contributes to pulmonary arterial cell migration and CH-induced PH.

Although inflammation was previously believed to play little role in PH due to CH, these findings indicate otherwise. From these studies we can conclude that the adaptive immune system, through IL-6 trans signaling and TH17 cell polarization and recruitment, contribute to CH-induced PH. These mechanisms likely contribute to CH-induced PH through enhanced PASMC migration, as seen when these cells are exposed to the TH17 cell-specific cytokine, IL-17, along with PASMC exposure to IL-6/sIL-6R complex.

Keywords

COPD, Pulmonary Hypertension, Th17, Chronic Hypoxia, CD4

Sponsors

Laura Gonzalez Bosc

Document Type

Dissertation

Language

English

Degree Name

Biomedical Sciences

Level of Degree

Doctoral

Department Name

Biomedical Sciences Graduate Program

First Committee Member (Chair)

Laura Gonzalez Bosc

Second Committee Member

Thomas Resta

Third Committee Member

Oscar Bizzozero

Fourth Committee Member

Nikki Jernigan

Fifth Committee Member

Judy Cannon

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