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Investigating the role of the immune system in the development of fibrosis in the lung

Final Report Summary - LUNG FIBROSIS (Investigating the role of the immune system in the development of fibrosis in the lung)

Summary description of the project objectives
Progressive loss of organ function due to fibrosis contributes significantly to the ever-growing burden of chronic disease in the world. It was recently estimated that 45% of deaths in the United States can now be attributed to diseases where fibrosis plays a major aetiological role. In addition, because progression in these diseases occurs over several years, they account for enormous morbidity within society, major health resource utilisation and loss of working days and tax revenues.
Fibrosis causes excessive collagen and extra-cellular matrix deposition in an organ or tissue as part of an attempted reparative process following injury. It may represent an aberrant response to a single major injury but more commonly is a response to a persistent or repetitive injury. Fibrosis can affect any organ including the lung, skin, heart, kidney and liver yet despite obvious aetiological differences in these diseases; it is hypothesized that the mechanisms leading to organ fibrosis may represent a common final cellular pathway. The association of fibrosis with many chronic inflammatory conditions suggests that the beneficial reparative processes that restore tissue homeostasis in physiological healing continue unchecked and instead result in pathological damage and loss of organ function.
Therapies to limit or reverse fibrosis in the lung have thus far proved unsuccessful further highlighting the need for a better understanding of the basic mechanisms driving fibrosis and in particular the link between fibrosis and inflammation. This project aimed to investigate the hypothesis that manipulation of lung macrophage phenotype may influence the ability of the lung to repair effectively and provide a novel therapeutic target to limit fibrosis. The project was conducted between the National Institutes of Health - NIH, Bethesda and Newcastle University.

Description of the work performed since the beginning of the project
CD11b-DTR (FVB-Tg(ITGAM-DTR/EGFP)34Lan/J) transgenic mice were obtained from The Jackson Laboratory and crossed with wild-type C57Bl6 mice (Taconic) to produce hemizygous transgenic mice for experiments. Selective depletion of macrophages by diphtheria toxin (intraperitoneal injection - 25ng/g) treatment of CD11b-DTR transgenic mice was confirmed as described previously (Duffield et al, J Clin Invest. 2005 Jan;115(1):56-65).
We sought to determine the roles of macrophages in an immune response where lung inflammation causes fibrosis. When injected intravenously, S. mansoni eggs become trapped in the lung vasculature and provoke Th2-dependent formation of spherical eosinophil-rich granulomas ringed by collagen-producing myofibroblasts. Lung inflammation, granuloma volume, and cytokine production peak at day 7 and then decline. IL-13 produced by CD4 T cells is the primary stimulus to activate the fibroblasts and cause fibrosis. By using CD11b-DTR transgenic mice we depleted macrophages at the start of granuloma formation, before the inflammatory peak, or as the granulomas began to resolve and collagen accumulated.
Early loss of macrophages reduced granuloma volume and collagen deposition in the lung, indicating that macrophages initially contribute to granuloma formation. Suprisingly, later depletion (eliminating macrophages only after granulomas formed and reached their peak size) also reduced granuloma volume and collagen deposition. This result implied that macrophages played an important role in both establishing and maintaining the lung fibrosis provoked by S. mansoni eggs. This contrasts with results from liver and kidney injury models where macrophages promote recovery at later stages.
S.mansoni egg induced lung granuloma formation is a strongly Th2-dependant process. Therefore one explanation for reduced granuloma volume and fibrosis could be that loss of macrophages changed the pattern of cytokines in a way that antagonized Th2 immunity. No effect on Th1 cytokines or IL-10 was observed in the absence of macrophages however macrophage depletion significantly attenuated the Th2 response in the lungs at all time points. Specifically, Th2 cytokines including IL-13 and IL-5 and Th2 responsive genes including Fizz-1, Ym-1, and Arg1 were reduced at all time points.
Next we determined whether macrophage depletion impaired T cell activation, differentiation, and homing. In the lung-draining mediastinal lymph nodes, loss of macrophages did not interfere with the percentages, numbers, or level of expression of IL-13 and IL-4 by CD4 T cells at any point of our experiments. Instead we found macrophage depletion led to a large drop in the numbers of IL-13+ and IL-4+ CD4 T cells in the lung, and also marginally diminished cytokine production on a per-cell basis. In contrast, loss of macrophages did not change the IFN-γ+ CD4 T cell population in either the lung or draining lymph nodes. We delivered CFSE-labeled, in vitro activated CD4 T cells by intravenous injection to test if macrophage depletion impaired effector T cells from homing to the lungs. We recovered equal numbers of donor cells from the blood and uninvolved inguinal lymph nodes but found loss of macrophages reduced effector T cell homing to, and retention in, the lungs and mediastinal lymph nodes. Our results implied that T cell priming did not depend on macrophages in this model but, once activated and differentiated, lung macrophages played a critical role in recruiting and/or restimulating effector Th2 cells.
The successful priming of CD4 T cells argued that antigen presentation in lymph nodes was not impaired in our experiments and, therefore, dendritic cells were present and not sufficient to drive granuloma formation. To confirm and directly test this conclusion, we generated bone marrow chimeras where wild-type recipients received wild-type control (no effect of DTX), CD11b-DTR (macrophages depleted), CD11c-DTR (dendritic cells depleted), or CD11b/c-DTR double-transgenic (both macrophages and dendritic cells depleted) donor bone marrow. DTX treatment of CD11c-DTR chimeras did not reduce granuloma volume or lung fibrosis induced by S.mansoni eggs. DTX-treated CD11b-DTR chimeras developed similarly reduced granuloma volume and lung fibrosis to non-chimeric CD11b-DTR mice. Strikingly, CD11b/c-DTR doubly-depleted chimeras showed no additional decline in granuloma volume or lung fibrosis compared to CD11b-DTR chimeras. We concluded from these data that dendritic cell depletion in CD11c-DTR chimeras is insufficient to impair the egg-induced Th2 response in the lungs. In contrast, the macrophages depleted in CD11b-DTR mice are required to maintain Th2-dependent lung inflammation and maximize fibrosis.
In additon to the above results, macrophage depletion also significantly impaired expulsion, increased egg production, and weakened Th2 immunity against helminth infection (Nippostrongylus brasiliensis) and weakened lung mucus, inflammatory, and cytokine responses to allergy (House dust mite).

Expected final results and their potential impact and use
Thus, in this model of a Th2-mediated immune response, macrophages play a key role in both forming and maintaining granulomas and act to increase inflammation and promote fibrosis in the lung even after the granulomas begin to resolve. Our findings suggest that therapies targeting macrophages may aid in resolving chronic immune-mediated pathology.