The inflammatory microenvironment of OP in human and murine lungs is still not well
understood. Previous analyses have for the most part focused on OP-related lung diseases,
in particular BO (Tab. 4). In these studies, similarly to our present study, TGFB1/Tgfb1,
TIMP1/Timp1, COL3A1/Col3a1 and CXCL12/Cxcl12 were analyzed in human patients who suffered
from BO following lung transplantation as well as in heterotopic tracheal transplantation
(“BO-like”) and “IPF-like” bleomycin mouse models (Table 4) 10], 16]–21]. Here, we aimed to investigate which inflammation-related factors expressed in our
OP mouse model are comparable with the corresponding results in human patients. Notably,
in both OP mouse and human tissues from OP patients we found comparable expression
levels of pivotal genes, such as TGFB1/Tgfb1, TIMP1/Timp1, TIMP2/Timp2, COL3A1/Col3a1,
CXCL12/Cxcl12, MMP2/Mmp2 and IL6/Il6. These results allow us to conclude that the
transgenic CCL2 mouse model not only shows pathogenomic, molecular and morphological
features of human OP but also exhibits a similar inflammatory profile.
Table 4. Literature review of the investigated inflammation- and fibrosis-associated genes
in bronchiolitis obliterans (BO), idiopathic pulmonary fibrosis (IPF) and organizing
pneumonia (OP) humans and mice
It has previously been shown that CCL2 Tg mice are prone to develop fibrotic pulmonary
lesions in terms of OP, following bacterial infection 9]. In the current study, we showed that human CCL2 overexpression adds to the endogenous
murine Ccl2 expression in CCL2 Tg mice. CCL2 is a major monocyte chemoattractant produced
by macrophages, lymphocytes, basophils, epithelial cells, endothelial cells and (myo)fibroblasts
25], and is involved in the pathogenesis of a variety of inflammatory lung diseases 4], 26]. In the context of bacterial pneumonia, Ccl2 knockout mice have been shown to exhibit
a decreased monocytic immune response 27]. In our previous analyses, we found that CCL2 Tg mice exhibited an improved pneumococcal
clearance after infection with S. pneumoniae, but at the same time developed OP lesions. Specific blockade of the CCL2 receptor
CCR2 with function-blocking antibody MC21 resulted in both reduced bacterial clearance
and abrogation of OP manifestations 9]. Therefore, combined CCL2/Ccl2 expression in lungs of transgenic mice is most likely
associated with manifestation of OP lesions in the small airways and adjacent alveoli
along with a complementary and multifocal accumulation of inflammatory cells.
The fact that the CCL2 transgenic animals without S. pneumonia infection did not developed OP, points to the importance of the infections in the
disease’s development. On the other hand, infected CCL2 transgenic animals developing
OP showed significantly higher expression of CCL2 than those infected CCL2 animals
that did not develop OP. Hence, our data suggested a direct relationship between S. pneumonia infection, high expression of CCL2 and the development of OP.
In OP lesions, increased expression of proinflammatory mediators indicates that leukocytes
and stromal cells generate a specific microenvironment, which in turn helps to recruit
more cells to sites of inflammation. We could demonstrate these compartment-specific
differences in gene expression by analyzing and comparing laser-microdissected OP
lesions and adjacent non-remodeled areas within the same animals. We found that the
molecular profile in murine and human OP lesions was characterized by increased expression
of proinflammatory signaling factors (Cxcl12/Cxcr4 in mice, CXCL12 in humans) and
profibrotic mediators (murine/human Tgfb1/TGFB1 and Timps/TIMPs). Cxcl12 is produced
and secreted by fibroblasts, while its complementary receptor Cxcr4 is expressed by
leukocytes 10], 11]. In a murine heterotopic tracheal transplant model with tracheal obliteration by
granulation tissue and other murine bleomycin-associated fibrosis models, Cxcl12-inhibition
resulted in an attenuation of fibroblastic trafficking/migration, which underlines
the importance of the Cxcl12/Cxcr4 axis for generating a profibrotic pulmonary microenvironment
18], 28], 29]. Profibrotic Thbs1/THBS1, Smads/SMADs and Ptk2/PTK2 were expressed in OP lesions
and these factors can, although not upregulated on the transcript level, contribute
to an inflammatory microenvironment. In human patients with progressive pulmonary
fibrosis, TGFB1-associated expression of fibroblastic PTK2 has been reported 20], 30] and experiments in a bleomycin-induced pulmonary fibrosis mouse model indicated that
this kinase is related to fibroblastic proliferation, activation and collagen production
31].
Tgfb1 as well as Il6 are cytokines which can be produced both by leukocytes and stroma
cells and both can induce an aberrant wound healing response resulting in (over)activation,
migration and proliferation of (myo)fibroblasts 32]–34]. In particular, Tgfb1 also induces the differentiation of fibroblast into myofibroblasts,
which promotes ECM production and collagen accumulation 10], 32], 34]. Smad3 is linked to Tgfb1 signaling while Smad1 is associated with Bmp signaling
pathways 35]–38]. In contrast to Tgfb1, downstream Smad1 and Smad3 were not increased in murine OP
lesions and both Bmp4 and its receptor Bmpr1b, were downregulated. Similar to the
regulation in mice, TGFB1 was increased in human OP lesions—but in contrast to the
murine setting, these factors were deregulated in an inverse fashion with increased
SMADs and BMP4/BMPR1B. Bmp4 is known to inhibit fibroblastic activation and proliferation
on its own, while together with Tgfb1, it further promotes differentiation of lung
fibroblasts into myofibroblasts 32], 34], 39]. Therefore, myofibroblastic activation in human OP is most likely mediated by the
TGFB1/SMAD3 axis, as well as by BMP4/SMAD1 signaling, while in mice an OP-restricted
decrease of murine Bmp4/Bmpr1b could contribute to aggregation of myofibroblasts within
the lesions.
In bronchoalveolar lavage fluids from human patients with OP, IL6 was shown to be
increased 40], while in other studies in lung-transplanted humans, alveolar macrophage-derived
IL6 was increased at the time of onset of acute rejection, but not in patients who
developed chronic lung allograft dysfunction (CLAD) 16]. In our analyses of mice and humans, the local expression levels of IL6 were comparable
in remodeled and non-remodeled segments of the lung. This indicates an unspecific
reaction to bacterial infection, suggesting that IL6 expression was in response to
infection, but not in an OP-specific manner, which affects all compartments of the
lung. However, the major driver of inflammatory processes within the murine and human
OP lesions was not Il6/IL6 but Tgfb1/TGFB1. Tgfb1 is secreted in the form of an inactive
complex which becomes activated by latency-associated protein (LAP) and Tgfb1/LMP
dissociation, which in turn can be induced by several factors, such as thrombospondins
(THBS) and Mmps 35], 41]. Thbs1, as well as Mmp inhibitors Timp1 and Timp2 are in turn typically associated
with Tgfb1-induced fibrotic tissue remodeling 32], 41]. Our analyses revealed that Timps/TIMPs were increased in murine and human OP lesions
alike, which was also paralleled by an increased expression of collagens, thus resulting
in accumulation of ECM. Matrix cleavage enzyme Mmp2 was significantly upregulated
in human and to a lesser extent murine OP. Since Mmp2/MMP2 can be inhibited by Timps/TIMPs,
accumulation of ECM components is increased in both systems. Furthermore, the matricellular
profibrotic factor Thbs1 was upregulated in murine—but not in human—OP lesions. In
human BO lesions, THBS1 has been shown to be overexpressed, which might indicate a
different mechanism of THBS1-mediated fibrosis in human OP and BO 10].