Introduction
Idiopathic pulmonary fibrosis (IPF) is an irreversible, fatal lung disease, marked by progressive deterioration of lung function due to an uncontrolled repair process [
1]. The pathogenesis of IPF is not completely understood, which is reflected in the lack of successful treatments and good diagnostic markers. Although it is generally accepted that IPF comprises an inflammatory and regenerative phase that results in excessive collagen deposition distorting normal lung architecture and function, the importance of inflammation in fibrosis has been challenged [
2]. The majority of investigations aimed at dissecting the pathogenesis of fibrosis have been conducted in animal models, which have strongly suggested that the dysregulated healing process is sustained by chronic inflammation [
3]. The inflammatory cells recruited to the lung and the factors they secrete dictate the nature, duration and severity of the inflammatory response. Chemokines and chemokine receptors are the primary regulators of the inflammatory cells that are (1) recruited to sites of injury (2) elicit wound-healing responses from the tissue resident cells.
C-C chemokine receptor (CCR)7 is a chemokine receptor that is expressed strongly on mature dendritic cells (DCs), naive and memory T cells, fibrocytes and structural cells comprising the fibroblastic foci in the lungs of patients with IPF [
4‐
6]. The CCR7 ligands, chemokine (C-C motif) ligand (CCL)19 and 21, are constitutively expressed in the secondary lymphoid organs and lymphatic vessels, and are central to the homing and trafficking of T cells. CCR7 expression allows these cells to home to the lymph nodes (LNs), where they expand in response to antigen stimulation and suppress effector cell responses. The T cells traditionally associated with attenuating immune cell activation are the T regulatory cells (Tregs), but their role in fibrotic responses is not clear. The importance of CCR7 in proper Treg mobilization has been demonstrated by studies showing that Tregs lacking CCR7 accumulate in the lung and fail to traffic to the LNs [
7,
8].
We have previously demonstrated that CCR7 and CCL21 are key regulators in the pathogenesis of PF [
6,
9,
10]. In the study we report here, we targeted CCR7 in a bleomycin (BLM) model of PF, which has been useful in dissecting the roles of the chemokine/cytokine network in both the inflammatory and fibrogenic phases of lung fibrosis. We conducted our investigation in the CCR7
-/- mouse, which possesses bronchus-associated lymphoid tissue (BALT) and demonstrates impaired homing of Tregs [
7,
8]. BALT is often described as a tertiary lymphoid tissue rich in B and T cells. Although its exact function is not clearly understood, several murine studies have implicated BALT in facilitating primary immune responses via local priming of lymphocytes and in serving as a reservoir for viruses during their latency period [
11,
12]. The importance of lymphocytes in the pathogenesis IPF remains controversial; however, lymphocyte infiltration in areas of active fibroblastic proliferation in the IPF have been identified and shown to exhibit an elevated CD4/CD8 T cell ratio that correlates positively with survival of these patients [
13‐
15].
In this study, we hypothesized that CCR7-/- mice would be protected from the deleterious pulmonary effects of BLM. We assessed the histologic effects of BLM in the lungs, and investigated the chemokine/cytokine milieu in the lungs of wild type (WT) and CCR7-/- mice. Our data suggest that the increased levels of inflammatory cell infiltrates in the BALT of CCR7-/- mice may protect against fibrosis. Further, these infiltrates correlated with the impaired trafficking of Tregs between the lungs and LNs during the inflammatory and fibrogenic phases of BLM-induced PF. We found Tregs accumulating in the lungs of both CCR7 gene-deleted mice. Our data support the further investigation of the underappreciated role of T cells and inflammation in the pathogenesis and maintenance of PF.
Discussion
The purpose of our study was to determine whether the absence of CCR7 attenuated PF in mice that were challenged intratracheally with BLM. We report that CCR7-/- mice have decreased sensitivity to BLM-induced injury in the lung because they fail to exhibit the severe pulmonary remodeling typically associated with BLM-induced PF. In this study we present a complete profile of cytokine expression in WT and CCR7-/- mice at baseline and after BLM challenge, and an examination of fibrocytes and immune cells into the lung. Although we acknowledge that significant differences were not quantifiable under our experimental conditions, we are confident about the striking, reproducible trends we have observed.
Although CCR7 is strongly expressed in various bone marrow-derived cells, most notably DCs, we focused our study on T cell trafficking in BLM-induced PF because an intrinsic characteristic of CCR7
-/- mice is the presence of BALT in their lungs. BALT is a tertiary lymphoid structure that functions as a local reservoir for priming T lymphocytes and is involved in the impaired homing of Tregs in CCR7
-/- mice [
7,
8,
12]. We observed that the BALT in CCR7
-/- mice was altered during the course of BLM-induced injury, and may represent the dynamics of Treg trafficking during BLM-induced pulmonary remodeling.
Treg cells represent a conservative 1-10% of total CD4
+ T cells, yet their immunosuppressive functions are indispensable for immune homeostasis [
23]. Our study demonstrates that Treg-promoting genes (IL-2 and IDO) are upregulated in the lungs of BLM-treated CCR7
-/- mice, which suggests that the lung environment in CCR7
-/- mice is favorable to the maintenance of a Treg cell population. Furthermore, we found that CD4
+CD25
hi+FoxP3
+ Treg cells are retained in the lungs of BLM-treated CCR7
-/- mice, which is consistent with an ameliorated remodeling response to BLM-induced injury in the lung. Preliminary studies using CCR7
-/- bone-marrow chimeras confirmed the localization of CCR7
-/- Tregs to the lung, which we consider is due to a CCR7 deficiency in hematopoietic cells and not to altered functions of the structural cells within the lungs of CCR7
-/- mice (GT, AJH, CMH, unpublished data). These ongoing studies also indicated an appreciable increase in the total number of CD4
+ cells in the lungs of chimeric mice (CCR7
-/-→WT) during the fibrotic phase at 21 days after BLM injection (GT, AJH, CMH, unpublished data), which is consistent with that in the intact CCR7 knockout mice. The Treg population in the lungs of chimeric mice (CCR7
-/-→WT) was also increased compared with WT controls (WT→WT), an effect previously observed in BLM-treated CCR7
-/- mice, and Tregs were present in the LNs of CCR7
-/- chimeric mice 21 days after BLM injection challenge. Taken together, our results present novel evidence for an underappreciated role for CCR7 in the context of Treg trafficking during the pathogenesis of PF.
We observed an increase of BALT in the lungs of CCR7
-/- during BLM-induced PF and we believe that this promotes protection from BLM-induced PF. Tertiary lymphoid structures in the lung have been implicated in providing sustained protective immunity to viruses and chronic asthma, and in lung cancers, tumor-induced BALT has been associated with increased survival [
11,
21,
24]. Similarly, lymphoid aggregates resembling lymphoid follicles have been identified in fibrosing diseases, including IPF [
25‐
27], although its exact function and origin is not known. We found that the BALT is a dynamic structure within the lungs of CCR7
-/- mice, and its function during BLM-induced PF may be related to the retention of Tregs in the lung.
A recurring observation in our study that warrants further investigation is CCR7-independent trafficking during PF. Firstly, CCR7 has been widely shown to function as a potent stimulus for chemotaxis of bone marrow-derived Col 1-positive cells (fibrocytes) [
18]. Our study did not indicate significant diminution of fibrocytes in BLM-treated CCR7
-/- mice despite their protection from fibrosis. These data were not entirely surprising, because recruitment of fibrocytes to murine lungs has been shown to be mediated by other receptors including chemokine CXC motif receptor (CXCR)4, CCR2 and CCR5 [
28‐
30]. Although we did not analyze the expression of these receptors in our system, it is plausible that they may function as a compensatory mechanism to recruit fibrocytes to the lungs of CCR7
-/- mice. Moreover, we cannot discount the possibility that fibrocyte proliferation in the lung may also account for the comparable levels of fibrocytes observed in WT and CCR7
-/- mice.
Second, contrary to our expected results and those previously reported by Kocks
et al., we consistently detected relatively high levels of CD4
+CD25
hi+FoxP3
+ Tregs in the brLN of CCR7
-/- mice on days 3 and 7, and in fact a higher percentage of CD4
+ and CD4
+CD25
hi+FoxP3
+ Tregs in CCR7
-/- mice (compared with WT) 21 days after BLM injection. These results could be indicative of compensatory, CCR7-independent mechanisms that regulate the trafficking of Tregs from the lungs to the brLNs. Such mechanisms have been proposed in the context of the trafficking of CD4
+ T cell to the brLNs of CCR7-deficient mice during influenza infection, the presence of CD11b
+CD11c
+ DCs in the mediastinal LNs of CCR7-deficient mice during the late phase of
Mycobacterium tuberculosis infection, and the homing of central memory CD8
+ T cells to peripheral LNs in
plt/plt (CCL19 and CCL21-Ser-gene deleted) mice [
31‐
33]. In addition, Zou
et al. previously demonstrated that human CD4
+CD25
+ Tregs traffic and are retained in the bone marrow through CXCR4/CXCL12 (CXC chemokine ligand 12) [
34]. Therefore, we cannot exclude the possibility that our detection of CD4
+CD25
hi+FoxP3
+ Tregs in brLNs of BLM-treated CCR7
-/- mice in this study is related to these CCR7-independent pathways.
During the preparation of this manuscript, a study by Kotsianidis
et al. was published, which reported a dramatic decrease in the number and function of Tregs in both bronchoalveolar lavage and peripheral blood from patients with IPF [
35]. The data presented in our current study contribute to the current understanding of the role of Tregs during PF. T cell expansion has clinical potential for treating a variety of diseases such as autoimmunity and chronic asthma, and can be manipulated to promote graft-specific tolerance during organ transplantation. Therapeutic injections of IL-2 monoclonal antibodies have been shown to expand the Treg population
in vivo without inducing proliferation of CD8
+ and NK cells [
36]. Moreover, therapeutic transfer of regulatory T cells prevents development of allergic airway remodeling [
37]. Our study provides a rationale for targeting CCR7 in IPF and for the further investigation of therapies to favor the expansion and retention of the Treg population in the IPF lung.
Methods
The Animal Use Committee at the University of Michigan (Ann Arbor, MI, USA) approved all protocols and experiments described in this report.
Mice
Specific-pathogen-free male C57BL/6 (WT; CCR7
+/+) mice (6 to 8 weeks of age) were purchased from Taconic (Germantown, NY, USA). CCR7
-/- mice were generated as previously described[
38], and were bred and housed under specific pathogen-free conditions.
BLM model of pulmonary inflammation and fibrosis
WT (CCR7+/+), CCR7-/- mice, or bone-marrow chimeras were given 0.05 U of sterile BLM sulfate (Blenoxane, Bristol-Meyers Pharmaceuticals, Evansville, IN, USA) dissolved in phosphate-buffered saline (approximately 1.7 U/kg body weight) via an intratracheal injection. Groups of WT (CCR7+/+), CCR7-/- mice (n = 5-10 per timepoint) and bone-marrow chimeras were monitored for survival. Mice in other groups were killed and their lung tissues analyzed at days 1, 3, 7 and 21 after BLM injection. Untreated mice (n = 5) did not receive BLM, and this time point was designated as day 0.
Hydroxyproline assay
Left lobe samples from WT (CCR7
+/+) and CCR7
-/- mice (n = 5 per group for each time point in each experiment) before (that is, day 0) and at day 21 after BLM challenge were analyzed for hydroxyproline using a previously described assay [
16,
39].
Cytokine and chemokine ELISA analysis
Screening for the chemokines/cytokines C10, interferon (IFN)-γ, IFN-inducible protein-10, monocyte chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)1α, IL-4, IL-10, IL-12, IL-13, CCL17, CXCL1, tumor necrosis factor-α and transforming growth factor-β was carried out on 50 μl samples of cell-free supernatants from whole lung homogenates using a standardized sandwich ELISA technique (R&D Systems, Minneapolis, MN, USA). Cytokine and chemokine levels in each sample were normalized to the protein present in cell-free preparation of each sample, as measured by the Bradford protein assay.
Flow cytometry analysis
Lung cells were isolated after collagenase dispersion to obtain single-cell suspensions, as previously described [
40], and single cell suspensions of lung-draining LNs were prepared by isolation of bronchial LNs, pushing cells through a 40 μm nylon mesh using a syringe. Cells were stained with the indicated antibodies after 5 min of preincubation with Fc receptor blocking reagent (Fc Block; BD Biosciences, San Jose, CA, USA) fixed overnight with 4% formalin and run on a Cytomics FC-500 (Beckman Coulter, Brea, CA, USA). Samples were analyzed using FlowJo software (Tree Star, Inc., Ashland, OR, USA).
Real-time TaqMan PCR analysis
Total RNA was prepared from whole lung samples using TRIzol reagent according to the manufacturer's directions (Invitrogen, Carlsbad, CA, USA). A total of 1.0 μg from whole lung RNA was reverse transcribed into cDNA using Moloney murine leukemia virus reverse transcriptase as described previously (Invitrogen) [
16]. TaqMan gene expression reagents were used to assay IL-2 and IDO (Applied Biosystems, Foster City, CA, USA). Whole lung gene expression in WT (CCR7
+/+) and CCR7
-/- mice was expressed as a fold increase in transcript expression in BLM-challenged lung compared with unchallenged lung. The fold difference in mRNA expression between treatment groups was determined by software developed by Applied Biosystems.
Statistical analysis
All results are expressed as mean ± SEM. The means between groups at different time points were compared by two-way ANOVA. Individual differences were further analyzed using the unpaired t-test with Welch correction or the Tukey-Kramer multiple comparisons test where indicated. Values of P < 0.1*, P < 0.01** and P < 0.001*** were considered significant.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
GT and AJH carried out the experiments and data analysis. GT, AJH and CMH designed the experiments and interpreted the data, and GT wrote the final manuscript. All authors read and approved the final manuscript.