CXCR4⁺ cells are increased in lung tissue of patients with idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive interstitial lung disease (ILD) with few effective drug therapies [1, 2]. An early event in IPF is recurrent epithelial injury resulting in activation of extracellular matrix (ECM)-producing fibroblasts [3]. CXCR4 is a transmembrane protein receptor that is constitutively expressed on epithelial and myeloid cells and is involved in stem cell migration [4]. CXCR4 is activated by the chemokine CXCL12, also known as stromal derived factor (SDF)-1 and in the bone marrow, CXCL12 is constitutively produced in order to maintain hemopoietic stem cells in the bone marrow environment [5].

In the lung, CXCL12 is upregulated upon epithelial injury [6]. Other CXCR4 expressing cells include fibrocytes which are bone marrow-derived, circulating progenitor cells that express the marker CD45, traffic to the lung via CXCR4/CXCL12 signalling and have been implicated in fibrotic ILD [7]. However, an analysis of the specific cell types expressing CXCR4 in IPF has not been attempted. As a result, there is still some uncertainty as to the origin of CXCR4 expressing cells in the diseased lung. In particular, whether CXCR4⁺ cells are derived from lung resident cells perhaps of epithelial origin or do they originate from the peripheral circulation remains elusive.

In a previous study, the i-body CXCR4 antagonist, AD-114, reduced experimental lung fibrosis and decreased IPF lung fibroblast invasion in-vitro [8], which supports the targeting of CXCR4 as a therapeutic option in IPF. CXCR4 is best known for its role in diverse disorders such as HIV-1 infection [9], tumour development [10], and acute myeloid leukemia [11]. In the lung, CXCR4 expression is thought to contribute to the cellular trafficking from the vessels into the interstitium and contribute to a number of fibrotic disorders including asthma and pulmonary fibrosis [12]. However, it is not known if circulating cells expressing only CXCR4 could be potential biomarkers of disease progression in IPF. Therefore, the first aim of this study was to investigate whether numbers of CXCR4⁺ cells in the circulation of patients with IPF were elevated compared to age-matched normal controls and if there was any correlation with clinical parameters of disease.

Radiologically, honeycomb lung is defined by clusters of sub-pleural basal cystic air spaces seen on high-resolution computed tomography (HRCT) scans in patients with end-stage fibrotic ILD, and in particular those with the usual interstitial pneumonia (UIP) pattern of fibrosis. Patients with ILDs that share a radiologic and histological pattern of UIP do worse than those that have other patterns such as non-specific interstitial pneumonia (NSIP) [13]. Histologically, honeycomb cysts are lined with cuboidal or ciliated cells that express a variety of epithelial markers such as e-cadherin and basal cell-specific keratins, supporting the hypothesis that honeycomb cysts are derived from the distal airway [14] but further investigation into the cells of the honeycomb cyst is necessary before anti-CXCR4 therapies in IPF can be fully realised.

We have previously published that CXCR4 expression is increased in IPF lung tissue, particularly in airway and honeycomb-associated epithelial cells [8]. However, it is not known whether these cells also express CXCL12, which would imply that an autocrine CXCR4-CXCL12 pathway could be continuously activated in IPF. Therefore, we then aimed to investigate the CXCR4/CXCL12 expression in the lung tissue of patients with IPF, non-IPF fibrotic ILD and NDC.

The first aim of this study was to investigate the utility of circulating CXCR4⁺ PMBCs as a potential biomarker in idiopathic pulmonary fibrosis (IPF). The second aim was to then quantify and characterize two populations of interstitial CXCR4⁺ cells in lung tissue of patients with end-stage pulmonary fibrosis, including IPF.

The precise pathophysiological pathways that drive idiopathic pulmonary fibrosis (IPF) are yet to be fully understood. Earlier work suggests that the CXCR4 pathway may be a therapeutic target [17, 18]. We have identified two distinct populations of CXCR4⁺ cell, one of epithelial origin (CXCR4⁺/e-cadherin⁺) and one of myeloid origin (CXCR4⁺/CD45⁺), that were increased in lung tissue of patients with IPF compared to non-diseased control (NDC) donors. Furthermore, we now provide more evidence that dysregulation of CXCR4 expression is not unique to IPF and therefore our findings are relevant to patients with fibrotic ILDs as well.

Fibrocytes are thought to be one source of fibroblasts in IPF by differentiating into matrix-producing myofibroblasts [19] after trafficking into the lung from the circulation. One study reported that most circulating fibrocytes express CXCR4 [20] and importantly, fibrocytes are not found in the tissue of normal lungs [21], therefore, lower levels of CXCR4⁺ cells in IPF patient circulation compared to NDC donors was unexpected and out of line with the literature [22]. It was not previously known if monitoring circulating cells by using only CXCR4⁺ as an identifier could be a surrogate marker of active fibrosis. Our study suggests that there exists a normal circulating level of CXCR4⁺ cells in healthy individuals which may be reduced as a result of active fibrosis in the lung tissue. These cells may be migrating from the circulation to the interstitial tissue through a chemical gradient produced by CXCL12-expressing cells, such as fibroblasts, or via activation in the circulation through elevated plasma CXCL12 levels.

Plasma CXCL12 concentration is elevated in chronic hypersensitivity pneumonitis, another related fibrotic ILD, and may be epithelial cell-derived [23]. The previous studies that have observed increased numbers of CXCR4⁺ cells in fibrotic lung tissue have primarily focused on the fibrocyte subtype which express haemopoietic markers like CD45 and Collagen 1 in addition to CXCR4 [19‐21, 24]. Relatively few studies have examined the contribution of CXCR4⁺ epithelial cells to pulmonary fibrosis [4, 25, 26]. During wound healing events, CXCR4 expression on alveolar type II (ATII) cells is important to drive epithelial cell migration and proliferation [4, 25, 27]. Both CXCR4 and CXCL12 expression is induced in ATII cells following lung injury [28] and both genes are regulated by a hypoxic microenvironment [29] such as in fibrotic tissue. Our study suggests that in the fibrotic lung, chronic injury and hypoxia drives constant upregulation of CXCR4 and CXCL12 in resident epithelial cells at the epithelial-alveolar interface, leading to overproliferation and migration of neighbouring ATII cells and driving alterations in the tissue structure.

The initiating event that leads to the formation of usual interstitial pneumonia (UIP) remains unknown but hyperplastic ATII cells are often seen at areas of active fibrosis in patients with fibrotic ILD [30] and there is some evidence to suggest that microscopic honeycomb cyst formation has a distal airway origin driven by dysregulation of the mucin gene MUC5B [14]. Recently, Chen and colleagues demonstrated the therapeutic potential of targeting honeycomb cyst formation via MUC5B inhibition in IPF [31]. Similarly, our study suggests that targeting epithelial CXCR4⁺ cells may prevent honeycomb cyst formation in IPF and other fibrotic ILDs that share the UIP pattern. It was outside the scope of this study to determine if there were differences in CXCR4 expression between IPF and the other fibrotic ILDs but it can be concluded that the UIP phenotype includes alveolar destruction involving epithelial CXCR4⁺ cells. Furthermore, these epithelial CXCR4⁺ cells, unlike their lymphoid counterparts, the CXCR4⁺CD45⁺ cells, also expressed CXCL12, indicating that the epithelium may be the primary site of fibrotic activity driven by mechanisms involving the CXCR4/CXCL12 axis in IPF. Future studies in a larger cohort are needed to determine the precise cell type and investigate the role of CXCR4⁺ cells that do not express either e-cadherin or CD45.

The limitation of our study was that the donors from whom PMBCs were obtained were not the same donors who had provided normal plasma. Furthermore, the IPF patients who provided whole blood were not the same patients from whom lung tissue was investigated. Therefore, it is difficult to determine how the quantity of CXCR4⁺ PBMCs relates to CXCL12 plasma levels in normal donors or to numbers of CXCR4⁺ cells in IPF lung tissue. Our study did not show any differences in CXCR4⁺/CD19⁺ (B-cell origin) or CXCR4⁺/CD33⁺ (early myeloid origin) cells, and therefore further investigation with a larger panel of surface markers may be necessary to delineate whether other circulating CXCR4⁺ cells are clinically relevant in IPF.

While the number of circulating CXCR4⁺ cells alone may not be a sufficient marker of disease progression in IPF, this study adds to the body of evidence that CXCR4/CXCL12 axis is an important mechanism for development of fibrosis. This data suggests that targeting of epithelial CXCR4 may be an appropriate therapeutic strategy for pulmonary fibrosis, particularly in patients with IPF and potentially those with non-IPF ILD. Long term safety data for the first generation of CXCR4 antagonists used for patients with acute myeloid leukemia, where a high CXCR4 expression is linked to poor progression [11], demonstrates that CXCR4 inhibition is a well-tolerated approach. Further studies are required to determine what role epithelial-CXCR4⁺ cells have in the progression of IPF.