The term fibrotic lung disease can be utilized for diseases marked by mesenchymal cell infiltration and collagen deposition of the functional units of the lung. There is a large spectrum of fibrotic lung diseases with varied anatomic involvement and clinical presentations. Massive infiltration of the alveolar airspaces by mesenchymal cells marks diseases presenting more acutely such as cryptogenic organizing pneumonia [1]. Fibrosis is also the dreaded complication in patients with Acute Respiratory Distress Syndrome (ARDS) and contributes significantly to irreversibility of respiratory failure [2, 3]. Chronic fibrotic lung diseases such as idiopathic pulmonary fibrosis (IPF) have a more insidious presentation and contribute significantly to the disease burden of chronic lung diseases [4]. IPF and fibrotic lung diseases associated with connective tissue diseases are characterized by interstitial fibrosis with loss of alveolar units [5]. In contrast, other fibrotic lung diseases such as bronchiolitis obliterans (BO) predominantly target small airways and lead to chronic respiratory failure marked by airflow obstruction [6]. BO is a common presentation after lung or bone marrow transplantation, conditions characterized by immune mismatch. Furthermore, autoimmune diseases such as rheumatoid arthritis and environmental exposures can also be associated with BO. However, although not characterized as fibrotic lung diseases, it is important to note that fibrosis as a pathological process marks almost all chronic lung diseases including chronic obstructive pulmonary disease (COPD) [7].

Epithelial cell injury is a common histologic feature across various fibrotic diseases [8–10]. In IPF, the delicate air exchange units of alveoli are replaced by honeycomb cysts which are lined by cuboidal epithelium [11]. Dysmorphic epithelium overlies mesenchymal cells in fibrotic foci, the pathognomonic lesions of IPF. Similarly, airway fibrosis is marked by loss of normal epithelium with decreased numbers of club cell specific factor (CCSP) positive cells [12]. Fibrosis can hence be characterized as failure of epithelial repair focusing attention on epithelial progenitors in the lung [13]. Animal studies demonstrate that isolated loss of epithelial progenitors can in fact induce fibrosis. It has been shown that isolated targeted injury to type II alveolar epithelial cells (AEC-II), the putative stem cell that regenerates type 1 AECs (AEC-I) [14], results in pulmonary fibrosis [15]; and that exogenous transplantation of AEC-II has protective effects against fibrotic insult induced by bleomycin [16].

In the airways, loss of basal cells precedes bronchiolitis obliterans -like pathological changes in a murine model of chlorine gas inhalation [17], and conditional deletion of the progenitor club cells induces peribronchiolar fibrosis characteristic of many airway fibrotic lung diseases [18]. That loss of endogenous stem cells can skew a lung towards fibrosis after an insult has also been recently suggested by studies of fulminant viral injury where selective ablation of distal airway epithelial cells, characterized as expressing Trp63 (p63) and keratin 5, prevented normal regeneration and promoted fibrotic responses [19]. These murine studies solidify the hypothesis that epithelial progenitors have a significant role in fibrotic diseases, and their inability to maintain epithelial integrity is an important pathogenic step in ensuing fibroproliferation.

The mesenchymal cell is the primary effector cell of fibrosis and hence its origin is of key significance in developing anti-fibrotic strategies targeting these cells [20]. In a fibrotic lung , mesenchymal cells demonstrate evidence of proliferation and differentiation into myofibroblasts, identified by α-smooth muscle actin (SMA) expression and increased matrix secretory function [21]. In this section, we review the various mesenchymal progenitors that can potentially participate in fibroproliferative responses and the present knowledge on their contribution to fibrotic diseases of human lungs.

Lung -resident mesenchymal progenitors: The term mesenchymal stem cell (MSC) is used to denote mesenchymal progenitor cells which are identified in vitro after plastic adherence and demonstrate an ability to differentiate into multiple mesenchymal lineages [22]. Multipotent human MSCs were first characterized from the bone marrow (BM) [23], and it was believed that BM is a source of these precursor cells for other non-hematopoietic tissues and organs such as the lung [24, 25]. However, human studies have conclusively demonstrated that MSCs in solid organs are resident cells and not derived from the bone marrow. These studies have utilized the ability to differentiate local versus hematopoietic origin of the cells on the basis of donor versus recipient status in transplanted organs. Single-cell cytogenetic studies of MSCs isolated from human lung grafts in gender mismatched transplantations demonstrated donor origin of lung-resident MSC at even greater than 10 years post-transplantation [26]. Similar findings were replicated in the studies of transplanted human heart and kidneys with MSCs derived from these organs demonstrating donor origin [27, 28].

Lung -resident MSCs demonstrate a gene expression profile which suggests that they are remnants of embryonic lung mesenchyme. Expression of Forkhead box gene FOXF1 in the splanchnic mesoderm during organogenesis [29] is essential for lung development [30, 31], and lung-derived MSCs demonstrate 30,000 higher fold expression of FOXF1 than bone marrow-resident MSCs [32]. Similarly, the HOX gene expression in lung-derived MSCs [32] mirrors that noted in embryonic mesenchyme of the developing lung [33, 34]. Regionally restricted spatial expression of HOX genes has been shown to be critical in region-specific patterning [35]. While this work establishes the presence of a lung-specific endogenous mesenchymal cell population in the human lung, it is important to recognize that there are likely going to be further subsets of mesenchymal progenitor cells within the adult lung with specialized structural functions. In fact, an ABCG2 positive MSC population associated with the alveolar capillary network has been described in human lungs which likely represent a pericyte precursor [36]. A recent investigation of murine lung mesenchyme during embryogenesis using advanced cellular labeling techniques to label mesenchymal progenitors at a single-cell resolution supports this notion and demonstrates that each differentiated mesenchymal cell type has a distinct mode of progenitor recruitment and regulated boundaries [37]. Such investigation in adult lungs and in context of injury will likely shed more light on lungs endogenous MSC populations.

The mesenchymal epithelial unit—implications for fibrosis : Fibrosis develops in context of tissue response to injury, and resident mesenchymal progenitor cells are likely to be important cellular players in this failed/overactive reparative process. However, investigation of this role requires knowledge of functions of mesenchymal progenitors within the lung microenvironment during normal homeostasis. In the bone marrow, MSCs are an important component of the hematopoietic stem cell niche and regulate hematopoiesis by their interaction with other resident cells via secretory molecules as well as by direct contact [38]. The requisite role of embryonic mesenchyme in defining epithelial cell fate during lung morphogenesis [39, 40], and similar unique lung-specific gene expression in adult lung-derived MSCs [26, 32], also suggests a tissue-specific function. In situ hybridization studies of human lung tissue demonstrate that FOXF1 expressing mesenchymal cells lie in close contact with AEC-II in the alveolar corners and communicate with AECs via gap junctions [41] suggesting that these mesenchymal progenitors potentially play a significant role in regulating the AEC-II cellular niche. The ABCG2 positive MSC population lies in contact with microvascular epithelial cells and AEC-I and also demonstrates communication with these cell types via gap junctions [42]. Mesenchymal epithelial interaction is also important in the airways, where murine investigations reiterate the role of parabronchial smooth muscle cells in supporting bronchial epithelial stem cells [43]. These mesenchymal progenitors are in turn also regulated by their niche as suggested by recent work demonstrating that changes in control signals can disrupt the differentiation and patterning of mesenchymal progenitor cells in a developing murine lung [37]. Hence, investigating the dynamics of crosstalk between the lung’s endogenous resident mesenchymal and epithelial progenitors is crucial in deciphering the evolution of fibrosis.

Endogenous lung –resident mesenchymal progenitors in lung fibrosis : Mobilization/recruitment and differentiation of mesenchymal cells are well recognized events in fibrosis. Recent investigations in human lung transplant recipients have demonstrated that lung-resident MSCs are mobilized in lung during injury and participate in fibrogenesis in the allograft [44]. MSC numbers, quantitated as the number of mesenchymal colony-forming units (CFUs) in bronchoalveolar lavage, are high early post-lung transplant, a time of intense ischemia–reperfusion and immunological insults. The number of MSCs then decreases and remains low in quiescent lungs free of chronic rejection. An increase in the number of MSCs later precedes development of chronic rejection or BO. Bioactive lipid lysophosphatidic acid (LPA) plays a principal role in the migration of human lung-resident MSCs through a signaling pathway involving LPA1-induced β-catenin activation [45]. Lung -resident MSCs can be induced into myofibroblast differentiation by pro-fibrotic cytokines, and in situ hybridization demonstrates FOXF1 expression in myofibroblasts in human fibrotic lesions suggesting that they are a lung-resident mesenchymal cell population [32]. FOXF1 is mesenchyme specific and is lacking in lung epithelial cells [29]. Furthermore, it has been shown that FOXF1 expression is not induced by in vitro epithelial mesenchymal transformation [32], underscoring the utility of FOXF1 in delineating local mesenchymal origin of cells.

Another methodology which can be utilized in humans to discriminate between pulmonary versus extrapulmonary derivation of cells is the investigation of donor versus recipient status in tissues in gender mismatched lung transplants. Such studies were employed initially to study chimerism in the epithelium , and although not the focus of that study, fibrotic tissue was suggested to be of donor origin [46]. Two subsequent studies have since addressed this in fibrotic BO lesions in transplanted lungs. While the initial study focused on delineating recipient contribution and suggested the presence of some cells with recipient karyotype in fibrotic lesions [47], a recent report utilizing whole explanted lungs, and stringent criteria to exclude inflammatory cells, has demonstrated that myofibroblasts in fibroproliferative lesions of BO are largely of donor origin [48]. Thus, these recent human investigations demonstrating donor origin of MSCs isolated from transplanted human lungs [26] as well as donor origin of myofibroblasts in situ [48] provide evidence for a crucial role for resident mesenchymal progenitors during lung fibrogenesis.

Animal models have also been utilized to address this important question of the origin of myofibroblasts and effector mesenchymal cells in fibrotic lungs. However, the majority of these studies have addressed the possibility of epithelial mesenchymal transformation, or the potential contribution of bone marrow-derived progenitors to generate mesenchymal cells in the fibrotic lung [49–54]. The lack of studies directly investigating the lung-resident mesenchymal cells can be attributed to lack of specific markers for these cells. The bleomycin lung injury model is most commonly utilized in these studies. This model has several shortcomings including a rather mixed inflammatory fibrotic response, and the reversibility of fibrosis over time [55]. Unlike human IPF where fibrotic foci with a distinct collection of myofibroblasts are present, fibrotic areas in bleomycin treated lungs, although recognizable by trichrome collagen staining, contain scattered myofibroblasts with inflammatory cells, thus making analysis of individual cells harder. In complex fibrotic tissue, staining for myofibroblasts markers such as α-SMA can give the appearance of co-localization in apposing cells. However, several recent investigations utilizing confocal imaging have now clarified the role of local mesenchymal cell populations in fibrotic responses by demonstrating no evidence of epithelial–mesenchymal transformation [56]. In the lung, lineage tracing of surfactant protein C positive AEC-II has shown no significant contribution of these cells to myofibroblasts in a bleomycin lung [57]. Furthermore, lineage tracing of ABCG2 positive mesenchymal progenitor cells in a murine bleomycin fibrosis model has revealed their contribution to vascular remodeling by differentiating into NG2 positive, α-SMA expressing cells [58].

FOXD1 is another marker which has been used to analyze the origin of myofibroblasts in fibrosis . FOXD1 is essential for renal morphogenesis, and FOXD1 lineage tracing has previously been utilized to demonstrate the local mesenchymal origin of myofibroblasts in murine models of renal fibrosis [56]. FOXD1 positive cells thought to represent pericytes in the lung were also recently investigated in a murine bleomycin lung injury model. FOXD1 progenitor-derived pericytes were shown to expand after bleomycin lung injury, and activate expression of collagen-I(α)1 and αSMA in fibrotic foci [59].

In summary, recent human and animal investigations have demonstrated an essential and primary role of lung -resident mesenchymal cell populations in lung fibrosis , underscoring the need to delineate subsets of mesenchymal progenitors and the modulation of their differentiation.

It has been suggested that bone marrow can be a source of progenitor cells for solid organs [60]. The extrapulmonary origin of fibroblasts in a fibrotic lung has also been an area of active investigations [50, 61]. An important question is whether bone marrow-derived populations, fibrocytes or MSCs, differentiate to myofibroblasts and contribute to collagen deposition during lung fibrosis .

Fibrocytes and pulmonary fibrosis : The term fibrocyte was coined to describe a circulating leukocyte subpopulation which was found to express some fibroblast markers, especially type I collagen. It was proposed that fibrocytes, recruited to sites of tissue injury, contribute to pathologic fibrotic responses [62]. Fibrocytes have been investigated in animal models of pulmonary fibrosis [52, 53] and have also been detected in the blood of patients with pulmonary fibrosis [63]. However, no significant functional contribution of hematopoietic cell-derived type I collagen to lung fibrogenesis has been demonstrated in recent investigations utilizing a double-transgenic mouse model in which the type I collagen gene was specifically and permanently deleted in hematopoietic cells by crossing mice carrying the Vav-Cre transgene with col1a1^fl/fl mice [64]. These investigations suggest that the intracellular collagen detected in fibrocytes is predominantly due to the uptake of collagen from neighboring collagen-secreting cells [64]. Thus, while fibrocytes likely play an important role in fibrosis by their interactions with resident mesenchymal cells, their potential role in collagen turnover [65], and their paracrine actions [66], they are not the precursor cell to myofibroblasts in the lung or a significant source of collagen during fibrosis.

Bone marrow MSCs and pulmonary fibrosis : Bone marrow MSCs (BM-MSCs) exhibit myofibroblast differentiation potential and hence can potentially contribute to fibrosis. BM-MSCs also demonstrate immunoregulatory and anti-inflammatory functions in vitro and in vivo after exogenous administration and have been shown to ameliorate fibrosis in murine models [67, 68]. However, there is an important distinction between endogenous and exogenous BM-MSCs in the context of a fibrotic lung . At present, there is no substantial evidence to support the hypothesis that endogenous MSCs are recruited from the bone marrow to the lung during injury or fibrosis, and that they contribute to pathogenesis of fibrotic lung diseases. As mesenchymal progenitors in the organs demonstrate tissue specificity, the role of BM-MSC in the human body during hemostasis and repair is likely local and involves the regulation of hematopoietic functions [69]. However, several characteristics of BM-MSCs make them suitable candidates for exogenous cellular therapy. These include their ability to be easily cultured and expanded; their immunoprivileged status enabling them to be utilized in HLA mismatched recipients; and most importantly, their ability to modulate the inflammatory milieu [70]. This has led to exploration of the ability of these cells to modulate fibrosis when administered exogenously in murine models. Interestingly, unlike lung-resident MSCs which demonstrate specific homing and long-term engraftment after intratracheal administration even in uninjured lungs [41], exogenous BM-MSC does not show any significant tissue engraftment after intravenous or intratracheal injections [71, 72]. Thus, MSCs derived from the bone marrow have not yet been shown to be present in normal or diseased human lungs and fail to reside in the lung even after exogenous administration. Hence, current data does not support a role for endogenous BM-MSCs as a direct cellular player in the lung milieu or as a source of mesenchymal progenitors in the lung.

Fibrotic diseases of the lung are a major cause of mortality and morbidity. Fibrosis evolves as normal homeostatic/reparative mechanisms fail. Hence, knowledge of the endogenous stem/progenitor cell niche (s) within the adult lung is essential in further unraveling the mechanism(s) of fibrosis . Recent studies have consolidated the key role of lung-resident epithelial and mesenchymal progenitor cells in fibrosis. Better understanding of mesenchymal progenitors and their niche, and their functions in an adult lung will help devise methodologies to restrain aberrant fibroproliferative responses and discover novel therapeutic options for fibrotic lung diseases.