In vitro co-culture studies and the crucial role of fibroblast-immune cell crosstalk in IPF pathogenesis

In vitro models used to examine fibroblast-immune crosstalk in IPF

To examine the role of fibroblast-immune cell interactions in IPF, various in vitro models have been utilized to mimic the microenvironment of the lung [22]. The simplest model used includes conditioned media-exposure experiments performed after culturing cells as traditional 2-dimensional (2D) monolayers before exposing one cell type (e.g., lung immune cells) to media harvested from the other cell type (e.g., lung fibroblasts) and vice versa [23]. This enables the investigation of the paracrine effects of mediators released from one cell type on the other exposed cell-type [24]. Other common models used are the in vitro co-cultures which have different variations (e.g., direct or transwell variations). The direct co-culture model involves culturing immune cells with lung fibroblasts directly in contact together in a tissue culture plate. In transwell co-cultures, lung immune cells are cultured in transwell inserts and lung fibroblasts grown in culture wells. Inserts with immune cells are then placed in wells with fibroblasts to establish transwell co-cultures [24, 25]. Additionally, 3D hydrogels established with natural (e.g., collagen I, gelatin) and artificial (e.g., polyethylene glycol, alginate) polymers have been used due to their ability to hold cells in a 3D spatial orientation and the ability to vary their matrix stiffness to mimic the in vivo tissue environment of the IPF lung more closely [26,27,28]. Here, fibroblasts and immune cells are either embedded together in 3D hydrogels or fibroblasts are embedded in hydrogels with immune cells seeded on top [28]. Co-culture models have been ideal for studying cell communication through the release of cellular mediators in the in vitro environment. However, these models are either established with plastic transwell inserts or reductionist natural ECM or artificial hydrogels. Hence, techniques have been developed that enables the decellularization of parts of the lung in vitro, which then serves as 3D scaffolds on which cells are cultured to assess their interactions in the natural pulmonary environment [29]. Further, lung tissue can also be used to generate thin slices about 100–500 μm thick, termed precision cut lung slices (PCLS) which is also used to study respiratory cell interactions in IPF [30]. PCLS are beneficial in vitro models because they can remain metabolically active while keeping the authentic structural integrity of the lung [30]. Ultimately, there is an abundance of in vitro models that can be employed and adapted to assess lung immune-fibroblast interactions in the pathobiology of IPF (see our reviews [25, 31, 32]. This review will integrate studies using these in vitro systems with specific emphasis on co-culture models to investigate the mechanisms and mediators behind the communication between fibroblasts and immune cells and how this contributes to the chronic lung tissue remodeling seen in IPF.

Fibroblast-immune cell crosstalk contributes to fibrotic mechanisms in IPF

IPF is characterized by chronic remodeling of the lung tissue that involves excessive fibrosis which results in the destruction of the lung parenchyma, subsequently disrupting gas exchange and concluding in respiratory failure [6, 33, 34]. Fibrosis in IPF entails an established milieu of fibroblast derived ECM protein deposition with increased growth factor and cytokine release due to recurrent epithelial injury [18, 35]. The signaling mediators derived from the epithelium and other lung cells result in the overproduction and degradation of ECM proteins from fibroblasts, contributing to increased lung mechanical stiffness [6, 35, 36]. In addition to epithelial cells, immune cells such as macrophages, mast cells, B and T cells have also been shown to communicate with fibroblasts to contribute to increased synthesis and degradation of ECM proteins, which is important for aberrant matrix turnover and deposition in IPF [19, 37].

In line with the role of defective fibroblast-immune cell interactions in aberrant matrix turnover [38, 39], Bagher and colleagues, directly cocultured the LAD2 mast cell-line with either control or IPF-derived primary human lung fibroblasts (PHLFs) or HFL-1 human lung fibroblast cell-lines in culture plates or on decellularized lung scaffolds. It was found that IPF-derived PHLFs released significantly more hepatocyte growth factor (HGF) compared to control-derived PHLFs after co-culture with mast cells [38]. Further, it was found that α-smooth muscle actin (α-SMA) was upregulated in lung fibroblasts stimulated with TGF-β after co-culture [38]. HGF is a pleiotropic growth factor that contributes to fibrotic mechanisms by preventing apoptosis of structural cells and thus contributing to their abnormal activation and aggregation during defective injury repair [39, 40]. In corroboration with this, another study also found that mast cell-fibroblast interactions contribute to increased ECM proteins in IPF [41]. Here, Wygrecka et al., isolated IPF-derived primary lung mast cells and fibroblasts. The cells were then directly cocultured together which caused an increase in fibroblast synthesis of fibronectin and collagen I, that was found to be largely due to the release of the enzyme tryptase (a serine protease) by mast cells [41]. Hence in summary, coculture studies show that fibroblast-mast cell interactions increase the release of enzymes (e.g., tryptase) by mast cells as well as growth factors (e.g., HGF), and ECM proteins (e.g., α-SMA, fibronectin and collagen I) from fibroblasts which may directly contribute to fibrosis of lung tissue in IPF.

Further, Novak et al., isolated IPF and control-derived primary alveolar macrophages (AMs) and lung fibroblasts. Various combinations of CM experiments, direct co-cultures in tissue culture plates, transwell and 3D collagen hydrogel cocultures were then set up to assess the interaction between different combinations of IPF- and control- derived lung fibroblasts and AMs [42]. After experiments, it was found that while coculturing control-derived AMs with control-derived fibroblasts led to a reduction in fibroblast-α-SMA expression, direct cocultures of control-derived AMs with IPF-derived fibroblasts resulted in increased fibronectin, collagen I and III as well as α-SMA gene expression pointing to potential myofibroblast differentiation [42]. Lastly, it was found that IPF-derived fibroblasts expressed more collagen I and III when cocultured with IPF-derived AMs compared to control-derived AMs cocultured with IPF-derived fibroblasts [42]. In connection with this, Qu et al., also co-cultured IPF-derived primary lung myofibroblasts with the THP1 macrophage cell-line [43]. The fibroblasts were originally cultured on either stiff or soft polyacrylamide hydrogels coated in rat tail collagen I and treated with the Fas ligand (FasL), a type II transmembrane protein, to induce apoptosis [43]. After a phagocytosis assay, it was found that macrophages were able to clear fibroblasts cultured on soft matrix substrates (mimicking healthy lungs) than fibroblasts cultured on stiffer matrixes (mimicking IPF mechanical lung environment) due to FasL-dependent apoptosis [43]. In addition, it was discovered that the expression of death domain 1α (DD1α), a receptor responsible for the crosstalk between macrophages and cells undergoing apoptosis, is induced by the activation of the p53 transcription factor and was dependent on the expression of the gene, mouse double minute 4 (MDM4), a human mouse homolog, in soft matrix conditions [43]. Collectively, these studies showed that macrophage-fibroblast crosstalk contributes to IPF by causing the overproduction of ECM proteins such as collagen I, collagen III and fibronectin as well as causing a defective clearance of apoptotic cells which further advances the stiffening and scarring of the lung tissue.

In addition to macrophages, neutrophils are also important innate immune cells with elevated numbers in the lungs of IPF patients [44,45,46]. Although understanding the contribution of neutrophil-fibroblast crosstalk in IPF pathogenesis will add to our understanding of more crucial multicellular mechanisms, there is a lack of studies in this area due to the complexity of culturing neutrophils in vitro as they need to be freshly isolated from blood for every experiment and have a relatively short lifespan [47]. To account for this, neutrophil derivatives are applied to fibroblasts to examine their potential contributions to fibrosis in the context of IPF [48]. As an example, Gregory and colleagues exposed LL47 human lung fibroblasts to the enzyme, neutrophil elastase (NE) which is a protease that is able to break down proteins, and found an increased α-SMA production by fibroblasts with significant increases in pSMAD3, independent of TGF-β [48]. Further, NE exposed fibroblasts were then embedded in rat tail collagen hydrogels, where NE was found to enhance fibroblast contractility. Hence, this proves a potential neutrophil-fibroblast interaction in IPF that may cause fibrotic changes in fibroblasts (e.g., α-SMA increase) to advance IPF pathogenesis.

The adaptive immunity has also been identified as an important contributor to fibrotic mechanisms in IPF [18, 19, 49, 50]. In addition to innate immune cell-fibroblast interactions that result in increased fibrotic protein secretion, adaptive immune cell-fibroblast interactions seem to result in both the production and degradation of ECM proteins [51,52,53]. In line with this, Ali et al., isolated B cells from blood samples of healthy control individuals and IPF patients before stimulating them with (or without) either β-glucan or CpG [51]. β-glucan and CpG are microbial antigens which activate B cells via their pattern recognition receptors (PRRs) and mimic the microbial load in respiratory exacerbations in IPF [51]. Ali and colleagues then exposed IPF-derived fibroblasts to the CM from the stimulated B cells. CpG pre-stimulated B cell CM resulted in increased α-SMA, fibronectin and plasminogen activator inhibitor-1 (PAI1) in IPF-derived fibroblasts, whereas stimulation with β-glucan did not induce an activated phenotype in fibroblasts [51]. In connection with this, Selman et al., exposed lung fibroblasts to CM from IPF-derived primary T cells and found a significant increase in collagen synthesis from fibroblasts which they speculated could be due to the release of prostaglandin E (PGE) an eicosanoid mediator of inflammation and remodeling [52]. Further, Lacy et al., also isolated IPF- and control -derived T lymphocytes and exposed these to CD3/CD28 beads in media supplemented with IL-2 to activate them without adding antigen presenting cells [53]. In contrast to previous studies, Lacy et al., found that the direct co-culture of healthy T cells with control- or IPF-derived fibroblasts significantly reduced TGF-β induced myofibroblast differentiation, which was marked by decreased calponin and α-SMA [53]. Further, in a direct co-culture of IPF-derived T cells with both control- or IPF-derived fibroblasts, it was found that IPF-derived T cells reduced TGF-β-induced myofibroblast differentiation in both healthy and IPF-derived fibroblasts. Additionally, co-culture conditions did not increase control- or IPF-derived fibroblast expression of poly (ADP-ribose) polymerase (PARP), an apoptosis marker [53], suggesting there was no induction of cell death [53]. All the results obtained were also found under indirect co-culture conditions using Millicell hanging inserts, suggesting they are independent of cell-cell contact [53]. To summarize, direct and indirect transwell co-cultures and conditioned medium studies show that B cell-fibroblast crosstalk in IPF results in the upregulation of fibrotic proteins (e.g., α -SMA, fibronectin and PAI1), whereas T cell-fibroblast interactions in IPF are diverse, with increased fibrotic markers on one hand and a potential protective mechanism that cause decreased fibrotic changes on the other hand. The differences in T-cell-fibroblast crosstalk reported may be due to different experimental conditions and require further investigation to clarify roles and when these contribute to mechanisms of IPF [51,52,53].

Taken together, the studies presented in this section demonstrate the importance of the innate and adaptive immunity in regulating mechanisms of immune cell-fibroblast crosstalk that may drive fibrotic changes in IPF. Innate immune cells such as mast cells, macrophages and neutrophils all interact with fibroblasts through the release of growth factors and enzymes (e.g., HGF, tryptase, NE) that leads to increased ECM and structural proteins such as collagen, fibronectin, and α-SMA [38, 41,42,43, 48, 54]. The contribution of adaptive immunity to fibrosis through immune cell-fibroblast crosstalk seems to be more diverse, with interactions between activated B cells and fibroblasts resulting in upregulated ECM proteins while T cell-fibroblast crosstalk result in both increased collagen and decreased myofibroblast expression of α-SMA and calponin (Fig. 1). These studies are crucial to understanding the underlying mediators of the nuanced fibrotic processes that occur in IPF and will aid in potentially finding novel therapeutic targets for the disease.

Fig. 1figure 1

Mechanisms of immune cell-fibroblast interactions and how they contribute to the pathogenesis of idiopathic pulmonary fibrosis as determined by in vitro co-culture and conditioned medium model studies. Environmental toxins are inhaled into the lungs and cause repetitive injury to the epithelial layer in IPF pathogenesis. Recurrent epithelial injury causes the release of mediators that over-activate fibroblasts and attract immune cells. Fibroblasts interact with several innate immune cells resulting in various aspects of IPF pathobiology. Stimulated B cells interact with fibroblasts to increased migration in fibroblasts as well as to increase the synthesis of fibronectin, PAI1 and α-SMA. The crosstalk between T cells and fibroblasts result in increased proliferation of fibroblasts and increased collagen production. T cell-fibroblast interaction also causes a decrease in calponin and α-SMA and myofibroblast differentiation. Mast cell-fibroblast interactions are largely dependent on tryptase release, which alter fibroblast phenotype by increasing their proliferation and migration, as well as enabling the increased synthesis and release of HGF, fibronectin, collagen I, α-SMA and IL-6. Neutrophil elastase causes fibroblasts to release increasing amounts of IL-8 while a bidirectional crosstalk between fibroblasts and macrophages causes an increased expression of collagen I and III as well as the increased the release of CCL18, CCL2, CX3CL1 and CXCL10. Thus, crosstalk between various immune cells and fibroblasts contribute to IPF remodeling by triggering the overactivation of fibroblasts leading to their increased migration and proliferation which gives rise to fibroblastic foci, while also causing the overproduction and degradation of ECM proteins and contributing to the progressive accumulation of scar tissue, as well as causing the release of classical chemoattractants for immune cells (Figure created in Biorender.com)

Fibroblast-immune cell crosstalk influences proliferation and migration and promotes apoptotic resistance in fibroblasts

The fibrotic mechanisms that characterise IPF have been shown to be impacted by the survival, proliferation, and migration of lung (myo)fibroblasts [55, 56]. In IPF, lung fibroblasts with abnormal (fibrotic) or myofibroblastic phenotypes often have prolonged survival rates due to processes that enable their resistance to apoptotic mechanisms [56, 57]. These mechanisms which include fibroblast-immune cell crosstalk, have also been shown to alter the proliferation and migration of defective lung fibroblasts/differentiated myofibroblasts which enables their accumulation in the IPF lung interstitium [58, 59].

To corroborate the role of lung fibroblast-immune cell crosstalk in mechanisms of fibroblast proliferation in IPF, Wygrecka et al., isolated IPF- and control -derived PHLFs and mast cells and co-cultured them directly [41]. The direct co-culture of IPF-derived human lung fibroblasts and mast cells rapidly induced increased proliferation of lung fibroblasts [41]. It was found that the high rate of fibroblast proliferation was largely due to an increase in the release of the enzyme tryptase by mast cells as the addition of the tryptase inhibitor, APC366, greatly attenuated the response [41]. The tryptase-mediated increase in lung fibroblast proliferation was also shown to be specifically mediated by the protease activated receptor-2 (PAR-2), which further induced the phosphorylation of protein kinase C-α (PKC-α), Raf-1, and p44/42. Hence, it was demonstrated that an interaction between the PKC-α/Raf-1/p44/42 pathway and PAR-2 receptor work together in advancing tryptase induced fibroblast proliferation due to mast cell regulation in IPF [41]. In connection with this, a study by Bagh

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