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1.2.3.2 The potential role of macrophage subsets in IPF

Macrophages may play a role in tissue injury and fibrosis through the release of a number of mediators, as described in the above section (section 1.2.3.1) in IPF patients. These different roles may be contributed by different macrophage subsets in response to signals from their immediate environment (Figure 1.5). Previous studies have suggested that M1 macrophages may participate in inflammation-induced tissue injury through its release of various ROS, RNS, pro-inflammatory cytokines, chemokines and tissue remodelling proteases. M2 and M2-like macrophages may contribute to the wound healing process, and if dysregulated the progression of pulmonary fibrosis through their release of pro-fibrotic mediators (Figure 1.5). On the other hand, M1, M2 and/or M2-like macrophages may also contribute to tissue repair and the resolution of fibrosis through the release of anti-fibrotic mediators²⁷⁸. However, as forementioned in section 1.2.1, tissue

macrophages interact with a number of priming or stimulatory signals, such that the phenotype of macrophage found in vivo are unlikely to be strictly associated with a distinct in vitro defined subset, but rather exist within the M1-M2 polarisation spectrum. It is therefore more likely than not that pro-inflammatory and pro-fibrotic macrophages observed in pulmonary fibrosis are M1- and M2-biased, rather than strictly adhering to a distinct subset.

Despite the shortfall on describing the complexity of macrophage heterogeneity in vivo, the M1-M2 paradigm provides a simple starting point for the assessment of complex macrophage polarisation in vivo. Previous studies of macrophage heterogeneity in IPF have based their classification along the M1-M2 paradigm, presumably due to the lack of technology to identify complexities in macrophage phenotypes. These include single-cell analysis to identify macrophage heterogeneity within a patient

Chapter 1 | General Introduction

76 sample, or genomic and proteomic analysis to identify subtle differences between in vivo and in vitro derived macrophage subsets.

In the following section, these previous studies on macrophage heterogeneity will be summarised in the context of the M1-M2 paradigm, with respect to the pro-inflammatory M1 macrophages, and pro-fibrotic M2 and M2-like macrophages as described in section 1.2.1. It should be noted that the macrophage-subsets described below are unlikely to be mutually exclusive and may coexist in the form of hybrid macrophages.

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1.2.3.2.1 The potential role of M1 macrophages in IPF

There has been one study indicating the presence of M1 macrophages in pulmonary fibrosis:

immunostaining analyses with anti-human or anti-mouse folate-receptor-beta (FR-β) monoclonal antibody revealed that FR-β positive macrophages were present in the fibrotic areas of IPF patients and mice with BLM-induced pulmonary fibrosis respectively⁵⁶⁵. These macrophages are likely to be M1 macrophages, as they have a pro-inflammatory phenotype and produce TNF-α and oxygen radicals⁵⁶⁶. Treatment by ablation of FR-β expressing macrophages by intranasal administration of a recombinant immunotoxin, consisting of Ig heavy and light chain Fv portions of an anti-mouse FR-β Figure 1.5 The potential contributions of various macrophage subsets to pulmonary fibrosis.M1

macrophages may promote inflammation-induced tissue injury and the initiation of pulmonary fibrosis through the release of various reactive oxygen species (ROS), reactive nitrogen species (RNS),

pro-inflammatory cytokines, chemokines and tissue remodelling proteases. M2 and M2-like macrophages may contribute to the wound healing process, but may promote the progression of fibrosis when this process becomes dysregulated when the irritation is persistent. Conversely, M1, M2 and M2-like macrophages also have the potential to resolve fibrosis through their release of anti-fibrotic mediators and phagocytosis of ECM components. Figure adapted from Boorsma CE, 2013²⁷⁸.

monoclonal antibody and truncated Pseudomonas exotoxin A, dosed from day 3 every other day, increased survival and reduced hydroxyproline (collagen precursor) and fibrosis in BLM-challenged mice at day 21⁵⁶⁵. Immunohistochemical analysis also revealed decreased numbers of TNF-α, CCL2 and CCL12 producing cells in the immunotoxin-treated group⁵⁶⁵. The authors concluded that FR-β-positive macrophages have a critical role in the initiation of pulmonary fibrosis, possibly through their release of pro-inflammatory mediators⁵⁶⁵. However, the pro-inflammatory role of FR-β positive macrophages in BLM-induced pulmonary fibrosis has to be confirmed by further flow cytometric analysis of pulmonary macrophages co-stained for FR-β and the aforementioned pro-inflammatory mediators. FR-β positive pulmonary macrophages can also be isolated by fluorescence activated cell sorting (FACS) and the levels of their protein products analysed by enzyme linked immunosorbent assay (ELISA) or meso scale discovery (MSD). M1 macrophages may contribute to inflammation induced tissue injury through the production of various pro-inflammatory mediators, including TNF-α, IL-1β and IL-12 p40, ROS and NO, and MMPs (MMP-7 and MMP-9), leading to fibrosis

development. TNF-α may contribute to pulmonary fibrosis through inducing promoting fibroblast proliferation, AEC1 apoptosis and/or propagating chronic inflammation. IL-1β may induce the release of other pro-inflammatory mediators (e.g. TNF-α and IL-6) to amplify inflammation⁵⁶⁷, pro-angiogenic mediators (e.g. CXCL1, CXCL2, CXCL8) to promote neovascularisation³⁷⁶ and also pro-fibrotic mediators (e.g. TGF-β and PDGF) to promote fibrosis⁵⁶⁷. IL-12 p40 may act as a

chemoattractant for macrophages as observed in vitro and in vivo^382,383. MMP-7 may promote epithelial cell migration by reducing the affinity of α2β1 integrin⁵¹⁴ and activate other pro-fibrotic proteases, including itself, pro-MMP1, -2 and -9⁵¹². MMP-9 is implicated in basement membrane disruption⁵¹⁵.

However, other evidence challenges the role of M1 macrophages in established fibrosis. TNF-α is also shown to inhibit collagen synthesis³⁶⁷ in myofibroblasts, and have a role in aiding epithelial cell recovery³⁶⁸ and treatment of TNF-α in established fibrosis contributes to resolution of BLM-induced pulmonary fibrosis in mice³⁶⁹. The pleiotropic effects of TNF-α observed in pulmonary fibrosis may result from different types of cells present at various stages of pulmonary fibrosis. TNF-α promotes inflammation-induced tissue injury, fibroblast proliferation and subsequent fibrosis in the inflammatory phase; on the other hand, fibroblast are activated into myofibroblasts in the fibrotic phases and their collagen-secreting activities are inhibited by TNF-α in a negative feedback loop.

Depletion of tissue macrophages and/or circulating inflammatory monocytes during the inflammatory phase of in vivo models does not affect the onset or degree of fibrosis developing later on³⁴⁹. On the other hand, it is equally possible that pro-inflammatory mediators that are associated with M1

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78 macrophages in the inflammatory phase are also released at a low level by M1/M2 hybrid macrophages in the fibrotic phases.

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1.2.3.2.2 The potential role of M2 macrophages in IPF

There is a body of evidence demonstrating that M2 macrophages accumulate in the lungs of IPF patients and also mouse models of pulmonary fibrosis. Markers found on or produced by M2 macrophages have been found to be increased in IPF patients, including Galectin-3 (Gal-3)^568,569, CCL18⁵⁷⁰, Chitinase 3 like 3 (Chi3l3)/ Tyrosine-Lysine-Leucine, 40 kDa (YKL-40)⁵⁷¹, CD163⁵⁷², IGF-I and PDGF^573–575. M2 markers are also increased in murine models of pulmonary fibrosis, including Arg-1⁵⁷⁶, Found In Inflammatory Zone (FIZZ1)/ Resistin-Like Molecule alpha (RELM-α)^577,578, and MRC1^337,579. There is also an increase in the M2-inducing cytokine IL-13 in IPF patients compared to controls, and AMɸ isolated from IPF patients produce more IL-13 than that from control lungs⁵⁸⁰. IL-13 may therefore participate in promoting the M2 macrophage phenotype in IPF patients. M2 macrophages may play a pro-fibrotic role through the release of their various associated mediators. For example, Arg-1 promotes the conversion of arginine into orthinine⁵⁸¹, the precursor of proline that is required for collagen synthesis could support fibrosis⁵⁸². FIZZ1 induces the differentiation and survival of myofibroblasts⁵⁸³, as well as increase ECM production in fibroblasts that would favour the development of fibrosis⁵⁷⁷. IGF-I and PDGF drive the proliferation of fibroblasts and their transformation to ECM-producing myofibroblasts¹²⁰. M2 macrophages are able to secrete the ECM component fibronectin¹¹⁹; Fibronectin is a major component of the fibrotic clot, and may also act as a scaffold for fibroblast migration^520,521.

Alternatively other studies have suggested that M2 macrophages have an anti-fibrotic role. For example, mannose receptors and the glycoprotein mfge8 are responsible for the uptake of ECM components, and both can attenuate fibrosis in different mouse models^358,584. Mannose receptors are associated with M2 macrophages, which may suggest that this subset may play a role in the resolution of fibrosis. These mechanisms for the clearance of ECM components may serve as a negative feedback mechanism for M2 macrophages to prevent excessive ECM accumulation. In the presence of a persistent irritant however, it is possible that the pro-wound healing activities of M2 macrophages may become dysregulated in a frustrated effort to repair damaged tissue, which is able to overcome negative feedback activities and lead to excessive ECM accumulation and fibrosis.

1.2.3.2.3 The potential role of M2-like macrophages in IPF

M2-like macrophages share many M2-associated proteins (e.g. Arg-1, FIZZ1, MRC1), but are distinguished by their high levels of IL-10 production. M2-like macrophages contribute to immunoregulation, matrix deposition, and tissue remodelling, and may be important during the

transition from inflammation towards wound healing following lung injury. Elevated levels of IL-10 have been observed in several fibrotic diseases, including IPF125,585,586 and systemic sclerosis patients with interstitial lung disease⁵⁷². The role of IL-10 in pulmonary fibrosis remains elusive. Some studies suggest that it has a protective role: IL-10 is an anti-inflammatory cytokine, and can suppress T-cell activation⁵⁸⁷, inhibit T cell activity³²⁰, and also suppress TNF-α and ROS production in macrophages following LPS stimulation⁵⁸⁸. IL-10 is essential for the regulation of acute and chronic inflammation: in IL-10-deficient mice infected with rapidly replicating pathogens, such as Trypanosoma cruzi and Toxoplasma gondii, the mice typically die rapidly but not from excessive growth of the pathogens, but of a massive inflammatory response^589,590. In the context of pulmonary fibrosis, IL-10 may prevent further tissue injury and fibrosis⁵⁹¹. However, this hypothesis is challenged by another study, where IL-10 knockout mice showed increased BLM-induced inflammation but not fibrosis⁵⁹². On the other hand, prophylactic and therapeutic gene delivery of IL-10 attenuated BLM-induced pulmonary fibrosis in mice through inhibiting the production of TGF-E in the lung, suggesting an anti-fibrotic role for IL-10⁵⁹³. IL-10 can have a pro-fibrotic role: long term lung-specific overexpression of IL-10 in mice induced pulmonary fibrosis through fibrocyte recruitment and M2-like macrophage activation through a CCR2/

CCL2 axis⁵⁹⁴. M2-like macrophages may also be producers of the pro-fibrotic cytokine TGF-E, which may promote fibrocyte recruitment, and fibroblast activation/differentiation into the ECM-producing myofibroblasts⁵⁵⁴.

In conclusion, there is evidence for both the contribution and inhibition by different macrophage subsets, including M1-, M2-, or M2-like macrophages, of pulmonary fibrosis. These pleiotropic effects of these macrophage subsets to promote or inhibit pulmonary fibrosis are more likely than not to represent the complexity in macrophage activation in vivo, with respect to the plasticity of macrophages to respond to multiple signals to give rise to hybrid macrophages, and the adaption of macrophages phenotype to the changing environment during disease progresson in IPF (i.e. temporal heterogeneity of IPF).