The peroxisome proliferator-activated receptor γ (PPARγ) is expressed in several organs and tissues [1–3] and is involved in the regulation of adipocyte differentiation and glucose homeostasis [4–7], being a regulator of energy homeostasis. PPARγ has been involved in lung maturation in mice [3, 8] and its expression was found in immune cells, like lymphocytes, macrophages, and granulocytes, the latter mainly involved in inflammatory reactions [9, 10]. PPARγ acts as a ligand-activated transcription factor . Prostaglandins [8, 12], but also synthetic and nonsteroidal anti-inflammatory substances [8, 13] activate the receptor. PPARγ activation has been shown to exhibit anti-inflammatory potential by inhibiting the activity of pro-inflammatory transcription factors such as e.g. the activator protein 1 (AP-1), signal transducer and activators of transcription (STATs), or the Nuclear factor kappa B (NF-κB), as shown in murine primary peritoneal macrophages [14–16].
In particular alveolar macrophages (AM) have increased levels of PPARγ  and are constantly bathed in lipid-rich surfactant  that consists of potential receptor activating ligands, or at least precursors of ligands [14, 18, 19]. This coexistence of high levels of PPARγ in an environment rich in lipophilic ligands is an important finding, since: i) resident AMs in the alveolus represent the first line of innate immune defence in the respiratory tract and ii) AM orchestrate inflammatory responses by recognizing tissue damage, promoting neutrophil recruitment for appropriate pathogen defence and finally leading to resolution of inflammation . This indispensable role in lung homeostasis makes the AM a promising target for the treatment of inflammatory lung diseases. In fact murine studies have revealed AM function requires upregulation of the expression of CD36, a PPARγ target. CD36 is a cell surface scavenger receptor and a key factor promoting phagocytosis of apoptotic neutrophils, lipids and unopsonized materials . Similarly, an increase in Fcγ receptor mediated phagocytosis of opsonized materials  seems to require PPARγ activation. This AM cell-mediated effector promoting resolution of inflammation depends on the PPARγ-induced molecular anti-inflammatory properties  as well as by factors of different lung structural cell types, thereby down-regulating pro-inflammatory mediators  like TNFα, neutrophil and monocyte-macrophage chemotactic factors IL-8, MCP-1, pro-oxidant enzyme iNOS, and MMP9 [23–25] while up-regulating expression of anti-inflammatory proteins like IL-10 (reviewed in ). These results suggest a potential therapeutic application of PPARγ activation to resolve lung inflammatory disorders. This is particularly relevant since AM play a critical role in pathogenesis of asthma, chronic obstructive pulmonary disease (COPD), lung fibrosis (IPF) and lung sarcoidosis (for review see ). Moreover PPARγ binding to the respective response elements in AMs is markedly reduced in chronic inflammatory pulmonary sarcoidosis and obstructive diseases [26, 27]. This suggests that the alveolar microenvironment might be immuno-suppressive in the absence of a specific stimulus , keeping the AM in a quiescent mode possibly supported by PPARγ function.
PPARγ knockout models have already revealed developmental airspace enlargement, and greater smoke-induced emphysema, with increased AM numbers [3, 8]. In agreement with this beneficial effects of ligand-induced PPARγ activation in the lung [8, 29] have been suggested, as indicated by the attenuation of pro-inflammatory cytokine release from activated AMs, eosinophils and type2 epithelial cells , and reduced smoke-induced epithelial mucin production . Improved pathophysiological states in models for asthma, COPD, IPF, and acute lung injury have also been found [29, 31–33]. In contrast, PPARγ deficiency or lack of receptor activation in macrophages resulted in increased atherosclerosis  and reduced CD36 expression [18, 35]. Take together all together, these findings highlight PPARγ as a promising target for the treatment of many inflammatory pathologies by promoting resolution of inflammation .
According to these anti-inflammatory effects in the lung and the fact that unresolved pulmonary inflammation may lead to chronic disease states, we tested the hypothesis that a diminished PPARγ function may result in an increased cellular and molecular inflammatory response, during acute inflammation and impaired resolution. With regard to an inflammatory stimulation of the lungs by particulate matter, so far PPARγ function has only been associated with exposure to cigarette smoke but not with environmental particles such as combustion derived nanoparticles. To address this hypothesis we investigated mice (C57BL/6J) carrying a dominant-negative point mutation (P465L) in the ligand-binding domain of the PPARγ receptor - a targeted mutation, equivalent to a rare mutation in humans (P467L) [5, 36–38]. Whereas human carriers of the mutation suffer from lipodystrophy, extreme insulin resistance, as well as hypertension, fatty liver, and lower adiponectin levels in circulation, humans with the homozygous for P465L die in utero. Mice with the same mutation developed apparently morphologically normal total amounts of adipose tissue - although displaying higher extra-abdominal fat mass - and were insulin sensitive [6, 7]. However, these animals recapitulated the human phenotype once challenged with positive energy balance . We favoured to use P465L/wt mutant mice over the more severely compromised PPARγ knock-out mice since it more reliably resembles the situation in chronic inflammatory lung diseases as described, in alveolar macrophages - like in asthma , pulmonary sarcoidosis [26, 27] and COPD  - or in epithelial cells like in cystic fibrosis , where PPARγ activation was found to be reduced, but not absent. Our rational was that if PPARγ contributes to an anti-inflammatory macrophage state and/or is involved in the resolution of inflammation, then PPARγ defective mice should show impaired resolution of particle induced lung inflammation, a model clearly involving alveolar macrophage function [41, 42].
To our knowledge, apart from cigarette smoke, yet no one has investigated PPARγ related effects in the context of particle related lung inflammation. Exposure to Printex 90 was primarily chosen as a surrogate for urban air pollution by combustion derived nanoparticles. However since in addition to its generation by combustion processes like from diesel engines, carbon black is a constituent of lots of products of modern societies, like inks and paints, rubber and plastic, and thus progressively becoming a more relevant anthropogenic source of ambient and indoor particulate matter. In fact more than 10 million tones are produced every year . But regardless of CNP ancestry, whether airborne, combustion derived or engineered, this sub-100 nm scaled particle class has gained toxicological interest due to their small dimensions, large surface area and high deposition efficiency in the lung being considered an important driver of adverse health effects linked to respiratory toxicity [44, 45]. It is widely accepted that particulate air pollution contributes to the adverse health effects in humans and that patients with metabolic syndrome (obesity, hypertension and diabetes mellitus) may be a more susceptible population. Thus the identification of underlying pathways linking the inflammatory responses induced by particle related health effects and susceptibility to metabolic diseases are of prime importance. In this respect we speculate that PPARγ might be one of the connections linking the regulation of lipid metabolism with alveolar inflammation.
In summary our aim was on to contribute to the understanding of the pathogenic role of PPARγ biology during pulmonary inflammation caused by non-infectious respirable stimuli as represented by carbonaceous particulate matter. We wanted to clarify whether the reduced availability of functional PPARγ in (P465L/wt) mutant mice increased the susceptibility towards acute inflammation and failed resolution in response to CNP-stimulus in comparison to PPARγ wild-type mice (wt/wt). Experiments were performed in adult, 12-14 weeks old, PPARγ wild-type (wt/wt) and P465L/wt mutant mice of both genders to account for sex-specific hormone levels [46, 47]. Animals were challenged using physically and chemically well characterized CNPs of moderate toxicity as described earlier .