Pleural mesothelial cells (PMCs), which are derived from the mesoderm, exhibit an extraordinary capacity to undergo phenotypic changes during development and disease. PMC transformation and trafficking has a newly defined role in idiopathic pulmonary fibrosis (IPF); however, the contribution of Wilms' tumor 1 (Wt1)-positive PMCs to the generation of pathognomonic myofibroblasts remains unclear. PMCs were obtained from IPF lung explants and healthy donor lungs that were not used for transplantation. Short hairpin Wt1-knockdown PMCs (sh Wt1) were generated with Wt1 shRNA, and morphologic and functional assays were performed in vitro. Loss of Wt1 abrogated the PMC phenotype and showed evidence of mesothelial-to-mesenchymal transition (MMT), with a reduced expression of E-cadherin and an increase in the profibrotic markers α-smooth muscle actin (α-SMA) and fibronectin, along with increased migration and contractility, compared with that of the control. Migration of PMCs in response to active transforming growth factor (TGF)-β1 was assessed by live-cell imaging with 2-photon microscopy and 3D imaging, of Wt1-EGFP transgenic mice. Lineage-tracing experiments to map the fate of Wt1(+) PMCs in mouse lung in response to TGF-β1 were also performed by using a Cre-loxP system. Our results, for the first time, demonstrate that Wt1 is necessary for the morphologic integrity of pleural membrane and that loss of Wt1 contributes to IPF via MMT of PMCs into a myofibroblast phenotype.

Non-tuberculous mycobacterial (NTM) infections occur in both immunocompromised and immunocompetent hosts and are an increasingly recognized cause of morbidity and mortality. The hallmark of pulmonary mycobacterial infections is the formation of granuloma in the lung. Our study focuses on the role of heme oxygenase-1 (HO-1), a cytoprotective enzyme, in the regulation of granuloma development and maturation following infection with Mycobacterium avium. We examined the role of HO-1 in regulating monocyte chemoattractant protein-1 (MCP-1) and chemokine receptor 2 (CCR2), two molecules involved in monocyte-macrophage cell trafficking after infection. We showed that RAW 264.7 mouse monocytes exposed to M. avium expressed HO-1 and MCP-1. Inhibition of heme oxygenase by zinc protoporphyrin-IX led to inhibition of MCP-1 and increased expression of CCR2, its cognate receptor. HO-1-/- mice did not develop organized granuloma in their lungs, had higher lung colony forming unit of M. avium when infected with intratracheal M. avium, and had loose collections of inflammatory cells in the lung parenchyma. Mycobacteria were found only inside defined granulomas but not outside granuloma in the lungs of HO-1+/+ mice. In HO-1-/- mice, mycobacteria were also found in the liver and spleen and showed increased mortality. Peripheral blood monocytes isolated from GFP+ mice and given intravenously to HO-1+/+ mice localized into tight granulomas, while in HO-1-/- mice they remained diffusely scattered in areas of parenchymal inflammation. Higher MCP-1 levels were found in bronchoalveolar lavage fluid (BALF) of M. avium infected HO-1-/- mice and CCR2 expression was higher in HO-1-/- alveolar macrophages when compared to HO-1+/+ mice. CCR2 expression localized to granuloma in HO-1+/+ mice but not in the HO-1-/- mice. These findings strongly suggest that HO-1 plays a protective role in the control of M. avium infection.

The origin of the myofibroblast in fibrotic lung disease is uncertain, and no effective medical therapy for fibrosis exists. We have previously demonstrated that transforming growth factor-β1 (TGF-β1) induces pleural mesothelial cell (PMC) transformation into myofibroblasts and haptotactic migration in vitro. Whether PMC differentiation and migration occurs in vivo, and whether this response can be modulated for therapeutic benefit, is unknown. Here, using mice recombinant for green fluorescent protein (GFP) driven by the Wilms tumor-1 (WT-1) promoter, we demonstrate PMC trafficking into the lung and differentiation into myofibroblasts. Carbon monoxide or the induction of heme oxygenase-1 (HO-1) inhibited the expression of myofibroblast markers, contractility, and haptotaxis in PMCs treated with TGF-β1. Intrapleural HO-1 induction inhibited PMC migration after intratracheal fibrogenic injury. PMCs from patients with idiopathic pulmonary fibrosis (IPF) exhibited increased expression of myofibroblast markers and enhanced contractility and haptotaxis, compared with normal PMCs. Carbon monoxide reversed this IPF PMC profibrotic phenotype. WT-1-expressing cells were present within fibrotic regions of the lungs in IPF subjects, supporting a role for PMC differentiation and trafficking as contributors to the myofibroblast population in lung fibrosis. Our findings also support a potential role for pleural-based therapies to modulate pleural mesothelial activation and parenchymal fibrosis progression.

UVB-induced DNA damage plays a critical role in development of photoimmunosuppression. The purpose of this study was to determine whether repair of UVB-induced DNA damage is regulated by Toll-like receptor-4 (TLR4). When TLR4 gene knockout (TLR4-/-) and TLR4 competent (TLR4+/+) mice were subjected to 90 mJ/cm2 UVB radiation locally, DNA damage in the form of CPD, were repaired more efficiently in the skin and bone marrow dendritic cells (BMDC) of TLR4-/- mice in comparison to TLR4+/+ mice. Expression of DNA repair gene XPA (Xeroderma pigmentosum complementation group A) was significantly lower in skin and BMDC of TLR4+/+ mice than TLR4-/- mice after UVB exposure. When cytokine levels were compared in these strains after UVB exposure, BMDC from UV-irradiated TLR4-/- mice produced significantly more interleukin (IL)-12 and IL-23 cytokines (p<0.05) than BMDC from TLR4+/+ mice. Addition of anti-IL-12 and anti-IL-23 antibodies to BMDC of TLR4-/- mice (before UVB exposure) inhibited repair of CPD, with a concomitant decrease in XPA expression. Addition of TLR4 agonist to TLR4+/+ BMDC cultures decreased XPA expression and inhibited CPD repair. Thus, strategies to inhibit TLR4 may allow for immunopreventive and immunotherapeutic approaches for managing UVB-induced cutaneous DNA damage and skin cancer.

Nonmelanoma skin cancers including basal and squamous cell carcinomas (SCC and BCC) represent a significant clinical problem due to their relatively high incidence, imposing an economic burden to healthcare systems around the world. It is accepted that ultraviolet radiation (UVR: λ = 290-400 nm) plays a crucial role in the initiation and promotion of BCC and SCC with UVB (λ = 290-320 nm) having a central role in this process. On the other hand, UVB is required for vitamin D3 (D3) production in the skin, which supplies >90% of the body's requirement for this prohormone. Prolonged exposure to UVB can also generate tachysterol and lumisterol. Vitamin D3 itself and its canonical ( 1,25(OH)2D3) and noncanonical (CYP11A1-intitated) D3 hydroxyderivatives show photoprotective functions in the skin. These include regulation of keratinocyte proliferation and differentiation, induction of anti-oxidative responses, inhibition of DNA damage and induction of DNA repair mechanisms, and anti-inflammatory activities. Studies in animals have demonstrated that D3 hydroxyderivatives can attenuate UVB or chemically induced epidermal cancerogenesis and inhibit growth of SCC and BCC. Genomic and nongenomic mechanisms of action have been suggested. In addition, vitamin D3 itself inhibits hedgehog signaling pathways which have been implicated in many cancers. Silencing of the vitamin D receptor leads to increased propensity to develop UVB or chemically induced epidermal cancers. Other targets for vitamin D compounds include 1,25D3-MARRS, retinoic orphan receptors α and γ, aryl hydrocarbon receptor, and Wnt signaling. Most recently, photoprotective effects of lumisterol hydroxyderivatives have been identified. Clinical trials demonstrated a beneficial role of vitamin D compounds in the treatment of actinic keratosis. In summary, recent advances in vitamin D biology and pharmacology open new exciting opportunities in chemoprevention and treatment of skin cancers.