Genetic deficiency of the transcription factor NFAT1 confers protection against fibrogenic responses independent of immune influx.

Idiopathic pulmonary fibrosis (IPF) is marked by unremitting matrix deposition and architectural distortion. Multiple profibrotic pathways contribute to the persistent activation of mesenchymal cells (MCs) in fibrosis, highlighting the need to identify and target common signaling pathways. The transcription factor nuclear factor of activated T cells 1 (NFAT1) lies downstream of second messenger calcium signaling and has been recently shown to regulate key profibrotic mediator autotaxin (ATX) in lung MCs. Herein, we investigate the role of NFAT1 in regulating fibroproliferative responses during the development of lung fibrosis. Nfat1-/--deficient mice subjected to bleomycin injury demonstrated improved survival and protection from lung fibrosis and collagen deposition as compared with bleomycin-injured wild-type (WT) mice. Chimera mice, generated by reconstituting bone marrow cells from WT or Nfat1-/- mice into irradiated WT mice (WT→WT and Nfat1-/-→WT), demonstrated no difference in bleomycin-induced fibrosis, suggesting immune influx-independent fibroprotection in Nfat1-/- mice. Examination of lung tissue and flow sorted lineageneg/platelet-derived growth factor receptor alpha (PDGFRα)pos MCs demonstrated decreased MC numbers, proliferation [↓ cyclin D1 and 5-ethynyl-2'-deoxyuridine (EdU) incorporation], myofibroblast differentiation [↓ α-smooth muscle actin (α-SMA)], and survival (↓ Birc5) in Nfat1-/- mice. Nfat1 deficiency abrogated ATX expression in response to bleomycin in vivo and MCs derived from Nfat1-/- mice demonstrated decreased ATX expression and migration in vitro. Human IPF MCs demonstrated constitutive NFAT1 activation, and regulation of ATX in these cells by NFAT1 was confirmed using pharmacological and genetic inhibition. Our findings identify NFAT1 as a critical mediator of profibrotic processes, contributing to dysregulated lung remodeling and suggest its targeting in MCs as a potential therapeutic strategy in IPF.NEW & NOTEWORTHY Idiopathic pulmonary fibrosis (IPF) is a fatal disease with hallmarks of fibroblastic foci and exuberant matrix deposition, unknown etiology, and ineffective therapies. Several profibrotic/proinflammatory pathways are implicated in accelerating tissue remodeling toward a honeycombed end-stage disease. NFAT1 is a transcriptional factor activated in IPF tissues. Nfat1-deficient mice subjected to chronic injury are protected against fibrosis independent of immune influxes, with suppression of profibrotic mesenchymal phenotypes including proliferation, differentiation, resistance to apoptosis, and autotaxin-related migration.

Modulating lung immune cells by pulmonary delivery of antigen-specific nanoparticles to treat autoimmune disease.

Antigen-specific particles can treat autoimmunity, and pulmonary delivery may provide for easier delivery than intravenous or subcutaneous routes. The lung is a "hub" for autoimmunity where autoreactive T cells pass before arriving at disease sites. Here, we report that targeting lung antigen-presenting cells (APCs) via antigen-loaded poly(lactide-co-glycolide) particles modulates lung CD4+ T cells to tolerize murine experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. Particles directly delivered to the lung via intratracheal administration demonstrated more substantial reduction in EAE severity when compared with particles delivered to the liver and spleen via intravenous administration. Intratracheally delivered particles were associated with lung APCs and decreased costimulatory molecule expression on the APCs, which inhibited CD4+ T cell proliferation and reduced their population in the central nervous system while increasing them in the lung. This study supports noninvasive pulmonary particle delivery, such as inhalable administration, to treat autoimmune disease.

Alveolar macrophage-derived extracellular vesicles inhibit endosomal fusion of influenza virus.

Alveolar macrophages (AMs) and epithelial cells (ECs) are the lone resident lung cells positioned to respond to pathogens at early stages of infection. Extracellular vesicles (EVs) are important vectors of paracrine signaling implicated in a range of (patho)physiologic contexts. Here we demonstrate that AMs, but not ECs, constitutively secrete paracrine activity localized to EVs which inhibits influenza infection of ECs in vitro and in vivo. AMs exposed to cigarette smoke extract lost the inhibitory activity of their secreted EVs. Influenza strains varied in their susceptibility to inhibition by AM-EVs. Only those exhibiting early endosomal escape and high pH of fusion were inhibited via a reduction in endosomal pH. By contrast, strains exhibiting later endosomal escape and lower fusion pH proved resistant to inhibition. These results extend our understanding of how resident AMs participate in host defense and have broader implications in the defense and treatment of pathogens internalized within endosomes.

Stem cell transplantation impairs dendritic cell trafficking and herpesvirus immunity.

Long-term survivors after hematopoietic stem cell transplantation are at high risk of infection, which accounts for one-third of all deaths related to stem cell transplantation. Little is known about the cause of inferior host defense after immune cell reconstitution. Here, we exploited a murine syngeneic BM transplantation (BMT) model of late infection with murine gammaherpesvirus 68 (MHV-68) to determine the role of conventional DC (cDC) trafficking in adaptive immunity in BMT mice. After infection, the expression of chemokine Ccl21 in the lung is reduced and the migration of cDCs into lung draining lymph nodes (dLNs) is impaired in BMT mice, limiting the opportunity for cDCs to prime Th cells in the dLNs. While cDC subsets are redundant in priming Th1 cells, Notch2 functions in cDC2s are required for priming increased Th17 responses in BMT mice, and cDC1s can lessen this activity. Importantly, Th17 cells can be primed both in the lungs and dLNs, allowing for increased Th17 responses without optimum cDC trafficking in BMT mice. Taken together, impaired cDC trafficking in BMT mice reduces protective Th1 responses and allows increased pathogenic Th17 responses. Thus, we have revealed a previously unknown mechanism for BMT procedures to cause long-term inferior immune responses to herpes viral infection.

Loss of myeloid-specific protein phosphatase 2A enhances lung injury and fibrosis and results in IL-10-dependent sensitization of epithelial cell apoptosis.

Protein phosphatase 2A (PP2A), a ubiquitously expressed Ser/Thr phosphatase is an important regulator of cytokine signaling and cell function. We previously showed that myeloid-specific deletion of PP2A (LysMcrePP2A-/-) increased mortality in a murine peritoneal sepsis model. In the current study, we assessed the role of myeloid PP2A in regulation of lung injury induced by lipopolysaccharide (LPS) or bleomycin delivered intratracheally. LysMcrePP2A-/- mice experienced increased lung injury in response to both LPS and bleomycin. LysMcrePP2A-/- mice developed more exuberant fibrosis in response to bleomycin, elevated cytokine responses, and chronic myeloid inflammation. Bone marrow-derived macrophages (BMDMs) from LysMcrePP2A-/- mice showed exaggerated inflammatory cytokine release under conditions of both M1 and M2 activation. Notably, secretion of IL-10 was elevated under all stimulation conditions, including activation of BMDMs by multiple Toll-like receptor ligands. Supernatants collected from LPS-stimulated LysMcrePP2A-/- BMDMs induced epithelial cell apoptosis in vitro but this effect was mitigated when IL-10 was also depleted from the BMDMs by crossing LysMcrePP2A-/- mice with systemic IL-10-/- mice (LysMcrePP2A-/- × IL-10-/-) or when IL-10 was neutralized. Despite these findings, IL-10 did not directly induce epithelial cell apoptosis but sensitized epithelial cells to other mediators from the BMDMs. Taken together our results demonstrate that myeloid PP2A regulates production of multiple cytokines but that its effect is most pronounced on IL-10 production. Furthermore, IL-10 sensitizes epithelial cells to apoptosis in response to myeloid-derived mediators, which likely contributes to the pathogenesis of lung injury and fibrosis in this model.

First-Onset Herpesviral Infection and Lung Injury in Allogeneic Hematopoietic Cell Transplantation.

Rationale: "Noninfectious" pulmonary complications are significant causes of morbidity and mortality after allogeneic hematopoietic cell transplant. Early-onset viral reactivations or infections are common after transplant. Whether the first-onset viral infection causes noninfectious pulmonary complications is unknown. Objectives: To determine whether the first-onset viral infection within 100 days after transplant predisposes to development of noninfectious pulmonary complications. Methods: We performed a retrospective review of 738 allogeneic hematopoietic cell transplant patients enrolled from 2005 to 2011. We also established a novel bone marrow transplantation mouse model to test whether herpesviral reactivation after transplant causes organ injury. Measurements and Main Results: First-onset viral infections with human herpesvirus 6 or Epstein-Barr virus within 100 days after transplant increase the risk of developing idiopathic pneumonia syndrome (adjusted hazard ratio [aHR], 5.52; 95% confidence interval [CI], 1.61-18.96; P = 0.007; and aHR, 9.21; 95% CI, 2.63-32.18; P = 0.001, respectively). First infection with human cytomegalovirus increases risk of bronchiolitis obliterans syndrome (aHR, 2.88; 95% CI, 1.50-5.55; P = 0.002) and grade II-IV acute graft-versus-host disease (aHR, 1.59; 95% CI, 1.06-2.39; P = 0.02). Murine roseolovirus, a homolog of human herpesvirus 6, can also be reactivated in the lung and other organs after bone marrow transplantation. Reactivation of murine roseolovirus induced an idiopathic pneumonia syndrome-like phenotype and aggravated acute graft-versus-host disease. Conclusions: First-onset herpesviral infection within 100 days after allogeneic hematopoietic cell transplant increases risk of pulmonary complications. Experimentally reactivating murine roseolovirus causes organ injury similar to phenotypes seen in human transplant recipients.

Influenza-induced immune suppression to methicillin-resistant Staphylococcus aureus is mediated by TLR9.

Bacterial lung infections, particularly with methicillin-resistant Staphylococcus aureus (MRSA), increase mortality following influenza infection, but the mechanisms remain unclear. Here we show that expression of TLR9, a microbial DNA sensor, is increased in murine lung macrophages, dendritic cells, CD8+ T cells and epithelial cells post-influenza infection. TLR9-/- mice did not show differences in handling influenza nor MRSA infection alone. However, TLR9-/- mice have improved survival and bacterial clearance in the lung post-influenza and MRSA dual infection, with no difference in viral load during dual infection. We demonstrate that TLR9 is upregulated on macrophages even when they are not themselves infected, suggesting that TLR9 upregulation is related to soluble mediators. We rule out a role for elevations in interferon-γ (IFNγ) in mediating the beneficial MRSA clearance in TLR9-/- mice. While macrophages from WT and TLR9-/- mice show similar phagocytosis and bacterial killing to MRSA alone, following influenza infection, there is a marked upregulation of scavenger receptor A and MRSA phagocytosis as well as inducible nitric oxide synthase (Inos) and improved bacterial killing that is specific to TLR9-deficient cells. Bone marrow transplant chimera experiments and in vitro experiments using TLR9 antagonists suggest TLR9 expression on non-hematopoietic cells, rather than the macrophages themselves, is important for regulating myeloid cell function. Interestingly, improved bacterial clearance post-dual infection was restricted to MRSA, as there was no difference in the clearance of Streptococcus pneumoniae. Taken together these data show a surprising inhibitory role for TLR9 signaling in mediating clearance of MRSA that manifests following influenza infection.

Natural Secretory Immunoglobulins Promote Enteric Viral Infections.

Noroviruses are enteric pathogens causing significant morbidity, mortality, and economic losses worldwide. Secretory immunoglobulins (sIg) are a first line of mucosal defense against enteric pathogens. They are secreted into the intestinal lumen via the polymeric immunoglobulin receptor (pIgR), where they bind to antigens. However, whether natural sIg protect against norovirus infection remains unknown. To determine if natural sIg alter murine norovirus (MNV) pathogenesis, we infected pIgR knockout (KO) mice, which lack sIg in mucosal secretions. Acute MNV infection was significantly reduced in pIgR KO mice compared to controls, despite increased MNV target cells in the Peyer's patch. Natural sIg did not alter MNV binding to the follicle-associated epithelium (FAE) or crossing of the FAE into the lymphoid follicle. Instead, naive pIgR KO mice had enhanced levels of the antiviral inflammatory molecules interferon gamma (IFN-γ) and inducible nitric oxide synthase (iNOS) in the ileum compared to controls. Strikingly, depletion of the intestinal microbiota in pIgR KO and control mice resulted in comparable IFN-γ and iNOS levels, as well as MNV infectious titers. IFN-γ treatment of wild-type (WT) mice and neutralization of IFN-γ in pIgR KO mice modulated MNV titers, implicating the antiviral cytokine in the phenotype. Reduced gastrointestinal infection in pIgR KO mice was also observed with another enteric virus, reovirus. Collectively, our findings suggest that natural sIg are not protective during enteric virus infection, but rather, that sIg promote enteric viral infection through alterations in microbial immune responses.IMPORTANCE Enteric virus, such as norovirus, infections cause significant morbidity and mortality worldwide. However, direct antiviral infection prevention strategies are limited. Blocking host entry and initiation of infection provides an established avenue for intervention. Here, we investigated the role of the polymeric immunoglobulin receptor (pIgR)-secretory immunoglobulin (sIg) cycle during enteric virus infections. The innate immune functions of sIg (agglutination, immune exclusion, neutralization, and expulsion) were not required during control of acute murine norovirus (MNV) infection. Instead, lack of pIgR resulted in increased IFN-γ levels, which contributed to reduced MNV titers. Another enteric virus, reovirus, also showed decreased infection in pIgR KO mice. Collectively, our data point to a model in which sIg-mediated microbial sensing promotes norovirus and reovirus infection. These data provide the first evidence of the proviral role of natural sIg during enteric virus infections and provide another example of how intestinal bacterial communities indirectly influence MNV pathogenesis.

Lung Dysbiosis, Inflammation, and Injury in Hematopoietic Cell Transplantation.

RATIONALE: Hematopoietic cell transplant (HCT) is a common treatment for hematological neoplasms and autoimmune disorders. Among HCT recipients, pulmonary complications are common, morbid, and/or lethal, and they have recently been associated with gut dysbiosis. The role of lung microbiota in post-HCT pulmonary complications is unknown. OBJECTIVES: To investigate the role of lung microbiota in post-HCT pulmonary complications using animal modeling and human BAL fluid. METHODS: For animal modeling, we used an established murine model of HCT with and without postengraftment herpes virus infection. For human studies, we characterized lung microbiota in BAL fluid from 43 HCT recipients. Lung bacteria were characterized using 16S ribosomal RNA gene sequencing and were compared with lung histology (murine) and with alveolar inflammation and pulmonary function testing (human). MEASUREMENTS AND MAIN RESULTS: Both HCT and viral infection independently altered the composition of murine lung microbiota, but they had no effect on lung microbial diversity. By contrast, combined HCT and viral infection profoundly altered lung microbiota, decreasing community diversity with an associated pneumonitis. Among human HCT recipients, increased relative abundance of the Proteobacteria phylum was associated with impaired pulmonary function, and lung microbiota were significantly associated with alveolar concentrations of inflammatory cytokines. CONCLUSIONS: In animal models and human subjects, lung dysbiosis is a prominent feature of HCT. Lung dysbiosis is correlated with histologic, immunologic, and physiologic features of post-HCT pulmonary complications. Our findings suggest the lung microbiome may be an unappreciated target for the prevention and treatment of post-HCT pulmonary complications.

IL-17A deficiency mitigates bleomycin-induced complement activation during lung fibrosis.

Interleukin 17A (IL-17A) and complement (C') activation have each been implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF). We have reported that IL-17A induces epithelial injury via TGF-ß in murine bronchiolitis obliterans; that TGF-ß and the C' cascade present signaling interactions in mediating epithelial injury; and that the blockade of C' receptors mitigates lung fibrosis. In the present study, we investigated the role of IL-17A in regulating C' in lung fibrosis. Microarray analyses of mRNA isolated from primary normal human small airway epithelial cells indicated that IL-17A (100 ng/ml; 24 h; n = 5 donor lungs) induces C' components (C' factor B, C3, and GPCR kinase isoform 5), cytokines (IL8, -6, and -1B), and cytokine ligands (CXCL1, -2, -3, -5, -6, and -16). IL-17A induces protein and mRNA regulation of C' components and the synthesis of active C' 3a (C3a) in normal primary human alveolar type II epithelial cells (AECs). Wild-type mice subjected to IL-17A neutralization and IL-17A knockout (il17a-/- ) mice were protected against bleomycin (BLEO)-induced fibrosis and collagen deposition. Further, BLEO-injured il17a-/- mice had diminished levels of circulating Krebs Von Den Lungen 6 (alveolar epithelial injury marker), local caspase-3/7, and local endoplasmic reticular stress-related genes. BLEO-induced local C' activation [C3a, C5a, and terminal C' complex (C5b-9)] was attenuated in il17a-/- mice, and IL-17A neutralization prevented the loss of epithelial C' inhibitors (C' receptor-1 related isoform Y and decay accelerating factor), and an increase in local TUNEL levels. RNAi-mediated gene silencing of il17a in fibrotic mice arrested the progression of lung fibrosis, attenuated cellular apoptosis (caspase-3/7) and lung deposition of collagen and C' (C5b-9). Compared to normals, plasma from IPF patients showed significantly higher hemolytic activity. Our findings demonstrate that limiting complement activation by neutralizing IL-17A is a potential mechanism in ameliorating lung fibrosis.-Cipolla, E., Fisher, A. J., Gu, H., Mickler, E. A., Agarwal, M., Wilke, C. A., Kim, K. K., Moore, B. B., Vittal, R. IL-17A deficiency mitigates bleomycin-induced complement activation during lung fibrosis.

Loss of CCR2 signaling alters leukocyte recruitment and exacerbates γ-herpesvirus-induced pneumonitis and fibrosis following bone marrow transplantation.

CCR2-expressing leukocytes are required for the progression of fibrosis in models of induced lung injury as well as models of bone marrow transplant (BMT)-related idiopathic pneumonia syndrome. Infection with murid γ-herpesvirus-68 (γHV-68) results in severe pneumonitis and pulmonary fibrosis following syngeneic BMT; however, the roles that various proinflammatory leukocyte populations play in this process remain unclear. Deletion of CCR2 in both non-BMT and BMT mice increased early lytic viral replication and resulted in a reduction in the numbers of lung-infiltrating GR1+,F4/80+ and CXCR1+ cells, while maintaining robust neutrophil infiltration. Similarly, in γHV-68-infected CCR2(-/-) BMT mice, recruitment of monocytes and lymphocytes were reduced whereas neutrophil recruitment was increased compared with wild-type (WT) BMT mice. Interestingly, levels of profibrotic IL-17 were increased in infected CCR2 BMT mice compared with WT BMT. Furthermore, an increase in lung-associated collagen was detected even though there was an overall decrease in the number of profibrotic CCR2+ fibrocytes detected in the lungs of CCR2(-/-) BMT mice. These data indicate that, contrary to most models of fibrosis, deletion of CCR2 offers no protection from γ-herpesvirus-induced pneumonitis and fibrosis, and, indeed, CCR2+ cells play a suppressive role during the development of pulmonary fibrosis following γ-herpesvirus infection post-BMT by limiting IL-7 and collagen production.

Targeting Inhibitor of Apoptosis Proteins Protects from Bleomycin-Induced Lung Fibrosis.

Accumulation of apoptosis-resistant fibroblasts is a hallmark of pulmonary fibrosis. We hypothesized that disruption of inhibitor of apoptosis protein (IAP) family proteins would limit lung fibrosis. We first show that transforming growth factor-ß1 and bleomycin increase X-linked IAP (XIAP) and cellular IAP (cIAP)-1 and -2 in murine lungs and mesenchymal cells. Functional blockade of XIAP and the cIAPs with AT-406, an orally bioavailable second mitochondria-derived activator of caspases (Smac) mimetic, abrogated bleomycin-induced lung fibrosis when given both prophylactically and therapeutically. To determine whether the reduction in fibrosis was predominantly due to AT-406-mediated inhibition of XIAP, we compared the fibrotic response of XIAP-deficient mice (XIAP(-/y)) with littermate controls and found no difference. We found no alterations in total inflammatory cells of either wild-type mice treated with AT-406 or XIAP(-/y) mice. AT-406 treatment limited CCL12 and IFN-γ production, whereas XIAP(-/y) mice exhibited increased IL-1ß expression. Surprisingly, XIAP(-/y) mesenchymal cells had increased resistance to Fas-mediated apoptosis. Functional blockade of cIAPs with AT-406 restored sensitivity to Fas-mediated apoptosis in XIAP(-/y) mesenchymal cells in vitro and increased apoptosis of mesenchymal cells in vivo, indicating that the increased apoptosis resistance in XIAP(-/y) mesenchymal cells was the result of increased cIAP expression. Collectively, these results indicate that: (1) IAPs have a role in the pathogenesis of lung fibrosis; (2) a congenital deficiency of XIAP may be overcome by compensatory mechanisms of other IAPs; and (3) broad functional inhibition of IAPs may be an effective strategy for the treatment of lung fibrosis by promoting mesenchymal cell apoptosis.

Prostaglandin E2 Production and T Cell Function in Mouse Adenovirus Type 1 Infection following Allogeneic Bone Marrow Transplantation.

Adenovirus infections are important complications of bone marrow transplantation (BMT). We demonstrate delayed clearance of mouse adenovirus type 1 (MAV-1) from lungs of mice following allogeneic BMT. Virus-induced prostaglandin E2 (PGE2) production was greater in BMT mice than in untransplanted controls, but BMT using PGE2-deficient donors or recipients failed to improve viral clearance, and treatment of untransplanted mice with the PGE2 analog misoprostol did not affect virus clearance. Lymphocyte recruitment to the lungs was not significantly affected by BMT. Intracellular cytokine staining of lung lymphocytes demonstrated impaired production of INF-γ and granzyme B by cells from BMT mice, and production of IFN-γ, IL-2, IL-4, and IL-17 following ex vivo stimulation was impaired in lymphocytes obtained from lungs of BMT mice. Viral clearance was not delayed in untransplanted INF-γ-deficient mice, suggesting that delayed viral clearance in BMT mice was not a direct consequence of impaired IFN-γ production. However, lung viral loads were higher in untransplanted CD8-deficient mice than in controls, suggesting that delayed MAV-1 clearance in BMT mice is due to defective CD8 T cell function. We did not detect significant induction of IFN-ß expression in lungs of BMT mice or untransplanted controls, and viral clearance was not delayed in untransplanted type I IFN-unresponsive mice. We conclude that PGE2 overproduction in BMT mice is not directly responsible for delayed viral clearance. PGE2-independent effects on CD8 T cell function likely contribute to the inability of BMT mice to clear MAV-1 from the lungs.

CD8+ T Cell Response to Gammaherpesvirus Infection Mediates Inflammation and Fibrosis in Interferon Gamma Receptor-Deficient Mice.

Idiopathic pulmonary fibrosis (IPF), one of the most severe interstitial lung diseases, is a progressive fibrotic disorder of unknown etiology. However, there is growing appreciation for the role of viral infection in disease induction and/or progression. A small animal model of multi-organ fibrosis, which involves murine gammaherpesvirus (MHV68) infection of interferon gamma receptor deficient (IFNγR-/-) mice, has been utilized to model the association of gammaherpesvirus infections and lung fibrosis. Notably, several MHV68 mutants which fail to induce fibrosis have been identified. Our current study aimed to better define the role of the unique MHV68 gene, M1, in development of pulmonary fibrosis. We have previously shown that the M1 gene encodes a secreted protein which possesses superantigen-like function to drive the expansion and activation of Vß4+ CD8+ T cells. Here we show that M1-dependent fibrosis is correlated with heightened levels of inflammation in the lung. We observe an M1-dependent cellular infiltrate of innate immune cells with most striking differences at 28 days-post infection. Furthermore, in the absence of M1 protein expression we observed reduced CD8+ T cells and MHV68 epitope specific CD8+ T cells to the lungs-despite equivalent levels of viral replication between M1 null and wild type MHV68. Notably, backcrossing the IFNγR-/- onto the Balb/c background, which has previously been shown to exhibit weak MHV68-driven Vß4+ CD8+ T cell expansion, eliminated MHV68-induced fibrosis-further implicating the activated Vß4+ CD8+ T cell population in the induction of fibrosis. We further addressed the role that CD8+ T cells play in the induction of fibrosis by depleting CD8+ T cells, which protected the mice from fibrotic disease. Taken together these findings are consistent with the hypothesized role of Vß4+ CD8+ T cells as mediators of fibrotic disease in IFNγR-/- mice.

Transforming growth factor-ß induces microRNA-29b to promote murine alveolar macrophage dysfunction after bone marrow transplantation.

Hematopoietic stem cell transplantation (HSCT) is complicated by pulmonary infections that manifest posttransplantation. Despite engraftment, susceptibility to infections persists long after reconstitution. Previous work using a murine bone marrow transplant (BMT) model implicated increased cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) in promoting impaired alveolar macrophage (AM) responses. However, mechanisms driving COX-2 overexpression remained elusive. Previously, transforming growth factor-ß (TGF-ß) signaling after BMT was shown to promote hypomethylation of the COX-2 gene. Here, we provide mechanistic insight into how this occurs and show that TGF-ß induces microRNA (miR)-29b while decreasing DNA methyltransferases (DNMT)1, DNMT3a, and DNMT3b in AMs after BMT. De novo DNMT3a and DNMT3b were decreased upon transient transfection of miR-29b, resulting in decreased methylation of the COX-2 promoter and induction of COX-2. As a consequence, miR-29b-driven upregulation of COX-2 promoted AM dysfunction, and transfection of BMT AMs with a miR-29b inhibitor rescued the bacterial-killing defect. MiR-29b-mediated defects in BMT AMs were dependent on increased levels of PGE2, as miR-29b-transfected AMs treated with a novel E prostanoid receptor 2 antagonist abrogated the impaired bacterial killing. We also demonstrate that patients that have undergone HSCT exhibit increased miR-29b; thus these studies highlight miR-29b in driving defective AM responses and identify this miRNA as a potential therapeutic target.

Periostin promotes fibrosis and predicts progression in patients with idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease without effective therapeutics. Periostin has been reported to be elevated in IPF patients relative to controls, but its sources and mechanisms of action remain unclear. We confirm excess periostin in lungs of IPF patients and show that IPF fibroblasts produce periostin. Blood was obtained from 54 IPF patients (all but 1 with 48 wk of follow-up). We show that periostin levels predict clinical progression at 48 wk (hazard ratio = 1.47, 95% confidence interval = 1.03-2.10, P < 0.05). Monocytes and fibrocytes are sources of periostin in circulation in IPF patients. Previous studies suggest that periostin may regulate the inflammatory phase of bleomycin-induced lung injury, but periostin effects during the fibroproliferative phase of the disease are unknown. Wild-type and periostin-deficient (periostin(-/-)) mice were anesthetized and challenged with bleomycin. Wild-type mice were injected with bleomycin and then treated with OC-20 Ab (which blocks periostin and integrin interactions) or control Ab during the fibroproliferative phase of disease, and fibrosis and survival were assessed. Periostin expression was upregulated quickly after treatment with bleomycin and remained elevated. Periostin(-/-) mice were protected from bleomycin-induced fibrosis. Instillation of OC-20 during the fibroproliferative phase improved survival and limited collagen deposition. Chimeric mouse studies suggest that hematopoietic and structural sources of periostin contribute to lung fibrogenesis. Periostin was upregulated by transforming growth factor-ß in lung mesenchymal cells, and periostin promoted extracellular matrix deposition, mesenchymal cell proliferation, and wound closure. Thus periostin plays a vital role in late stages of pulmonary fibrosis and is a potential biomarker for disease progression and a target for therapeutic intervention.

COX-2 expression is upregulated by DNA hypomethylation after hematopoietic stem cell transplantation.

Hematopoietic stem cell transplant therapy is limited by pulmonary infections. Mice with fully reconstituted hematopoietic compartments, including alveolar macrophages (AMs), after bone marrow transplantation (BMT) have impaired host defense against Gram-negative Pseudomonas aeruginosa. Impaired innate immunity is related to increased production of PGE(2) by AMs. Cyclooxygenase (COX)-2 is the rate-limiting enzyme for synthesis of PGE(2) from arachidonic acid, and COX-2 expression is elevated in AMs post-BMT. We hypothesized that epigenetic mechanisms may be responsible for upregulation of COX-2 in AMs. Using bisulfite sequencing, we observed the 5'-untranslated region and exon 1 of the COX-2 gene is hypomethylated in the AMs of BMT mice compared with control. COX-2 expression was increased in primary AMs and in the AM cell line (MHS) after treatment with 5-aza-2'-deoxycytidine (a methyltransferase inhibitor). Methylation by SssI methyltransferase of a 698-bp region of the COX-2 promoter including the beginning of exon 1 driving a luciferase reporter silenced luciferase expression. Because TGF-ß1 is elevated in lungs post-BMT, we tested whether TGF-ß1 could promote expression of COX-2 in a hypermethylated COX-2 vector, and observed TGF-ß1-induced modest expression of COX-2, suggesting an ability to demethylate the promoter. Finally, BMTs performed with marrow from mice expressing a dominant-negative form of the TGF-ßRII on CD11c-expressing cells (which includes AMs) demonstrated improved host defense and AM function. Our findings suggest impaired innate immunity and PGE(2) elevation post-BMT are due to hypomethylation of the COX-2 gene, which is at least partly regulated by TGF-ß1.

Pulmonary fibrosis induced by γ-herpesvirus in aged mice is associated with increased fibroblast responsiveness to transforming growth factor-ß.

Young (4 month) and aged (15-18 months) mice were given intranasal saline or γ--herpesvirus-68 infection. After 21 days, aged, but not young mice, showed significant increases in collagen content and fibrosis. There were no differences in viral clearance or inflammatory cells (including fibrocytes) between infected aged and young mice. Enzyme-linked immunosorbent assays showed increased transforming growth factor-ß in whole lung homogenates of infected aged mice compared with young mice. When fibroblasts from aged and young mice were infected in vitro, aged, but not young, fibroblasts upregulate alpha-smooth muscle actin and collagen I protein. Infection with virus in vivo also demonstrates increased alpha-smooth muscle actin and collagen I protein and collagen I, collagen III, and fibronectin messenger RNA in aged fibroblasts. Furthermore, evaluation revealed that aged fibroblasts at baseline have increased transforming growth factor-ß receptor 1 and 2 levels compared with young fibroblasts and are resistant to apoptosis. Increased responsiveness to transforming growth factor-ß was verified by increased collagen III and fibronectin messenger RNA after treatment in vitro with transforming growth factor-ß.

Severe gammaherpesvirus-induced pneumonitis and fibrosis in syngeneic bone marrow transplant mice is related to effects of transforming growth factor-ß.

Pulmonary infections and pneumonitis occur frequently after hematopoietic stem cell transplantation. Using a syngeneic mouse model of bone marrow transplantation (BMT), we have previously demonstrated that BMT mice are more susceptible to acute gammaherpesvirus 68 (MHV-68) replication at day 7 after infection. By day 21, the virus is latent in lungs of BMT and control mice, and there is no difference in viral load. Despite similar latent viral load, BMT mice develop severe pneumonitis associated with reduced oxygen saturation, fibrosis, peripheral inflammation, hyaline membranes, and foamy alveolar macrophages, a phenotype that persists for 7 weeks after infection. BMT mice demonstrate increased bronchoalveolar lavage (BAL) cells, and this population is enriched in neutrophils and T cells. Alternatively, activated macrophages appear earlier than do classically activated macrophages. BAL fluid from BMT mice at day 21 after infection contains increased levels of hydrogen peroxide, nitrite, and transforming growth factor-ß (TGF-ß). Mice expressing the dominant-negative transgene dn-TGFßRII in multiple cell types were used as BMT donors. BMT mice with T-cell dnTGFßRII are largely protected from the pneumonitis phenotype, whereas mice with CD11c-dnTGFßRII BMT mice are only modestly protected from pneumonitis. Protection in BMT mice with T-cell dnTGFßRII is associated with decreased TGF-ß derived from parenchymal cells in the BAL fluid, lower nitrite levels, and reduced apoptosis, whereas alternatively activated macrophage markers are unchanged.

TLR9-induced interferon ß is associated with protection from gammaherpesvirus-induced exacerbation of lung fibrosis.

BACKGROUND: We have shown previously that murine gammaherpesvirus 68 (γHV68) infection exacerbates established pulmonary fibrosis. Because Toll-like receptor (TLR)-9 may be important in controlling the immune response to γHV68 infection, we examined how TLR-9 signaling effects exacerbation of fibrosis in response to viral infection, using models of bleomycin- and fluorescein isothiocyanate-induced pulmonary fibrosis in wild-type (Balb/c) and TLR-9-/- mice. RESULTS: We found that in the absence of TLR-9 signaling, there was a significant increase in collagen deposition following viral exacerbation of fibrosis. This was not associated with increased viral load in TLR-9-/- mice or with major alterations in T helper (Th)1 and Th2 cytokines. We examined alveolar epithelial-cell apoptosis in both strains, but this could not explain the altered fibrotic outcomes. As expected, TLR-9-/- mice had a defect in the production of interferon (IFN)-ß after viral infection. Balb/c fibroblasts infected with γHV68 in vitro produced more IFN-ß than did infected TLR-9-/- fibroblasts. Accordingly, in vitro infection of Balb/c fibroblasts resulted in reduced proliferation rates whereas infection of TLR-9-/- fibroblasts did not. Finally, therapeutic administration of CpG oligodeoxynucleotides ameliorated bleomycin-induced fibrosis in wild-type mice. CONCLUSIONS: These results show a protective role for TLR-9 signaling in murine models of lung fibrosis, and highlight differences in the biology of TLR-9 between mice and humans.