Julia L. Nugent

Severe airway epithelial injury, aberrant repair and bronchiolitis obliterans develops after diacetyl instillation in rats.

Background Bronchiolitis obliterans (BO) is a fibrotic lung disease that occurs in a variety of clinical settings, including toxin exposures, autoimmunity and lung or bone marrow transplant. Despite its increasing clinical importance, little is known regarding the underlying disease mechanisms due to a lack of adequate small animal BO models. Recent epidemiological studies have implicated exposure to diacetyl (DA), a volatile component of artificial butter flavoring, as a cause of BO in otherwise healthy factory workers. Our overall hypothesis is that DA induces severe epithelial injury and aberrant repair that leads to the development of BO. Therefore, the objectives of this study were 1) to determine if DA, delivered by intratracheal instillation (ITI), would lead to the development of BO in rats and 2) to characterize epithelial regeneration and matrix repair after ITI of DA. Methods and Main Results Male Sprague-Dawley rats were treated with a single dose of DA (125 mg/kg) or sterile water (vehicle control) by ITI. Instilled DA resulted in airway specific injury, followed by rapid epithelial regeneration, and extensive intraluminal airway fibrosis characteristic of BO. Increased airway resistance and lung fluid neutrophilia occurred with the development of BO, similar to human disease. Despite rapid epithelial regeneration after DA treatment, expression of the normal phenotypic markers, Clara cell secretory protein and acetylated tubulin, were diminished. In contrast, expression of the matrix component Tenascin C was significantly increased, particularly evident within the BO lesions. Conclusions We have established that ITI of DA results in BO, creating a novel chemical-induced animal model that replicates histological, biological and physiological features of the human disease. Furthermore, we demonstrate that dysregulated epithelial repair and excessive matrix Tenacin C deposition occur in BO, providing new insights into potential disease mechanisms and therapeutic targets.

Protective Role of T-bet and Th1 Cytokines in Pulmonary Graft-versus-Host Disease and Peribronchiolar Fibrosis

T-box expressed in T cells (T-bet) is a critical transcription factor for T helper (Th) 1 responses. Although Th1 cells are thought to contribute to certain alloimmune responses, their role in pulmonary graft-versus-host disease (GVHD) is uncertain. We have established a murine model of acute pulmonary GVHD after hematopoietic cell transplant (HCT) and inhaled LPS exposure. We tested the hypothesis that pulmonary GVHD can occur independent of Th1 cells using T-bet-deficient donors. B10.BR(H2(k)) mice underwent allogeneic (Allo) or syngeneic (Syn) HCT with cells from either C57Bl/6J(H2(b)) mice (Allo wild-type [WT] or SynWT) or C57Bl/6J mice lacking T-bet (AlloTbet(-/-) or SynTbet(-/-)). After HCT, mice were exposed daily to aerosolized LPS and subsequently bronchoalveolar lavage and lung tissue were analyzed for cytokines, lymphocytic inflammation, pathology, and fibrosis. Independent of LPS exposure, AlloTbet(-/-) mice developed pulmonary GVHD manifested by lymphocytic inflammation. Furthermore, AlloTbet(-/-) mice developed features of chronic pulmonary GVHD, including increased peribronchiolar fibrosis and collagen content. LPS exposure increased neutrophil recruitment and decreased static compliance in AlloTbet(-/-) mice as compared with LPS-exposed AlloWT mice or LPS-exposed SynTbet(-/-) mice. In addition, LPS-exposed AlloTbet(-/-) mice had increased pulmonary IL-17, IL-13, and Th17 cells, and diminished regulatory T cells compared with the other groups. Our results demonstrate that Th1 cytokines are dispensable in pulmonary GVHD. In the absence of T-bet, there is increased production of Th17 and Th2 cytokines that is associated with peribronchiolar fibrosis and is further enhanced by LPS. These results suggest that the interplay between local innate immunity and non-Th1 T cell subsets contribute to chronic pulmonary GVHD.

Diacetyl induces amphiregulin shedding in pulmonary epithelial cells and in experimental bronchiolitis obliterans.

Diacetyl (DA), a component of artificial butter flavoring, has been linked to the development of bronchiolitis obliterans (BO), a disease of airway epithelial injury and airway fibrosis. The epidermal growth factor receptor ligand, amphiregulin (AREG), has been implicated in other types of epithelial injury and lung fibrosis. We investigated the effects of DA directly on the pulmonary epithelium, and we hypothesized that DA exposure would result in epithelial cell shedding of AREG. Consistent with this hypothesis, we demonstrate that DA increases AREG by the pulmonary epithelial cell line NCI-H292 and by multiple independent primary human airway epithelial donors grown under physiologically relevant conditions at the air-liquid interface. Furthermore, we demonstrate that AREG shedding occurs through a TNF-α-converting enzyme (TACE)-dependent mechanism via inhibition of TACE activity in epithelial cells using the small molecule inhibitor, TNF-α protease inhibitor-1, as well as TACE-specific small inhibitor RNA. Finally, we demonstrate supportive in vivo results showing increased AREG transcript and protein levels in the lungs of rodents with DA-induced BO. In summary, our novel in vitro and in vivo observations suggest that further study of AREG is warranted in the pathogenesis of DA-induced BO.

Surfactant protein-A suppresses eosinophil-mediated killing of Mycoplasma pneumoniae in allergic lungs.

Surfactant protein-A (SP-A) has well-established functions in reducing bacterial and viral infections but its role in chronic lung diseases such as asthma is unclear. Mycoplasma pneumoniae (Mp) frequently colonizes the airways of chronic asthmatics and is thought to contribute to exacerbations of asthma. Our lab has previously reported that during Mp infection of non-allergic airways, SP-A aides in maintaining airway homeostasis by inhibiting an overzealous TNF-alpha mediated response and, in allergic mice, SP-A regulates eosinophilic infiltration and inflammation of the airway. In the current study, we used an in vivo model with wild type (WT) and SP-A−/− allergic mice challenged with the model antigen ovalbumin (Ova) that were concurrently infected with Mp (Ova+Mp) to test the hypothesis that SP-A ameliorates Mp-induced stimulation of eosinophils. Thus, SP-A could protect allergic airways from injury due to release of eosinophil inflammatory products. SP-A deficient mice exhibit significant increases in inflammatory cells, mucus production and lung damage during concurrent allergic airway disease and infection (Ova+Mp) as compared to the WT mice of the same treatment group. In contrast, SP-A deficient mice have significantly decreased Mp burden compared to WT mice. The eosinophil specific factor, eosinophil peroxidase (EPO), which has been implicated in pathogen killing and also in epithelial dysfunction due to oxidative damage of resident lung proteins, is enhanced in samples from allergic/infected SP-A−/− mice as compared to WT mice. In vitro experiments using purified eosinophils and human SP-A suggest that SP-A limits the release of EPO from Mp-stimulated eosinophils thereby reducing their killing capacity. These findings are the first to demonstrate that although SP-A interferes with eosinophil-mediated biologic clearance of Mp by mediating the interaction of Mp with eosinophils, SP-A simultaneously benefits the airway by limiting inflammation and damage.

Innate immune activation by the viral PAMP poly I:C potentiates pulmonary graft-versus-host disease after allogeneic hematopoietic cell transplant

Respiratory viral infections cause significant morbidity and increase the risk for chronic pulmonary graft-versus-host disease (GVHD) after hematopoietic cell transplantation (HCT). Our overall hypothesis is that local innate immune activation potentiates adaptive alloimmunity. In this study, we hypothesized that a viral pathogen-associated molecular pattern (PAMP) alone can potentiate pulmonary GVHD after allogeneic HCT. We, therefore, examined the effect of pulmonary exposure to polyinosinic:polycytidylic acid (poly I:C), a viral mimetic that activates innate immunity, in an established murine HCT model. Poly I:C-induced a marked pulmonary T cell response in allogeneic HCT mice as compared to syngeneic HCT, with increased CD4+ cells in the lung fluid and tissue. This lymphocytic inflammation persisted at 2 weeks post poly I:C exposure in allogeneic mice and was associated with CD3+ cell infiltration into the bronchiolar epithelium and features of epithelial injury. In vitro, poly I:C enhanced allospecific proliferation in a mixed lymphocyte reaction. In vivo, poly I:C exposure was associated with an early increase in pulmonary monocyte recruitment and activation as well as a decrease in CD4+FOXP3+ regulatory T cells in allogeneic mice as compared to syngeneic. In contrast, intrapulmonary poly I:C did not alter the extent of systemic GVHD in either syngeneic or allogeneic mice. Collectively, our results suggest that local activation of pulmonary innate immunity by a viral molecular pattern represents a novel pathway that contributes to pulmonary GVHD after allogeneic HCT, through a mechanism that includes increased recruitment and maturation of intrapulmonary monocytes.

Novel role for surfactant protein A in gastrointestinal graft-versus-host disease.

Graft-versus-host disease (GVHD) is a severe and frequent complication of allogeneic bone marrow transplantation (BMT) that involves the gastrointestinal (GI) tract and lungs. The pathobiology of GVHD is complex and involves immune cell recognition of host Ags as foreign. We hypothesize a central role for the collectin surfactant protein A (SP-A) in regulating the development of GVHD after allogeneic BMT. C57BL/6 (H2b; WT) and SP-A–deficient mice on a C57BL/6 background (H2b; SP-A−/−) mice underwent allogeneic or syngeneic BMT with cells from either C3HeB/FeJ (H2k; SP-A–deficient recipient mice that have undergone an allogeneic BMT [SP-A−/−alloBMT] or SP-A–sufficient recipient mice that have undergone an allogeneic BMT) or C57BL/6 (H2b; SP-A–deficient recipient mice that have undergone a syngeneic BMT or SP-A–sufficient recipient mice that have undergone a syngeneic BMT) mice. Five weeks post-BMT, mice were necropsied, and lung and GI tissue were analyzed. SP-A−/− alloBMT or SP-A–sufficient recipient mice that have undergone an allogeneic BMT had no significant differences in lung pathology; however, SP-A−/−alloBMT mice developed marked features of GI GVHD, including decreased body weight, increased tissue inflammation, and lymphocytic infiltration. SP-A−/−alloBMT mice also had increased colon expression of IL-1β, IL-6, TNF-α, and IFN-γ and as well as increased Th17 cells and diminished regulatory T cells. Our results demonstrate the first evidence, to our knowledge, of a critical role for SP-A in modulating GI GVHD. In these studies, we demonstrate that mice deficient in SP-A that have undergone an allogeneic BMT have a greater incidence of GI GVHD that is associated with increased Th17 cells and decreased regulatory T cells. The results of these studies demonstrate that SP-A protects against the development of GI GVHD and establishes a role for SP-A in regulating the immune response in the GI tract.

Impaired CD8+ T cell immunity after allogeneic bone marrow transplantation leads to persistent and severe respiratory viral infection☆

RATIONALE Bone marrow transplant (BMT) recipients experience frequent and severe respiratory viral infections (RVIs). However, the immunological mechanisms predisposing to RVIs are uncertain. Therefore, we hypothesized that antiviral T cell immunity is impaired as a consequence of allogeneic BMT, independent of pharmacologic immunosuppression, and is responsible for increased susceptibility to RVI. METHODS Bone marrow and splenocytes from C57BL/6(H2(b)) mice were transplanted into B10.BR(H2(k)) (Allo) or C57BL/6(H2(b)) (Syn) recipients. Five weeks after transplantation, recipient mice were inoculated intranasally with mouse parainfluenza virus type 1 (mPIV-1), commonly known as Sendai virus (SeV), and monitored for relevant immunological and disease endpoints. MAIN RESULTS Severe and persistent airway inflammation, epithelial injury, and enhanced mortality are found after viral infection in Allo mice but not in control Syn and non-transplanted mice. In addition, viral clearance is delayed in Allo mice as evidenced by prolonged detection of viral transcripts at Day 15 post-inoculation (p.i.) but not in control mice. In concert with these events, we also detected decreased levels of total and virus-specific CD8(+) T cells, as well as increased T cellexpression of inhibitory receptor programmed death-1 (PD-1), in the lungs of Allo mice at Day 8 p.i. Adoptive transfer of CD8(+) T cells from non-transplanted mice recovered from SeV infection into Allo mice at Day 8 p.i. restored normal levels of viral clearance, epithelial repair, and lung inflammation. CONCLUSIONS Taken together these results indicate that allogeneic BMT results in more severe RVI based on the failure to develop an appropriate pulmonary CD8(+) T cell response, providing an important potential mechanism to target in improving outcomes of RVI after BMT.

Role of C-C Motif Ligand 2 and C-C Motif Receptor 2 in Murine Pulmonary Graft-versus-Host Disease after Lipopolysaccharide Inhalations

Environmental exposures are a potential trigger of chronic pulmonary graft-versus-host disease (pGVHD) after successful recovery from hematopoietic cell transplant (HCT). We hypothesized that inhalations of LPS, a prototypic environmental stimulus, trigger pGVHD via increased pulmonary recruitment of donor-derived antigen-presenting cells (APCs) through the C-C motif ligand 2 (CCL2)-C-C motif receptor 2 (CCR2) chemokine axis. B10.BR(H2(k)) and C57BL/6(H2(b)) mice underwent allogeneic (Allo) or syngeneic (Syn) HCT with wild-type (WT) C57BL/6, CCL2(-/-), or CCR2(-/-) donors. After 4 weeks, recipient mice received daily inhaled LPS for 5 days and were killed at multiple time points. Allo mice exposed to repeated inhaled LPS developed prominent lymphocytic bronchiolitis, similar to human pGVHD. The increase in pulmonary T cells in Allo mice after LPS exposures was accompanied by increased CCL2, CCR2, and Type-1 T-helper cytokines as well as by monocytes and monocyte-derived dendritic cells (moDCs) compared with Syn and nontransplanted controls. Using CCL2(-/-) donors leads to a significant decrease in lung DCs but to only mildly reduced CD4 T cells. Using CCR2(-/-) donors significantly reduces lung DCs and moDCs but does not change T cells. CCL2 or CCR2 deficiency does not alter pGVHD pathology but increases airway hyperreactivity and IL-5 or IL-13 cytokines. Our results show that hematopoietic donor-derived CCL2 and CCR2 regulate recruitment of APCs to the Allo lung after LPS exposure. Although they do not alter pathologic pGVHD, their absence is associated with increased airway hyperreactivity and IL-5 and IL-13 cytokines. These results suggest that the APC changes that result from CCL2-CCR2 blockade may have unexpected effects on T cell differentiation and physiologic outcomes in HCT.