Christopher Bauer

αβ T Cells and a Mixed Th1/Th17 Response are Important in Organic Dust-Induced Airway Disease

BACKGROUND Organic dust exposure in agricultural environments induces an inflammatory response that attenuates over time, yet repetitive dust exposures result in chronic lung diseases. Animal models resembling this chronic lung inflammatory response have been developed, yet the underlying cellular mechanisms are not well defined. OBJECTIVE Because mice repetitively exposed to organic dust extracts (DE) display increased CD3+ T cell lung infiltrates, we sought to determine the phenotype and importance of these cells. METHODS Mice received swine confinement DE repetitively for 3 weeks by established intranasal inhalation protocol. Studies were conducted with peptidoglycan (PGN) because it is a major DE component in large animal farming environments and has shared similar biologic effects with DE. Enumeration of T cells and intracellular cytokine profiles were conducted by flow cytometry techniques. Whole lung homogenate cytokines were analyzed by multiplex immunoassay. T cell receptor (TCR) αβ knockouts were used to determine the functional importance of αβ-expressing T cells. RESULTS DE increased lung-associated CD3+CD4+ T cells and interleukin (IL)-17 (but not IL-4, interferon [IFN]-γ, IL-10) producing CD4+ T cells. PGN treatment resulted in increased IL-17 and IFN-γ producing CD4+ T cells and IFN-γ producing CD8+ T cells. Both DE and PGN augmented expression of cytokines associated with Th1 and Th17 polarization in lung homogenates. DE-induced lung mononuclear aggregates and bronchiolar compartment inflammation were significantly reduced in TCR knockout animals; however, neutrophil influx and alveolar compartment inflammation were not affected. CONCLUSION Studies demonstrated that DE and PGN exposure promote a Th1/Th17 lung microenvironment and that αβ-expressing T cells are important in mediating DE-induced lung pathologic conditions.

CD11c+/CD11b+ Cells Are Critical for Organic Dust–Elicited Murine Lung Inflammation

Organic dust exposure in the agricultural industry results in significant lung disease. Macrophage infiltrates are increased in the lungs after organic dust exposures, yet the phenotype and functional importance of these cells remain unclear. Using an established intranasal inhalation murine model of dust-induced lung inflammation, animals were treated once or daily for 3 weeks with swine confinement organic dust extract (DE). Repetitive DE treatment for 3 weeks resulted in significant increases in CD11c(+)/CD11b(+) macrophages in whole lung-associated tissue. These cells displayed increased costimulatory molecule (CD80 and CD86) expression, enhanced phagocytic ability, and an increased production of IL-6, CXCL1, and CXCL2. Similar findings were observed with the CD11c(+)/CD11b(+) macrophage infiltrate after repetitive exposure to peptidoglycan, a major DE component. To determine the functional importance of macrophages in mediating DE-induced airway inflammation, lung macrophages were selectively depleted using a well-established intranasal clodronate liposome depletion/suicide strategy. First, macrophage depletion by clodronate liposomes resulted in significant reductions in airway neutrophil influx and TNF-α and IL-6 production after a single exposure to DE. In contrast, after repetitive 3-week exposure to DE, airway lavage fluid and lung tissue neutrophils were significantly increased in clodronate liposome-treated mice compared with control mice. A histological examination of lung tissue demonstrated striking increases in alveolar and bronchiolar inflammation, as well as in the size and distribution of cellular aggregates in clodronate-liposome versus saline-liposome groups repetitively exposed to DE. These studies demonstrate that DE elicits activated CD11c(+)/CD11b(+) macrophages in the lung, which play a critical role in regulating the outcome of DE-induced airway inflammation.

Myeloid Differentiation Factor 88–Dependent Signaling Is Critical for Acute Organic Dust–Induced Airway Inflammation in Mice

Organic dust exposure within agricultural environments results in airway diseases. Toll-like receptor 2 (TLR2) and TLR4 only partly account for the innate response to these complex dust exposures. To determine the central pathway in mediating complex organic dust-induced airway inflammation, this study targeted the common adaptor protein, myeloid differentiation factor 88 (MyD88), and investigated the relative contributions of receptors upstream from this adaptor. Wild-type, MyD88, TLR9, TLR4, IL-1 receptor I (RI), and IL-18R knockout (KO) mice were challenged intranasally with organic dust extract (ODE) or saline, according to an established protocol. Airway hyperresponsiveness (AHR) was assessed by invasive pulmonary measurements. Bronchoalveolar lavage fluid was collected to quantitate leukocyte influx and cytokine/chemokine (TNF-α, IL-6, chemokine [C-X-C motif] ligands [CXCL1 and CXCL2]) concentrations. Lung tissue was collected for histopathology. Lung cell apoptosis was determined by a terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, and lymphocyte influx and intercellular adhesion molecule-1 (ICAM-1) expression were assessed by immunohistochemistry. ODE-induced AHR was significantly attenuated in MyD88 KO mice, and neutrophil influx and cytokine/chemokine production were nearly absent in MyD88 KO animals after ODE challenges. Despite a near-absent airspace inflammatory response, lung parenchymal inflammation was increased in MyD88 KO mice after repeated ODE exposures. ODE-induced epithelial-cell ICAM-1 expression was diminished in MyD88 KO mice. No difference was evident in the small degree of ODE-induced lung-cell apoptosis. Mice deficient in TLR9, TLR4, and IL-18R, but not IL-1IR, demonstrated partial protection against ODE-induced neutrophil influx and cytokine/chemokine production. Collectively, the acute organic dust-induced airway inflammatory response is highly dependent on MyD88 signaling, and is dictated, in part, by important contributions from upstream TLRs and IL-18R.

Organic Dust, Lipopolysaccharide, and Peptidoglycan Inhalant Exposures Result in Bone Loss/Disease

Skeletal health consequences associated with chronic inflammatory respiratory disease, and particularly chronic obstructive pulmonary disease (COPD), contribute to overall disease morbidity. Agricultural environmental exposures induce significant airway diseases, including COPD. However, animal models to understand inhalant exposure-induced lung injury and bone disease have not been described. Using micro-computed tomography (micro-CT) imaging technology and histology, bone quantity and quality measurements were investigated in mice after repetitive intranasal inhalation exposures to complex organic dust extracts (ODEs) from swine confinement facilities. Comparison experiments with LPS and peptidoglycan (PGN) alone were also performed. After 3 weeks of repetitive ODE inhalation exposure, significant loss of bone mineral density and trabecular bone volume fraction was evident, with altered morphological microarchitecture changes in the trabecular bone, compared with saline-treated control animals. Torsional resistance was also significantly reduced. Compared with saline treatment, ODE-treated mice demonstrated decreased collagen and proteoglycan content in their articular cartilage, according to histopathology. Significant bone deterioration was also evident after repetitive intranasal inhalant treatment with LPS and PGN. These findings were not secondary to animal distress, and not entirely dependent on the degree of induced lung parenchymal inflammation. Repetitive LPS treatment demonstrated the most pronounced changes in bone parameters, and PGN treatment resulted in the greatest lung parenchymal inflammatory changes. Collectively, repetitive inhalation exposures to noninfectious inflammatory agents such as complex organic dust, LPS, and PGN resulted in bone loss. This animal model may contribute to efforts toward understanding the mechanisms and evaluating the therapeutics associated with adverse skeletal health consequences after subchronic airway injury.

Maresin-1 reduces airway inflammation associated with acute and repetitive exposures to organic dust

Agriculture industry workers are at a higher risk for chronic bronchitis and obstructive pulmonary diseases, and current therapeutics are not entirely effective. We previously found that the specialized proresolving lipid mediator maresin-1 (MaR1) reduced proinflammatory cytokine release and intracellular adhesion molecule-1 (ICAM-1) expression in bronchial epithelial cells exposed to extracts of organic dust (DE) derived from swine confinement facilities in vitro. The objective of this study was to determine whether MaR1 is effective at limiting lung inflammation associated with acute and repetitive exposures to DE in an established murine model of inhalant dust exposures. C57Bl/6 mice were treated with MaR1 or vehicle control and intranasally instilled with DE once or daily for 3 weeks. Bronchioalveolar lavage fluid was analyzed for total and differential cell counts and proinflammatory cytokine levels, and lung tissues were assessed for histopathology and ICAM-1 expression. In both single and repetitive DE exposure studies, MaR1 significantly decreased bronchoalveolar lavage neutrophil infiltration, interleukin 6, tumor necrosis factor α, and chemokine C-X-C motif ligand 1 levels without altering repetitive DE-induced bronchioalveolar inflammation or lymphoid aggregate formation. Lung tissue ICAM-1 expression was also reduced in both single and repetitive exposure studies. These data suggest that MaR1 might contribute to an effective strategy to reduce airway inflammatory diseases induced by agricultural-related organic dust environmental exposures.

Expression of Suppressor of Cytokine Signaling 1 (SOCS1) Impairs Viral Clearance and Exacerbates Lung Injury during Influenza Infection

Suppressor of cytokine signaling (SOCS) proteins are inducible feedback inhibitors of cytokine signaling. SOCS1−/− mice die within three weeks postnatally due to IFN-γ-induced hyperinflammation. Since it is well established that IFN-γ is dispensable for protection against influenza infection, we generated SOCS1−/−IFN-γ−/− mice to determine whether SOCS1 regulates antiviral immunity in vivo. Here we show that SOCS1−/−IFN-γ−/− mice exhibited significantly enhanced resistance to influenza infection, as evidenced by improved viral clearance, attenuated acute lung damage, and consequently increased survival rates compared to either IFN-γ−/− or WT animals. Enhanced viral clearance in SOCS1−/−IFN-γ−/− mice coincided with a rapid onset of adaptive immune responses during acute infection, while their reduced lung injury was associated with decreased inflammatory cell infiltration at the resolution phase of infection. We further determined the contribution of SOCS1-deficient T cells to antiviral immunity. Anti-CD4 antibody treatment of SOCS1−/−IFN-γ−/− mice had no significant effect on their enhanced resistance to influenza infection, while CD8+ splenocytes from SOCS1−/−IFN-γ−/− mice were sufficient to rescue RAG1−/− animals from an otherwise lethal infection. Surprisingly, despite their markedly reduced viral burdens, RAG1−/− mice reconstituted with SOCS1−/−IFN-γ−/− adaptive immune cells failed to ameliorate influenza-induced lung injury. In conclusion, in the absence of IFN-γ, the cytoplasmic protein SOCS1 not only inhibits adaptive antiviral immune responses but also exacerbates inflammatory lung damage. Importantly, these detrimental effects of SOCS1 are conveyed through discrete cell populations. Specifically, while SOCS1 expression in adaptive immune cells is sufficient to inhibit antiviral immunity, SOCS1 in innate/stromal cells is responsible for aggravated lung injury.

Nox2-derived oxidative stress results in inefficacy of antibiotics against post-influenza S. aureus pneumonia

Phagocyte oxidative burst is the primary source of lethal lung injury during influenza and MRSA coinfection.

Vitamin D treatment modulates organic dust-induced cellular and airway inflammatory consequences.

Exposure to organic dusts elicits airway inflammatory diseases. Vitamin D recently has been associated with various airway inflammatory diseases, but its role in agricultural organic dust exposures is unknown. This study investigated whether vitamin D reduces organic dust–induced inflammatory outcomes in cell culture and animal models. Organic dust extracts obtained from swine confinement facilities induced neutrophil chemokine production (human IL‐8, murine CXCL1/CXCL2). Neutrophil chemokine induction was reduced in human blood monocytes, human bronchial epithelial cells, and murine lung slices pretreated with 1,25‐(OH)2D3. Intranasal inhalation of organic dust extract induced neutrophil influx, and CXCL1/CXCL2 release was also decreased in mice fed a relatively high vitamin D diet as compared to mice fed a low vitamin D diet. These findings were associated with reduced tracheal epithelial cell PKCα and PKCε activity and whole lung TLR2 and TLR4 gene expression. Collectively, vitamin D plays a role in modulating organic dust–induced airway inflammatory outcomes.

Resistance to Acute Macrophage Killing Promotes Airway Fitness of Prevalent Community-Acquired Staphylococcus aureus Strains

The incidence of methicillin-resistant Staphylococcus aureus (MRSA) pneumonia in otherwise healthy individuals is increasing. To investigate the mechanism underlying the epidemiological success of predominant community-associated (CA)-MRSA strains, we examined their fitness traits during the initial interaction between bacteria and the host occurring in the lower airway. Using a mouse respiratory infection model, we show that clinical isolates often responsible for CA infections are highly resistant to clearance from healthy airways, whereas S. aureus strains not as prevalent or traditionally associated with hospital-associated infections are relatively susceptible. Mechanistically, the competitive fitness of S. aureus is a result of both agr-dependent and -independent resistance to innate bacterial killing. Furthermore, we show that rather than evasion from neutrophil-dependent bactericidal process, the observed S. aureus fitness in the lower airways is due to its intrinsic resistance to resident alveolar macrophage–mediated intracellular killing. Importantly, we demonstrate that the virulence determinants responsible for bacterial persistence in immune-competent mice are dispensable in mice with predisposing conditions such as influenza infection. Taken together, these novel findings of the improved competence of predominant CA-MRSA strains to survive innate killing in healthy hosts, particularly at the very beginning stage of infection, provide a unique insight into their epidemiological success.

IL-1 signaling prevents alveolar macrophage depletion during influenza and streptococcus pneumoniae coinfection

Influenza and bacterial coinfection is a significant cause of hospitalization and death in humans during influenza epidemics and pandemics. However, the fundamental protective and pathogenic mechanisms involved in this complex virus–host–bacterium interaction remain incompletely understood. In this study, we have developed mild to lethal influenza and Streptococcus pneumoniae coinfection models for comparative analyses of disease pathogenesis. Specifically, wild-type and IL-1R type 1–deficient (Il1r1−/−) mice were infected with influenza virus and then superchallenged with noninvasive S. pneumoniae serotype 14 (Spn14) or S. pneumoniae serotype 19A (Spn19A). The coinfections were followed by comparative analyses of inflammatory responses and animal protection. We found that resident alveolar macrophages are efficient in the clearance of both pneumococcal serotypes in the absence of influenza infection; in contrast, they are essential for airway control of Spn14 infection but not Spn19A infection. In agreement, TNF-α and neutrophils play a compensatory protective role in secondary bacterial infection associated with Spn19A; however, the essential requirement for alveolar macrophage–mediated clearance significantly enhances the virulence of Spn14 during postinfluenza pneumococcal infection. Furthermore, we show that, although IL-1 signaling is not required for host defense against pneumococcal infection alone, it is essential for sustaining antibacterial immunity during postinfluenza pneumococcal infection, as evidenced by significantly aggravated bacterial burden and animal mortality in Il1r1−/− mice. Mechanistically, we show that through preventing alveolar macrophage depletion, inflammatory cytokine IL-1 signaling is critically involved in host resistance to influenza and pneumococcal coinfection.