Division of Pulmonary and Critical Care

Permanent URI for this collectionhttps://digital.lib.washington.edu/handle/1773/15683

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Immunomodulation of the Innate Host Response by Mesenchymal-Derived Versican during Influenza A Virus Infection
(2025-07-31) Brune, Jourdan E.; Chang, Mary Y.; Tang, Fengying; Lopez-Martinez, Cecilia; Reeves, Stephen R.; Chan, Christina K.; Waldron, Peter; Boyd, David F.; Gharib, Sina A.; Thomas, Paul G.; Altemeier, William A.; Frevert , Charles W.
Viral and bacterial lung infections place a significant burden on public health. Versican, an extracellular matrix (ECM) chondroitin sulfate proteoglycan, coordinates the innate immune response in multiple experimental models. Versican’s potential as an immunomodulatory molecule makes it a promising therapeutic target for controlling the host’s immune response to lung infection. However, versican’s contribution to lung inflammation, injury, and immune cell activity during influenza A virus (IAV) infection represents a critical knowledge gap. To address our central hypothesis that mesenchymal-derived versican is pro-inflammatory and enhances the innate immune response to IAV infection, we generated a tamoxifen-inducible mouse deficient in mesenchymal-derived versican (B6. Col1a2-CreERT+/-/Vcantm1.1Cwf, Col1a2/Vcan-/-). We report that mesenchymal-derived versican plays a critical role in neutrophil, monocyte, and dendritic cell migration into the lungs and airways early in IAV infection. Intriguingly, mesenchymal-derived versican deficiency had the most substantial negative impact on neutrophil emigration into the lungs. We found that neutrophils were less adhesive to the ECM of Col1a2/Vcan-/- mouse lung fibroblasts (mLFs), which had a significant decrease in versican compared to wild-type mLFs. Additionally, Col1a2/Vcan-/- mLFs treated with poly(I:C) in vitro have reduced cell-associated hyaluronan. These findings suggest that fibroblast-derived versican is necessary for adhesion to lung fibroblasts by neutrophils as they transit into the lung interstitium and airways from the pulmonary vasculature. Our findings demonstrate that mesenchymal-derived versican is a key regulator of the early host immune responses to IAV.
Regulation of Versican Expression in Macrophages is Mediated by Canonical Type I Interferon Signaling via ISGF3
(2024) Chang, Mary; Chan, Christina; Brune, Jourdan; Manicone, Anne; Bomsztyk, Karol; Frevert, Charles; Altemeier, William
Growing evidence supports a role for versican as an important component of the inflammatory response, with both pro- and anti-inflammatory roles depending on the specific context of the system or disease under investigation. Our goal is to understand the regulation of macrophage-derived versican and the role it plays in innate immunity. In previous work, we showed that LPS triggers a signaling cascade involving TLR4, the Trif adaptor, type I interferons, and the type I interferon receptor, leading to increased versican expression by macrophages. In the present study, we used a combination of chromatin immunoprecipitation, siRNA, chemical inhibitors, and mouse model approaches to investigate the regulatory events downstream of the type I interferon receptor to better define the mechanism controlling versican expression. Results indicate that transcriptional regulation by canonical type I interferon signaling via the heterotrimeric transcription factor, ISGF3, controls versican expression in macrophages exposed to LPS. This pathway is not dependent on MAPK signaling, which has been shown to regulate versican expression in other cell types. The stability of versican mRNA may also contribute to prolonged versican expression in macrophages. These findings strongly support a role for macrophage-derived versican as a type I interferon-stimulated gene and further our understanding of versican’s role in regulating inflammation.
Supplemental Materials - Multi-Compartmental Analysis of the Murine Pulmonary Immune Response by Spectral Flow Cytometry
(2023) Chang, Mary; Brune, Jourdan; Altemeier, William; Black, Michele; Frevert, Charles
Studies of pulmonary inflammation require unique considerations due to the complex structure and composition of the lungs. The lungs have multiple compartments, and diverse immune cell populations, with inherently high autofluorescence, and are involved in the host response to pulmonary pathogens and environmental factors. Here we describe a protocol that accounts for these factors through a novel combination of methodologies – in vivo compartmental analysis and spectral flow cytometry allowing for a broad panel of antibodies and the ability to minimize autofluorescence in immune cells. In vivo compartmental analysis enables the precise localization of immune cells within the marginated vasculature, the lung interstitium, the non-lavageable airways, and the lavageable airways of the lungs, as well as within the pulmonary lymph nodes. Spectral flow cytometry maximizes the information that can be obtained regarding the diverse leukocyte subpopulations involved in the pulmonary response. A broad panel of antibodies supports an unbiased exploratory approach to investigating diverse immune cell populations during pulmonary inflammation. Most importantly, spectral flow utilizes cellular autofluorescence to aid in the resolution and identification of immune cell populations. This methodology enables the acquisition of high-quality data compatible with informed gating and dimensionality reduction algorithms. Additionally, our protocol emphasizes considerations for compartmentalization of the inflammatory response, spectral flow panel design, and autofluorescence spectra analysis. The methodologies employed by this protocol, including an unbiased approach, are critical for increasing the rigor of pulmonary research. We apply this protocol for the precise characterization and localization of immune cells within the lungs of C57BL6/J mice during the transition from the innate to an adaptive immune response to the influenza A virus. We demonstrate that implementing this protocol improves the quantification and localization of alveolar macrophages within the airways. The methodology is modifiable and expandable to allow for further characterization of immune cell populations of particular interest. We also present considerations for applying this methodology to studies in other mouse models and with other agonists, including lipopolysaccharide, bleomycin, and house dust mite allergens.
Multi-Compartmental Analysis of the Murine Pulmonary Immune Response by Spectral Flow Cytometry-Supplemental Materials
(2023) Chang, Mary; Brune, Jourdan; William, Altemeier; Black, Michele; Frevert, Charles
Studies of pulmonary inflammation require unique considerations due to the complex structure and composition of the lungs. The lungs have multiple compartments, and diverse immune cell populations, with inherently high autofluorescence, and are involved in the host response to pulmonary pathogens and environmental factors. Here we describe a protocol that accounts for these factors through a novel combination of methodologies – in vivo compartmental analysis and spectral flow cytometry allowing for a broad panel of antibodies and the ability to minimize autofluorescence in immune cells. In vivo compartmental analysis enables the precise localization of immune cells within the marginated vasculature, the lung interstitium, the non-lavageable airways, and the lavageable airways of the lungs, as well as within the pulmonary lymph nodes. Spectral flow cytometry maximizes the information that can be obtained regarding the diverse leukocyte subpopulations involved in the pulmonary response. A broad panel of antibodies supports an unbiased exploratory approach to investigating diverse immune cell populations during pulmonary inflammation. Most importantly, spectral flow utilizes cellular autofluorescence to aid in the resolution and identification of immune cell populations. This methodology enables the acquisition of high-quality data compatible with informed gating and dimensionality reduction algorithms. Additionally, our protocol emphasizes considerations for compartmentalization of the inflammatory response, spectral flow panel design, and autofluorescence spectra analysis. The methodologies employed by this protocol, including an unbiased approach, are critical for increasing the rigor of pulmonary research. We apply this protocol for the precise characterization and localization of immune cells within the lungs of C57BL6/J mice during the transition from the innate to an adaptive immune response to the influenza A virus. We demonstrate that implementing this protocol improves the quantification and localization of alveolar macrophages within the airways. The methodology is modifiable and expandable to allow for further characterization of immune cell populations of particular interest. We also present considerations for applying this methodology to studies in other mouse models and with other agonists, including lipopolysaccharide, bleomycin, and house dust mite allergens.
Activation of Toll-like receptors by Burkholderia pseudomallei
(2008) West, T. Eoin; Ernst, Robert K.; Jansson-Hutson, Malinka J.; Skerrett, Shawn J.
Background: Melioidosis, a lethal tropical infection that is endemic in southeast Asia and northern Australia, is caused by the saprophytic Gram-negative bacterium Burkholderia pseudomallei. Overall mortality approaches 40% yet little is known about mechanisms of host defense. Toll-like receptors (TLRs) are host transmembrane receptors that recognize conserved pathogen molecular patterns and induce an inflammatory response. The lipopolysaccharide (LPS) of Gram-negative bacteria is a potent inducer of the host innate immune system. TLR4, in association with MD-2, is the archetype receptor for LPS although B. pseudomallei LPS has been previously identified as a TLR2 agonist. We examined TLR signaling induced by B. pseudomallei, B. pseudomallei LPS, and B. pseudomallei lipid A using gain-of-function transfection assays of NF-?B activation and studies of TLR-deficient macrophages. Results: In HEK293 cells transfected with murine or human TLRs, CD14, and MD-2, heat-killed B. pseudomallei activated TLR2 (in combination with TLR1 or TLR6) and TLR4. B. pseudomallei LPS and lipid A activated TLR4 and this TLR4-mediated signaling required MD-2. In TLR2-/- macrophages, stimulation with heat-killed B. pseudomallei augmented TNF-a and MIP-2 production whereas in TLR4-/- cells, TNF-a, MIP-2, and IL-10 production was reduced. Cytokine production by macrophages stimulated with B. pseudomallei LPS or lipid A was entirely dependent on TLR4 but was increased in the absence of TLR2. TLR adaptor molecule MyD88 strongly regulated TNF-a production in response to heat-killed B. pseudomallei. Conclusion: B. pseudomallei activates TLR2 and TLR4. In the presence of MD-2, B. pseudomallei LPS and lipid A are TLR4 ligands. Although the macrophage cytokine response to B. pseudomallei LPS or lipid A is completely dependent on TLR4, in TLR2-/- macrophages stimulated with B. pseudomallei, B. pseudomallei LPS or lipid A, cytokine production is augmented. Other MyD88-dependent signaling pathways may also be important in the host response to B. pseudomallei infection. These findings provide new insights into critical mechanisms of host defense in melioidosis.

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