Laurie P. Shornick

Cutting Edge: B and T Lymphocyte Attenuator and Programmed Death Receptor-1 Inhibitory Receptors Are Required for Termination of Acute Allergic Airway Inflammation

T cell activation is regulated by coordinate interaction of the T cell Ag receptor and costimulatory signals. Although there is considerable insight into processes that regulate the initiation of inflammation, less is known about the signals that terminate immune responses. We have examined the role of the inhibitory receptors programmed death receptor-1 and B and T lymphocyte attenuator in the regulation of allergic airway inflammation. Our results demonstrate that there is a temporally regulated expression of both the receptors and their ligands during the course of allergic airway inflammation. Following a single inhaled challenge, sensitized wild-type mice exhibit peak inflammation on day 3, which resolves by day 10. In contrast, mice deficient in the expression of programmed death receptor-1 or B and T lymphocyte attenuator have persistent inflammation out to 15 days following challenge. Thus, these receptors are critical determinants of the duration of allergic airway inflammation.

Airway Epithelial versus Immune Cell Stat1 Function for Innate Defense against Respiratory Viral Infection

The epithelial surface is often proposed to actively participate in host defense, but evidence that this is the case remains circumstantial. Similarly, respiratory paramyxoviral infections are a leading cause of serious respiratory disease, but the basis for host defense against severe illness is uncertain. Here we use a common mouse paramyxovirus (Sendai virus) to show that a prominent early event in respiratory paramyxoviral infection is activation of the IFN-signaling protein Stat1 in airway epithelial cells. Furthermore, Stat1−/− mice developed illness that resembled severe paramyxoviral respiratory infection in humans and was characterized by increased viral replication and neutrophilic inflammation in concert with overproduction of TNF-α and neutrophil chemokine CXCL2. Poor control of viral replication as well as TNF-α and CXCL2 overproduction were both mimicked by infection of Stat1−/− airway epithelial cells in culture. TNF-α drives the CXCL2 response, because it can be reversed by TNF-α blockade in vitro and in vivo. These findings pointed to an epithelial defect in Stat1−/− mice. Indeed, we next demonstrated that Stat1−/− mice that were reconstituted with wild-type bone marrow were still susceptible to infection with Sendai virus, whereas wild-type mice that received Stat1−/− bone marrow retained resistance to infection. The susceptible epithelial Stat1−/− chimeric mice also exhibited increased viral replication as well as excessive neutrophils, CXCL2, and TNF-α in the airspace. These findings provide some of the most definitive evidence to date for the critical role of barrier epithelial cells in innate immunity to common pathogens, particularly in controlling viral replication.

Chlorinated lipid species in activated human neutrophils: Lipid metabolites of 2-chlorohexadecanal

Neutrophils are important in the host response against invading pathogens. One chemical defense mechanism employed by neutrophils involves the production of myeloperoxidase (MPO)-derived HOCl. 2-Chlorohexadecanal (2-ClHDA) is a naturally occurring lipid product of HOCl targeting the vinyl ether bond of plasmalogens. Previous studies have shown that exogenously-added 2-ClHDA is oxidized to 2-chlorohexadecanoic acid (2-ClHA) and reduced to 2-chlorohexadecanol (2-ClHOH) by endothelial cells. These studies show that both 2-ClHA and 2-ClHOH are produced in activated neutrophils in an MPO- and time-dependent manner and are released by neutrophils into media. 2-ClHDA levels peak following 30 min of phorbol 12-myristate-13-acetate stimulation. In contrast, 2-ClHA and 2-ClHOH levels steadily increased over 60 min, suggesting a precursor-product relationship between 2-ClHDA and both 2-ClHA and 2-ClHOH. Additional experiments using wild-type CHO.K1 and CHO.K1 cells deficient in fatty aldehyde dehydrogenase (FALDH), FAA.K1A, demonstrated that 2-ClHDA oxidation to 2-ClHA is dependent on FALDH activity. Furthermore, mice exposed to intranasal Sendai virus displayed lung neutrophil recruitment, as well as elevated 2-ClHA levels in plasma and bronchoalveolar lavage compared with control-treated mice. Taken together, these data demonstrate, for the first time, that metabolites of 2-ClHDA are produced both in vivo as well as in isolated human neutrophils.

Nonhematopoietic Expression of Janus Kinase 3 Is Required for Efficient Recruitment of Th2 Lymphocytes and Eosinophils in OVA-Induced Airway Inflammation

Tyrosine kinases of the Janus kinase (Jak) family transduce signals from the type I and type II cytokine receptors. Jak3 is unique in this family because its expression must be induced and is predominantly limited to cells of the lymphoid and myeloid lineages. Deficient expression of Jak3 interferes with normal development and function of T, B, and NK cells. Using irradiated Jak3-deficient (Jak3−/−) mice reconstituted with normal bone marrow (Jak3−/− chimeric mice), we have investigated possible actions of Jak3 outside of the hematopoietic system. We show that efficient recruitment of inflammatory cells to the airways of OVA-sensitized mice challenged with aerosolized OVA requires the expression of Jak3 in radioresistant nonhematopoietic cells. Failure to develop eosinophil-predominant airway inflammation in Jak3−/− chimeric mice is not due to failure of T cell sensitization, because Jak3−/− chimeric mice showed delayed-type hypersensitivity responses indistinguishable from wild-type chimeric mice. Jak3−/− chimeric mice, however, express less endothelial-associated VCAM-1 after airway Ag challenge. Given the key role of VCAM-1 in recruitment of Th2 cells and eosinophils, our data suggest that Jak3 in airway-associated endothelial cells is required for the expression of eosinophilic airway inflammation. This requirement for nonhematopoietic expression of Jak3 represents the first demonstration of a physiological function of Jak3 outside of the lymphoid lineages.

Hit-and-run effects of paramyxoviruses as a basis for chronic respiratory disease

Background: The traditional scheme for asthma pathogenesis depends on increased T helper type 2 (Th2) over T helper type 1 (Th1) responses to allergic and nonallergic stimuli and consequent airway inflammation, hyperreactivity and hypersecretion. Here we question whether the innate immune system, including airway epithelial cells, and the adaptive one may manifest an aberrant antiviral response as an additional basis for chronic inflammatory diseases, including asthma. Methods: We focused on the signal transduction and genetic basis for mucosal immunity, inflammation and remodeling, especially in relation to airway diseases. We concentrated on the response to paramyxoviruses because these agents are closely associated with common acute and chronic airway diseases. We used viral, cellular and mouse models, as well as human subjects, for study and made comparisons among these systems. Our approach aims to answer 2 major questions: (1) what are the factors that control acute paramyxoviral infection; and (2) how can these transient infections cause long term airway disease? Conclusions: Our studies show that antiviral defense depends on a special network of epithelial immune response genes that signal to the adaptive immune system. Viruses ordinarily trigger this network, but it is also permanently activated in asthma, even in the absence of viral infection. In addition, we find that, in susceptible genetic backgrounds, respiratory viruses cause a “hit-and-run” phenomenon indicated by the development of an asthmatic phenotype long after the infection has cleared. On the basis of this information, we developed a new scheme for asthma pathogenesis that includes epithelial, viral and allergic components and allows viral reprogramming of host behavior.

Maternal inflammation modulates infant immune response patterns to viral lung challenge in a murine model

Background:Chorioamnionitis, an inflammatory gestational disorder, commonly precedes preterm delivery. Preterm infants may be at particular risk for inflammation-related morbidity related to infection, although the pathogenic mechanisms are unclear. We hypothesized that maternal inflammation modulates immune programming to drive postnatal inflammatory processes.Methods:We used a novel combined murine model to treat late gestation dams with low-dose lipopolysaccharide (LPS) and to secondarily challenge exposed neonates or weanlings with Sendai virus (SeV) lung infection. Multiple organs were analyzed to characterize age-specific postnatal immune and inflammatory responses.Results:Maternal LPS treatment enhanced innate immune populations in the lungs, livers, and/or spleens of exposed neonates or weanlings. Secondary lung SeV infection variably affected neutrophil, macrophage, and dendritic cell proportions in multiple organs of exposed pups. Neonatal lung infection induced brain interleukin (IL)-4 expression, although this response was muted in LPS-exposed pups. Adaptive immune cells, including lung, lymph node, and thymic lymphocytes and lung CD4 cells expressing FoxP3, interferon (IFN)-γ, or IL-17, were variably prominent in LPS-exposed pups.Conclusion:Maternal inflammation modifies postnatal immunity and augments systemic inflammatory responses to viral lung infection in an age-specific manner. We speculate that inflammatory modulation of the developing immune system contributes to chronic morbidity and mortality in preterm infants.

Reduced inflammation and altered innate response in neonates during paramyxoviral infection

BackgroundHuman infants are frequently hospitalized due to infection with the paramyxovirus respiratory syncytial virus (RSV). However, very little is known about the neonatal response to paramyxoviral infection. Here, a neonatal model of paramyxoviral infection is developed using the mouse pathogen Sendai virus (SeV).ResultsAdult mice infected with SeV developed a predominantly neutrophilic inflammatory cell influx and a concomitant reduction in lung function, as determined by oxygen saturation. In contrast, neonates with SeV had significantly reduced inflammation and normal lung function. Surprisingly, infected neonates had similar viral loads as adult mice. A reduced neutrophil influx in the neonates may be due in part to reduced expression of both CXCL2 and intracellular adhesion molecule-1 (ICAM-1). Expression of IFN-γ and TNF-α increased in a dose-dependent manner in adult lungs, but neonates did not increase expression of either of these cytokines, even at the highest doses. Importantly, the expression of the RIG-I-like receptors (RLRs) was delayed in the neonatal mice, which might have contributed to their reduced inflammation and differential cytokine expression.ConclusionsNeonatal mice developed similar SeV titers and cleared the virus with similar efficiency despite developing a dramatically lower degree of pulmonary inflammation compared to adults. This suggests that inflammation in the lung may not be required to control viral replication. Future studies will be needed to determine any effect the reduced inflammation may have on the development of a protective memory response in neonates.

Comparison of silk fibroin electrospun scaffolds with poloxamer and honey additives for burn wound applications

A primary aim in wound-healing research is to construct an inexpensive, biodegradable dermal regeneration template with heightened moisture retention and permeability properties. The presence of moisture is important for optimal burn wound healing as it creates an environment for re-epithelialization and minimizes the risk of infections. Permeability can be achieved through a process known as electrospinning. This scaffold fabrication technique creates a mat of randomly oriented nanofibers that can readily mimic native extracellular matrix. Novel electrospun silk fibroin scaffolds were fabricated with poloxamer 407 (P407) and Manuka honey additives for a burn wound dermal regeneration template application. Enhanced human dermal fibroblast adhesion and cell infiltration were observed with the inclusion of P407, and scaffolds incorporated with Manuka honey demonstrated increased water uptake and a higher cell density within the scaffolds at the end of a 28-day period. Overall, this study established that both the silk fibroin/P407 and silk fibroin/Manuka honey scaffolds have the potential to be successful dermal regeneration templates, with P407 increasing surface wettability and Manuka honey modulating moisture retention.

Using Electrospun Scaffolds to Promote Macrophage Phenotypic Modulation and Support Wound Healing

Abstract The development of pressure ulcers in spinal cord injury patients is extremely common, often requiring extensive surgical procedures. Macrophages (MACs) play a crucial role in the innate immune system, contributing to wound healing and overall regeneration. MACs have been found to possess the potential to be activated by external factors from their M0 inactive state to an M1 proinflammatory or M2 regenerative state. This study conducted a comprehensive evaluation of MAC phenotype in response to electrospun scaffolds of varying material fiber/pore diameter, fiber stiffness, and +/− inclusion of platelet-rich plasma (PRP). Generally, itwas found that the addition of PRP resulted in decreased pore size, where 5 silk fibroin (SF) had the stiffest fibers. Furthermore, PRP scaffolds demonstrated an increased production of VEGF and chemotaxis. The polycaprolactone (PCL) and SF scaffolds had the largest cell infiltration and proliferation. Overall, it was found that 5% SF had both ideal fiber and pore structure, allowing for cell infiltration further enhanced by the presence of PRP. Additionally, this scaffold led to a reasonable production of VEGF while still allowing fibroblast proliferation to occur. These results suggest that such a scaffold could provide an off-the-shelf product capable of modifying the local MAC response.

Defining and adjusting divergent host responses to viral infection.

Our laboratory focuses on the signal-transduction basis for mucosal immunity, inflammation, and remodeling, especially in relation to respiratory viral infection. Our approach aims to answer two major questions: (1) What are the mechanisms that control common viral infections? and (2) How can these transient infections cause longterm diseases, such as asthma? Our studies show that antiviral defense depends critically on a specialized network of mucosal epithelial cells and macrophages. When this network is compromised, the host is highly susceptible to infection, but when it is engineered to be broadly hyperresponsive to interferon, the host is markedly resistant to otherwise lethal viral infections. Similar but less effective hyperresponsiveness appears in asthma, suggesting that evolving attempts to improve antiviral defense may instead cause inflammatory disease. Indeed, in susceptible genetic backgrounds, respiratory viruses can also cause a hit-and-run phenomenon that is manifest by the development of a permanent asthmatic phenotype long after the infection has been cleared. This complex phenotype can be segregated into individual traits using pharmacologic, immunologic, and genetic strategies to achieve more precise definition of just how viruses can reprogram host behavior. Evidence of reprogramming is manifest by persistent abnormalities in epithelial cell survival and macrophage activation that when corrected can prevent the development of disease phenotypes. Our results led us to pursue the hypothesis that specific components of the innate immune system may manifest an aberrant antiviral response as a basis for chronic inflammatory diseases and that adjusting this response can improve short-and long-term outcomes after viral infection.