Michael A. Pazos

Cutting Edge: Stealth Influenza Virus Replication Precedes the Initiation of Adaptive Immunity

A timely immune response is crucial for the effective control of virus infection. The influenza virus NS1 protein interferes with the expression of key proinflammatory cytokines from infected cells in vitro. To investigate the effect of NS1 during the onset of immunity in vivo, we systematically studied the early events that occur in the lungs and draining lymph nodes upon infection with influenza virus. Strikingly, no sign of innate immunity was detected in the lungs for almost 2 days after infection until a sudden inflammatory burst, including IFNs, cytokines, and chemokines, occurred. This burst preceded the robust dendritic cell migration and T cell activation in the lymph nodes. An NS1-deficient virus triggered rapid inflammation in the lungs whereas a wild-type virus did not. Thus, we demonstrate that, in vivo, influenza virus uses the NS1 protein to replicate for almost 2 days after infection before detection by the immune system.

p53 Serves as a Host Antiviral Factor That Enhances Innate and Adaptive Immune Responses to Influenza A Virus

Several direct target genes of the p53 tumor suppressor have been identified within pathways involved in viral sensing, cytokine production, and inflammation, suggesting a potential role of p53 in antiviral immunity. The increasing need to identify immune factors to devise host-targeted therapies against pandemic influenza A virus (IAV) led us to investigate the role of endogenous wild-type p53 on the immune response to IAV. We observed that the absence of p53 resulted in delayed cytokine and antiviral gene responses in lung and bone marrow, decreased dendritic cell activation, and reduced IAV-specific CD8+ T cell immunity. Consequently, p53−/− mice showed a more severe IAV-induced disease compared with their wild-type counterparts. These findings establish that p53 influences the antiviral response to IAV, affecting both innate and adaptive immunity. Thus, in addition to its established functions as a tumor suppressor gene, p53 serves as an IAV host antiviral factor that might be modulated to improve anti-IAV therapy and vaccines.

Identification of the Salmonella enterica serotype Typhimurium SipA domain responsible for inducing neutrophil recruitment across the intestinal epithelium

In human intestinal disease induced by Salmonella enterica serotype Typhimurium (S. typhimurium) transepithelial migration of polymorphonuclear leukocytes (PMNs) rapidly follows attachment of the bacteria to the epithelial apical membrane. Previously, we have shown that the S. typhimurium effector protein, SipA, plays a pivotal role in signalling epithelial cell responses that lead to the transepithelial migration of PMNs. Thus, the objective of this study was to determine the functional domain of SipA that regulates this signalling event. SipA was divided into two fragments: the SipAb C‐terminal fragment426−684 (259 AA), which binds actin, and the SipAa fragment2−425 (424 AA), which a role has yet to be described. In both in vitro and in vivo models of S. typhimurium‐induced intestinal inflammation the SipAa fragment exhibited a profound ability to induce PMN transmigration, whereas the SipAb actin‐binding domain failed to induce PMN transmigration. Subsequent mapping of the SipAa domain identified a 131‐amino‐acid region (SipAa3294−424) responsible for modulating PMN transepithelial migration. Interestingly, neither intracellular translocation nor actin association of SipA was necessary for its ability to induce PMN transepithelial migration. As these results indicate SipA has at least two separate functional domains, we speculate that during infection S. typhimurium requires delivery of SipA to both extracellular and intracellular spaces to maximize pro‐inflammatory responses and mechanisms of bacterial invasion.

Distinct Isoforms of Phospholipase A2 Mediate the Ability of Salmonella enterica Serotype Typhimurium and Shigella flexneri To Induce the Transepithelial Migration of Neutrophils

ABSTRACT Salmonella spp. and Shigella spp. are responsible for millions of cases of enteric disease each year worldwide. While these pathogens have evolved distinct strategies for interacting with the human intestinal epithelium, they both induce significant proinflammatory responses that result in massive transepithelial migration of neutrophils across the intestinal mucosa. It has previously been shown with Salmonella enterica serotype Typhimurium that the process of neutrophil transmigration is mediated in part by the secretion of hepoxilin A3 (HXA3; 8-hydroxy-11,12-epoxy-eicosatetraenoic acid), a potent neutrophil chemoattractant, from the apical surface of infected model intestinal epithelium. This study confirms that HXA3 is also secreted in response to infection by Shigella flexneri, that it is produced by a pathway involving 12/15-lipoxygenase (12/15-LOX), and that S. enterica serovar Typhimurium and S. flexneri share certain elements in the mechanism(s) that underlies the otherwise separate signal transduction pathways that are engaged to induce polymorphonuclear leukocyte (PMN) transepithelial migration (protein kinase C and extracellular signal-regulated kinases 1 and 2, respectively). PMN transepithelial migration in response to infection with S. flexneri was dependent on 12/15-LOX activity, the enzyme responsible for the initial metabolism of arachidonic acid to HXA3. Probing further into this pathway, we also found that S. enterica serovar Typhimurium and S. flexneri activate different subtypes of phospholipase A2, a critical enzyme involved in the liberation of arachidonic acid from cellular membranes. Thus, although S. enterica serovar Typhimurium and S. flexneri utilize different mechanisms for triggering the induction of PMN transepithelial migration, we found that their reliance on 12/15-LOX is conserved, suggesting that enteric pathogens may ultimately stimulate similar pathways for the synthesis and release of HXA3.

Estrogen Mediates Innate and Adaptive Immune Alterations to Influenza Infection in Pregnant Mice

Pregnancy is a leading risk factor for severe complications during an influenza virus infection. Women infected during their second and third trimesters are at increased risk for severe cardiopulmonary complications, premature delivery, and death. Here, we establish a murine model of aerosolized influenza infection during pregnancy. We find significantly altered innate antiviral responses in pregnant mice, including decreased levels of IFN-β, IL-1α, and IFN-γ at early time points of infection. We also find reduced cytotoxic T cell activity and delayed viral clearance. We further demonstrate that pregnancy levels of the estrogen 17-β-estradiol are able to induce key anti-inflammatory phenotypes in immune responses to the virus independently of other hormones or pregnancy-related stressors. We conclude that elevated estrogen levels result in an attenuated anti-viral immune response, and that pregnancy-associated morbidities occur in the context of this anti-inflammatory phenotype.

Multidrug Resistance-Associated Transporter 2 Regulates Mucosal Inflammation by Facilitating the Synthesis of Hepoxilin A3

Neutrophil transmigration across mucosal surfaces contributes to dysfunction of epithelial barrier properties, a characteristic underlying many mucosal inflammatory diseases. Thus, insight into the directional movement of neutrophils across epithelial barriers will provide important information relating to the mechanisms of such inflammatory disorders. The eicosanoid hepoxilin A3, an endogenous product of 12-lipoxygenase activity, is secreted from the apical surface of the epithelial barrier and establishes a chemotactic gradient to guide neutrophils from the submucosa across epithelia to the luminal site of an inflammatory stimulus, the final step in neutrophil recruitment. Currently, little is known regarding how hepoxilin A3 is secreted from the intestinal epithelium during an inflammatory insult. In this study, we reveal that hepoxilin A3 is a substrate for the apical efflux ATP-binding protein transporter multidrug resistance-associated protein 2 (MRP2). Moreover, using multiple in vitro and in vivo models, we show that induction of intestinal inflammation profoundly up-regulates apical expression of MRP2, and that interfering with hepoxilin A3 synthesis and/or inhibition of MRP2 function results in a marked reduction in inflammation and severity of disease. Lastly, examination of inflamed intestinal epithelia in human biopsies revealed up-regulation of MRP2. Thus, blocking hepoxilin A3 synthesis and/or inhibiting MRP2 may lead to the development of new therapeutic strategies for the treatment of epithelial-associated inflammatory conditions.

Distinct Cellular Sources of Hepoxilin A3 and Leukotriene B4 Are Used To Coordinate Bacterial-Induced Neutrophil Transepithelial Migration

Neutrophilic infiltration is a leading contributor to pathology in a number of pulmonary disease states, including cystic fibrosis. Hepoxilin A3 (HXA3) is a chemotactic eicosanoid shown to mediate the transepithelial passage of neutrophils in response to infection in several model systems and at multiple mucosal surfaces. Another well-known eicosanoid mediating general neutrophil chemotaxis is leukotriene B4 (LTB4). We sought to distinguish the roles of each eicosanoid in the context of infection of lung epithelial monolayers by Pseudomonas aeruginosa. Using human and mouse in vitro transwell model systems, we used a combination of biosynthetic inhibitors, receptor antagonists, as well as mutant sources of neutrophils to assess the contribution of each chemoattractant in driving neutrophil transepithelial migration. We found that following chemotaxis to epithelial-derived HXA3 signals, neutrophil-derived LTB4 is required to amplify the magnitude of neutrophil migration. LTB4 signaling is not required for migration to HXA3 signals, but LTB4 generation by migrated neutrophils plays a significant role in augmenting the initial HXA3-mediated migration. We conclude that HXA3 and LTB4 serve independent roles to collectively coordinate an effective neutrophilic transepithelial migratory response.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms. Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils. This process has the potential to be destructive to tissues if excessive or held in an unresolved state. Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration. This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil. Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface. The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1). Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000). The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls. This in vitro model system can be manipulated and applied to other mucosal surfaces. Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections. A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

Commensal Bacteria-Induced Inflammasome Activation in Mouse and Human Macrophages Is Dependent on Potassium Efflux but Does Not Require Phagocytosis or Bacterial Viability

Gut commensal bacteria contribute to the pathogenesis of inflammatory bowel disease, in part by activating the inflammasome and inducing secretion of interleukin-1ß (IL-1ß). Although much has been learned about inflammasome activation by bacterial pathogens, little is known about how commensals carry out this process. Accordingly, we investigated the mechanism of inflammasome activation by representative commensal bacteria, the Gram-positive Bifidobacterium longum subspecies infantis and the Gram-negative Bacteroides fragilis. B. infantis and B. fragilis induced IL-1ß secretion by primary mouse bone marrow-derived macrophages after overnight incubation. IL-1ß secretion also occurred in response to heat-killed bacteria and was only partly reduced when phagocytosis was inhibited with cytochalasin D. Similar results were obtained with a wild-type immortalized mouse macrophage cell line but neither B. infantis nor B. fragilis induced IL-1ß secretion in a mouse macrophage line lacking the nucleotide-binding/leucine-rich repeat pyrin domain containing 3 (NLRP3) inflammasome. IL-1ß secretion in response to B. infantis and B. fragilis was significantly reduced when the wild-type macrophage line was treated with inhibitors of potassium efflux, either increased extracellular potassium concentrations or the channel blocker ruthenium red. Both live and heat-killed B. infantis and B. fragilis also induced IL-1ß secretion by human macrophages (differentiated THP-1 cells or primary monocyte-derived macrophages) after 4 hours of infection, and the secretion was inhibited by raised extracellular potassium and ruthenium red but not by cytochalasin D. Taken together, our findings indicate that the commensal bacteria B. infantis and B. fragilis activate the NLRP3 inflammasome in both mouse and human macrophages by a mechanism that involves potassium efflux and that does not require bacterial viability or phagocytosis.

Development of a Primary Human Co-Culture Model of Inflamed Airway Mucosa

Neutrophil breach of the mucosal surface is a common pathological consequence of infection. We present an advanced co-culture model to explore neutrophil transepithelial migration utilizing airway mucosal barriers differentiated from primary human airway basal cells and examined by advanced imaging. Human airway basal cells were differentiated and cultured at air-liquid interface (ALI) on the underside of 3 µm pore-sized transwells, compatible with the study of transmigrating neutrophils. Inverted ALIs exhibit beating cilia and mucus production, consistent with conventional ALIs, as visualized by micro-optical coherence tomography (µOCT). µOCT is a recently developed imaging modality with the capacity for real time two- and three-dimensional analysis of cellular events in marked detail, including neutrophil transmigratory dynamics. Further, the newly devised and imaged primary co-culture model recapitulates key molecular mechanisms that underlie bacteria-induced neutrophil transepithelial migration previously characterized using cell line-based models. Neutrophils respond to imposed chemotactic gradients, and migrate in response to Pseudomonas aeruginosa infection of primary ALI barriers through a hepoxilin A3-directed mechanism. This primary cell-based co-culture system combined with µOCT imaging offers significant opportunity to probe, in great detail, micro-anatomical and mechanistic features of bacteria-induced neutrophil transepithelial migration and other important immunological and physiological processes at the mucosal surface.