Cherrie-Lee Small

Influenza infection leads to increased susceptibility to subsequent bacterial superinfection by impairing NK cell responses in the lung.

Influenza viral infection is well-known to predispose to subsequent bacterial superinfection in the lung but the mechanisms have remained poorly defined. We have established a murine model of heterologous infections by an H1N1 influenza virus and Staphylococcus aureus. We found that indeed prior influenza infection markedly increased the susceptibility of mice to secondary S. aureus superinfection. Severe sickness and heightened bacterial infection in flu and S. aureus dual-infected animals were associated with severe immunopathology in the lung. We further found that flu-experienced lungs had an impaired NK cell response in the airway to subsequent S. aureus bacterial infection. Thus, adoptive transfer of naive NK cells to the airway of prior flu-infected mice restored flu-impaired antibacterial host defense. We identified that TNF-α production of NK cells played an important role in NK cell-mediated antibacterial host defense as NK cells in flu-experienced lungs had reduced TNF-α expression and adoptive transfer of TNF-α–deficient NK cells to the airway of flu-infected mice failed to restore flu-impaired antibacterial host defense. Defected NK cell function was found to be an upstream mechanism of depressed antibacterial activities by alveolar macrophages as contrast to naive wild-type NK cells, the NK cells from flu-infected or TNF-α–deficient mice failed to enhance S. aureus phagocytosis by alveolar macrophages. Together, our study identifies the weakened NK cell response in the lung to be a novel critical mechanism for flu-mediated susceptibility to bacterial superinfection.

NK Cells Play a Critical Protective Role in Host Defense against Acute Extracellular Staphylococcus aureus Bacterial Infection in the Lung

Staphylococcus aureus remains a common cause of nosocomial bacterial infections and are often antibiotic resistant. The role of NK cells and IL-15 and their relationship in host defense against extracellular bacterial pathogens including S. aureus remain unclear. We have undertaken several approaches to address this issue using wild type (WT), IL-15 gene knock-out (KO), and NK cell-depleted mouse models. Upon pulmonary staphylococcal infection WT mice had markedly increased activated NK cells, but not NKT or γδ T cells, in the airway lumen that correlated with IL-15 production in the airway and with alveolar macrophages. In vitro exposure to staphylococcal products and/or coculture with lung macrophages directly activated NK cells. In contrast, lung macrophages better phagocytosed S. aureus in the presence of NK cells. In sharp contrast to WT controls, IL-15 KO mice deficient in NK cells were found to be highly susceptible to pulmonary staphylococcal infection despite markedly increased neutrophils and macrophages in the lung. In further support of these findings, WT mice depleted of NK cells were similarly susceptible to staphylococcal infection while they remained fully capable of IL-15 production in the lung at levels similar to those of NK-competent WT hosts. Our study thus identifies a critical role for NK cells in host defense against pulmonary extracellular bacterial infection and suggests that IL-15 is involved in this process via its indispensable effect on NK cells, but not other innate cells. These findings hold implication for the development of therapeutics in treating antibiotic-resistant S. aureus infection.

Immunopathology in influenza virus infection: uncoupling the friend from foe.

Influenza epidemics and pandemics cause significant morbidity and mortality worldwide associated with severe immunopathology in the lung, and the mechanisms of such immunopathogenesis still remain poorly understood. While human studies help to understand influenza immunopathology, they provide only limited mechanistic information. On the other hand, recent studies using experimental animal models have significantly enhanced our understanding of the complex mechanisms involved in the immunopathogenesis during primary influenza or influenza-associated bacterial superinfection. This includes the involvement of acute inflammatory responses (macrophages, neutrophils, dendritic cells, toll-like receptors, cytokines, chemokines), CD4 and CD8 T cells, tissue remodeling processes, and contribution of bacterial superinfection. In particular, progress has been made in uncoupling the mechanisms that are involved in both anti-viral host defense and in immunopathogenesis from those that solely contribute to lung immunopathology. Uncoupling such events will facilitate the discovery of new intervention strategies to treat pulmonary immunopathology associated with influenza infection.

Murine airway luminal antituberculosis memory CD8 T cells by mucosal immunization are maintained via antigen-driven in situ proliferation, independent of peripheral T cell recruitment.

RATIONALE The airway luminal memory CD8 T cells induced by respiratory mucosal immunization in a murine model have been found to be critical to antituberculosis immunity. However, the mechanisms of their maintenance on airway mucosal surface still remain poorly understood. OBJECTIVES Using a model of adenovirus-based intranasal immunization we investigated the immune property and the mechanisms of maintenance of airway luminal CD8 T cells. METHODS Immune properties of airway luminal Mycobacterium tuberculosis antigen-specific CD8 T cells were examined. Proliferation of airway luminal CD8 T cells was determined by in vivo T cell-labeling techniques. The role of peripheral T cell recruitment in maintaining airway luminal CD8 T cells was investigated by blocking lymphocyte trafficking from lymphoid and peripheral tissues. The requirement of M. tuberculosis antigens for in situ T cell proliferation was evaluated using a T cell transfer approach. An airway M. tuberculosis challenge model was used to study the relationship between CD8 T cell-mediated protection and peripheral T cell recruitment. MEASUREMENTS AND MAIN RESULTS Intranasal immunization leads to elicitation of persisting M. tuberculosis antigen-specific CD8 T cells in the airway lumen, which display an activated effector memory phenotype different from those in peripheral tissues. Airway luminal T cells continuously proliferate in an antigen-dependent manner, and can be maintained even in the absence of peripheral T cell recruitment. The lungs equipped with such CD8 T cells are protected from airway M. tuberculosis challenge independent of both peripheral T cell supply and CD4 T cells. CONCLUSIONS Vaccine-inducible airway luminal antituberculosis memory CD8 T cells are self-renewable in an antigen-dependent manner, and can be maintained independent of peripheral T cell supply.

Persistent infection with Crohn’s disease-associated adherent-invasive Escherichia coli leads to chronic inflammation and intestinal fibrosis

Crohn’s disease is a chronic inflammatory condition of the gastrointestinal tract in which alterations to the bacterial community contribute to disease. Adherent-invasive E. coli (AIEC) are associated with human Crohn’s disease, however their role in intestinal immunopathology is unclear due to the lack of an animal model compatible with chronic timescales. Here we establish chronic AIEC infection in streptomycin-treated conventional mice (CD-1, DBA/2, C3HeN, 129e, C57BL/6), enabling the study of host response and immunopathology. AIEC induces an active Th17 response, heightened levels of proinflammatory cytokines and fibrotic growth factors, with transmural inflammation and fibrosis. Depletion of CD8+ T cells increases cecal bacterial load, pathology and intestinal fibrosis in C57BL/6 mice suggesting a protective role. Our findings provide evidence that chronic AIEC infections result in immunopathology similar to that seen in Crohn’s disease. With this model, research into the host and bacterial genetics associated with AIEC-induced disease becomes more widely accessible.

Negative Regulation of Lung Inflammation and Immunopathology by TNF-α during Acute Influenza Infection

Lung immunopathology is the main cause of influenza-mediated morbidity and death, and much of its molecular mechanisms remain unclear. Whereas tumor necrosis factor-α (TNF-α) is traditionally considered a proinflammatory cytokine, its role in influenza immunopathology is unresolved. We have investigated this issue by using a model of acute H1N1 influenza infection established in wild-type and TNF-α-deficient mice and evaluated lung viral clearance, inflammatory responses, and immunopathology. Whereas TNF-α was up-regulated in the lung after influenza infection, it was not required for normal influenza viral clearance. However, TNF-α deficiency led not only to a greater extent of illness but also to heightened lung immunopathology and tissue remodeling. The severe lung immunopathology was associated with increased inflammatory cell infiltration, anti-influenza adaptive immune responses, and expression of cytokines such as monocyte chemoattractant protein-1 (MCP-1) and fibrotic growth factor, TGF-β1. Thus, in vivo neutralization of MCP-1 markedly attenuated lung immunopathology and blunted TGF-β1 production following influenza infection in these hosts. On the other hand, in vivo transgenic expression of MCP-1 worsened lung immunopathology following influenza infection in wild-type hosts. Thus, TNF-α is dispensable for influenza clearance; however, different from the traditional belief, this cytokine is critically required for negatively regulating the extent of lung immunopathology during acute influenza infection.

Immunization With a Bivalent Adenovirus-vectored Tuberculosis Vaccine Provides Markedly Improved Protection Over Its Monovalent Counterpart Against Pulmonary Tuberculosis

Recombinant virus-vectored vaccines hold great promise for tuberculosis (TB) vaccination strategies. However, there is a lack of side-by-side comparative investigations to dissect the functional differences and support the advantage of multivalent virus-vectored vaccine over its monovalent counterpart. We previously successfully developed a monovalent adenovirus (Ad)-vectored vaccine expressing Ag85a (AdAg85a) and demonstrated its superior protective efficacy in models of pulmonary TB. In this study, we have developed a bivalent Ad TB vaccine expressing Ag85a and TB10.4 antigens as a fusion protein (AdAg85a:TB10.4) and compared its T-cell-activating and immune protective efficacy with that by monovalent AdAg85a. A single intranasal (i.n.) administration of AdAg85a:TB10.4 induced robust T-cell responses toward the respective antigens within the airway lumen and spleen, although the level of Ag85a-specific T-cell responses in the airway lumen triggered by bivalent AdAg85a:TB10.4 was lower than that by its monovalent counterpart at earlier time points. Thus, a single i.n. delivery of AdAg85a:TB10.4 conferred a markedly improved and sustained level of protection in the lung against Mycobacterium tuberculosis (M.tb) challenge over that by AdAg85a or by conventional BCG immunization with similarly induced levels of protection in the spleen. Our results indicate a unique advantage of multivalent viral-vectored TB vaccines for immunization against pulmonary TB.

CD11c+ antigen presenting cells from the alveolar space, lung parenchyma and spleen differ in their phenotype and capabilities to activate naïve and antigen-primed T cells

BackgroundThe lung is divided into two major compartments: the alveolar space and the parenchyma. The alveolar macrophages are the first line of leukocytes in the lung taking up incoming microbes or microbial antigens whereas the parenchymal dendritic cells (DCs) are believed to be the sole potent antigen presenting cells (APCs) in the lung. Both resting alveolar macrophages and parenchymal DCs express CD11c. Several important questions remain to be elucidated: 1] to which extent the alveolar space and lung parenchymal CD11c+ APCs differ in their phenotype and ability to activate naïve T cells; 2] whether they differ in their ability to activate antigen-experienced or -primed T cells; and 3] whether these lung CD11c+ APC populations differ from the splenic CD11c+ APCs which have been commonly used for understanding APC biology.ResultsCD11c+ APCs from the alveolar space, lung parenchyma, and the spleen display differential co-stimulatory molecule expression and cytokine responsiveness upon stimulation. Alveolar space APCs are weak activators of naïve T cells compared to lung parenchymal and splenic CD11c+ APC populations. However, alveolar space APCs are able to potently activate the in vivo microbial antigen-primed T cells to a similar extent as lung parenchymal and splenic APCs.ConclusionTogether our findings indicate that alveolar CD11c+ APCs have a specialized T cell-activating function, capable of activating antigen-primed, but not naïve, T cells whereas lung CD11c+ APCs are capable of activating both the naïve and antigen-primed T cell populations.

Mucosally Delivered Dendritic Cells Activate T Cells Independently of IL-12 and Endogenous APCs

In vitro manipulated dendritic cells (DC) have increasingly been used as a promising vaccine formulation against cancer and infectious disease. However, improved understanding of the immune mechanisms is needed for the development of safe and efficacious mucosal DC immunization. We have developed a murine model of respiratory mucosal immunization by using a genetically manipulated DC vaccine. Within 24 h of intranasal delivery, the majority of vaccine DCs migrated to the lung mucosa and draining lymph nodes and elicited a significant level of T cells capable of IFN-γ secretion and CTL in the airway lumen as well as substantial T cell responses in the spleen. And such T cell responses were associated with enhanced protection against respiratory mucosal intracellular bacterial challenge. In comparison, parenteral i.m. DC immunization did not elicit marked airway luminal T cell responses and immune protection regardless of strong systemic T cell activation. Although repeated mucosal DC delivery boosted Ag-specific T cells in the airway lumen, added benefits to CD8 T cell activation and immune protection were not observed. By using MHC-deficient vaccine DCs, we further demonstrated that mucosal DC immunization-mediated CD8 and CD4 T cell activation does not require endogenous DCs. By using IL-12-deficient vaccine DCs, we also observed that IL-12−/− DCs failed to migrate to the lymph nodes but remained capable of T cell activation. Our observations indicate that mucosal delivery of vaccine DCs represents an effective approach to enhance mucosal T cell immunity, which may operate independent of vaccine IL-12 and endogenous DCs.

Pulmonary Mycobacterial Granuloma: Increased IL-10 Production Contributes to Establishing a Symbiotic Host–Microbe Microenvironment

The granuloma, a hallmark of host defense against pulmonary mycobacterial infection, has long been believed to be an active type 1 immune environment. However, the mechanisms regarding why granuloma fails to eliminate mycobacteria even in immune-competent hosts, have remained largely unclear. By using a model of pulmonary Mycobacterium bovis Bacillus Calmette-Guerin (BCG) infection, we have addressed this issue by comparing the immune responses within the airway luminal and granuloma compartments. We found that despite having a similar immune cellular profile to that in the airway lumen, the granuloma displayed severely suppressed type 1 immune cytokine but enhanced chemokine responses. Both antigen-presenting cells (APCs) and T cells in granuloma produced fewer type 1 immune molecules including tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and nitric oxide. As a result, the granuloma APCs developed a reduced capacity to phagocytose mycobacteria and to induce T-cell proliferation. To examine the molecular mechanisms, we compared the levels of immune suppressive cytokine IL-10 in the airway lumen and granuloma and found that both granuloma APCs and T cells produced much more IL-10. Thus, IL-10 deficiency restored type 1 immune activation within the granuloma while having a minimal effect within the airway lumen. Hence, our study provides the first experimental evidence that, contrary to the conventional belief, the BCG-induced lung granuloma represents a symbiotic host-microbe microenvironment characterized by suppressed type 1 immune activation.