An Unexpected Role for Hypoxic Response: Oxygenation and Inflammation

The eradication of invading microorganisms depends initially on innate immunity mechanisms that preexist in all individuals and act within minutes of infection. Pathogen spread is often countered by an inflammatory response that recruits more effector molecules and cells of the innate immune system from local blood vessels, while inducing clotting farther downstream so that pathogens cannot spread throughout the blood. If a microorganism crosses an epithelial barrier and begins to replicate in the tissues of the host, it is, in some cases, immediately recognized by the mononuclear phagocytes, or macrophages, that reside in tissues. Macrophages mature continuously from circulating monocytes that leave the circulation to migrate into tissues throughout the body. The second major family of phagocytes, the neutrophils or polymorphonuclear leukocytes (PMNs) are short-lived cells that are abundant cells in the blood but are not present in healthy tissues. Both phagocytic cell types play a key role in innate immunity because they can recognize, ingest and destroy many pathogens without the aid of an adaptive immune response. This infiltration of neutrophils and later macrophages to the site of bacterial infection is tightly linked with the need of these immune defense cells to respond to the tissue microenvironment.     

An unexpected role for hypoxic response: oxygenation and inflammation

The eradication of invading microorganisms depends initially on innate immunity mechanisms that preexist in all individuals and act within minutes of infection. Pathogen spread is often countered by an inflammatory response that recruits more effector molecules and cells of the innate immune system from local blood vessels, while inducing clotting farther downstream so that pathogens cannot spread throughout the blood. If a microorganism crosses an epithelial barrier and begins to replicate in the tissues of the host, it is, in some cases, immediately recognized by the mononuclear phagocytes, or macrophages, that reside in tissues. Macrophages mature continuously from circulating monocytes that leave the circulation to migrate into tissues throughout the body. The second major family of phagocytes, the neutrophils or polymorphonuclear leukocytes (PMNs) are short-lived cells that are abundant cells in the blood but are not present in healthy tissues. Both phagocytic cell types play a key role in innate immunity because they can recognize, ingest and destroy many pathogens without the aid of an adaptive immune response. This infiltration of neutrophils and later macrophages to the site of bacterial infection is tightly linked with the need of these immune defense cells to respond to the tissue microenvironment.     

Modulation of phagocyte apoptosis by bacterial pathogens

Phagocytic leukocytes such as neutrophils and macrophages are essential for the innate immune response against invading bacteria. Binding and ingestion of bacteria by these host cells triggers potent anti-microbial activity, including production of reactive oxygen species. Although phagocytes are highly adept at destroying bacteria, modulation of leukocyte apoptosis or cell death by bacteria has emerged as a mechanism of pathogenesis. Whereas induction of macrophage apoptosis by pathogens may adversely affect the host immune response to infection, acceleration of neutrophil apoptosis following phagocytic interaction with bacteria appears essential for the resolution of infection. This idea is supported by the finding that some bacterial pathogens alter normal phagocytosis-induced neutrophil apoptosis to survive and cause disease. This review summarizes what is currently known about modulation of phagocyte apoptosis by bacteria and describes a paradigm whereby bacteria-induced neutrophil apoptosis plays a role in the resolution of infection.     

c-di-GMP Enhances Protective Innate Immunity in a Murine Model of Pertussis

Innate immunity represents the first line of defense against invading pathogens in the respiratory tract. Innate immune cells such as monocytes, macrophages, dendritic cells, NK cells, and granulocytes contain specific pathogen-recognition molecules which induce the production of cytokines and subsequently activate the adaptive immune response. c-di-GMP is a ubiquitous second messenger that stimulates innate immunity and regulates biofilm formation, motility and virulence in a diverse range of bacterial species with potent immunomodulatory properties. In the present study, c-di-GMP was used to enhance the innate immune response against pertussis, a respiratory infection mainly caused by Bordetella pertussis. Intranasal treatment with c-di-GMP resulted in the induction of robust innate immune responses to infection with B. pertussis characterized by enhanced recruitment of neutrophils, macrophages, natural killer cells and dendritic cells. The immune responses were associated with an earlier and more vigorous expression of Th1-type cytokines, as well as an increase in the induction of nitric oxide in the lungs of treated animals, resulting in significant reduction of bacterial numbers in the lungs of infected mice. These results demonstrate that c-di-GMP is a potent innate immune stimulatory molecule that can be used to enhance protection against bacterial respiratory infections. In addition, our data suggest that priming of the innate immune system by c-di-GMP could further skew the immune response towards a Th1 type phenotype during subsequent infection. Thus, our data suggest that c-di-GMP might be useful as an adjuvant for the next generation of acellular pertussis vaccine to mount a more protective Th1 phenotype immune response, and also in other systems where a Th1 type immune response is required.     

Comparative Genomic Analysis of Buffalo (Bubalus bubalis) NOD1 and NOD2 Receptors and Their Functional Role in In-Vitro Cellular Immune Response

Nucleotide binding and oligomerization domain (NOD)-like receptors (NLRs) are innate immune receptors that recognize bacterial cell wall components and initiate host immune response. Structure and function of NLRs have been well studied in human and mice, but little information exists on genetic composition and role of these receptors in innate immune system of water buffalo—a species known for its exceptional disease resistance. Here, a comparative study on the functional domains of NOD1 and NOD2 was performed across different species. The NOD mediated in-vitro cellular responses were studied in buffalo peripheral blood mononuclear cells, resident macrophages, mammary epithelial, and fibroblast cells. Buffalo NOD1 (buNOD1) and buNOD2 showed conserved domain architectures as found in other mammals. The domains of buNOD1 and buNOD2 showed analogy in secondary and tertiary conformations. Constitutive expressions of NODs were ubiquitous in different tissues. Following treatment with NOD agonists, peripheral lymphocytes showed an IFN-γ response along-with production of pro-inflammatory cytokines. Alveolar macrophages and mammary epithelial cells showed NOD mediated in-vitro immune response through NF-κB dependent pathway. Fibroblasts showed pro-inflammatory cytokine response following agonist treatment. Our study demonstrates that both immune and non-immune cells could generate NOD-mediated responses to pathogens though the type and magnitude of response depend on the cell types. The structural basis of ligand recognition by buffalo NODs and knowledge of immune response by different cell types could be useful for development of non-infective innate immune modulators and next generation anti-inflammatory compounds.     

Living in the danger zone: innate immunity to Salmonella

Phagocytic cells, including macrophages, neutrophils and dendritic cells, are critical components of the innate immune response to bacterial pathogens such as Salmonella typhimurium. These cells can have several roles during the early stage of an infection including controlling bacterial replication and producing cytokines and chemokines that activate and recruit additional cells. Macrophages, neutrophils and dendritic cells increase in number early after oral Salmonella infection and produce cytokines important in host survival such as tumor necrosis factor alpha (TNF-alpha). All three phagocytic cell types also harbor bacteria during infection. Natural killer cells, natural killer T cells and T cell receptor alpha beta T cells also respond rapidly to infection and are early sources of interferon-gamma during infection with Salmonella. Studies using infection models with Salmonella are providing a picture of the innate response to bacteria and insight into the role of defined cell types and cytokines important in the transition from innate to adaptive immunity.     

Modeling Granulomas in Response to Infection in the Lung

Alveolar macrophages play a large role in the innate immune response of the lung. However, when these highly immune-regulatory cells are unable to eradicate pathogens, the adaptive immune system, which includes activated macrophages and lymphocytes, particularly T cells, is called upon to control the pathogens. This collection of immune cells surrounds, isolates and quarantines the pathogen, forming a small tissue structure called a granuloma for intracellular pathogens like Mycobacterium tuberculosis (Mtb). In the present work we develop a mathematical model of the dynamics of a granuloma by a system of partial differential equations. The ‘strength’ of the adaptive immune response to infection in the lung is represented by a parameter α, the flux rate by which T cells and M1 macrophages that immigrated from the lymph nodes enter into the granuloma through its boundary. The parameter α is negatively correlated with the ‘switching time’, namely, the time it takes for the number of M1 type macrophages to surpass the number of infected, M2 type alveolar macrophages. Simulations of the model show that as α increases the radius of the granuloma and bacterial load in the granuloma both decrease. The model is used to determine the efficacy of potential host-directed therapies in terms of the parameter α, suggesting that, with fixed dosing level, an infected individual with a stronger immune response will receive greater benefits in terms of reducing the bacterial load.     

Recent insights into the role of the innate immune system in the development of alcoholic liver disease

The innate immune system is responsible for the rapid, initial response of the organism to potentially dangerous stresses, including pathogens, tissue injury, and malignancy. Pattern-recognition receptors of the toll-like receptor (TLR) family expressed by macrophages provide a first line of defense against microbial invasion. Activation of these receptors results in a stimulus-specific expression of genes required to control the infection, including the production of inflammatory cytokines and chemokines, followed by the recruitment of neutrophils to the site of infection. The early stages in the development of alcoholic liver disease (ALD) follow a pattern characteristic of an innate immune response. Kupffer cells, the resident macrophages in the liver, are activated in response to bacterial endotoxins (lipopolysaccharide, LPS), leading to the production of inflammatory and fibrogenic cytokines, reactive oxygen species, as well as the recruitment of neutrophils to the liver. One mechanism by which chronic ethanol can turn the highly regulated innate immune response into a pathway of disease is by disrupting the signal transduction cascades mediating the innate immune response. Recent studies have identified specific modules in the TLR-4 signaling cascade that are disrupted after chronic ethanol exposure, including CD14 and the mitogen-activated protein kinase family members, ERK1/2 and p38. Enhanced activation of these TLR-4 dependent signaling pathways after chronic ethanol likely contributes to the development of alcoholic liver disease.     

Mechanisms and consequences of bacterial targeting by the autophagy pathway

Autophagy is a key component of our immune response to invading pathogens. Autophagic targeting of intracellular bacteria within vacuolar compartments or the cytosol helps to control bacterial replication in the host cell. The mechanism by which these invading pathogens are selectively targeted for degradation is of particular interest. Recently, several signaling factors have been shown to play roles in the specific targeting of bacteria by the autophagy pathway including: pattern recognition receptors, reactive oxygen species, ubiquitin and diacylglycerol. Here, we discuss these signaling factors and the consequences of bacterial targeting by autophagy during infection of host cells.     

Cutting edge: heat shock protein (HSP) 60 activates the innate immune response: CD14 is an essential receptor for HSP60 activation of mononuclear cells

Heat shock proteins (HSP), highly conserved across species, are generally viewed as intracellular proteins thought to serve protective functions against infection and cellular stress. Recently, we have reported the surprising finding that human and chlamydial HSP60, both present in human atheroma, can activate vascular cells and macrophages. However, the transmembrane signaling pathways by which extracellular HSP60 may activate cells remains unclear. CD14, the monocyte receptor for LPS, binds numerous microbial products and can mediate activation of monocytes/macrophages and endothelial cells, thus promoting the innate immune response. We show here that human HSP60 activates human PBMC and monocyte-derived macrophages through CD14 signaling and p38 mitogen-activated protein kinase, sharing this pathway with bacterial LPS. These findings provide further insight into the molecular mechanisms by which extracellular HSP may participate in atherosclerosis and other inflammatory disorders by activating the innate immune system.     

Increases in calmodulin abundance and stabilization of activated inducible nitric oxide synthase mediate bacterial killing in RAW 264.7 macrophages

The rapid activation of macrophages in response to bacterial antigens is central to the innate immune system that permits the recognition and killing of pathogens to limit infection. To understand regulatory mechanisms underlying macrophage activation, we have investigated changes in the abundance of calmodulin (CaM) and iNOS in response to the bacterial cell wall component lipopolysaccharide (LPS) using RAW 264.7 macrophages. Critical to these measurements was the ability to differentiate free iNOS from the CaM-bound (active) form of iNOS associated with nitric oxide generation. We observe a rapid 2-fold increase in CaM abundance during the first 30 min that is blocked by inhibition of either NFkappaB nuclear translocation or protein synthesis. A similar 2-fold increase in the abundance of the complex between CaM and iNOS is observed with the same time dependence. In contrast, there are no detectable increases in the CaM-free (i.e., inactive) form of iNOS within the first 2 h; it remains at a very low abundance during the initial phase of macrophage activation. Increasing cellular CaM levels in stably transfected macrophages results in a corresponding increase in the abundance of the CaM/iNOS complex that promotes effective bacterial killing following infection by Salmonella typhimurium. Thus, LPS-dependent increases in CaM abundance function in the stabilization and activation of iNOS on the rapid time scale associated with macrophage activation and bacterial killing. These results explain how CaM and iNOS coordinately function to form a stable complex that is part of a rapid host response that functions within the first 30 min following bacterial infection to upregulate the innate immune system involving macrophage activation.     

Autophagy and innate immunity: Triggering,targeting and tuning

Autophagy is a conserved catabolic stress response pathway that is increasingly recognized as an important component of both innate and acquired immunity to pathogens. The activation of autophagy during infection not only provides cell-autonomous protection through lysosomal degradation of invading pathogens (xenophagy), but also regulates signaling by other innate immune pathways. This review will focus on recent advances in our understanding of three major areas of the interrelationship between autophagy and innate immunity, including how autophagy is triggered during infection, how invading pathogens are targeted to autophagosomes, and how the autophagy pathway participates in “tuning” the innate immune response.     

STAT4 is a critical mediator of early innate immune responses against pulmonary Klebsiella infection

Bacterial pneumonia is a leading cause of morbidity and mortality in the U.S. An effective innate immune response is critical for the clearance of bacteria from the lungs. IL-12, a key T1 cytokine in innate immunity, signals through STAT4. Thus, understanding how STAT4 mediates pulmonary immune responses against bacterial pathogens will have important implications for the development of rational immunotherapy targeted at augmenting innate immunity. We intratracheally administered Klebsiella pneumoniae to wild-type BALB/c and STAT4 knockout (STAT4-/-) mice. Compared with wild-type controls, STAT4-/- mice had decreased survival following intratracheal Klebsiella administration, which was associated with a higher lung and blood bacterial burden. STAT4-/- animals also displayed impaired pulmonary IFN-gamma production and decreased levels of proinflammatory cytokines, including the ELR- CXC chemokines IFN-gamma-inducible protein-10 and monokine induced by IFN-gamma. Although total lung leukocyte populations were similar between STAT4-/- and wild-type animals following infection, alveolar macrophages isolated from infected STAT4-/- mice had decreased production of proinflammatory cytokines, including IFN-gamma, compared with infected wild-type mice. The intrapulmonary overexpression of IFN-gamma concomitant with the systemic administration of IFN-gamma partially reversed the immune deficits observed in STAT4-/- mice, resulting in improved bacterial clearance from the blood. Collectively, these studies demonstrate that STAT4 is required for the generation of an effective innate host defense against bacterial pathogens of the lung.     

Ambient ozone primes pulmonary innate immunity in mice

Exposure to ozone in air pollution in urban environments is associated with increases in pulmonary-related hospitalizations and mortality. Because ozone also alters clearance of pulmonary bacterial pathogens, we hypothesized that inhalation of ozone modifies innate immunity in the lung. To address our hypothesis, we exposed C57BL/6J mice to either free air or ozone, and then subsequently challenged with an aerosol of Escherichia coli LPS. Pre-exposure to ozone resulted in enhanced airway hyperreactivity, higher concentrations of both total protein and proinflammatory cytokines in lung lavage fluid, enhanced LPS-mediated signaling in lung tissue, and higher concentrations of serum IL-6 following inhalation of LPS. However, pre-exposure to ozone dramatically reduced inflammatory cell accumulation to the lower airways in response to inhaled LPS. The reduced concentration of cells in the lower airways was associated with enhanced apoptosis of both lung macrophages and systemic circulating monocytes. Moreover, both flow cytometry and confocal microscopy indicate that inhaled ozone causes altered distribution of TLR4 on alveolar macrophages and enhanced functional response to endotoxin by macrophages. These observations indicate that ozone exposure increases both the pulmonary and the systemic biologic response to inhaled LPS by priming the innate immune system.     

An alloy of zinc and innate immunity: Galvanising host defence against infection

Innate immune cells such as macrophages and neutrophils initiate protective inflammatory responses and engage antimicrobial responses to provide frontline defence against invading pathogens. These cells can both restrict the availability of certain transition metals that are essential for microbial growth and direct toxic concentrations of metals towards pathogens as antimicrobial responses. Zinc is important for the structure and function of many proteins, however excess zinc can be cytotoxic. In recent years, several studies have revealed that innate immune cells can deliver toxic concentrations of zinc to intracellular pathogens. In this review, we discuss the importance of zinc status during infectious disease and the evidence for zinc intoxication as an innate immune antimicrobial response. Evidence for pathogen subversion of this response is also examined. The likely mechanisms, including the involvement of specific zinc transporters that facilitate delivery of zinc by innate immune cells for metal ion poisoning of pathogens are also considered. Precise mechanisms by which excess levels of zinc can be toxic to microorganisms are then discussed, particularly in the context of synergy with other antimicrobial responses. Finally, we highlight key unanswered questions in this emerging field, which may offer new opportunities for exploiting innate immune responses for anti‐infective development.