Gayathriy Balamayooran

Mechanisms of Neutrophil Accumulation in the Lungs Against Bacteria

Bacterial lung diseases are a major cause of morbidity and mortality both in immunocompromised and in immunocompetent individuals. Neutrophil accumulation, a pathological hallmark of bacterial diseases, is critical to host defense, but may also cause acute lung injury/acute respiratory distress syndrome. Toll-like receptors, nucleotide-binding oligomerization domain (NOD)-like receptors, transcription factors, cytokines, and chemokines play essential roles in neutrophil sequestration in the lungs. This review highlights our current understanding of the role of these molecules in the lungs during bacterial infection and their therapeutic potential. We also discuss emerging data on cholesterol and ethanol as environmentally modifiable factors that may impact neutrophil-mediated pulmonary innate host defense. Understanding the precise molecular mechanisms leading to neutrophil influx in the lungs during bacterial infection is critical for the development of more effective therapeutic and prophylactic strategies to control the excessive host response to infection.

Monocyte Chemoattractant Protein 1 Regulates Pulmonary Host Defense via Neutrophil Recruitment during Escherichia coli Infection

ABSTRACT Neutrophil accumulation is a critical event to clear bacteria. Since uncontrolled neutrophil recruitment can cause severe lung damage, understanding neutrophil trafficking mechanisms is important to attenuate neutrophil-mediated damage. While monocyte chemoattractant protein 1 (MCP-1) is known to be a monocyte chemoattractant, its role in pulmonary neutrophil-mediated host defense against Gram-negative bacterial infection is not understood. We hypothesized that MCP-1/chemokine (C-C motif) ligand 2 is important for neutrophil-mediated host defense. Reduced bacterial clearance in the lungs was observed in MCP-1−/− mice following Escherichia coli infection. Neutrophil influx, along with cytokines/chemokines, leukotriene B4 (LTB4), and vascular cell adhesion molecule 1 levels in the lungs, was reduced in MCP-1−/− mice after infection. E. coli-induced activation of NF-κB and mitogen-activated protein kinases in the lung was also reduced in MCP-1−/− mice. Administration of intratracheal recombinant MCP-1 (rMCP-1) to MCP-1−/− mice induced pulmonary neutrophil influx and cytokine/chemokine responses in the presence or absence of E. coli infection. Our in vitro migration experiment demonstrates MCP-1-mediated neutrophil chemotaxis. Notably, chemokine receptor 2 is expressed on lung and blood neutrophils, which are increased upon E. coli infection. Furthermore, our findings show that neutrophil depletion impairs E. coli clearance and that exogenous rMCP-1 after infection improves bacterial clearance in the lungs. Overall, these new findings demonstrate that E. coli-induced MCP-1 causes neutrophil recruitment directly via chemotaxis as well as indirectly via modulation of keratinocyte cell-derived chemokine, macrophage inflammatory protein 2, and LTB4.

Intrapulmonary Administration of Leukotriene B 4 Augments Neutrophil Accumulation and Responses in the Lung to Klebsiella Infection in CXCL1 Knockout Mice

In prior studies, we demonstrated that 1) CXCL1/KC is essential for NF-κB and MAPK activation and expression of CXCL2/MIP-2 and CXCL5/LPS-induced CXC chemokine in Klebsiella-infected lungs, and 2) CXCL1 derived from hematopoietic and resident cells contributes to host immunity against Klebsiella. However, the role of CXCL1 in mediating neutrophil leukotriene B4 (LTB4), reactive oxygen species (ROS), and reactive nitrogen species (RNS) production is unclear, as is the contribution of these factors to host immunity. In this study, we investigated 1) the role of CXCL1 in LTB4, NADPH oxidase, and inducible NO synthase (iNOS) expression in lungs and neutrophils, and 2) whether LTB4 postinfection reverses innate immune defects in CXCL1−/− mice via regulation of NADPH oxidase and iNOS. Our results demonstrate reduced neutrophil influx, attenuated LTB4 levels, and decreased ROS and iNOS production in the lungs of CXCL1−/− mice after Klebsiella pneumoniae infection. Using neutrophil depletion and repletion, we found that neutrophils are the predominant source of pulmonary LTB4 after infection. To treat immune defects in CXCL1−/− mice, we intrapulmonarily administered LTB4. Postinfection, LTB4 treatment reversed immune defects in CXCL1−/− mice and improved survival, neutrophil recruitment, cytokine/chemokine expression, NF-κB/MAPK activation, and ROS/RNS production. LTB4 also enhanced myeloperoxidase, H2O2, RNS production, and bacterial killing in K. pneumoniae-infected CXCL1−/− neutrophils. These novel results uncover important roles for CXCL1 in generating ROS and RNS in neutrophils and in regulating host immunity against K. pneumoniae infection. Our findings suggest that LTB4 could be used to correct defects in neutrophil recruitment and function in individuals lacking or expressing malfunctional CXCL1.

Review: Toll-like receptors and NOD-like receptors in pulmonary antibacterial immunity.

Lung diseases caused by bacteria are a leading cause of death in both immunocompromised and immunocompetent individuals as well as in children. Although neutrophil recruitment is critical to augment the host defence, excessive neutrophil accumulation results in life-threatening diseases, such as acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Therefore, it is important to modulate excessive neutrophil influx in ALI/ARDS to mitigate lung damage and mortality. A better understanding of the basic mechanisms underlying neutrophil influx is crucial to designing novel and innovative treatment strategies for ALI/ARDS. Recognition of bacteria in the lung is the critical first step leading to neutrophil influx. Pattern recognition receptors, such as Toll-like receptors and NOD-like receptors, play an important role in the recognition of bacterial pathogens. Understanding the molecular and cellular mechanisms associated with the recognition of bacterial pathogens by the host is critical for the development of effective therapeutic strategies to control parenchymal damage via modulating neutrophil accumulation in the lung.

Intrapulmonary G-CSF Rescues Neutrophil Recruitment to the Lung and Neutrophil Release to Blood in Gram-Negative Bacterial Infection in MCP-1−/− Mice

We previously demonstrated that MCP-1 is important for E. coli–induced neutrophil migration to the lungs. However, E. coli neither disseminates nor induces death in mice. Furthermore, the cell types and the host defense mechanisms that contribute to MCP-1–dependent neutrophil trafficking have not been defined. In this study, we sought to explore the cell types and the mechanisms associated with Klebsiella pneumoniae–mediated MCP-1–dependent neutrophil influx. MCP-1−/− mice are more susceptible to pulmonary K. pneumoniae infection and show higher bacterial burden in the lungs and dissemination. MCP-1−/− mice also display attenuated neutrophil influx, cytokine/chemokine production, and activation of NF-κB and MAPKs following intratracheal K. pneumoniae infection. rMCP-1 treatment in MCP-1−/− mice following K. pneumoniae infection rescued impairment in survival, bacterial clearance, and neutrophil accumulation in the lung. Neutrophil numbers in the blood of MCP-1−/− mice were associated with G-CSF concentrations in bronchoalveolar lavage fluid and blood. Bone marrow or resident cell–derived MCP-1 contributed to bacterial clearance, neutrophil accumulation, and cytokine/chemokine production in the lungs following infection. Furthermore, exogenous MCP-1 dose dependently increased neutrophil counts and G-CSF concentrations in the blood. Intriguingly, administration of intratracheal rG-CSF to MCP-1−/− mice after K. pneumoniae infection rescued survival, bacterial clearance and dissemination, and neutrophil influx in MCP-1−/− mice. Collectively, these novel findings unveil an unrecognized role of MCP-1 in neutrophil-mediated host immunity during K. pneumoniae pneumonia and illustrate that G-CSF could be used to rescue impairment in host immunity in individuals with absent or malfunctional MCP-1.

Nuclear Factor-κB: a Key Regulator in Health and Disease of Lungs

Rel/NF-κB transcription factors play a key role in modulating the response of immunoregulatory genes including cytokines and chemokines, cell adhesion molecules, acute phase proteins, and anti-microbial peptides. Furthermore, an array of genes important for angiogenesis, tumor invasion and metastasis is also regulated by nuclear factor-κB (NF-κB). Close association of NF-κB with inflammation and tumorigenesis makes it an attractive target for basic research as well as for pharmaceutical industries. Studies involving various animal and cellular models have revealed the importance of NF-κB in pathobiology of lung diseases. This review (a) describes structures, activities, and regulation of NF-κB family members; (b) provides information which implicates NF-κB in pathogenesis of pulmonary inflammation and cancer; and (c) discusses information about available synthetic and natural compounds which target NF-κB or specific components of NF-κB signal transduction pathway and which may provide the foundation for development of effective therapy for lung inflammation and bronchogenic carcinomas.

Receptor-Interacting Protein 2 Controls Pulmonary Host Defense to Escherichia coli Infection via the Regulation of Interleukin-17A

ABSTRACT Recognition of microbial patterns by host receptors is the first step in a multistep sequence leading to neutrophil-dependent host resistance. Although the role of membrane-bound sensors in bacterial recognition has been examined in detail, the importance of cytosolic sensors in the lungs is largely unexplored. In this context, there is a major lack of understanding related to the downstream signaling mediators, such as cells and/or molecules, during acute extracellular Gram-negative bacterial pneumonia. In order to determine the role of NOD-like receptors (NLRs), we used an experimental Escherichia coli infection model using mice deficient in the gene coding for the NLR adaptor, receptor-interacting protein 2 (RIP2). RIP2−/− mice with E. coli infection displayed higher bacterial burden and reduced neutrophil recruitment and tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), macrophage inflammatory protein 2 (MIP-2), and CXCL5/LIX expression, along with attenuated histopathological changes in the lungs. Decreased IL-17A levels were observed, along with lower numbers of IL-17A-producing T cells, in RIP2−/− mice after infection. RIP2−/− mice also show reduced IL-6 and IL-23 levels in the lungs, along with decreased activation of STAT3 after infection. Furthermore, activation of NF-κB and mitogen-activated protein kinases (MAPKs) and expression of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) in the lungs of infected RIP2−/− mice were attenuated following infection. Although neutrophil mobilization to the blood was impaired in RIP2−/− mice following infection, the expression of CD62P, CD11a/18, CD11b, and CXCR2 on blood and lung neutrophils was not altered between infected wild-type (WT) and RIP2−/− mice. Thus, RIP2 contributes to neutrophil-dependent host defense against an extracellular Gram-negative pathogen via (i) IL-17A regulation and (ii) neutrophil mobilization to the blood.

Role of CXCL5 in Leukocyte Recruitment to the Lungs during Secondhand Smoke Exposure

Chronic obstructive pulmonary disease (COPD) is the third leading cause of mortality in the United States. The major cause of COPD is cigarette smoking. Extensive leukocyte influx into the lungs, mediated by chemokines, is a critical event leading to COPD. Although both resident and myeloid cells secrete chemokines in response to inflammatory stimuli, little is known about the role of epithelial-derived chemokines, such as CXC chemokine ligand (CXCL)5, in the pathogenesis of cigarette smoke-induced inflammation. To explore the role of CXCL5, we generated CXCL5 gene-deficient mice and exposed them to secondhand smoke (SHS) for 5 hours/day for 5 days/week up to 3 weeks (subacute exposure). We observed a reduced recruitment of leukocytes to the lungs of CXCL5(-/-) mice compared with their wild-type (WT) counterparts, and noted that macrophages comprised the predominant leukocytes recruited to the lungs. Irradiation experiments performed on CXCL5(-/-) or WT mice transplanted with WT or CXCL5(-/-) bone marrow revealed that resident but not hematopoietic cell-driven CXCL5 is important for mediating SHS-induced lung inflammation. Interestingly, we observed a significant reduction of monocyte chemotactic protein-1 (MCP-1/CC chemokine ligand 2) concentrations in the lungs of CXCL5(-/-) mice. The instillation of recombinant MCP-1 in CXCL5(-/-) mice reversed macrophage recruitment. Our results also show the reduced activation of NF-κB/p65 in the lungs, as well as the attenuated activation of C-Jun N-terminal kinase, p42/44, and p38 mitogen-activated protein kinases and the expression of intercellular adhesion molecule-1 in the lungs of SHS-exposed CXCL5(-/-) mice. Our findings suggest an important role for CXCL5 in augmenting leukocyte recruitment in SHS-induced lung inflammation, and provide novel insights into CXCL5-driven pathogenesis.

NOD2 Signaling Contributes to Host Defense in the Lungs against Escherichia coli Infection

ABSTRACT Bacterial pneumonia remains a significant cause of mortality in the United States. The innate immune response is the first line of defense against invading bacteria. Neutrophil recruitment to the lungs is the first step in a multistep sequence leading to bacterial clearance. Ligand interaction with pattern-recognizing receptors (PRRs) leads to chemokine production, which drives neutrophils to the site of infection. Although we demonstrated that RIP2 is important for host defense in the lungs against Escherichia coli, the individual roles of NOD1 and NOD2 in pulmonary defense have not been addressed. Here, we explored the role of NOD2 in neutrophil-mediated host defense against an extracellular pathogen, E. coli. We found enhanced bacterial burden and reduced neutrophil and cytokine/chemokine levels in the lungs of NOD2−/− mice following E. coli infection. Furthermore, we observed reduced activation of NF-κB and mitogen-activated protein kinases (MAPKs) in the lungs of NOD2−/− mice upon E. coli challenge. Moreover, NOD2−/− neutrophils show impaired intracellular bacterial killing. Using NOD2/RIP2−/− mice, we observed bacterial burden and neutrophil accumulation in the lungs similar to those seen with NOD2−/− mice. In addition, bone marrow-derived macrophages obtained from NOD2/RIP2−/− mice demonstrate a reduction in activation of NF-κB and MAPKs similar to that seen with NOD2−/− mice in response to E. coli. These findings unveil a previously unrecognized role of the NOD2-RIP2 axis for host defense against extracellular Gram-negative bacteria. This pathway may represent a novel target for the treatment of lung infection/inflammation.

The Armadillo as a Model for Peripheral Neuropathy in Leprosy

Leprosy (also known as Hansens Disease) is a chronic infectious disease caused by Mycobacterium leprae that primarily targets the peripheral nervous system; skin, muscle, and other tissues are also affected. Other than humans, nine-banded armadillos (Dasypus novemcinctus) are the only natural hosts of M. leprae, and they are the only laboratory animals that develop extensive neurological involvement with this bacterium. Infection in the armadillo closely recapitulates many of the structural, physiological, and functional aspects of leprosy seen in humans. Armadillos can be useful models of leprosy for basic scientific investigations into the pathogenesis of leprosy neuropathy and its associated myopathies, as well as for translational research studies in piloting new diagnostic methods or therapeutic interventions. Practical and ethical constraints often limit investigation into human neuropathies, but armadillos are an abundant source of leprotic neurologic fibers. Studies with these animals may provide new insights into the mechanisms involved in leprosy that also might benefit the understanding of other demyelinating neuropathies. Although there is only a limited supply of armadillo-specific reagents, the armadillo whole genomic sequence has been completed, and gene expression studies can be employed. Clinical procedures, such as electrophysiological nerve conduction testing, provide a functional assessment of armadillo nerves. A variety of standard histopathological and immunopathological procedures including Epidermal Nerve Fiber Density (ENFD) analysis, Schwann Cell Density, and analysis for other conserved cellular markers can be used effectively with armadillos and will be briefly reviewed in this text.