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Intra-tracheal Administration of <em>Haemophilus influenzae</em> in Mouse Models to Study Airway Inflammation

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We ...

BAYLOR RESEARCH INSTITUTE

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.

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

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 B(4) (LTB(4)), 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 LTB(4).

Monocyte chemoattractant protein 1 regulates pulmonary host defense via neutrophil recruitment during Escherichia coli infection.

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.

Receptor-interacting protein 2 controls pulmonary host defense to Escherichia coli infection via the regulation of interleukin-17A.

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.

NOD2 signaling contributes to host defense in the lungs against Escherichia coli infection.

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.

Intrapulmonary G-CSF rescues neutrophil recruitment to the lung and neutrophil release to blood in Gram-negative bacterial infection in MCP-1-/- mice.

Deficiency in the E3 ubiquitin ligase Itch causes a skin-scratching phenotype in mice. We found that there was increased phosphorylation and activation of the mitogen-activated protein kinase p38α in spontaneous and experimentally induced skin lesions of Itch-deficient (Itch-/-) mice. Itch bound directly to the TGF-β-activated kinase 1-binding protein 1 (Tab1) through a conserved PPXY motif and inhibited the activation of p38α. Knockdown of Tab1 by short hairpin RNA attenuated the prolonged p38α phosphorylation exhibited by Itch-/- cells. Similarly, reconstitution of Itch-/- cells with wild-type Itch, but not the ligase-deficient Itch-C830A mutant, inhibited the phosphorylation and activation of p38α. Compared to the skin of wild-type mice, the skin of Itch-/- mice contained increased amounts of the mRNAs of proinflammatory cytokines, including tumor necrosis factor (TNF), interleukin-6 (IL-6), IL-1β, IL-11, and IL-19. Inhibition of p38 or blocking the interaction between p38α and Tab1 with a cell-permeable peptide substantially attenuated skin inflammation in Itch-/- mice. These findings provide insight into how Itch-mediated regulatory mechanisms prevent chronic skin inflammation, which could be exploited therapeutically.

Balamayooran Theivanthiran

Baylor Institute for Immunology Research

BAYLOR RESEARCH INSTITUTE

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

Balamayooran Theivanthiran

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BAYLOR RESEARCH INSTITUTE

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

Baylor Institute for Immunology Research