Nikolaos M. Nikolaidis

Identification of a Cooperative Mechanism Involving Interleukin-13 and Eotaxin-2 in Experimental Allergic Lung Inflammation

Pulmonary eosinophilia, a hallmark pathologic feature of allergic lung disease, is regulated by interleukin-13 (IL-13) as well as the eotaxin chemokines, but the specific role of these cytokines and their cooperative interaction are only partially understood. First, we elucidated the essential role of IL-13 in the induction of the eotaxins by comparing IL-13 gene-targeted mice with wild type control mice by using an ovalbumin-induced model of allergic airway inflammation. Notably, ovalbumin-induced expressions of eotaxin-1 and eotaxin-2 mRNA in the lungs were almost completely dependent upon IL-13. Second, in order to address the specific role of eotaxin-2 in IL-13-induced pulmonary eosinophilia, we generated eotaxin-2 gene-deficient mice by homologous recombination. Notably, in contrast to observations made in eotaxin-1-deficient mice, eotaxin-2-deficient mice had normal base-line eosinophil levels in the hematopoietic tissues and gastrointestinal tract. However, following intratracheal IL-13 administration, eotaxin-2-deficient mice showed a profound reduction in airway eosinophilia compared with wild type mice. Most interestingly, the level of peribronchial lung tissue eosinophils in IL-13-treated eotaxin-2-deficient mice was indistinguishable from wild type mice. Furthermore, IL-13 lung transgenic mice genetically engineered to be deficient in eotaxin-2 had a marked reduction of luminal eosinophils. Mechanistic analysis identified IL13-induced eotaxin-2 expression by macrophages in a distinct lung compartment (luminal inflammatory cells) compared with eotaxin-1, which was expressed solely in the tissue. Taken together, these results demonstrate a cooperative mechanism between IL-13 and eotaxin-2. In particular, IL-13 mediates allergen-induced eotaxin-2 expression, and eotaxin-2 mediates IL-13-induced airway eosinophilia.

Transcript signatures in experimental asthma: identification of STAT6-dependent and -independent pathways.

The analysis of polygenic diseases such as asthma poses a challenging problem. In an effort to provide unbiased insight into disease pathogenesis, we took an empirical approach involving transcript expression profiling of lung tissue from mice with experimental asthma. Asthmatic responses were found to involve sequential induction of 4.7% of the tested genome; notably, there was ectopic expression of a series of genes not previously implicated in allergic or pulmonary responses. Genes were widely distributed throughout all chromosomes, but preferentially included genes involved in immunity, development, and homeostasis. When asthma was induced by two independent experimental regimens, unique gene transcript profiles were found depending upon the mode of disease induction. However, the majority of genes were common to both models representing an asthma signature genome. Analysis of STAT6-deficient mice revealed that an unexpectedly large segment of the asthma genes were STAT6 independent; this correlated with sustained inflammatory events in these mice. Notably, induction of asthma in STAT6-deficient mice resulted in gene induction not seen in wild-type mice. These results raise concern that therapeutic blockade of STAT6 in the asthmatic setting may reprogram the genetic signature, resulting in alternative lung pathology, which we indeed observed in STAT6-deficient mice. These results provide unprecedented insight into the complex steps involved in the pathogenesis of allergic airway responses; as such, these results have significant therapeutic and clinical implications.

RON RECEPTOR TYROSINE KINASE NEGATIVELY REGULATES TNFα PRODUCTION IN ALVEOLAR MACROPHAGES BY INHIBITING NF-κB ACTIVITY AND ADAM17 PRODUCTION

The Ron receptor tyrosine kinase (TK) plays a regulatory role in the inflammatory response to acute lung injury induced by intranasal administration of bacterial LPS. Previously, we have shown that mice with a targeted deletion of the TK signaling domain of the Ron receptor exhibited more severe lung injury in response to intranasal LPS administration as evidenced by an increased leakage of albumin in the lungs and a greater thickening of the alveolar septa compared with wild-type mice. In addition, lung injury in the Ron TK-deficient (TK−/−) mice was associated with increased activation of the transcription factor, nuclear factor-&kgr;B (NF-&kgr;B), and significantly increased intrapulmonary expression of TNF&agr;. TNF&agr;, a multifunctional proinflammatory cytokine, is a central mediator in several disease states, including rheumatoid arthritis and sepsis. On the basis of the observation that TNF&agr; production is increased in the Ron TK−/− mice and that macrophages are a major source of this cytokine, we hypothesized that the alterations observed in the Ron TK−/− mice may be due, in part, to Ron signaling, specifically in alveolar macrophages. To test this hypothesis, we used the wild-type and Ron TK−/− primary alveolar macrophages and the murine alveolar macrophage cell line, MH-S, to examine the effects of Ron activation on LPS-induced TNF&agr; production and NF-&kgr;B activity. Here, we reported that Ron is expressed on alveolar macrophages and MH-S cells. Activation of Ron by its ligand, hepatocyte growth factor-like protein, decreases TNF&agr; production in alveolar macrophages after LPS challenge. Decreased TNF&agr; is associated with hepatocyte growth factor-like protein-induced decreases in NF-&kgr;B activation and increases in the NF-&kgr;B inhibitory protein, I&kgr;B. We also provided the first evidence for Ron as a negative regulator of Adam17, the metalloprotease involved in TNF&agr; processing. These results indicate that Ron plays a critical role in regulation of alveolar macrophage signaling and validates this receptor as a target in TNF&agr;-mediated pulmonary pathologies.ABBREVIATIONS-HGFL-hepatocyte growth factor-like protein; TK-tyrosine kinase; NF-&kgr;B-nuclear factor &kgr;B; Adam17-a disintegrin and metalloprotease

Mutagenesis of Surfactant Protein D Informed by Evolution and X-ray Crystallography Enhances Defenses against Influenza A Virus in Vivo

Background: SP-D plays important roles in the defense against influenza A. Results: A recombinant SP-D with combinatorial mutations shows enhanced interactions with hemagglutinin-associated glycans and augmented antiviral activity in vitro and in vivo. Conclusion: Exogenous forms of recombinant SP-D can rescue mice from a lethal challenge with influenza. Significance: It may be possible to develop collectin-based interventions for influenza. The recognition of influenza A virus (IAV) by surfactant protein D (SP-D) is mediated by interactions between the SP-D carbohydrate recognition domains (CRD) and glycans displayed on envelope glycoproteins. Although native human SP-D shows potent antiviral and aggregating activity, trimeric recombinant neck+CRDs (NCRDs) show little or no capacity to influence IAV infection. A mutant trimeric NCRD, D325A/R343V, showed marked hemagglutination inhibition and viral neutralization, with viral aggregation and aggregation-dependent viral uptake by neutrophils. D325A/R343V exhibited glucose-sensitive binding to Phil82 hemagglutinin trimer (HA) by surface plasmon resonance. By contrast, there was very low binding to the HA trimer from another virus (PR8) that lacks glycans on the HA head. Mass spectrometry demonstrated the presence of high mannose glycans on the Phil82 HA at positions known to contribute to IAV binding. Molecular modeling predicted an enhanced capacity for bridging interactions between HA glycans and D325A/R343V. Finally, the trimeric D325A/R343V NCRD decreased morbidity and increased viral clearance in a murine model of IAV infection using a reassortant A/WSN/33 virus with a more heavily glycosylated HA. The combined data support a model in which altered binding by a truncated mutant SP-D to IAV HA glycans facilitates viral aggregation, leading to significant viral neutralization in vitro and in vivo. These studies demonstrate the potential utility of homology modeling and protein structure analysis for engineering effective collectin antivirals as in vivo therapeutics.

G-CSF Drives a Posttraumatic Immune Program That Protects the Host from Infection

Traumatic injury is generally considered to have a suppressive effect on the immune system, resulting in increased susceptibility to infection. Paradoxically, we found that thermal injury to the skin induced a robust time-dependent protection of mice from a lethal Klebsiella pneumoniae pulmonary challenge. The protective response was neutrophil dependent and temporally associated with a systemic increase in neutrophils resulting from a reprioritization of hematopoiesis toward myeloid lineages. A prominent and specific activation of STAT3 in the bone marrow preceded the myeloid shift in that compartment, in association with durable increases in STAT3 activating serum cytokines G-CSF and IL-6. Neutralization of the postburn increase in serum G-CSF largely blocked STAT3 activation in marrow cells, reversing the hematopoietic changes and systemic neutrophilia. Daily administration of rG-CSF was sufficient to recapitulate the changes induced by injury including hematopoietic reprioritization and protection from pulmonary challenge with K. pneumoniae. Analysis of posttraumatic gene expression patterns in humans reveals that they are also consistent with a role for G-CSF as a switch that activates innate immune responses and suppresses adaptive immune responses. Our findings suggest that the G-CSF STAT3 axis constitutes a key protective mechanism induced by injury to reduce the risk for posttraumatic infection.

Allergen induced TFF2 is expressed by mucus-producing airway epithelial cells but is not a major regulator of inflammatory responses in the murine lung.

Asthma is a complex pulmonary disorder characterized by reversible airflow obstruction, airway hyperresponsiveness, mucus cell metaplasia, and inflammation. Employing animal models of pulmonary inflammation induced by different allergens and Th2 cytokines, the authors have previously described the up-regulation of trefoil factor 2 (TFF2) in the lung. Given the known biological role of trefoil factors in epithelial restitution, it has been postulated that allergen-induced TFF2 might have an important role in asthmatic responses. Here the authors show that TFF2 is induced early and maintained for 2 weeks following allergen challenge in the mouse lung. In situ mRNA hybridization demonstrated expression of TFF2 primarily in a subset of bronchial epithelial cells and TFF2 immunohistochemistry identified expression in alcian blue–positive bronchial epithelial cells. TFF2 gene–deleted mice inoculated with allergen displayed a 10-fold increase in total cellularity compared with saline controls. Although this response was modestly attenuated compared to wild type controls, the loss of TFF2 did not affect gross levels of tissue inflammation. Furthermore, the loss of TFF2 did not affect induction or resolution of mucus cell metaplasia as measured by periodic acid–Schiff (PAS) or alcian blue staining. Thus, TFF2 is an allergen-induced gene, which is expressed in mucus-positive airways, but is not a major contributor to allergen-induced goblet cell metaplasia, mucus production, or inflammatory responses in the lung.

Modeling pulmonary alveolar microlithiasis by epithelial deletion of the Npt2b sodium phosphate cotransporter reveals putative biomarkers and strategies for treatment

Epithelial deletion of Npt2b results in a tractable mimic of pulmonary alveolar microlithiasis. Casting the first stone for lung disease Pulmonary alveolar microlithiasis (PAM) is a rare lung disease characterized by spherical deposits of calcium phosphate. PAM is thought to be a genetic disorder, and mutations in the gene encoding the NPT2b sodium-dependent phosphate cotransporter have been implicated. Now, Saito et al. delete Npt2b in epithelial cells in mice and observe a disease that mimics human PAM. Whole-lung EDTA can reduce the burden of stones in the lungs, and a low-phosphate diet prevents stone formation. These data support a causative role of Npt2b in PAM and suggest strategies for treatment. Pulmonary alveolar microlithiasis (PAM) is a rare, autosomal recessive lung disorder associated with progressive accumulation of calcium phosphate microliths. Inactivating mutations in SLC34A2, which encodes the NPT2b sodium-dependent phosphate cotransporter, has been proposed as a cause of PAM. We show that epithelial deletion of Npt2b in mice results in a progressive pulmonary process characterized by diffuse alveolar microlith accumulation, radiographic opacification, restrictive physiology, inflammation, fibrosis, and an unexpected alveolar phospholipidosis. Cytokine and surfactant protein elevations in the alveolar lavage and serum of PAM mice and confirmed in serum from PAM patients identify serum MCP-1 (monocyte chemotactic protein 1) and SP-D (surfactant protein D) as potential biomarkers. Microliths introduced by adoptive transfer into the lungs of wild-type mice produce marked macrophage-rich inflammation and elevation of serum MCP-1 that peaks at 1 week and resolves at 1 month, concomitant with clearance of stones. Microliths isolated by bronchoalveolar lavage readily dissolve in EDTA, and therapeutic whole-lung EDTA lavage reduces the burden of stones in the lungs. A low-phosphate diet prevents microlith formation in young animals and reduces lung injury on the basis of reduction in serum SP-D. The burden of pulmonary calcium deposits in established PAM is also diminished within 4 weeks by a low-phosphate diet challenge. These data support a causative role for Npt2b in the pathogenesis of PAM and the use of the PAM mouse model as a preclinical platform for the development of biomarkers and therapeutic strategies.

Ron receptor deficient alveolar myeloid cells exacerbate LPS-induced acute lung injury in the murine lung

Previous studies have shown that the Ron receptor tyrosine kinase is an important regulator of the acute lung inflammatory response induced by intranasal administration of bacterial LPS. Compared to wild-type mice, complete loss of the Ron receptor in all cell types in vivo was associated with increased lung damage as determined by histological analyses and several markers of lung injury including increases in pro-inflammatory cytokines such as TNF-α. Tumor-necrosis factor-α is a multifunctional cytokine secreted by macrophages, which plays a major role in inflammation and is a central mediator of several disease states including rheumatoid arthritis and sepsis. Based on increased TNF-α production observed in the Ron-deficient mice, we hypothesized that Ron receptor function in the inflammatory cell compartment is essential for the regulating lung injury in vivo. To test this hypothesis, we generated myeloid lineage-specific Ron-deficient mice. In this study, we report that loss of Ron signaling selectively in myeloid cells results in increased lung injury following intranasal administration of LPS as measured by increases in TNF-α production, ensuing neutrophil accumulation and increased lung histopathology. These findings corroborate the role of Ron receptor tyrosine kinase as a negative regulator of inflammation and further demonstrate the in vivo significance of Ron signaling selectively in myeloid cells as a major regulator of this response in vivo. These data authenticate Ron as a potential target in innate immunity and TNF-α-mediated pathologies.

Mutations flanking the carbohydrate binding site of surfactant protein D confer antiviral activity for pandemic influenza A viruses.

We recently reported that a trimeric neck and carbohydrate recognition domain (NCRD) fragment of human surfactant protein D (SP-D), a host defense lectin, with combinatorial substitutions at the 325 and 343 positions (D325A+R343V) exhibits markedly increased antiviral activity for seasonal strains of influenza A virus (IAV). The NCRD binds to glycan-rich viral envelope proteins including hemagglutinin (HA). We now show that replacement of D325 with serine to create D325S+R343V provided equal or increased neutralizing activity compared with D325A+R343V. The activity of the double mutants was significantly greater than that of either single mutant (D325A/S or R343V). D325A+R343V and D325S+R343V also strongly inhibited HA activity, and markedly aggregated, the 1968 pandemic H3N2 strain, Aichi68. D325S+R343V significantly reduced viral loads and mortality of mice infected with Aichi68, whereas wild-type SP-D NCRD did not. The pandemic H1N1 strains of 1918 and 2009 have only one N-linked glycan side on the head region of the HA and are fully resistant to inhibition by native SP-D. Importantly, we now show that D325A+R343V and D325S+R343V inhibited Cal09 H1N1 and related strains, and reduced uptake of Cal09 by epithelial cells. Inhibition of Cal09 was mediated by the lectin activity of the NCRDs. All known human pandemic strains have at least one glycan attachment on the top or side of the HA head, and our results indicate that they may be susceptible to inhibition by modified host defense lectins.

Keratinocyte growth factor supports pulmonary innate immune defense through maintenance of alveolar antimicrobial protein levels and macrophage function.

Keratinocyte growth factor (KGF) is an epithelial mitogen that has been reported to protect the lungs from a variety of toxic and infectious insults. In prior studies we found that recombinant human KGF accelerates clearance of bacteria from the murine lung by augmenting the function of alveolar macrophages (AM). In this study we tested the hypothesis that endogenous KGF plays a role in the maintenance of innate pulmonary defense against gram-negative bacterial infections. KGF-deficient mice exhibited delayed clearance of Escherichia coli from the lungs, attenuated phagocytosis by AM, and decreased antimicrobial activity in bronchoalveolar lavage (BAL) fluid, due in part to reductions in levels of surfactant protein A, surfactant protein D, and lysozyme. These immune deficits were accompanied by lower alveolar type II epithelial cell counts and reduced alveolar type II epithelial cell expression of collectin and lysozyme genes on a per cell basis. No significant between-group differences were detected in selected inflammatory cytokines or BAL inflammatory cell populations at baseline or after bacterial challenge in the wild-type and KGF-deficient mice. A single intranasal dose of recombinant human KGF reversed defects in bacterial clearance, AM function, and BAL fluid antimicrobial activity. We conclude that KGF supports alveolar innate immune defense through maintenance of alveolar antimicrobial protein levels and functions of AM. Together these data demonstrate a role for endogenous KGF in maintenance of normal pulmonary innate immune function.