Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.     

5-Lipoxygenase is a key determinant of acute myocardial inflammation and mortality during Trypanosoma cruzi infection

This study provides evidence supporting the idea that although inflammatory cells migration to the cardiac tissue is necessary to control the growth of Trypanosoma cruzi, the excessive influx of such cells during acute myocarditis may be deleterious to the host. Production of lipid mediators of inflammation like leukotrienes (LTs) along with cytokines and chemokines largely influences the severity of inflammatory injury in response to tissue parasitism. T. cruzi infection in mice deficient in 5-lipoxygenase (5-LO), the enzyme responsible for the synthesis of LTs and other lipid inflammatory mediators, resulted in transiently increased parasitemia, and improved survival rate compared with WT mice. Myocardia from 5-LO^?/? mice exhibited reduced inflammation, collagen deposition, and migration of CD4⁺, CD8⁺, and IFN-γ-producer cells compared with WT littermates. Moreover, decreased amounts of TNF-α, IFN-γ, and nitric oxide synthase were found in the hearts of 5-LO^?/? mice. Interestingly, despite of early higher parasitic load, 5-LO^?/? mice survived, and controlled T. cruzi infection. These results show that efficient parasite clearance is possible in a context of moderate inflammatory response, as occurred in 5-LO^?/? mice, in which reduced myocarditis protects the animals during T. cruzi infection.     

Painful stimulation suppresses joint inflammation by inducing shedding of L-selectin from neutrophils.

Although the inflammatory response is essential for protecting tissues from injury and infection, unrestrained inflammation can cause chronic inflammatory diseases such as arthritis, colitis and asthma. Physiological mechanisms that downregulate inflammation are poorly understood. Potent control might be achieved by regulating early stages in the inflammatory response, such as accumulation of neutrophils at the site of injury, where these cells release chemical mediators that promote inflammatory processes including plasma extravasation, bacteriocide and proteolysis. To access an inflammatory site, neutrophils must first adhere to the vascular endothelium in a process mediated in part by the leukocyte adhesion molecule L-selectin. This adhesion is prevented when L-selectin is shed from the neutrophil membrane. Although shedding of L-selectin is recognized as a potentially important mechanism for regulating neutrophils, its physiological function has not been demonstrated. Shedding of L-selectin may mediate endogenous downregulation of inflammation by limiting neutrophil accumulation at inflammatory sites. Here we show that activation of nociceptive neurons induces shedding of L-selectin from circulating neutrophils in vivo and that this shedding suppresses an ongoing inflammatory response by inhibiting neutrophil accumulation. These findings indicate a previously unknown mechanism for endogenous feedback control of inflammation. Failure of this mechanism could contribute to the etiology of chronic inflammatory disease.     

Galectins as inflammatory mediators

Over the last decade a vast amount of reports have shown that galectin-1 and galectin-3 are important mediators of inflammation. In this review we describe how the galectins may be involved in several parts of the inflammatory process, including the recruitment of neutrophils into an infected tissue and the recognition and killing of bacteria by activation of the tissue destructive phagocytic respiratory burst. During bacterial infection or aseptic inflammatory processes, galectins are produced and released by e.g. infected epithelium, activated tissue-resident macrophages and endothelial cells. These extracellular galectins may facilitate binding of neutrophils to the endothelium by cross-linking carbohydrates on the respective cells. Further the galectins improve binding of the neutrophil to the extracellular matrix proteins laminin and fibronectin, and are potential chemotactic factors, inducing migration through the extracellular matrix towards the inflammatory focus. When the cells encounter bacteria, galectin-3 could function as an opsonin, cross-linking bacterial lipopolysaccharide or other carbohydrate-containing surface structures to phagocyte surface glycoconjugates. Both galectin-1 and galectin-3 have the capacity to induce a respiratory burst in neutrophils, provided that the cells have been primed by degranulation and receptor upregulation. The reactive oxygen species produced may be destructive to the invading micro-organisms as well as to the surrounding host tissue, pointing out the possible role of galectins, not only in defence toward infection, but also in inflammatory-induced tissue destruction. Published in 2004.     

Galectin-3 Functions as an Alarmin: Pathogenic Role for Sepsis Development in Murine Respiratory Tularemia

Sepsis is a complex immune disorder with a mortality rate of 20–50% and currently has no therapeutic interventions. It is thus critical to identify and characterize molecules/factors responsible for its development. We have recently shown that pulmonary infection with Francisella results in sepsis development. As extensive cell death is a prominent feature of sepsis, we hypothesized that host endogenous molecules called alarmins released from dead or dying host cells cause a hyperinflammatory response culminating in sepsis development. In the current study we investigated the role of galectin-3, a mammalian β-galactoside binding lectin, as an alarmin in sepsis development during F. novicida infection. We observed an upregulated expression and extracellular release of galectin-3 in the lungs of mice undergoing lethal pulmonary infection with virulent strain of F. novicida but not in those infected with a non-lethal, attenuated strain of the bacteria. In comparison with their wild-type C57Bl/6 counterparts, F. novicida infected galectin-3 deficient (galectin-3^−/−) mice demonstrated significantly reduced leukocyte infiltration, particularly neutrophils in their lungs. They also exhibited a marked decrease in inflammatory cytokines, vascular injury markers, and neutrophil-associated inflammatory mediators. Concomitantly, in-vitro pre-treatment of primary neutrophils and macrophages with recombinant galectin-3 augmented F. novicida-induced activation of these cells. Correlating with the reduced inflammatory response, F. novicida infected galectin-3^−/− mice exhibited improved lung architecture with reduced cell death and improved survival over wild-type mice, despite similar bacterial burden. Collectively, these findings suggest that galectin-3 functions as an alarmin by augmenting the inflammatory response in sepsis development during pulmonary F. novicida infection.     

Aberrant Innate Immune Activation following Tissue Injury Impairs Pancreatic Regeneration

Normal tissue architecture is disrupted following injury, as resident tissue cells become damaged and immune cells are recruited to the site of injury. While injury and inflammation are critical to tissue remodeling, the inability to resolve this response can lead to the destructive complications of chronic inflammation. In the pancreas, acinar cells of the exocrine compartment respond to injury by transiently adopting characteristics of progenitor cells present during embryonic development. This process of de-differentiation creates a window where a mature and stable cell gains flexibility and is potentially permissive to changes in cellular fate. How de-differentiation can turn an acinar cell into another cell type (such as a pancreatic β-cell), or a cell with cancerous potential (as in cases of deregulated Kras activity) is of interest to both the regenerative medicine and cancer communities. While it is known that inflammation and acinar de-differentiation increase following pancreatic injury, it remains unclear which immune cells are involved in this process. We used a combination of genetically modified mice, immunological blockade and cellular characterization to identify the immune cells that impact pancreatic regeneration in an in vivo model of pancreatitis. We identified the innate inflammatory response of macrophages and neutrophils as regulators of pancreatic regeneration. Under normal conditions, mild innate inflammation prompts a transient de-differentiation of acinar cells that readily dissipates to allow normal regeneration. However, non-resolving inflammation developed when elevated pancreatic levels of neutrophils producing interferon-γ increased iNOS levels and the pro-inflammatory response of macrophages. Pancreatic injury improved following in vivo macrophage depletion, iNOS inhibition as well as suppression of iNOS levels in macrophages via interferon-γ blockade, supporting the impairment in regeneration and the development of chronic inflammation arises from aberrant activation of the innate inflammatory response. Collectively these studies identify targetable inflammatory factors that can be used to influence the development of non-resolving inflammation and pancreatic regeneration following injury.     

Galectins in parasite infection and allergic inflammation

Galectins are increasingly recognised as important immunological mediators of homeostasis and disease regulation. This paper gives an overview of current knowledge of galectin involvement in parasite infection and allergic inflammation, two very different but immunologically linked phenomena. Galectins are produced by both the parasite and the host and appear to be intimately involved in parasite establishment, as well as directing the course of infection and the immune response. Host galectins have also been shown to be active participants in the recruitment of cells to sites of inflammation and modulating the effector function of mast cells, neutrophils and eosinophils. Moreover, the ability of galectins to induce differential expression of cytokine genes in leukocytes suggests that they are able to direct the nature of an adaptive immune response, in particular towards a T2-type allergic response. Published in 2004.     

The heterogenic properties of monocytes/macrophages and neutrophils in inflammatory response in diabetes

Inflammation is a complicated biological process in response to harmful stimuli, which involves the cooperation of immune system and vascular system. Upon pathogen invasion or tissue injury, resident innate immune cells such as macrophages and dendritic cells are activated and release inflammatory mediators, which result in the vasodilation and recruitment of leukocytes, mainly monocytes and neutrophils. As two of the most important inflammation-mediating immune cells, macrophages and neutrophils have long been regarded to have a pro-inflammatory effect. However, increasing evidences suggest the role of macrophage and neutrophil in inflammation is more complicated and diversified than we thought. Differently activated macrophages and neutrophils lead to diverse even opposite activities. Precise understanding of the role of different subpopulations is critical to achieve the effective treatment for inflammatory diseases. In this review, we discuss the two potentially distinct activation routes of macrophages and neutrophils in obesity and diabetes.     

Alarmin Function of Galectin-9 in Murine Respiratory Tularemia

Sepsis is a complex immune disorder that is characterized by systemic hyperinflammation. Alarmins, which are multifunctional endogenous factors, have been implicated in exacerbation of inflammation in many immune disorders including sepsis. Here we show that Galectin-9, a host endogenous β-galactoside binding lectin, functions as an alarmin capable of mediating inflammatory response during sepsis resulting from pulmonary infection with Francisella novicida, a Gram negative bacterial pathogen. Our results show that this galectin is upregulated and is likely released during tissue damage in the lungs of F. novicida infected septic mice. In vitro, purified recombinant galectin-9 exacerbated F. novicida-induced production of the inflammatory mediators by macrophages and neutrophils. Concomitantly, Galectin-9 deficient (Gal-9^-/-) mice exhibited improved lung pathology, reduced cell death and reduced leukocyte infiltration, particularly neutrophils, in their lungs. This positively correlated with overall improved survival of F. novicida infected Gal-9^-/- mice as compared to their wild-type counterparts. Collectively, these findings suggest that galectin-9 functions as a novel alarmin by augmenting the inflammatory response in sepsis development during pulmonary F. novicida infection.     

A search for endogenous mechanisms of anti-inflammation uncovers novel chemical mediators: missing links to resolution

Multicellular responses to infection, injury, or inflammatory stimuli lead to the formation and release of a wide range of local chemical mediators by the host. The integrated response of the host is essential in health and disease, thus it is important to achieve a more complete understanding of the local cellular and molecular events that govern the formation and actions of local mediators that can serve as endogenous counter-regulatory functions in effector cells of the immune system or endogenous local mediators of resolution. Since these compounds in theory and in experimental models of inflammation appear to control the duration and magnitude of inflammation, knowledge of their elucidation could provide new avenues for appreciating the molecular phenotypes of many inflammatory diseases. The first of these endogenous local counter-regulators recognized were the lipoxins, which are trihydroxytetraene-containing lipid mediators that can be formed during cell–cell interactions via transcellular biosynthesis. Since this circuit of lipoxin formation and action appears to be of physiological relevance for the resolution of inflammation, therapeutic modalities targeted at this system are likely to have fewer unwanted side effects acting as agonists than the inhibitor approach currently used in anti-inflammatory therapies. This chapter provides an overview of the recent knowledge about the biosynthesis and bioactions of the novel anti-inflammatory lipid mediators, resolvins, docosatrienes, and neuroprotectins, and their aspirin-triggered counterparts. These novel families of lipid-derived mediators, which carry anti-inflammatory, pro-resolving, and protective properties, were originally isolated during spontaneous resolution. These new pathways open new opportunities for appreciating the role of neutrophils in the generation of potent protective lipid mediators and protective host signaling.     

Signal integration in forward and reverse neutrophil migration: Fundamentals and emerging mechanisms

Neutrophils migrate to sites of tissue damage, where they protect the host against pathogens. Often, the cost of these neutrophil defenses is collateral damage to healthy tissues. Thus, the immune system has evolved multiple mechanisms to regulate neutrophil migration. One of these mechanisms is reverse migration — the process whereby neutrophils leave the source of inflammation. In vivo, neutrophils arrive and depart the wound simultaneously — indicating that neutrophils dynamically integrate conflicting signals to engage in forward and reverse migration. This finding is seemingly at odds with the established chemoattractant hierarchy in vitro, which places wound-derived signals at the top. Here we will discuss recent work that has uncovered key players involved in retaining and dispersing neutrophils from wounds. These findings offer the opportunity to integrate established and emerging mechanisms into a holistic model for neutrophil migration in vivo.     

Impaired innate immune response and enhanced pathology during Citrobacter rodentium infection in mice lacking functional P‐selectin

The selectin family of adhesion molecules mediates recruitment of immune cells to sites of inflammation which is critical for host resistance against infection. To characterize the role of selectins in host defence against Citrobacter rodentium infection, wild‐type (WT) mice and mice lacking P‐selectin glycoprotein ligand‐1 (PSGL‐1), P‐, E‐ and L‐selectin were infected using a Citrobacter‐induced colitis model. Infected mice lacking PSGL‐1 or P‐selectin showed a more pronounced morbidity associated with higher bacterial load, elevated IL‐12 p70, TNF‐α, IFN‐γ, MCP‐1 and IL‐6 production, more severe inflammation and surprisingly higher leucocyte infiltration in the guts than WT control. Recruitment of neutrophils and macrophages and caecal inflammation were drastically reduced in infected P‐selectin knockout mice receiving blocking monoclonal antibodies to ICAM‐1 or LFA‐1, indicating that these adhesion molecules may compensate for the loss of selectins in leucocyte recruitment. Furthermore, the adaptive immune response in mice lacking PSGL‐1 or P‐selectin remained functional since these infected mice were capable of eradicating the bacteria and being protected upon re‐challenge with C. rodentium. These data demonstrate a definitive phenotypic impairment of innate response in mice lacking PSGL‐1 or P‐selectin, and suggest that these adhesion molecules are important in host innate immune response against Citrobacter infection.     

Live Brugia malayi Microfilariae Inhibit Transendothelial Migration of Neutrophils and Monocytes

Lymphatic filariasis is a major tropical disease caused by the parasite Brugia malayi. Microfilariae (Mf) circulate in the peripheral blood for 2–3 hours in synchronisation with maximal feeding of the mosquito vector. When absent from the peripheral blood, Mf sequester in the capillaries of the lungs. Mf are therefore in close contact with vascular endothelial cells (EC) and may induce EC immune function and/or wound repair mechanisms such as angiogenesis. In this study, Mf were co-cultured with human umbilical vein EC (HUVEC) or human lung microvascular EC (HLMVEC) and the transendothelial migration of leukocyte subsets was analysed. In addition, the protein and/or mRNA expression of chemokine, cytokine and angiogenic mediators in endothelial cells in the presence of live microfilariae were measured by a combination of cDNA arrays, protein arrays, ELISA and fluorescence antibody tests.Surprisingly, our findings indicate that Mf presence partially blocked transendothelial migration of monocytes and neutrophils, but not lymphocytes. However, Mf exposure did not result in altered vascular EC expression of key mediators of the tethering stage of extravasation, such as ICAM-1, VCAM-1 and various chemokines. To further analyse the immunological function of vascular EC in the presence of Mf, we measured the mRNA and/or protein expression of a number of pro-inflammatory mediators. We found that expression levels of the mediators tested were predominantly unaltered upon B. malayi Mf exposure. In addition, a comparison of angiogenic mediators induced by intact Mf and Wolbachia-depleted Mf revealed that even intact Mf induce the expression of remarkably few angiogenic mediators in vascular EC. Our study suggests that live microfilariae are remarkably inert in their induction and/or activation of vascular cells in their immediate local environment. Overall, this work presents important insights into the immunological function of the vascular endothelium during an infection with B. malayi.     

The role of neutrophils and inflammation in gastric mucosal injury

Gastric inflammation is a highly complex biochemical protective response to cellular/tissue injury. When this process occurs in an uncontrolled manner, the result is excessive cellular/tissue damage that results chronic inflammation and destruction of normal tissue. Current evidence suggests that Helicobacter pylori (H. pylori) infection and nonsteroidal anti-inflammatory drug (NSAID) ingestion are major causative factors in the pathogenesis of gastric mucosal injury in humans. In response to H. pylori infection or NSAID, neutrophils are recruited to the site of inflammation and generate reactive oxygen and nitrogen species and proteases. However, neutrophils are not able to kill the bacteria that live in the gastric mucus, and compounds produced by activated neutrophils themselves may be potentially harmful for normal tissue. It has been shown that leukocyte-vascular endothelial cell interaction is regulated by various cell adhesion molecules, and that this interaction is directly or indirectly modified by many factors, the origin of which is H. pylori and NSAIDs. This review describes the potential role of neutrophils and neutrophil-associated inflammation for gastric oxidative stress and injury induced by H. pylori and/or NSAID.     

The complex roles of neutrophils in APAP‐induced liver injury

Acetaminophen (APAP) is a widely applied drug for the alleviation of pain and fever, which is also a dose‐depedent toxin. APAP‐induced acute liver injury has become one of the primary causes of liver failure which is an increasingly serious threat to human health. Neutrophils are the major immune cells in human serving as the first barrier against the invasion of pathogen. It has been reported that neutrophils patriciate in the occurrence and development of APAP‐induced liver injury. However, evolving evidences suggest that neutrophils also contribute to tissue repair and actively orchestrate resolution of inflammation. Here, we addressed the complex roles in APAP‐induced liver injury on the basis of brief introduction of neutrophil''s activation, recruitment and migration.

A lot of factors deeply influence the neutrophil''s activation, recruitment and migration in APAP‐induced liver injury. DAMPs like HMGB1 and ATP are released from damaged hepatocytes, recognized by RAGE, P2Y2 receptors, respectively, inducing the activation and recruitment of neutrophils. But the TLR9 expressed on neutrophils recognize mtDNA and then neutrophils could release miR‐223, which inhibits the neutrophil''s activation and recruitment. As for other immune cells, they are divided into cells that promote and inhibit the activation and recruitment of neutrophils. Firstly, macrophages express OPN after APAP treatment and then attract neutrophils. Moreover, HMGB1 could recognize TLR4 receptor expressed on macrophages, release IL‐23 to induce the release of IL‐17A in γδ T cells and finally activate and recruit neutrophils. However, Ly6C^hi monocytes inhibit the activation and recruitment of neutrophils via CCR2 and M‐CSF pathways. Macrophages could also generate MerTK to inhibit neutrophils. Furthermore, TNF‐α/LPS MDSCs could express iNOS to decrease the intrahepatic neutrophils infiltration and induce the apoptosis of activated neutrophil. Activated and recruited neutrophils then migrate to the site of injury to alleviate the inflammation and induce tissue repair. GRPR antagonist could downregulate the expression of CD11b/CD66b via activating MAPKs pathways, which significantly influence the migration of neutrophils to the injury site.     

Cells, cytokines and cellular immunity in the pathogenesis of fibroproliferative vasculopathies

Atherosclerosis and restenosis are the result of vascular injury followed by an inflammatory and fibroproliferative response that involves a large number of growth factors, cytokines, and cellular elements. Platelet activation and leukocyte recruitment into the arterial intima play a crucial role, initiating a whole spectrum of reactions leading to vascular smooth muscle cell hyperplasia and intimal migration. The roles of macrophages and lymphocytes and mast cells as mediators of inflammation and immune response is discussed, as are the roles of growth factors and cytokines. New light on the 'old' problems will help us to devise newer and better therapeutic strategies to combat these clinical entities.     

Effects of Porphyromonas gingivalis LipopolysaccharideTolerized Monocytes on Inflammatory Responses in Neutrophils

Periodontitis is a chronic inflammatory disease induced by bacteria. Exposure of the host to periodontal pathogens and their virulence factors induces a state of hyporesponsiveness to subsequent stimulations, which is termed endotoxin tolerance. The role and mechanism of lipopolysaccharide (LPS)–tolerized monocytes in inflammatory responses in neutrophils are currently unclear. Here, conditioned supernatants were collected from THP-1 cells treated with or without repeated 1 μg/ml Porphyromonas gingivalis (P.gingivalis) LPS. The chemotactic response of freshly isolated neutrophils recruited by supernatants was determined by a transwell migration assay, which demonstrated a reduced migration of neutrophils stimulated with supernatants from tolerized THP-1 cells in comparison to non-tolerized THP-1 cells. In addition, there was a marked increase in reactive oxygen species (ROS) generation and a significant decrease in Caspase 3 activities in neutrophils treated with supernatants from THP-1 cells that were treated repeatedly with P.gingivalis LPS in comparison to single treatment. A cytokine antibody array was then used to assess cytokine expression patterns in THP-1 cells. In tolerized THP-1 cells, 43 cytokine (43/170) expression levels were decreased, including chemokine ligand 23 (CCL23) and IFN-γ, while 11 cytokine (11/170) expression levels were increased, such as death receptor 6 (DR6). Furthermore, there was decreased production of IFN-γ and epithelial neutrophil activating peptide-78 (ENA-78) in THP-1 cells after stimulation with repeated P. gingivalis LPS in comparison to single challenge, which was confirmed by ELISA. Therefore, P.gingivalis LPS- tolerized THP-1 cells were able to depress neutrophil chemotaxis and apoptosis, and contribute to respiratory burst, which might be related to the changes in cytokine expression patterns in THP-1 cells.     

Functional features of lymphocytes recovered from a human renal allograft

Lymphocytes recovered from a rejected human renal allograft were cultured in vitro for their ability to produce several soluble mediators associated with cellular hypersensitivity as well as a procoagulantlike material. In addition, their response in mixed lymphocyte culture was tested. These lymphocytes were of recipient origin by sex karyotyping. An alteration in their proliferative capacity could be demonstrated by an earlier response in mixed lymphocyte cultures although peak response was essentially unchanged. Cultured supernatants from recipient lymphocytes (recovered from the rejected kidney) contained several mediator activities—macrophage migration inhibitory factor, chemotactic factors for neutrophils and macrophages, a factor mitogenic for lymphocytes, as well as a procoagulant material. These findings extend previous work of others who have demonstrated the presence of lymphocytes infiltrating allografts by showing that these cells are immunologically reactive in vitro.     

Dynamic optimization reveals alveolar epithelial cells as key mediators of host defense in invasive aspergillosis

Aspergillus fumigatus is an important human fungal pathogen and its conidia are constantly inhaled by humans. In immunocompromised individuals, conidia can grow out as hyphae that damage lung epithelium. The resulting invasive aspergillosis is associated with devastating mortality rates. Since infection is a race between the innate immune system and the outgrowth of A. fumigatus conidia, we use dynamic optimization to obtain insight into the recruitment and depletion of alveolar macrophages and neutrophils. Using this model, we obtain key insights into major determinants of infection outcome on host and pathogen side. On the pathogen side, we predict in silico and confirm in vitro that germination speed is an important virulence trait of fungal pathogens due to the vulnerability of conidia against host defense. On the host side, we found that epithelial cells, which have been underappreciated, play a role in fungal clearance and are potent mediators of cytokine release. Both predictions were confirmed by in vitro experiments on established cell lines as well as primary lung cells. Further, our model affirms the importance of neutrophils in invasive aspergillosis and underlines that the role of macrophages remains elusive. We expect that our model will contribute to improvement of treatment protocols by focusing on the critical components of immune response to fungi but also fungal virulence traits.     

The two-component sensor response regulator RoxS/RoxR plays a role in Pseudomonas aeruginosa interactions with airway epithelial cells

Pseudomonas aeruginosa is an opportunistic pathogen that infects the lungs of patients with cystic fibrosis causing aberrant and destructive neutrophil (PMN)-dominated inflammation of airways. Interaction of P. aeruginosa with the lung epithelial barrier resulting in trans-epithelial PMN migration likely represents a key event during PMN recruitment. To investigate bacterial factors involved in interactions with lung epithelial cells, a mutant library of two-component system response regulators was evaluated to identify mutants exhibiting defects in the ability to induce PMN trans-epithelial migration. Of forty-eight mutants, five reproducibly demonstrated a reduced PMN trans-epithelial migration response. All five mutants also exhibited a decreased ability to interact with lung epithelial cells. One mutant identified lacks the response regulator gene roxR, which has not previously been reported to be involved regulating factors that facilitate interactions with lung epithelial cells. This finding suggests that RoxR likely regulates genes with relevance to P. aeruginosa mediated lung disease.