Selenomonas sputigena Interactions with Gingival Epithelial Cells That Promote Inflammation.

Increased prevalence and abundance of Selenomonas sputigena have been associated with periodontitis, a chronic inflammatory disease of tooth-supporting tissues, for more than 50 years. Over the past decade, molecular surveys of periodontal disease using 16S and shotgun metagenomic sequencing approaches have confirmed the disease association of classically recognized periodontal pathogens such as Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia while highlighting previously underappreciated organisms such as Filifactor alocis and S. sputigena. Despite abundant clinical association between S. sputigena and periodontal disease, we have little to no understanding of its pathogenic potential, and virulence mechanisms have not been studied. In this study, we sought to characterize the response of gingival epithelial cells to infection with S. sputigena. Here, we show that S. sputigena attaches to gingival keratinocytes and induces expression and secretion of cytokines and chemokines associated with inflammation and leukocyte recruitment. We demonstrate that S. sputigena induces signaling through Toll-like receptor 2 (TLR2) and TLR4 but evades activation of TLR5. Cytokines released from S. sputigena-infected keratinocytes induced monocyte and neutrophil chemotaxis. These results show that S. sputigena-host interactions have the potential to contribute to bacterially driven inflammation and tissue destruction, the hallmark of periodontitis. Characterization of previously unstudied pathogens may provide novel approaches to develop therapeutics to treat or prevent periodontal disease.

Tobacco smoke exacerbates Filifactor alocis pathogenicity.

AIM: Filifactor alocis has recently emerged as a periodontal pathobiont that appears to thrive in the oral cavity of smokers. We hypothesized that identification of smoke-responsive F. alocis genes would provide insight into adaptive strategies and that cigarette smoke would enhance F. alocis pathogenesis in vivo. MATERIALS AND METHODS: F. alocis was grown in vitro and cigarette smoke extract-responsive genes determined by RNAseq. Mice were exposed, or not, to mainstream 1R6F research cigarette smoke and infected with F. alocis, or not, in an acute ligature model of periodontitis. Key clinical, infectious, and immune data were collected. RESULTS: In culture, F. alocis growth was unaffected by smoke conditioning and only a small number of genes were specifically regulated by smoke exposure. Reduced murine mass, differences in F. alocis-cognizant antibody production, and altered immune profiles as well as altered alveolar bone loss were all attributable to smoke exposure and/or F. alocis infection in vivo. CONCLUSIONS: F. alocis is well-adapted to tobacco-rich conditions and its pathogenesis is enhanced by tobacco smoke exposure. A smoke-exposed ligature model of periodontitis shows promise as a tool with which to further unravel mechanisms underlying tobacco-enhanced, bacteria-induced disease.

Whole Transcriptome Analysis Reveals That Filifactor alocis Modulates TNFα-Stimulated MAPK Activation in Human Neutrophils.

Periodontitis is an irreversible, bacteria-induced, chronic inflammatory disease that compromises the integrity of tooth-supporting tissues and adversely affects systemic health. As the immune system's first line of defense against bacteria, neutrophils use their microbicidal functions in the oral cavity to protect the host against periodontal disease. However, periodontal pathogens have adapted to resist neutrophil microbicidal mechanisms while still propagating inflammation, which provides essential nutrients for the bacteria to proliferate and cause disease. Advances in sequencing technologies have recognized several newly appreciated bacteria associated with periodontal lesions such as the Gram-positive anaerobic rod, Filifactor alocis. With the discovery of these oral bacterial species, there is also a growing need to assess their pathogenic potential and determine their contribution to disease progression. Currently, few studies have addressed the pathogenic mechanisms used by oral bacteria to manipulate the neutrophil functional responses at the level of the transcriptome. Thus, this study aims to characterize the global changes at the gene expression level in human neutrophils during infection with F. alocis. Our results indicate that the challenge of human neutrophils with F. alocis results in the differential expression of genes involved in multiple neutrophil effector functions such as chemotaxis, cytokine and chemokine signaling pathways, and apoptosis. Moreover, F. alocis challenges affected the expression of components from the TNF and MAPK kinase signaling pathways. This resulted in transient, dampened p38 MAPK activation by secondary stimuli TNFα but not by fMLF. Functionally, the F. alocis-mediated inhibition of p38 activation by TNFα resulted in decreased cytokine production but had no effect on the priming of the respiratory burst response or the delay of apoptosis by TNFα. Since the modulatory effect was characteristic of viable F. alocis only, we propose this as one of F. alocis' mechanisms to control neutrophils and their functional responses.

TLR4 induced Wnt3a-Dvl3 restrains the intensity of inflammation and protects against endotoxin-driven organ failure through GSK3ß/ß-catenin signaling.

BACKGROUND: Accumulating evidence suggests a regulatory role of Wnt proteins in innate immune responses. However, the effects of Wnt3a signaling on TLR4-mediated inflammatory responses are controversial and the signaling crosstalk between TLR4 and Wnt3a remains uncertain. METHODS: Gain- and Loss- of function approaches were utilized to determine the function of Wnt3a signaling in TLR4-mediated inflammatory responses. Cytokine production at protein and mRNA levels and phosphorylation of signaling molecules were measured by ELISA, qRT-PCR, and Western Blot, respectively. Endotoxemia mouse model was employed to assess the effect of Wnt3a on systemic inflammatory cytokine levels and neutrophil infiltration. RESULTS: LPS stimulation leads to an increase of Wnt3a expression and its downstream molecule, Dvl3, in primary monocytes. Inhibition or silence of Wnt3a or Dvl3 significantly increases the production of pro-inflammatory cytokines (IL-12, IL-6, TNFα), robustly reduces ß-catenin accumulation, and enhances the phosphorylation of NF-κB P65 and its DNA binding activity. These results were confirmed by multiple gain- and loss- of function approaches including specific siRNA and ectopic expression of Dvl3, GSK3ß, and ß-catenin in monocytes. Moreover, in vivo relevance was established in a murine endotoxin model, in which Wnt3a inhibition enhances the inflammatory responses by augmenting the systemic pro-inflammatory cytokine levels and neutrophil infiltration. CONCLUSIONS: TLR4 activation promotes Wnt3a-Dvl3 signaling, which acts as rheostats to restrain the intensity of inflammation through regulating GSK3ß-ß-catenin signaling and NF-κB activity. GENERAL SIGNIFICANCE: Wnt3a-Dvl3-ß-catenin signaling axis could be a potential interventional target for manipulating the direction and intensity of inflammatory responses.

The roles of NADPH oxidase in modulating neutrophil effector responses.

Neutrophils are phagocytic innate immune cells essential for killing bacteria via activation of a wide variety of effector responses and generation of large amounts of reactive oxygen species (ROS). Majority of the ROS in neutrophils is generated by activation of the superoxide-generating enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Independent of their anti-microbial function, NADPH oxidase-derived ROS have emerged as key regulators of host immune responses and neutrophilic inflammation. Data from patients with inherited defects in the NADPH oxidase subunit alleles that ablate its enzyme function as well as mouse models demonstrate profound dysregulation of host inflammatory responses, neutrophil hyper-activation and tissue damage in response to microbial ligands or tissue trauma. A large body of literature now demonstrates how oxidants function as essential signaling molecules that are essential for the regulation of neutrophil responses during priming, degranulation, neutrophil extracellular trap formation, and apoptosis, independent of their role in microbial killing. In this review we summarize how NADPH oxidase-derived oxidants modulate neutrophil function in a cell intrinsic manner and regulate host inflammatory responses. In addition, we summarize studies that have elucidated possible roles of oxidants in neutrophilic responses within the oral mucosa and periodontal disease.

Inflammasome-Independent Leukotriene B4 Production Drives Crystalline Silica-Induced Sterile Inflammation.

Silicosis is a lung inflammatory disease caused by chronic exposure to crystalline silica (CS). Leukotriene B4 (LTB4) plays an important role in neutrophilic inflammation, which drives silicosis and promotes lung cancer. In this study, we examined the mechanisms involved in CS-induced inflammatory pathways. Phagocytosis of CS particles is essential for the production of LTB4 and IL-1ß in mouse macrophages, mast cells, and neutrophils. Phagosomes enclosing CS particles trigger the assembly of lipidosome in the cytoplasm, which is likely the primary source of CS-induced LTB4 production. Activation of the JNK pathway is essential for both CS-induced LTB4 and IL-1ß production. Studies with bafilomycin-A1- and NLRP3-deficient mice revealed that LTB4 synthesis in the lipidosome is independent of inflammasome activation. Small interfering RNA knockdown and confocal microscopy studies showed that GTPases Rab5c, Rab40c along with JNK1 are essential for lipidosome formation and LTB4 production. BI-78D3, a JNK inhibitor, abrogated CS-induced neutrophilic inflammation in vivo in an air pouch model. These results highlight an inflammasome-independent and JNK activation-dependent lipidosome pathway as a regulator of LTB4 synthesis and CS-induced sterile inflammation.

Filifactor alocis manipulates human neutrophils affecting their ability to release neutrophil extracellular traps induced by PMA.

Neutrophils operate at the site of injury or inflammation in the periodontal pocket to ensure periodontal health and clearance of bacterial pathogens. Filifactor alocis is recently identified as a potential periodontal pathogen, and in this study, we assessed the formation of neutrophil extracellular traps (NETs), in response to the presence of the organism . NET formation by human neutrophils was not induced when challenged with F. alocis, independent of opsonization, viability, time, or bacterial dose. F. alocis also failed to induce NETs from TNF-α-primed neutrophils and did not induce the release of extracellular neutrophil elastase. However, significant NET induction was observed when neutrophils were challenged with Streptococcus gordonii or Peptoanaerobacter stomatis, In addition, co-infection studies revealed that the presence of F. alocis with S. gordonii or P. stomatis does not enhance or reduce NETs. Additionally, F. alocis failed to impact pre-formed NETs induced by either S. gordonii or P. stomatis. Pretreatment with F. alocis prior to stimulation with phorbol 12-myristate 13-acetate (PMA), S. gordonii, or P. stomatis revealed that the bacterium is capable of reducing only PMA but not S. gordonii or P. stomatis NET formation. These results indicate that F. alocis manipulates neutrophils, inhibiting the triggering of NET induction.

Frontline Science: Tumor necrosis factor-α stimulation and priming of human neutrophil granule exocytosis.

Neutrophil granule exocytosis plays an important role in innate and adaptive immune responses. The present study examined TNF-α stimulation or priming of exocytosis of the 4 neutrophil granule subsets. TNF-α stimulated exocytosis of secretory vesicles and gelatinase granules and primed specific and azurophilic granule exocytosis to fMLF stimulation. Both stimulation and priming of exocytosis by TNF-α were dependent on p38 MAPK activity. Bioinformatic analysis of 1115 neutrophil proteins identified by mass spectrometry as being phosphorylated by TNF-α exposure found that actin cytoskeleton regulation was a major biologic function. A role for p38 MAPK regulation of the actin cytoskeleton was confirmed experimentally. Thirteen phosphoproteins regulated secretory vesicle quantity, formation, or release, 4 of which-Raf1, myristoylated alanine-rich protein kinase C (PKC) substrate (MARCKS), Abelson murine leukemia interactor 1 (ABI1), and myosin VI-were targets of the p38 MAPK pathway. Pharmacologic inhibition of Raf1 reduced stimulated exocytosis of gelatinase granules and priming of specific granule exocytosis. We conclude that differential regulation of exocytosis by TNF-α involves the actin cytoskeleton and is a necessary component for priming of the 2 major neutrophil antimicrobial defense mechanisms: oxygen radical generation and release of toxic granule contents.

A Pseudomonas aeruginosa hepta-acylated lipid A variant associated with cystic fibrosis selectively activates human neutrophils.

Pseudomonas aeruginosa (PA) infection in cystic fibrosis (CF) lung disease causes airway neutrophilia and hyperinflammation without effective bacterial clearance. We evaluated the immunostimulatory activities of lipid A, the membrane anchor of LPS, isolated from mutants of PA that synthesize structural variants, present in the airways of patients with CF, to determine if they correlate with disease severity and progression. In a subset of patients with a severe late stage of CF disease, a unique hepta-acylated lipid A, hepta-1855, is synthesized. In primary human cell cultures, we found that hepta-1855 functioned as a potent TLR4 agonist by priming neutrophil respiratory burst and stimulating strong IL-8 from monocytes and neutrophils. hepta-1855 also had a potent survival effect on neutrophils. However, it was less efficient in stimulating neutrophil granule exocytosis and also less potent in triggering proinflammatory TNF-α response from monocytes. In PA isolates that do not synthesize hepta-1855, a distinct CF-specific adaptation favors synthesis of a penta-1447 and hexa-1685 LPS mixture. We found that penta-1447 lacked immunostimulatory activity but interfered with inflammatory IL-8 synthesis in response to hexa-1685. Together, these observations suggest a potential contribution of hepta-1855 to maintenance of the inflammatory burden in late-stage CF by recruiting neutrophils via IL-8 and promoting their survival, an effect presumably amplified by the absence of penta-1447. Moreover, the relative inefficiency of hepta-1855 in triggering neutrophil degranulation may partly explain the persistence of PA in CF disease, despite extensive airway neutrophilia.

Resolvin D1, resolvin D2 and maresin 1 activate the GSK3ß anti-inflammatory axis in TLR4-engaged human monocytes.

Pro-resolving, docosahexaenoic acid-derived mediators have recently emerged as important potential therapeutic agents for the amelioration of complications arising from inflammation, such as vascular disease, asthma, acute lung injury and colitis. While resolvin D1 (RVD1), resolvin D2 (RVD2) and maresin 1 (MaR1) are established pro-resolvins, their mechanisms of action remain unclear. Here we show that, in LPS-stimulated primary human monocytes, RVD1, RVD2 and MaR1 each suppress the release of pro-inflammatory cytokines (TNF, IL-1ß, IL-8) and the innate/adaptive bridging cytokine, IL-12 p40, while simultaneously augmenting the production of the anti-inflammatory cytokine, IL-10. Such resolving activity is accompanied by the increased phosphorylation (enhanced anti-inflammatory state) of glycogen synthase kinase 3ß (GSK3ß) along with increased phosphorylation (activation) of Akt, SGK1 and CREB but not MAPK-related molecules. Gain and loss of function experiments confirm a key role for GSK3ß and CREB in the anti-inflammatory actions of resolvins. These results suggest that induction of the GSK3ß anti-inflammatory axis is a common mechanism of action for RVD1, RVD2 and MaR1.

Macrolide Use and Neutrophil Function/Cytokine Levels in Hospitalized Patients with Community-Acquired Pneumonia: A Pilot Study.

PURPOSE: The objective of this study was to measure plasma cytokine levels and blood neutrophil functions as well as clinical outcomes in hospitalized patients with community-acquired pneumonia (CAP) treated with or without macrolide use--a known modulator of inflammatory response. METHODS: Subjects with CAP had peripheral blood analyzed for some neutrophil functions (degranulation of secretory vesicles and specific granules, respiratory burst response and phagocytosis) and ten cytokine levels measured in serum and sputum supernatants. Neutrophil function in healthy volunteers was also measured for reference. Values were measured on the day of enrollment, days 2-4 and 5-7, depending on a patient's length of stay. Early and late clinical outcomes were also evaluated. All values were compared between those treated with or without a macrolide. RESULTS: A total of 40 subjects were in this study; 14 received macrolide treatment, and 26 did not. Neutrophil function in the macrolide group was not significantly different compared to the non-macrolide group. None of the median cytokine levels or IQRs were statistically significant between the groups. However, a trend toward decreased IL-6, IL-8, and IFN-γ levels, and favorable clinical outcomes were present in the macrolide group. CONCLUSIONS: This pilot study showed no statistical difference between cytokine levels or neutrophil activity for CAP patients prescribed a macrolide containing regimen. Considering the trend of lower cytokine levels in the macrolide group when comparing the 5- to 7-day time period with the non-macrolide group, a full study with an appropriate sample size may be warranted.

Filifactor alocis infection and inflammatory responses in the mouse subcutaneous chamber model.

Recent microbiome studies have implicated a role for Filifactor alocis in periodontal disease. In this study, we investigated the colonization and survival properties of F. alocis in a mouse subcutaneous chamber model of infection and characterized host innate immune responses. An infection of 10(9) F. alocis successfully colonized all chambers; however, the infection was cleared after 72 h. F. alocis elicited a local inflammatory response with neutrophils recruited into the chambers at 2 h postinfection along with an increase in levels of the proinflammatory cytokines interleukin 1ß (IL-1ß), IL-6, and tumor necrosis factor (TNF). F. alocis also induced apoptosis in chamber epithelial cells and neutrophils. Consistent with resolution of infection, neutrophil numbers and cytokine levels returned to baseline by 72 h. Fluorescent in situ hybridization (FISH) and quantitative PCR demonstrated that F. alocis exited the chambers and spread to the spleen, liver, lung, and kidney. Massive neutrophil infiltration was observed in the spleen and lungs, and the recruited neutrophils were in close proximity to the infecting bacteria. Significant epithelial injury was observed in the kidneys. Infection of all tissues was resolved after 7 days. This first in vivo study of the pathogenicity of F. alocis shows that in the chamber model the organism can establish a proinflammatory, proapoptotic local infection which is rapidly resolved by the host concordant with neutrophil influx. Moreover, F. alocis can spread to, and transiently infect, remote tissues where neutrophils can also be recruited.

The pore-forming toxin listeriolysin O is degraded by neutrophil metalloproteinase-8 and fails to mediate Listeria monocytogenes intracellular survival in neutrophils.

The pore-forming toxin listeriolysin O (LLO) is a major virulence factor secreted by the facultative intracellular pathogen Listeria monocytogenes. This toxin facilitates L. monocytogenes intracellular survival in macrophages and diverse nonphagocytic cells by disrupting the internalization vesicle, releasing the bacterium into its replicative niche, the cytosol. Neutrophils are innate immune cells that play an important role in the control of infections, yet it was unknown if LLO could confer a survival advantage to L. monocytogenes in neutrophils. We report that LLO can enhance the phagocytic efficiency of human neutrophils and is unable to protect L. monocytogenes from intracellular killing. To explain the absence of L. monocytogenes survival in neutrophils, we hypothesized that neutrophil degranulation leads to the release of LLO-neutralizing molecules in the forming phagosome. In support of this, L. monocytogenes is a potent inducer of neutrophil degranulation, since its virulence factors, such as LLO, facilitate granule exocytosis. Within the first few minutes of interaction with L. monocytogenes, granules can fuse with the plasma membrane at the bacterial interaction site before closure of the phagosome. Furthermore, granule products directly degrade LLO, irreversibly inhibiting its activity. The matrix metalloproteinase-8, stored in secondary granules, was identified as an endoprotease that degrades LLO, and blocking neutrophil proteases increased L. monocytogenes intracellular survival. In conclusion, we propose that LLO degradation by matrix metalloproteinase-8 during phagocytosis protects neutrophil membranes from perforation and contributes to maintaining L. monocytogenes in a bactericidal phagosome from which it cannot escape.

Suppression of T-cell chemokines by Porphyromonas gingivalis.

Porphyromonas gingivalis is a major pathogen in periodontal disease and is associated with immune dysbiosis. In this study, we found that P. gingivalis did not induce the expression of the T-cell chemokine IP-10 (CXCL10) from neutrophils, peripheral blood mononuclear cells (PBMCs), or gingival epithelial cells. Furthermore, P. gingivalis suppressed gamma interferon (IFN-γ)-stimulated release of IP-10, ITAC (CXCL11), and Mig (CXCL9) from epithelial cells and inhibited IP-10 secretion in a mixed infection with the otherwise stimulatory Fusobacterium nucleatum. Inhibition of chemokine expression occurred at the level of gene transcription and was associated with downregulation of interferon regulatory factor 1 (IRF-1) and decreased levels of Stat1. Ectopic expression of IRF-1 in epithelial cells relieved P. gingivalis-induced inhibition of IP-10 release. Direct contact between P. gingivalis and epithelial cells was not required for IP-10 inhibition. These results highlight the immune-disruptive potential of P. gingivalis. Suppression of IP-10 and other Th1-biasing chemokines by P. gingivalis may perturb the balance of protective and destructive immunity in the periodontal tissues and facilitate the pathogenicity of oral microbial communities.

Exocytosis of neutrophil granule subsets and activation of prolyl isomerase 1 are required for respiratory burst priming.

This study tested the hypothesis that priming the neutrophil respiratory burst requires both granule exocytosis and activation of the prolyl isomerase Pin1. Fusion proteins containing the TAT cell permeability sequence and either the SNARE domain of syntaxin-4 or the N-terminal SNARE domain of SNAP-23 were used to examine the role of granule subsets in TNF-mediated respiratory burst priming using human neutrophils. Concentration-inhibition curves for exocytosis of individual granule subsets and for priming of fMLF-stimulated superoxide release and phagocytosis-stimulated H2O2 production were generated. Maximal inhibition of priming ranged from 72 to 88%. Linear regression lines for inhibition of priming versus inhibition of exocytosis did not differ from the line of identity for secretory vesicles and gelatinase granules, while the slopes or the y-intercepts were different from the line of identity for specific and azurophilic granules. Inhibition of Pin1 reduced priming by 56%, while exocytosis of secretory vesicles and specific granules was not affected. These findings indicate that exocytosis of secretory vesicles and gelatinase granules and activation of Pin1 are independent events required for TNF-mediated priming of neutrophil respiratory burst.

Granule exocytosis contributes to priming and activation of the human neutrophil respiratory burst.

The role of exocytosis in the human neutrophil respiratory burst was determined using a fusion protein (TAT-SNAP-23) containing the HIV transactivator of transcription (TAT) cell-penetrating sequence and the N-terminal SNARE domain of synaptosome-associated protein-23 (SNAP-23). This agent inhibited stimulated exocytosis of secretory vesicles and gelatinase and specific granules but not azurophil granules. GST pulldown showed that TAT-SNAP-23 bound to the combination of vesicle-associated membrane protein-2 and syntaxin-4 but not to either individually. TAT-SNAP-23 reduced phagocytosis-stimulated hydrogen peroxide production by 60% without affecting phagocytosis or generation of HOCl within phagosomes. TAT-SNAP-23 had no effect on fMLF-stimulated superoxide release but significantly inhibited priming of this response by TNF-α and platelet-activating factor. Pretreatment with TAT-SNAP-23 inhibited the increase in plasma membrane expression of gp91(phox) in TNF-α-primed neutrophils, whereas TNF-α activation of ERK1/2 and p38 MAPK was not affected. The data demonstrate that neutrophil granule exocytosis contributes to phagocytosis-induced respiratory burst activity and plays a critical role in priming of the respiratory burst by increasing expression of membrane components of the NADPH oxidase.

Effects of fluoroquinolones on the migration of human phagocytes through Chlamydia pneumoniae-infected and tumor necrosis factor alpha-stimulated endothelial cells.

The anti-inflammatory activities of three quinolones, levofloxacin, moxifloxacin, and gatifloxacin, were investigated with an in vitro model of transendothelial migration (TEM). Human umbilical vein endothelial cells (HUVEC) were seeded in Transwell inserts, treated with serial dilutions of antibiotics, infected with Chlamydia pneumoniae, or stimulated with tumor necrosis factor alpha (TNF-alpha). Neutrophils or monocytes were also preincubated with serial dilutions of each antibiotic. TEM was assessed by light microscopic examination of the underside of the polycarbonate membrane, and levels of interleukin-8 (IL-8) and monocyte chemotactic protein 1 (MCP-1) were measured by enzyme-linked immunosorbent assay. In HUVEC infected with C. pneumoniae or stimulated with TNF-alpha, all fluoroquinolones significantly decreased neutrophil and monocyte TEM, compared to antibiotic-free controls. Moxifloxacin and gatifloxacin produced a significant decrease in IL-8 in C. pneumoniae-infected and TNF-alpha-stimulated HUVEC; however, moxifloxacin was the only fluoroquinolone that produced a significant decrease in MCP-1 levels under both conditions. Results from this study indicate similarities in the anti-inflammatory activities of these fluoroquinolones, although no statistically significant decrease in chemokine secretion was observed when levofloxacin was used. Mechanisms of neutrophil and monocyte TEM inhibition by fluoroquinolone antibiotics are unknown but may be partially due to inhibition of IL-8 and MCP-1 production, respectively.

Effect of macrolide antibiotics on human endothelial cells activated by Chlamydia pneumoniae infection and tumor necrosis factor-alpha.

This study investigated the potential anti-inflammatory activity of 3 macrolide antibiotics, clarithromycin, roxithromycin, and azithromycin, in an in vitro model of transendothelial migration (TEM). Human umbilical vein endothelial cells (HUVECs) were seeded in Transwell inserts, treated with serial dilutions of the antibiotics, and infected with Chlamydia pneumoniae or stimulated with tumor necrosis factor (TNF)-alpha. In HUVECs infected with C. pneumoniae or stimulated with TNF-alpha, both azithromycin and roxithromycin caused significant decreases in neutrophil and monocyte TEM, compared with antibiotic-free controls. Clarithromycin had no detectable effect in either group. Azithromycin caused significant decreases in interleukin (IL)-8 and monocyte chemotactic protein (MCP)-1, whereas roxithromycin significantly decreased IL-8. This study indicates heterogeneity in the anti-inflammatory activity of these antibiotics. Mechanisms of monocyte and neutrophil TEM inhibition by azithromycin and roxithromycin are unclear but may be partially due to inhibition of IL-8 and MCP-1 production.