Respiratory Chlamydia Infection Induce Release of Hepoxilin A3 and Histamine Production by Airway Neutrophils

Background: Hepoxilins are biologically active metabolites of arachidonic acid that are formed through the 12-lipoxygenase pathway. Hepoxilin A3 is now known to be an important regulator of mucosal inflammation in response to infection by bacterial pathogens and was recently identified as a potent neutrophil chemoattractant in the intestinal mucosa. Our goal in this study was to determine if airway infection with Chlamydia in a murine model of allergic airway disease (AAD) induces hepoxilin secretion along with airway neutrophilia.Methods: We utilized an AAD adult Balb/c mouse model to evaluate airway pathology and immune response by assaying bronchoalveolar lavage (BAL) fluid cytokine, cellularity, histidine decarboxylase (HDC) as well as histamine released in response to in-vivo chlamydial antigen stimulation of purified airway neutrophils. Hepoxilin A3 production was determined by Western blot identification of 12-lipoxygenase precursor (12-LO).Results: Chlamydial infection induced increased production of IL-2, IL-12, TNF-α, and IFN-γ in BAL fluid compared to uninfected animals. Chlamydia-infected mice responded with robust airway neutrophil infiltration and upon induction of AAD increased their production of IL-4, IL-5, and IL-13 by >3 fold compared to unsensitized groups. In addition, 12-LO mRNA was upregulated in infected, but not in uninfected AAD mice, suggesting the production of hepoxilin A3. mRNA expression of HDC was induced only in neutrophils from the airways of Chlamydia-infected mice, but was not seen in AAD only or uninfected controls. When purified neutrophils from infected animals were challenged with chlamydial antigen in vitro there was significant histamine release.Conclusions: Our data confirms the production and release of hepoxilin A3 in the murine airways concomitant with airway neutrophilia in response to chlamydial infection. We further confirmed that Chlamydia provokes the production and release of histamine by these neutrophils. These findings suggest that neutrophils, provoked by Chlamydia infection can synthesize and release histamine, thereby contributing directly to airway inflammation

Correction: Detection of Chlamydia in the peripheral blood cells of normal donors using in vitro culture, immunofluorescence microscopy and flow cytometry techniques

BACKGROUND: Chlamydia trachomatis (Ct) and Chlamydia pneumoniae (Cp) are medically significant infectious agents associated with various chronic human pathologies. Nevertheless, specific roles in disease progression or initiation are incompletely defined. Both pathogens infect established cell lines in vitro and polymerase chain reaction (PCR) has detected Chlamydia DNA in various clinical specimens as well as in normal donor peripheral blood monocytes (PBMC). However, Chlamydia infection of other blood cell types, quantification of Chlamydia infected cells in peripheral blood and transmission of this infection in vitro have not been examined. METHODS: Cp specific titers were assessed for sera from 459 normal human donor blood (NBD) samples. Isolated white blood cells (WBC) were assayed by in vitro culture to evaluate infection transmission of blood cell borne chlamydiae. Smears of fresh blood samples (FB) were dual immunostained for microscopic identification of Chlamydia-infected cell types and aliquots also assessed using Flow Cytometry (FC). RESULTS: ELISA demonstrated that 219 (47.7%) of the NBD samples exhibit elevated anti-Cp antibody titers. Imunofluorescence microscopy of smears demonstrated 113 (24.6%) of samples contained intracellular Chlamydia and monoclonals to specific CD markers showed that in vivo infection of neutrophil and eosinophil/basophil cells as well as monocytes occurs. In vitro culture established WBCs of 114 (24.8%) of the NBD samples harbored infectious chlamydiae, clinically a potentially source of transmission, FC demonstrated both Chlamydia infected and uninfected cells can be readily identified and quantified. CONCLUSION: NBD can harbor infected neutrophils, eosinophil/basophils and monocytes. The chlamydiae are infectious in vitro, and both total, and cell type specific Chlamydia carriage is quantifiable by FC

Caveolin-2 associates with intracellular chlamydial inclusions independently of caveolin-1

BACKGROUND: Lipid raft domains form in plasma membranes of eukaryotic cells by the tight packing of glycosphingolipids and cholesterol. Caveolae are invaginated structures that form in lipid raft domains when the protein caveolin-1 is expressed. The Chlamydiaceae are obligate intracellular bacterial pathogens that replicate entirely within inclusions that develop from the phagocytic vacuoles in which they enter. We recently found that host cell caveolin-1 is associated with the intracellular vacuoles and inclusions of some chlamydial strains and species, and that entry of those strains depends on intact lipid raft domains. Caveolin-2 is another member of the caveolin family of proteins that is present in caveolae, but of unknown function. METHODS: We utilized a caveolin-1 negative/caveolin-2 positive FRT cell line and laser confocal immunofluorescence techniques to visualize the colocalization of caveolin-2 with the chlamydial inclusions. RESULTS: We show here that in infected HeLa cells, caveolin-2, as well as caveolin-1, colocalizes with inclusions of C. pneumoniae (Cp), C. caviae (GPIC), and C. trachomatis serovars E, F and K. In addition, caveolin-2 also associates with C. trachomatis serovars A, B and C, although caveolin-1 did not colocalize with these organisms. Moreover, caveolin-2 appears to be specifically, or indirectly, associated with the pathogens at the inclusion membranes. Using caveolin-1 deficient FRT cells, we show that although caveolin-2 normally is not transported out of the Golgi in the absence of caveolin-1, it nevertheless colocalizes with chlamydial inclusions in these cells. However, our results also show that caveolin-2 did not colocalize with UV-irradiated Chlamydia in FRT cells, suggesting that in these caveolin-1 negative cells, pathogen viability and very likely pathogen gene expression are necessary for the acquisition of caveolin-2 from the Golgi. CONCLUSION: Caveolin-2 associates with the chlamydial inclusion independently of caveolin-1. The function of caveolin-2, either in the uninfected cell or in the chlamydial developmental cycle, remains to be elucidated. Nevertheless, this second caveolin protein can now be added to the small number of host proteins that are associated with the inclusions of this obligate intracellular pathogen

Infection-mediated asthma: etiology, mechanisms and treatment options, with focus on Chlamydia pneumoniae and macrolides

Abstract Asthma is a chronic respiratory disease characterized by reversible airway obstruction and airway hyperresponsiveness to non-specific bronchoconstriction agonists as the primary underlying pathophysiology. The worldwide incidence of asthma has increased dramatically in the last 40 years. According to World Health Organization (WHO) estimates, over 300 million children and adults worldwide currently suffer from this incurable disease and 255,000 die from the disease each year. It is now well accepted that asthma is a heterogeneous syndrome and many clinical subtypes have been described. Viral infections such as respiratory syncytial virus (RSV) and human rhinovirus (hRV) have been implicated in asthma exacerbation in children because of their ability to cause severe airway inflammation and wheezing. Infections with atypical bacteria also appear to play a role in the induction and exacerbation of asthma in both children and adults. Recent studies confirm the existence of an infectious asthma etiology mediated by Chlamydia pneumoniae (CP) and possibly by other viral, bacterial and fungal microbes. It is also likely that early-life infections with microbes such as CP could lead to alterations in the lung microbiome that significantly affect asthma risk and treatment outcomes. These infectious microbes may exacerbate the symptoms of established chronic asthma and may even contribute to the initial development of the clinical onset of the disease. It is now becoming more widely accepted that patterns of airway inflammation differ based on the trigger responsible for asthma initiation and exacerbation. Therefore, a better understanding of asthma subtypes is now being explored more aggressively, not only to decipher pathophysiologic mechanisms but also to select treatment and guide prognoses. This review will explore infection-mediated asthma with special emphasis on the protean manifestations of CP lung infection, clinical characteristics of infection-mediated asthma, mechanisms involved and antibiotic treatment outcomes

BAL cellularity during chlamydial infection.

<p>Elevated total WBCs and neutrophil levels were seen in infected neonatal and adult groups (A, C). Infected adults had elevated macrophage/monocytes (panel B), whereas infected neonates were characterized by significantly elevated eosinophil levels on day 28 pi compared to uninfected adults (P = 0.0449, panel D).</p

Mediastinal lymph node cell stimulation during chlamydial Infection.

<p>WBCs harvested from mediastinal lymph nodes were stimulated with heat killed chlamydial antigen and resulting cytokine secretions revealed that cells from infected adult mice secreted elevated levels of IFN-g and IL-2 when compared to infected neonates and controls (A, B). Cells harvested from infected neonatal mice secreted predominantly Th2 (IL-5, 4, 10, and 13) cytokines in response to chlamydial antigen compared to infected adults and control groups (C-F). WBCs from infected neonatal mice 14 days and 28 days post infection produced significantly elevated levels of IL-5 (P = 0.0353, 0.0456 respectively, C) and IL-4 (P = 0.0444, D) when compared to WBCs from infected adults. Cells were also stimulated with PBS and J774A.1 proteins as controls (data not shown).</p

Mediastinal lymph node cell stimulation during allergic airway disease.

<p>WBCs harvested from mediastinal lymph nodes were stimulated as described previously described (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083453#pone-0083453-g005" target="_blank">Figure 5</a>). Stimulation revealed that cells from infected neonatal mice with active infections (Groups 1 and 3) secreted elevated levels of IFN-g and IL-2 when compared to infected adults and controls (A, B). Cells harvested from infected neonatal mice secreted predominantly Th2 (IL-5, 4, 10, and 13) cytokines in response to chlamydial antigen compared to infected adults and control groups (C-F). Infected neonates with AAD (group 1) produced significantly more IL-5, IL-4, and IL-13 compared to corresponding adult groups (P = 0.0302, 0.0437, and 0.0362 respectively C, D, F). Infected neonates which did not have AAD were also able to produce significantly elevated levels of IL-4 and IL-13 compared to adults groups as well (P = 0.0456, 0.0075 respectively C, F). Cells were also stimulated with PBS and J774A.1 proteins as controls (data not shown).</p

BAL Ova-Specific IgE During Allergic Airway Induction.

<p>Allergic airways were induced using ovalbumin. Groups which were both sensitized and challenged with ova had significantly elevated levels of ova-specific IgE antibodies present in BAL fluid (A) and serum (B), demonstrating successful allergic airway induction. Although not significant, infected neonatal group produced more ova-IgE than comparable adult group (Group 1).</p

BAL cytokines during chlamydial infection.

<p>Infected adults displayed a more pronounced Th1 cytokine response (IFN-γ and IL-2) than infected neonates (A, B). Infected neonates responded to chlamydial infection with a robust Th2 cytokine response (IL-4, IL-10, IL-5, and IL-13) compared to their adult counterparts (C-F). Specifically, neonatal mice produced significantly elevated levels of IL-4 compared to infected adults 14 and 28 days post infection (P = 0.0312, 0.0248 respectively, D) as well as IL-10 28 days post infection (P = 0.0434 E).</p