Differential Expression of mRNA Encoding Cytokines and Chemokines in the Reproductive Tract after Infection of Mice with \u3cem\u3eChlamydia trachomatis\u3c/em\u3e

Infection with Chlamydia trachomatis targets epithelial cells within the genital tract which respond by secreting chemokines and cytokines. Persistent inflammation can lead to fibrosis, tubal infertility and/or ectopic pregnancy; many infections are asymptomatic. Most studies have investigated the inflammatory response in the initial stages of infection, less is known about the later stages of infection, especially with a low, potentially asymptomatic, bacterial load. Our objective was to determine the inflammatory mediators involved in clearance of low-grade infection and the potential involvement in chronic inflammation. Six to eight week old C3H/HeJ mice were pretreated with 2.5 mg medroxyprogesterone acetate on day -10 and -3 before infection. Mice (n=3 for 28 d, n=3 for 35 d) were infected with 5 × 102 inclusion-forming units of C. trachomatis, serovar D; vaginal cultures were obtained weekly to monitor infection. Control mice (n=3 for 28 d, n=3 for 35 d) were sham infected. Mice were killed on day 28 (experiment 1) and day 35 (experiment 2) post-infection and vaginal tissue, uterine horns and oviducts collected for analysis of mRNAs encoding inflammatory cytokines and chemokines. Total RNA was isolated and a superarray analysis performed using mouse Cytokines and Chemokines PCR arrays (Qiagen, Valencia, CA). Statistical differences in gene expression were determined using a paired Students t-test. At 28 days after infection, the expression of mRNA encoding 6, 35 and 3 inflammatory genes differed from controls in vaginal, uterine and oviductal tissues, respectively (P \u3c 0.05). At 35 days after infection, the expression of mRNA encoding 16, 38 and 14 inflammatory genes differed from controls in vaginal, uterine and oviductal tissues, respectively (P \u3c 0.05). Understanding the mechanisms involved in the inflammatory response at later stages of infection should aid in the development of treatment options that minimize the development of asymptomatic, chronic inflammation-induced infertility

Dendritic Cells from Aged Subjects Display Enhanced Inflammatory Responses to Chlamydophila pneumoniae

Chlamydophila pneumoniae (CPn) is a common respiratory pathogen that causes a chronic and persistent airway infection. The elderly display an increased susceptibility and severity to this infection. However, the underlying mechanisms are not well understood. Dendritic cells (DCs) are the initiators and regulators of immune responses. Therefore, we investigated the role of DCs in the age-associated increased CPn infection in vitro in humans. Though the expression of activation markers was comparable between the two age groups, DCs from aged subjects secreted enhanced levels of proinflammatory mediators such as TNF-α and CXCL-10 in response to CPn. In contrast, the secretion of IL-10 and innate interferons, IFN-α and IFN-λ, was severely impaired in DCs from aged donors. The increased activation of DCs from aged subjects to CPn also resulted in enhanced proliferation of CD4 and CD8 T cells in a DC-T coculture. Furthermore, T cells primed with CPn-stimulated DCs from aged subjects secreted increased levels of IFN-γ and reduced levels of IL-10 compared to DCs obtained from young subjects. In summary, DCs from the elderly displayed enhanced inflammatory response to CPn which may result in airway remodeling and increase the susceptibility of the elderly to respiratory diseases such as asthma

Autophagy Limits Inflammasome During Chlamydia pneumoniae Infection

Autophagy can either antagonize or promote intracellular bacterial growth, depending on the pathogen. Here, we investigated the role of autophagy during a pulmonary infection with the obligate intracellular pathogen, Chlamydia pneumoniae (CP). In mouse embryonic fibroblasts (MEFs) or macrophages, deficiency of autophagy pathway components led to enhanced CP replication, suggesting that autophagy exerts a bactericidal role. However, in vivo, mice with myeloid-specific deletion of the autophagic protein ATG16L1 suffered increased mortality during CP infection, neutrophilia, and increased inflammasome activation despite no change in bacterial burden. Induction of autophagy led to reduced CP replication in vitro, but impaired survival in CP-infected mice, associated with an initial reduction in IL-1β production, followed by enhanced neutrophil recruitment, defective CP clearance, and later inflammasome activation and IL-1β production, which drove the resulting mortality. Taken together, our data suggest that a delicate interplay exists between autophagy and inflammasome activation in determining the outcome of CP infection, perturbation of which can result in inflammatory pathology or unrestricted bacterial growth

Chlamydia pneumoniae Infection Induced Allergic Airway Sensitization Is Controlled by Regulatory T-Cells and Plasmacytoid Dendritic Cells

Chlamydia pneumoniae (CP) is associated with induction and exacerbation of asthma. CP infection can induce allergic airway sensitization in mice in a dose- and time-dependent manner. Allergen exposure 5 days after a low dose (mild-moderate), but not a high dose (severe) CP infection induces antigen sensitization in mice. Innate immune signals play a critical role in controlling CP infection induced allergic airway sensitization, however these mechanisms have not been fully elucidated. Wild-type, TLR2−/−, and TLR4−/− mice were infected intranasally (i.n.) with a low dose of CP, followed by i.n. exposure to human serum albumin (HSA) and challenged with HSA 2 weeks later. Airway inflammation, immunoglobulins, eosinophils, and goblet cells were measured. Low dose CP infection induced allergic sensitization in TLR2−/− mice, but not in TLR4−/− mice, due to differential Treg responses in these genotypes. TLR2−/− mice had reduced numbers of Tregs in the lung during CP infection while TLR4−/− mice had increased numbers. High dose CP infection resulted in an increase in Tregs and pDCs in lungs, which prevented antigen sensitization in WT mice. Depletion of Tregs or pDCs resulted in allergic airway sensitization. We conclude that Tregs and pDCs are critical determinants regulating CP infection-induced allergic sensitization. Furthermore, TLR2 and TLR4 signaling during CP infection may play a regulatory role through the modulation of Tregs

The NOD/RIP2 Pathway Is Essential for Host Defenses Against Chlamydophila pneumoniae Lung Infection

Here we investigated the role of the Nod/Rip2 pathway in host responses to Chlamydophila pneumoniae–induced pneumonia in mice. Rip2−/− mice infected with C. pneumoniae exhibited impaired iNOS expression and NO production, and delayed neutrophil recruitment to the lungs. Levels of IL-6 and IFN-γ levels as well as KC and MIP-2 levels in bronchoalveolar lavage fluid (BALF) were significantly decreased in Rip2−/− mice compared to wild-type (WT) mice at day 3. Rip2−/− mice showed significant delay in bacterial clearance from the lungs and developed more severe and chronic lung inflammation that continued even on day 35 and led to increased mortality, whereas WT mice cleared the bacterial load, recovered from acute pneumonia, and survived. Both Nod1−/− and Nod2−/− mice also showed delayed bacterial clearance, suggesting that C. pneumoniae is recognized by both of these intracellular receptors. Bone marrow chimera experiments demonstrated that Rip2 in BM-derived cells rather than non-hematopoietic stromal cells played a key role in host responses in the lungs and clearance of C. pneumoniae. Furthermore, adoptive transfer of WT macrophages intratracheally was able to rescue the bacterial clearance defect in Rip2−/− mice. These results demonstrate that in addition to the TLR/MyD88 pathway, the Nod/Rip2 signaling pathway also plays a significant role in intracellular recognition, innate immune host responses, and ultimately has a decisive impact on clearance of C. pneumoniae from the lungs and survival of the infectious challenge

CHROMagar Candida Medium for Direct Susceptibility Testing of Yeast from Blood Cultures

An evaluation was performed on 95 blood cultures positive for Candida spp. to determine the correlation of direct susceptibility testing of fluconazole versus both standardized disk diffusion and MIC methods. For direct testing, an aliquot taken from BD BACTEC Plus and/or BD BACTEC Lytic/10 bottles (Becton Dickinson [BD], Sparks, MD) positive by gram stain for yeast was subcultured to CHROMagar Candida (BD), and a 25-μg fluconazole disk (BD) was placed on the plate. The area of growth inhibition surrounding the disk was measured at 24 and 48 h. In addition, a subculture of the isolate was tested by a microdilution MIC using YeastOne (TREK Diagnostics Systems Inc., OH) and disk diffusion (NCCLS M44-A) using a standardized inoculum plated onto CHROMagar Candida as well as Mueller-Hinton agar to which 2% glucose and 0.5 μg/ml methylene blue dye was added (MH-GMB). The categorical interpretation derived from the MIC was used as the reference to which the disk diffusion results were compared. There were a total of 41 Candida albicans, 23 Candida glabrata, 20 Candida parapsilosis, 9 Candida tropicalis, and 1 each of Candida krusei and Candida lusitaniae tested. At 24 h there was full agreement among the methods for all C. albicans, C. tropicalis, C. lusitaniae, and C. krusei isolates. For the C. parapsilosis isolates at 24 h there was one very major discrepancy using the direct CHROMagar and one major error with the standardized MH-GMB. The majority of the errors were seen at 24 h with the C. glabrata isolates. Of the 23 C. glabrata isolates at 24 h by direct CHROMagar, there were 10 minor and 1 very major error; by MH-GMB there were 12 minor and 2 very major errors; and by standardized CHROMagar Candida there were 13 minor and 2 major errors. There were no very major errors with C. glabrata when all plates were read at 48 h. At 24 h by the direct and standardized CHROMagar the majority of C. glabrata isolates were more resistant, whereas by MH-GMB they were more susceptible than the reference MIC interpretation. In summary, subculturing yeast directly from blood cultures onto CHROMagar to which a fluconazole disk has been added may provide a presumptive identification at 24 h and, with the exception of C. glabrata, was able to predict the susceptibility to fluconazole with the majority of Candida isolates examined in this evaluation

Interaction between Components of the Type III Secretion System of Chlamydiaceae

Members of the family Chlamydiaceae possess at least 13 genes, distributed throughout the chromosome, that are homologous with genes of known type III secretion systems (TTS). The aim of this study was to use putative TTS proteins of Chlamydophila pneumoniae, whose equivalents in other bacterial TTS function as chaperones, to identify interactions between chlamydial proteins. Using the BacterioMatch Two-Hybrid Vector system (Stratagene, La Jolla, Calif.), lcrH-2 and sycE, positions 1021 and 0325, respectively, from C. pneumoniae CM-1 were used as “bait” to identify target genes (positions 0324, 0705, 0708, 0808 to 0810, 1016 to 1020, and 1022) in close proximity on the chromosome. Interaction between the products of the lcrH-2 (1021) and lcrE (copN) (0324) genes was detected and confirmed by pull-down experiments and enzyme immunoassays using recombinant LcrH-2 and LcrE. As further confirmation of this interaction, the homologous genes from Chlamydia trachomatis, serovar E, and Chlamydophila psittaci, Texas turkey, were also cloned in the two-hybrid system to determine if LcrH-2 and LcrE would interact with their orthologs in other species. Consistent with their genetic relatedness, LcrH-2 from C. pneumoniae interacted with LcrE produced from the three species of Chlamydiaceae; LcrH-2 from C. psittaci reacted with LcrE from C. pneumoniae but not from C. trachomatis; and C. trachomatis LcrH-2 did not react with LcrE from the other two species. Deletions from the N and C termini of LcrE from C. pneumoniae identified the 50 C-terminal amino acids as essential for the interaction with LcrH-2. Thus, it appears that in the Chlamydiaceae TTS, LcrH-2 interacts with LcrE, and therefore it may serve as a chaperone for this protein