Genevieve Goy

Histone deacetylase inhibitors impair innate immune responses to Toll-like receptor agonists and to infection

Regulated by histone acetyltransferases and deacetylases (HDACs), histone acetylation is a key epigenetic mechanism controlling chromatin structure, DNA accessibility, and gene expression. HDAC inhibitors induce growth arrest, differentiation, and apoptosis of tumor cells and are used as anticancer agents. Here we describe the effects of HDAC inhibitors on microbial sensing by macrophages and dendritic cells in vitro and host defenses against infection in vivo. HDAC inhibitors down-regulated the expression of numerous host defense genes, including pattern recognition receptors, kinases, transcription regulators, cytokines, chemokines, growth factors, and costimulatory molecules as assessed by genome-wide microarray analyses or innate immune responses of macrophages and dendritic cells stimulated with Toll-like receptor agonists. HDAC inhibitors induced the expression of Mi-2β and enhanced the DNA-binding activity of the Mi-2/NuRD complex that acts as a transcriptional repressor of macrophage cytokine production. In vivo, HDAC inhibitors increased the susceptibility to bacterial and fungal infections but conferred protection against toxic and septic shock. Thus, these data identify an essential role for HDAC inhibitors in the regulation of the expression of innate immune genes and host defenses against microbial pathogens.

Role of Chlamydia trachomatis in Miscarriage

TOC Summary: Women experiencing miscarriage should be screened for C. trachomatis.

Development of a real-time PCR for the specific detection of Waddlia chondrophila in clinical samples.

Waddlia chondrophila is considered as an emerging human pathogen likely involved in miscarriage and lower respiratory tract infections. Given the low sensitivity of cell culture to recover such an obligate intracellular bacteria, molecular-based diagnostic approaches are warranted. We thus developed a real-time PCR that amplifies Waddlia chondrophila DNA. Specific primers and probe were selected to target the 16S rRNA gene. The PCR specifically amplified W. chondrophila but did not amplify other related-bacteria such as Parachlamydia acanthamoebae, Simkania negevensis and Chlamydia pneumoniae. The PCR exhibited a good intra-run and inter-run reproducibility and a sensitivity of less than ten copies of the positive control. This real-time PCR was then applied to 32 nasopharyngeal aspirates taken from children with bronchiolitis not due to respiratory syncytial virus (RSV). Three samples revealed to be Waddlia positive, suggesting a possible role of this Chlamydia-related bacteria in this setting.

Waddlia chondrophila enters and multiplies within human macrophages

Waddlia chondrophila is an obligate intracellular bacterium of the Chlamydiales order. W. chondrophila has been isolated twice from aborted bovine foetuses and a serological study supported the abortigenic role of W. chondrophila in bovine species. Recently, we observed a strong association between the presence of anti-Waddlia antibodies and human miscarriage. To further investigate the pathogenic potential of W. chondrophila in humans, we studied the entry and the multiplication of this Chlamydia-like organism in human macrophages. Confocal and electron microscopy confirmed that W. chondrophila is able to enter human monocyte-derived macrophages. Moreover, W. chondrophila multiplied readily within macrophages. The proportion of infected macrophages increased from 13% at day 0 to 96% at day 4, and the mean number of bacteria per macrophage increased by 3logs in 24h. Intracellular growth of W. chondrophila was associated with a significant cytopathic effect. Thus, W. chondrophila may enter and grow rapidly within human macrophages, inducing lysis of infected cells. Since macrophages are one of the major components of the innate immune response, these findings indirectly suggest the possible human pathogenicity of W. chondrophila.

Role of Waddlia chondrophila placental infection in miscarriage.

Waddlia chondrophila is an intracellular bacterium suspected to cause human and bovine abortion. We confirmed an association between antibodies against W. chondrophila and human miscarriage and identified this organism in placenta or genital tract of women who had had miscarriages. These results suggest a possible role of W. chondrophila infection in miscarriage.

Antibiotic Susceptibility of Waddlia chondrophila in Acanthamoeba castellanii Amoebae

ABSTRACT Waddlia chondrophila is an emerging cause of miscarriage in bovines and humans. Given the strict intracellular growth of this Chlamydia-like organism, its antibiotic susceptibility was tested by amoebal coculture, cell culture, and real-time PCR. W. chondrophila was susceptible to doxycycline and azithromycin but resistant to β-lactams and fluoroquinolones.

Parachlamydia acanthamoebae infection and abortion in small ruminants.

To the Editor: Abortion in ruminants is of worldwide economic importance. Moreover, several abortigenic agents have a zoonotic potential, i.e., Brucella abortus, Coxiella burnetii, and Chlamydophila abortus. C. abortus, which causes ovine enzootic abortion, may also infect pregnant women who have had contact with C. abortus–infected sheep and goats, and such infection can lead to miscarriage (1). Parachlamydia acanthamoebae (2) is a Chlamydia-related organism considered as an emerging agent of pneumonia in humans. Recently, we reported its role in the setting of bovine abortion (3). Here, we investigated the prevalence of C. abortus and P. acanthamoebae infections in abortions in small ruminants. Formalin-fixed placenta, fetal lung and liver, or both, were available from abortion products from 144 goats and 86 sheep (n = 211). These specimens had previously been investigated for several abortigenic agents (4). Placentas and fetal organs were analyzed by histopathologic examination and by specific real-time PCR and immunohistochemical protocols that detect members of the Chlamydiaceae family and P. acanthamoebae. DNA from paraffin blocks was extracted as described (5) by using the DNeasy Tissue kit (QIAGEN, Hilden, Germany). The real-time PCR for Chlamydiaceae was conducted on an ABI 7500 (Applied Biosystems, Foster City, CA, USA) by using a modified version of Everett’s PCR (6). Primers Ch23S-F (5′-CTGAAACCAGTAGCTTATAAGCGGT-3′), Ch23S-R (5′-ACCTCGCCGTTTAACTTAACTCC-3′), and probe Ch23S-p (5′-FAM-CTCATCA TGCAAAAGGCACGCCG-TAMRA-3′) were used to amplify and detect a 111-bp product specific for members of the family Chlamydiaceae. Chlamydial species identification of real-time PCR positive cases was performed with the ArrayTube Microarray (Clondiag, Jena, Germany) as described (7). The Parachlamydia-specific real-time PCR was performed with the ABI Prism 7000 sequence detection system (Applied Biosystems), as reported (8). This PCR is genus-specific, as demonstrated by the absence of PCR positivity with DNA extracted from other Parachlamydiaceae (Protochlamydia spp./Neochlamydia hartmannellae). To confirm positive results, another specific PCR, which targeted the tlc gene, was performed (9). Paraffin sections from specimens positive in real-time PCR were further examined by immunohistochemical tests. A Chlamydiaceae-specific mouse monoclonal antibody directed against the chlamydial lipopolysaccharide (Progen, Heidelberg, Germany) and a specific mouse polyclonal antibody against Parachlamydia spp. was used as described (3,5,10). These antibodies were applied at dilutions of 1:200 and 1:1,000, respectively. Detection was performed with a detection kit (ChemMate; Dako, Glostrup, Denmark). Antigen retrieval was performed by enzyme digestion for 10 minutes (Pronase; Dako) for the Chlamydiaceae antibody and repeated microwave treatment in citrate buffer (ChemMate; Dako) for the Parachlamydia antibody, respectively. Double immunohistochemical labeling was performed on the sheep abortion specimen identified as simultaneously infected with Chlamydiaceae and Parachlamydia spp. Immunohistochemical analysis for both pathogens was performed subsequently by using diaminobenzidine as substrate for the Chlamydiaceae antibody (brown labeling) and by using 3-amino-9-ethylcarbazole as substrate for the Parachlamydia antibody (red labeling). Specificity of PCR and immunohistochemical tests for Chlamydiaceae and Parachlamydia spp., respectively, was assessed by using negative control placentas taken from 2 healthy ruminants (both specimens were negative in all tests). Results of real-time PCR showed that 55 (26.1%) of 211 specimens were positive for Chlamydiaceae. All 55 cases could be identified as C. abortus by ArrayTube Microarray (Clondiag). Of these, 42 (76.4%) could be confirmed by immunohistochemical analysis with the anti-Chlamydiaceae antibody. Of the 211 specimens, only 2 (0.9%) were positive for Parachlamydia spp. by real-time PCR, and both cases could be confirmed by immunohistochemical testing with the parachlamydial antibody. These 2 specimens were negative for other common abortigenic agents such as Toxoplasma gondii, C. burnetii, and border disease virus (data not shown). One case was recorded among the 144 goat samples investigated. This placenta displayed necrotizing placentitis and was positive for Parachlamydia spp. by 16S rRNA-specific real-time PCR (cycle threshold [Ct] 40.5) and immunohistochemical testing, but negative for Chlamydiaceae. Results of this PCR was confirmed by another PCR, targeting the tlc gene (Ct 36.7), which excluded false-positive results because of amplicon contamination. The second case was identified among the 86 sheep investigated. Placenta and fetal lung and liver exhibited necrotizing placentitis and vasculitis (Figure, panel A), interstitial pneumonia (Figure, panel B), and mixed cellular periportal hepatitis. Fetal liver was negative by parachlamydial 16S rRNA real-time PCR and immunohistochemical analysis, but the fetal lung was positive by parachlamydial 16S rRNA real-time PCR (Ct 40.7) and immunohistochemical tests (Figure, panel C), but negative with the tlc PCR. Fetal lung and liver were positive by real-time PCR for Chlamydiaceae (mean Ct for both organs 36.7), but negative by immunohistochemical tests. The placenta was positive for Chlamydiaceae by immunohistochemical tests and real-time PCR (mean Ct 23.3), and C. abortus was identified by ArrayTube Microarray. Brown (Chlamydiaceae) and red (Parachlamydia spp.) granular reaction was demonstrated within the necrotic lesions of the placenta by double immunohistochemical labeling (Figure, panel D). Figure A) Sheep placenta positive by real-time PCR and immunohistochemistry for Parachlamydia spp. and Chlamydiaceae. Chlamydophila abortus was identified by ArrayTube Microarray. Necrotizing placentitis and vasculitis are shown (hematoxylin and eosin stain; ... We report Parachlamydia infection in small ruminant abortion. C. abortus and Parachlamydia spp. were simultaneously present in an aborted sheep placenta. Parachlamydia spp. could be further detected in the lung of the aborted sheep fetus by real-time PCR and immunohistochemistry. Parachlamydia was also detected in a goat placenta. Thus, Parachlamydia spp. should be considered as a new abortigenic agent in sheep and goats. Persons in contact with small ruminants should be informed about the zoonotic potential of these abortigenic agents.

Roles of bovine Waddlia chondrophila and Chlamydia trachomatis in human preterm birth

Waddlia chondrophila and Chlamydia trachomatis are intracellular bacteria associated with human miscarriage. We investigated their role in human preterm birth. Whereas presence of Chlamydia trachomatis DNA in genital tract was associated with human preterm birth, Waddlia was not, despite being present in womens genital tracts.