| Abstract: | Parachlamydia acanthamoebae is a Chlamydia-related organism whose pathogenic role in pneumonia is supported by serological and molecular clinical studies and an experimental mouse model of lung infection. Toll-like receptors (TLRs) play a seminal role in sensing microbial products and initiating innate immune responses. The aim of this study was to investigate the roles of MyD88, TLR2, and TLR4 in the interaction of Parachlamydia with macrophages. Here, we showed that Parachlamydia entered bone-marrow derived macrophages (BMDMs) in a TLR-independent manner but did not multiply intracellularly. Interestingly, compared to live bacteria, heat-inactivated Parachlamydia induced the production of substantial amounts of tumor necrosis factor alpha (TNF), interleukin-6 (IL-6), and IL-12p40 by BMDMs and of TNF and IL-6 by peritoneal macrophages as well as RAW 264.7 and J774 macrophage cell lines. Cytokine production by BMDMs, which was partially inhibited upon trypsin treatment of Parachlamydia, was dependent on MyD88, TLR4, and, to a lesser extent, TLR2. Finally, MyD88−/−, TLR4−/−, and TLR2−/− mice were as resistant as wild-type mice to lung infection following the intratracheal instillation of Parachlamydia. Thus, in contrast to Chlamydia pneumoniae, Parachlamydia acanthamoebae weakly stimulates macrophages, potentially compensating for its low replication capacity in macrophages by escaping the innate immune surveillance.Parachlamydia acanthamoebae is a strict intracellular bacterium which naturally infects free-living amoebae. Like other members of the Chlamydiales order, it exhibits a two-stage developmental cycle with infectious elementary bodies and metabolically active replicating reticulate bodies (25). Several pieces of evidence support the role of P. acanthamoebae as a new agent of lower respiratory tract infection (reviewed in references 18 and 32). The first hint was provided by the recovery of P. acanthamoebae strain Hall''s coccus from the water of a humidifier associated with an outbreak of fever and the presence of anti-Parachlamydia antibodies among exposed individuals (2). Additional serological studies demonstrated a higher seropositivity rate among patients with pneumonia than among controls (20, 33). Furthermore, parachlamydial DNA was detected by PCR in mononuclear cells of a patient with bronchitis and in sputa and bronchoalveolar lavage samples from patients with lower respiratory tract infections (11, 12, 19, 37). Moreover, Parachlamydia infects human pneumocytes and macrophages in vitro (7, 23, 24). Finally, we recently developed an experimental model in which mice injected with living Parachlamydia showed signs of severe pneumonia and bacterial localization in cells that were likely pneumocytes and macrophages (9, 10).The sensing of invasive pathogens by innate immune cells relies on their capacity to sense microbial molecular motifs through pattern recognition receptors. Toll-like receptors (TLRs) expressed on the surface or in the endosomes of immune cells allow the detection of microbially derived molecular structures such as lipids, proteins, and nucleic acids. TLR4 is an obligate partner for the host response to bacterial lipopolysaccharide (LPS) (endotoxin) and most Gram-negative bacteria (1). TLR2, which generates heterodimers in combination with either TLR1 or TLR6, has been reported to recognize a broad range of microbial compounds, among which are lipopeptides, lipoproteins, peptidoglycan subcomponents, and β-glucans (29). The activation of the intracellular signaling pathways upon microbial recognition by TLRs engages several adaptor molecules. Myeloid differentiation primary response gene 88 (MyD88) specifies most TLRs and the TLR4 MyD88-dependent signaling pathway, which is involved in the activation of mitogen-activated protein kinases and nuclear factor κB and in the generation of proinflammatory cytokines and immune-related genes (35).Although Parachlamydia may represent an emerging agent of pneumonia (18, 26), very little is known about its recognition by innate immune cells. Unfortunately, the mechanisms involved in the sensing of Chlamydia are most likely irrelevant for Parachlamydia given the specificities of this bacterium, such as its ability to replicate within amoebae (22) and its genome size, which is more than twice that of Chlamydia (21). In addition, the bioinformatics-based annotation of the genome of the Parachlamydia-related symbiont UWE25 indicates that it likely possesses a truncated LPS and lacks most immunogenic outer membrane proteins present in the Chlamydiaceae (27). Considering the central role played by TLRs in microbial sensing, we studied the contributions of MyD88, TLR2, and TLR4 in the recognition of P. acanthamoebae by macrophages in vitro and in the outcome of parachlamydial infection in an experimental model of pneumonia in mice. Our results show that living P. acanthamoebae enters but does not multiply in macrophages and that heat-inactivated P. acanthamoebae stimulates cytokine production by macrophages in a MyD88/TLR4-dependent manner and to a lesser extend through TLR2. Furthermore, MyD88−/−, TLR4−/−, and TLR2−/− mice are resistant to P. acanthamoebae infection. Taken together, these data indicate that P. acanthamoebae weakly stimulates the innate immune system, which may allow the bacterium to survive in infected cells despite its low replication capacity. |
