Mycoplasma fermentansand TNF-β interact to amplify immune-modulating cytokines in human lung fibroblasts

Abstract

Mycoplasma can establish latent infections and are associated with arthritis, leukemia, and chronic lung disease. We developed an experimental model in which lung cells are deliberately infected with Mycoplasma fermentans. Human lung fibroblasts (HLF) were exposed to live M. fermentans and immune-modulating cytokine release was assessed with and without known inducers of cytokine production. M. fermentans increased IL-6, IL-8/CXCL8, MCP-1/CCL2, and Gro-α/CXCL1 production. M. fermentans interacted with TNF-β to release more IL-6, CXCL8, and CXCL1 than predicted by the responses to either stimulus alone. The effects of live infection were recapitulated by exposure to M. fermentans-derived macrophage-activating lipopeptide-2 (MALP-2), a Toll-like receptor-2-and receptor-6-specific ligand. The synergistic effect of combined stimuli was more pronounced with prolonged incubations. Preexposure to TNF-β sensitized the cells to subsequent MALP-2 challenge, but preexposure to MALP-2 did not alter the IL-6 response to TNF-β. Exposure to M. fermentans or MALP-2 did not enhance nuclear localization, DNA binding, or transcriptional activity of NF-κB and did not modulate early NF-κB activation in response to TNF-β. Application of specific inhibitors of various MAPKs suggested that p38 and JNK/stress-activated protein kinase were involved in early IL-6 release after exposure to TNF-β and M. fermentans, respectively. The combined response to M. fermentans and TNF-β, however, was uniquely sensitive to delayed application of SP-600125, suggesting that JNK/stress-activated protein kinase contributes to the amplification of IL-6 release. Thus M. fermentans interacts with stimuli such as TNF-β to amplify lung cell production of immune-modulating cytokines. The mechanisms accounting for this interaction can now be dissected with the use of this in vitro model. NIH Public Access Author ManuscriptAm J Physiol Lung Cell Mol Physiol. Author manuscript; available in PMC 2010 July 6. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript MYCOPLASMA (class Mollicute) are a class of microorganisms that need to be considered as a potential factor in the genesis of chronic inflammatory disease. These ubiquitous cell wall-free bacteria represent the simplest known self-replicating organisms and have adapted to a strict commensal/parasitic lifestyle in an intimate relationship with a variety of animal and human hosts. M. pneumoniae was identified as a causative agent for human tracheobronchitis and atypical pneumonia in 1962 (9); however, this and other species are now reemerging in their importance as covert and chronic infectious agents with potential to act as cofactors in the progression of chronic inflammatory disease (5,7). It was recently shown that M. pneumoniae and other mollicute species could be detected in the airways of subjects in the absence of symptoms of acute infection and that the incidence was greater in individuals with asthma (23).M. fermentans has been previously isolated from the human genitour...

Multiple protein kinase pathways mediate amplified IL-6 release by human lung fibroblasts co-exposed to nickel and TLR-2 agonist, MALP-2

“…We cannot, however, fully exclude the possibility that trace amounts of Ni remain within the cell even after vigorous washing and contribute to the response. We have previously observed a similar effect of facilitation of MALP-2-dependent IL-6 release following pre-exposure of HLF to TNF-β, an extracellular protein ligand that is likely more confidently removed with washing (Fabisiak et al , 2006). The addition of Ni (or TNF) along with the MALP-2 challenge, however, does further enhance the response indicating that maximal effects can be better realized with concurrent exposure to both agonists.…”

“…Individual cell lines prepared from previously frozen stocks were propagated in Minimal Essential Medium (MEM) supplemented with fetal bovine serum (10%, final concentration), glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 μg/ml) in a humidified incubator with 5% CO 2 /95% air as previously described (Fabisiak et al , 2006). All cultures were negative for mycoplasma as determined by fluorescent microscopy using Hoechst 33258 (Chen, 1977) or MycoAlert ™ mycoplasma detection kit (Cambrex BioScience, Rockland, ME).…”

Nickel Alterations of TLR2-Dependent Chemokine Profiles in Lung Fibroblasts Are Mediated by COX-2

“…Recent evidence suggests that colonization with species originally considered commensal and not causing overt disease (e.g., Mycoplasma fermentans) also has the potential to interact with other stimuli and/or alter biological responses in infected host cells (5,6). For example, we have previously shown that exposure of human lung fibroblast cells (HLF) to M. fermentans synergistically enhances the release of IL-6 by PM in the form of residual oil fly ash (ROFA) compared to either stimulus alone (7).…”

Animal model of Mycoplasma fermentans respiratory infection

“…This study showed the ability of Mycoplasma fermentans to persist in different organs for at least 50 days after inoculation, probably because they can evade the immune system and enter the cells where the antibodies and antibiotics cannot act and induce a chronic inflammation [18]. Intensive studies have been done in order to understand the strategy used by Mycoplasma fermentans to interact with host cells and evade the host protection system.…”

“…Mycoplasma fermentans serves as a stimulus for the production of several immune modulating cytokines and components of an inflammatory response. Mycoplasma may modulate inflammatory and immune processes within the lung as well as potentiate T cell dependent immunopathologies [18]. …”