Dataset Information

Treponema pallidum Dysregulates Monocytes and Promotes the Expression of IL-1? and Migration in Monocytes Through the mTOR Signaling Pathway.

ABSTRACT: Monocytes are widely involved in the body's defense response, and abnormally regulated monocyte subsets are closely related to the pathogenesis of various diseases. It is unclear whether Treponema pallidum (Tp) dysregulates monocyte subsets and impacts the functions of monocytes. This study aims to analyze the distribution of monocyte subsets in syphilis patients and the effect of Tp on monocyte functions to explore the pathogenesis of syphilis. Flow cytometry was employed to detect monocyte subsets. With or without pre-treatment with rapamycin, THP-1 cell migration stimulated by Tp was investigated by a Transwell migration assay, and THP-1 cell phagocytosis was studied using fluorescent microspheres. IL-1? and TNF-? expression was quantified by PCR and flow cytometry, while LC3 and mTOR were investigated in Tp-exposed THP-1 cells using western blotting. Tp infection led to an increase in the proportion of CD14⁺⁺CD16⁺ monocytes and a decrease in the proportion of CD14⁺⁺CD16^- monocytes. In addition, Tp promoted monocyte (THP-1) CD14 and CD16 expression in vitro, induced the expression of IL-1? and TNF-? in a dose-dependent manner and promoted the migration and autophagy of monocytes. Furthermore, mTOR phosphorylation on monocytes was stimulated by Tp, and the levels peaked at 30 min. Pre-treatment with rapamycin (mTOR inhibitor) attenuated the expression of IL-1? and migration in Tp-exposed THP-1 cells. Tp abnormally regulates monocyte subsets and promotes migration, autophagy, and the expression of IL-1? and TNF-? in THP-1 cells. Meanwhile, the mTOR affected the expression of IL-1? and migration in Tp-exposed THP-1 cells. This study is important as it sheds light on the mechanism by which monocytes interact with Tp during infection.

SUBMITTER: Liu WN

PROVIDER: S-EPMC7691244 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Publications

Treponemapallidum Dysregulates Monocytes and Promotes the Expression of IL-1β and Migration in Monocytes Through the mTOR Signaling Pathway.

Liu Wen-Na WN Jiang Xiao-Yong XY Xu Yan-Zhu YZ Sun Xiao-Han XH Wu Kai-Xuan KX Hu Xin-Lin XL Lin Yong Y Lin Li-Rong LR Tong Man-Li ML Liu Li-Li LL

Frontiers in cellular and infection microbiology 20201113

Monocytes are widely involved in the body's defense response, and abnormally regulated monocyte subsets are closely related to the pathogenesis of various diseases. It is unclear whether Treponema pallidum (Tp) dysregulates monocyte subsets and impacts the functions of monocytes. This study aims to analyze the distribution of monocyte subsets in syphilis patients and the effect of Tp on monocyte functions to explore the pathogenesis of syphilis. Flow cytometry was employed t ...[more]

PMID: 33282751

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Project description:Neuro-immune interaction during development is strongly implicated in the pathogenesis of neurodevelopmental disorders, but the mechanisms that cause neuronal circuit dysregulation are not well understood. We performed in vivo imaging of the developing retinotectal system in the larval zebrafish to characterize the effects of immune system activation on refinement of an archetypal sensory processing circuit. Acute inflammatory insult induced hyper-dynamic remodeling of developing retinal axons in larval fish and increased axon arbor elaboration over days. Using calcium imaging in GCaMP6s transgenic fish we showed that these morphological changes were accompanied by a shift toward decreased visual acuity in tectal cells. This finding was supported by poorer performance in a visually guided behavioral task. We further found that the pro-inflammatory cytokine, interleukin-1β (IL-1β) is upregulated by the inflammatory insult, and that down-regulation of IL-1β abrogated the effects of inflammation on axonal dynamics and growth. Moreover, baseline branching of the RGC arbors in IL-1β morphant animals was significantly different from that in control larvae, and their performance in a predation assay was impaired, indicating a role for this cytokine in normal neuronal development. This work establishes a simple and powerful non-mammalian model of developmental immune activation and demonstrates a role for IL-1β in mediating the pathological effects of inflammation on neuronal circuit development.SIGNIFICANCE STATEMENTMaternal immune activation (MIA) can increase the risk of neurodevelopmental disorders in offspring, however the mechanisms involved are not fully understood. Using a non-mammalian vertebrate model of developmental immune activation, we show that even brief activation of inflammatory pathways has immediate and long-term effects on the arborization of axons, and that these morphological changes have functional and behavioral consequences. Finally, we show that the pro-inflammatory cytokine IL-1β plays an essential role in both the effects of inflammation on circuit formation and normal axonal development. Our data add to a growing body of evidence supporting epidemiological studies linking immune activation to neurodevelopmental disorders, and help shed light on the molecular and cellular processes that contribute to the etiology of these disorders.

Project description:In response to danger signals, macrophages rapidly produce many inflammatory cytokines that trigger the cascade release of inflammatory mediators, leading to tissue damage, which is an important cause of clinical manifestations of syphilis at all stages. However, we still know very little about the specific mechanism of this process. Tp0768 is an infection-stage-dependent antigen that plays an important role in the infection of Treponema pallidum. In this study, we demonstrated that Tp0768 stimulation of macrophages can cause IL-1?, IL-6, and IL-8 mRNA expression levels to increase in a dose- and time-dependent manner. Further research showed that Tp0768 activated ER stress and the ROS/NF-?B pathway in macrophages. Inhibition of ER stress and the ROS/NF-?B pathway inhibited the expression of IL-1?, IL-6, and IL-8 induced by Tp0768. In addition, pretreatment with a PERK pathway inhibitor significantly reduced the expression of the NF-?B and JNK pathways, while also downregulating the expression of IL-1?, IL-6, and IL-8. Tp0768 stimulation can activate IRE1?/XBP-1 signaling and participate in the induction of inflammatory cytokines through the JNK pathway. These findings indicate that Tp0768 promotes the secretion of proinflammatory cytokines IL-1?, IL-6, and IL-8 by macrophages through ER stress and the ROS/NF-?B pathway, which are also involved in the activation of the NF-?B and JNK pathways that are induced by the PERK pathway and activation of IRE1?/XBP-1 signaling. KEY POINTS: • This study found for the first time that the recombinant Treponema pallidum protein Tp0768 promotes the production of IL-1?, IL-6, and IL-8 by macrophages through ER stress. • Recombinant Treponema pallidum protein Tp0768 regulates the ROS/NF-?B pathway through ER stress. • ER stress-related pathway PERK induces the expression of IL-1?, IL-6, and IL-8 by activating the NF-?B pathway and the JNK pathway. • IRE1? can induce the splicing of XBP-1mRNA and activate the JNK pathway.

Project description:Despite more than a century of research, genetic manipulation of Treponema pallidum subsp. pallidum (T. pallidum), the causative agent of syphilis, has not been successful. The lack of genetic engineering tools has severely limited understanding of the mechanisms behind T. pallidum success as a pathogen. A recently described method for in vitro cultivation of T. pallidum, however, has made it possible to experiment with transformation and selection protocols in this pathogen. Here, we describe an approach that successfully replaced the tprA (tp0009) pseudogene in the SS14 T. pallidum strain with a kanamycin resistance (kanR) cassette. A suicide vector was constructed using the pUC57 plasmid backbone. In the vector, the kanR gene was cloned downstream of the tp0574 gene promoter. The tp0574prom-kanR cassette was then placed between two 1-kbp homology arms identical to the sequences upstream and downstream of the tprA pseudogene. To induce homologous recombination and integration of the kanR cassette into the T. pallidum chromosome, in vitro-cultured SS14 strain spirochetes were exposed to the engineered vector in a CaCl2-based transformation buffer and let recover for 24 hours before adding kanamycin-containing selective media. Integration of the kanR cassette was demonstrated by qualitative PCR, droplet digital PCR (ddPCR), and whole-genome sequencing (WGS) of transformed treponemes propagated in vitro and/or in vivo. ddPCR analysis of RNA and mass spectrometry confirmed expression of the kanR message and protein in treponemes propagated in vitro. Moreover, tprA knockout (tprAko-SS14) treponemes grew in kanamycin concentrations that were 64 times higher than the MIC for the wild-type SS14 (wt-SS14) strain and in infected rabbits treated with kanamycin. We demonstrated that genetic manipulation of T. pallidum is attainable. This discovery will allow the application of functional genetics techniques to study syphilis pathogenesis and improve syphilis vaccine development.

Project description:Immune evasion and disease progression of Treponema pallidum subsp. pallidum are associated with sequence diversity in the hypervariable outer membrane protein TprK. Previous attempts to study variation within TprK have sequenced at depths insufficient to fully appreciate the hypervariable nature of the protein, failed to establish linkage between the protein's seven variable regions, or were conducted on isolates passed through rabbits. As a consequence, a complete profile of tprK during infection in the human host is still lacking. Furthermore, prior studies examining how T. pallidum subsp. pallidum uses its repertoire of genomic donor sites to generate diversity within the variable regions of the tprK have yielded a partial understanding of this process due to the limited number of tprK alleles examined. In this study, we used short- and long-read deep sequencing to directly characterize full-length tprK alleles from T. pallidum subsp. pallidum collected from early lesions of patients attending two sexually transmitted infection clinics in Italy. We demonstrate that strains collected from cases of secondary syphilis contain significantly more unique variable region sequences and full-length TprK sequences than those from cases of primary syphilis. Our data, combined with recent data available on Chinese T. pallidum subsp. pallidum specimens, show the near-complete absence of overlap in TprK sequences among the 41 specimens profiled to date. We further estimate that the potential antigenic variability carried by TprK rivals that of current estimates of the human adaptive immune system. These data underscore the immunoevasive ability of TprK that allows T. pallidum subsp. pallidum to establish lifelong infection.IMPORTANCE Syphilis continues to be a significant public health issue in both low- and high-income countries, including the United States where the rate of syphilis infection has increased over the past 5 years. Treponema pallidum subsp. pallidum, the causative agent of syphilis, carries the outer membrane protein TprK that undergoes segmental gene conversion to constantly create new sequences. We performed full-length deep sequencing of TprK to examine TprK diversity in clinical T. pallidum subsp. pallidum strains. We then combined our results with data from all samples for which TprK deep sequencing results were available. We found almost no overlap in TprK sequences between different patients. Moreover, our data allowed us to estimate the total number of TprK variants that T. pallidum subsp. pallidum can potentially generate. Our results support how the T. pallidum subsp. pallidum TprK antigenic variation system is an equal adversary of the human immune system leading to pathogen persistence in the host.

Dataset Information

Treponema pallidum Dysregulates Monocytes and Promotes the Expression of IL-1? and Migration in Monocytes Through the mTOR Signaling Pathway.

Publications

<i>Treponema</i><i>pallidum</i> Dysregulates Monocytes and Promotes the Expression of IL-1β and Migration in Monocytes Through the mTOR Signaling Pathway.

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