Progress 05/15/19 to 01/29/21
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?The results from this project have been incorporated into my Ph.D. dissertation andwill be published in academic journal by the end of this year. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
In this study, we studied the previously established pregnant mouse model and discovered factors mediating remarkable phenotypic differences in the placenta and the mononuclear phagocyte system during B. abortus infection. While macrophages, which are main target cells for B. abortus, usually survive during the infection, placental trophoblasts, which are important cells for maintaining pregnancy, are massively killed during B. abortus infection. We have demonstrated that this killing is dependent on the ER stress response triggered by an bacterial T4SS effector protein, VceC. Further, blockade of the ER stress response or tumor necrosis factor α (TNF-α) reduced placental inflammation and improved fetal viability. Deficiency of CHOP, a transcription factor induced by ER stress, could also prevent death of trophoblasts, reduce inflammation, and increase the survival of mouse pups. To gain more insight into how ER stress response is triggered during B. abortus infection, we characterized the molecular mechanism underlying the induction of ER stress signaling pathway by VceC. We showed that the proline-rich region of ER-located VceC contributes to perturbation of ER homeostasis, thereby decreasing of ATP levels in the ER, activating the UPR and triggering downstream pro- inflammatory cytokine production. Collectively, not only did we show that B. abortus uses its effector protein, VceC, to induce ER stress that triggers placental inflammation and abortion, we also characterize the molecular mechanisms by which VceC elicits ER stress-induced inflammation. Our work indicates a fine- tuned ER stress response that B. abortus modulates in different cell types to ensuring its survival, replication, and egress. The results of this dissertation research may have implications for understanding placental pathologies caused by other bacteria and may facilitate future functional and mechanistic studies of how a bacterial virulence factor can elicit ER stress-induced inflammation in the placenta.
Publications
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Progress 05/15/19 to 05/14/20
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?I will contibute todetermine the consequences of ER stress-mediated trophoblast cell death during placental infection for placentitis and abortio, as I proposed in aim 2.
Impacts
What was accomplished under these goals?
To interrogate the interactions between B. abortus and placental cells and tissues that trigger inflammatory responses leading to transmission, we discovered that the B. abortus Type IV secretion system (T4SS) effector VceC induces proinflammatory cytokine, tumor necrosis factor α (TNFα), which is crucial for B. abortus-induced fetal loss and placentitis. Further, blockade of ER stress-induced TNF-α reduced placental inflammation and improved fetal viability. B. abortus induced TNFα expression in placenta but not in spleen During the acute stage of Brucella infection in mice, the production of Th1 cytokines, including interferon γ (IFN-γ) and TNFα, are upregulated by the host immune system (1) . These pre-inflammatory cytokines have also been shown to induce abortion thus are detrimental to pregnancy. Interestingly, our recent studies have discovered a cell-type-specific pathological feature between splenic and placental tissue during Brucella infection. While no histologic evidence of cell death was observed in the infected spleen, moderate to severe cell death that eventually lead to abortion was seen in the infected placenta (2). These findings lead us to further investigate the role of TNFα in Brucella-induced abortion in the placenta. First, we measured the levels of TNFα in spleen and placenta from the infected pregnant mice by qRT-PCR. As shown in figure 1A, the expression level of TNFα from the infected placenta peaked at 13 days post infection, while the TNFα level from the infected spleen remained relatively low throughout the course of the experiment (Fig 1B). These data suggested that TNFα was induced tissue-specifically during Brucella infection. Blockade of TNFα prevents placental pathology One of the major symptoms caused by B. abortus in its natural host is the acute severe inflammatory response following abortion and infertility (3). Since TNFα was upregulated during Brucella infection, we investigated whether it mediates fetal loss in pregnant mice. We administered neutralizing antibodies against TNF-α to pregnant mice infected with B. abortus and measured the percent viability of pups. A partial rescue of fetal viability was observed in B. abortus-infected mice after TNF-α neutralization (Fig 2A). Moreover, as shown in figure 2B, placentas from wild-type B. abortus infected mice treated with TNFα antibody exhibited histologic evidence of mild or absent placentitis, suggesting TNFα blockade alleviate severe inflammatory response observed in placenta. Anti-TNFα treatment did not reduce fitness of B. abortus in the placental infection niche, as seen in the similar colony-forming units (CFU) recovered from the control or anti-TNFα treated infected placentas (Fig 2C). TNFα expression was depended on the induction of ER stress Our previous results indicated a role for VceC, a T4SS effector protein that induces the UPR, in abortion caused by B. abortus in mice, as mice infected with VceC mutant had decreased inflammatory response and increased viability of pups after infection (2, 4). Therefore, to investigate the role of VceC in TNFα induction, we infected the pregnant mice with wildtype B. abortus 2308 or isogenic mutants carrying deletions in vceC that has been previously reported (4). Placental expression of TNF-α and circulating TNF-α could be suppressed by the ER stress inhibitor ,TUDCA , suggesting that it was induced by ER stress (Fig 3A and 3B). Further, infection with the vceC mutant did not elicit TNF-α production in infected placenta, indicating VceC elicited induction of TNFα during Brucella infection (Fig 1A, Fig 3A and 3B). All together, these results indicated that in infected placenta, VceC plays a major role in the UPR-induced TNFα expression. TNFα blockade affects CHOP regulation during infection Since our recent studies indicated that the ER stress-induced transcription factor CHOP plays a crucial role in the cell death of infected placental trophoblasts that lead to abortion (2), we asked whether the TNFα blockade affected CHOP upregulation. Pregnant mice were treated with control isotype or with TNFα antibody during B. abortus infection. qRT-PCR results suggested that not only the pro-inflammatory gene, IL?8 homologues KC, was blunted by the TNFα antibody treatment (Fig 4A), the transcript levels of CHOP were also significantly reduced (Fig 4B). Together, these results show that VceC, by eliciting ER stress and, elicits an inflammatory response that contributes to placentitis and fetal loss during B. abortus infection. Summary Placental inflammation and fetal loss were blunted by deletion of VceC, by preventing ER stress, or by blocking the inflammatory cytokine TNF-α. Taken together, our results support a model in which VceC triggers an ER stress response leading toplacentitis, which in turn leads to fetal death. Figures and legends attached in the link below: http://drive.google.com/drive/folders/1O4rFVKrr_damKDSArxlJYl2QFwQ9YKw6?usp=sharing 1. Kim S, Lee DS, Watanabe K, Furuoka H, Suzuki H, Watarai M. Interferon-gamma promotes abortion due to Brucella infection in pregnant mice. BMC Microbiol. 2005;5:22. 2. Byndloss MX, Tsai AY, Walker GT, Miller CN, Young BM, English BC, Seyffert N, Kerrinnes T, de Jong MF, Atluri VL, Winter MG, Celli J, Tsolis RM. Brucella abortus Infection of Placental Trophoblasts Triggers Endoplasmic Reticulum Stress-Mediated Cell Death and Fetal Loss via Type IV Secretion System-Dependent Activation of CHOP. mBio. 2019;10(4). 3. Byndloss MX, Tsolis RM. Brucella spp. Virulence Factors and Immunity. Annu Rev Anim Biosci. 2016;4:111-27. 4. de Jong MF, Starr T, Winter MG, den Hartigh AB, Child R, Knodler LA, van Dijl JM, Celli J, Tsolis RM. Sensing of bacterial type IV secretion via the unfolded protein response. mBio. 2013;4(1):e00418-12.
Publications
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