Investigating roles of Topoisomerase 3b (TOP3B) in immune responses to infection by coronavirus and bacteria

National Institute On Aging

Investigators

Abstract

Our group has discovered that Topoisomerase 3b (TOP3B) is a dual activity enzyme that can change the topology of not only DNA, but also RNA (XU et al. 2013). TOP3B forms a complex with TDRD3; this complex interacts with Fragile X Mental Retardation Protein (FMRP). Our recent studies show that Top3b-TDRD3 regulates stimulus-induced transcription, mRNA translation and turnover, as well as siRNA-guided heterochromatin formation (LEE et al. 2018; SU et al. 2022). The deletion of Top3b in human is associated with neural diseases, such as schizophrenia and autism (STOLL et al. 2013; XU et al. 2013; AHMAD et al. 2017). Our studies also showed that Top3b-KO mice had neurodevelopment and behavioral impairments (JOO et al. 2020). Interestingly, TOP3B-KO mice have shortened lifespan and immune dysfunction, exemplified by increased serum autoantibodies and an increase in numerical aberrations of chromosomes in splenocytes and bone marrow cells (KWAN et al. 2007). However, the mechanism of how Top3B functions in immune system is unclear. This new proposal is to investigate how TOP3B regulates immune response to mouse coronavirus MHV (Mouse Hepatitis virus) infection in mouse, and to bacterial infection in Drosophila. Severe Acute Respiratory Syndrome Coronavirus2 (SARS-Cov2) has caused economic and life damages over the past few years. The host immune response to SARS-Cov2 is responsible for viral elimination and recovery (GARCIA 2020). On the other hand, the strong inflammation (termed cytokine storm) elicited by the virus can also cause severe sickness or even lethality to the host (GARCIA 2020). Anti-inflammation drugs are often used to suppress cytokine storms in SARS-Cov2 patient. Notably, inhibitors of Topoisomerase 1 (Top1) have been developed as one class of anti-inflammation drugs, and are now being examined in clinical trials to treat SARS-Cov2 patients (HO et al. 2021). TOP3B resembles TOP1 in that both can promote transcriptional activation in response to extra cellular stimulus. We propose to study the roles of TOP3B in immune responses to infection by coronavirus and bacteria. The data should provide insights on whether TOP3B inhibitors can work as drugs to control immune responses. Specific Aims: Aim1: Investigate the functions of TOP3B in T and B cell response to MHV infection in mouse. Aim2: Investigate the functions of TOP3B in cytokine response to MHV infection in mouse-models and cell lines. Aim3: Investigate whether and how Top3b-Tdrd3 contributes to regulation of immune signaling pathways using a Drosophila model. Progress update: 1. Top3b-KO mice display altered levels of Lymphocytes and Neutrophils responding to MHV. We started to test whether Top3b-KO mouse-models show altered levels of lymphocytes and neutrophils in response to MHV infection. Four groups of mice, two wild type and two Top3b-KO, were inoculated with 105 pfu MHV or DMEM as a negative control. Blood was collected at three time points: seven days prior to inoculation, two days post inoculation, and 5 days post inoculation. Blood cell analysis was performed to test which cells were most affected during MHV infection in wild types and Top3b-KO mouse-models. We found that MHV-infected Top3b-KO mice showed a significant increase in lymphocytes and a robust decrease in Neutrophils compared to MHV-infected WT mice by subtraction of day 2 from day 5 reads. Meanwhile, control groups did not show any significant change between wildtype and Top3b-KO. The data suggest that Top3b is required for normal immune response to MHV. 2. A family of chemokines are upregulated in the lungs of Top3b-KO mice infected by MHV. We preformed RNA-seq analysis of lungs harvested on day 5 post MHV inoculation. 2 male mice of each group (1. WT MHV, 2. Top3b-KO MHV) were used. We found inflammatory response, lymphocyte chemotaxis, and macrophage chemotaxis genes were upregulated in MHV infected Top3b-KO mice compared to WT mice. Among these genes, a family of chemokines, involved in immunoregulation, was found upregulated in MHV infected Top3b-KO and in WT. We checked the changes of mRNA levels Chemokines (ccl2, ccl7, and ccl12) and Top3b (as a control)by RT-qPCR in each groups (1. WT MHV, 2. Top3b-KO MHV, 3. WT DMEM, and 4. Top3b-KO DMEM). Their levels , were normalized with GAPDH. Chemokine mRNA levels were increased in MHV-infected WT and Top3b-KO samples when compared with DMEM-treated controls. Consistent with the RNA-seq data, chemokine mRNA levels in MHV infected Top3b-KO mice are higher than in MHV-infected WT mice. The data are consistent with blood cell findings that TOP3B is required for normal immune response to MHV infection. 3. Top3b-KO and TDRD3-KO flies have dysregulated immune responses to bacterial infection Our preliminary study utilizing the pathogenic bacteria Listeria monocytogenes infected Drosophila shows that in the absence of Top3b and Tdrd3, expression of many genes involved in immune signaling, pathogen recognition, and stress response are altered, based on RNA-seq. These gene expression changes may account for a noticeable melanization phenotype between WT, Top3bko, and Tdrd3ko flies. In Drosophila, melanization is a unique rapid wound response akin to clotting and scab formation in humans. Around the site of infection or injury, melanocytes produce increasing amounts of melanin pigment to create a physical barrier in preventing further spread of pathogens and cytotoxic materials (DE GREGORIO et al. 2002). Upon infection, Top3b-/- and Tdrd3-/- flies exhibit both increased area and intensity of pigmentation. Interestingly, we observed a diffusion-like spreading of melanin pigment radiating outward from the original site of infection, suggesting a possible dysregulation in the melanization response. Our RNA-sequencing data reveals specific patterns of gene expression unique to Top3bko, where an immunosuppressive cytokine, Diedel, is highly upregulated and edin, a key player in the humoral immune response, is downregulated (data not shown). Together with data from MHV-infected mice, these findings suggest that Top3b is important for maintaining normal immune response to pathogens.