Periodontitis and Gestational Diabetes Mellitus: A Potential Inflammatory Vicious Cycle

Periodontitis is a chronic inflammatory immune disease associated with a dysbiotic state, influenced by keystone bacterial species responsible for disrupting the periodontal tissue homeostasis. Furthermore, the severity of periodontitis is determined by the interaction between the immune cell response in front of periodontitis-associated species, which leads to the destruction of supporting periodontal tissues and tooth loss in a susceptible host. The persistent bacterial challenge induces modifications in the permeability and ulceration of the sulcular epithelium, which facilitates the systemic translocation of periodontitis-associated bacteria into distant tissues and organs. This stimulates the secretion of pro-inflammatory molecules and a chronic activation of immune cells, contributing to a systemic pro-inflammatory status that has been linked with a higher risk of several systemic diseases, such as type 2 diabetes mellitus (T2DM) and gestational diabetes mellitus (GDM). Although periodontitis and GDM share the common feature of systemic inflammation, the molecular mechanistic link of this association has not been completely clarified. This review aims to examine the potential biological mechanisms involved in the association between periodontitis and GDM, highlighting the contribution of both diseases to systemic inflammation and the role of new molecular participants, such as extracellular vesicles and non-coding RNAs, which could act as novel molecular intercellular linkers between periodontal and placental tissues.

New insights into the development of radiation-induced cardiac damage using a mouse model

Background The improvement of anticancer-therapy results in a greater amount of long-term survivors after radiotherapy. Therefore, the understanding of cardiotoxicity after irradiation is of increasing importance. Long-term adverse cardiovascular events may become evident years or decades after radiotherapy. The relative contribution of irradiation in relation to other cancer treatments can often only be estimated. Recent experimental and clinical evidence suggests that cardiovascular symptoms, including exertional dyspnoea, may be caused by heart failure with preserved ejection fraction (HFpEF), which remains incompletely understood in patients after radiation therapy. Purpose We aim to characterize the development of radiation-induced cardiomyopathy and elucidate underlying patho-mechanisms. Methods Mice received a single dose of whole thorax irradiation (12.5 Gy) and were sacrificed at 1 and 3 days or 3, 6, 12, 16, 20 and 25–30 weeks. Endothelial cells and immune cells at different time points were quantified using flow cytometry (FACS). Structural changes and localization of endothelial cell damage was imaged using light-sheet fluorescence microscopy (LSFM) with CD31 staining. Development of fibrosis was determined using qRT-PCR (fibronectin and TGFβ), western blot (collagen-1,α-smooth muscle) and (immune-)histological analyses. Functional analyses were conducted using echocardiography and pressure-volume-(PV-)catheterization. Results Endothelial damage was determined by significant reduction of CD31 expression in mouse hearts 6 weeks after irradiation compared to sham-treated control mice using FACS analyses. LSFM showed structural changes especially in the edge zone of left ventricle presented as less densely CD31 stained regions. Additionally, we investigated cardiac immune cell response regarding innate and adaptive immunity, showing specific response to tissue damage at different time points. Invasion of monocytes started 6 weeks after irradiation and highest level of monocytes and macrophages was measured at 12 weeks. Regarding cardiac long-term damage, myocardial fibrosis was detected on RNA- and protein-level as well as in histological analyses with significant changes 20 weeks after chest irradiation. This could be correlated with echocardiographic parameters for diastolic dysfunction (elevated isovolumic relaxation time/mitral valve deceleration time). Also functional reserve of irradiated mice was reduced, investigated by measurement of cardiac output and stroke volume after dobutamine injection in PV-catheterization. Conclusion We described a novel time-dependent endothelial cell damage and immune cell response after thoracic irradiation in mice, which could also be imaged using LSFM. Characterization of long-term damage showed cardiac fibrosis correlating with diastolic dysfunction and reduced contractile reserve. Furthermore, therapeutic approaches will be investigated using the established mouse model. FUNDunding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Dr. S.M. Mrotzek acknowledges the following funding source: IFORES research grant from the Medical Faculty, University Duisburg-Essen, Germany.

Identification of Somatic Mutation-Driven Immune Cells by Integrating Genomic and Transcriptome Data

Tumor somatic mutations in protein-coding regions may generate neoantigens which may trigger antitumor immune cell response. Increasing evidence supports that immune cell response may profoundly influence tumor progression. However, there are no calculated tools to systematically identify immune cells driven by specific somatic mutations. It is urgent to develop a calculated method to comprehensively detect tumor-infiltrating immune cells driven by the specific somatic mutations in cancer. We developed a novel software package (SMDIC) that enables the automated identification of somatic mutation-driven immune cell. SMDIC provides a novel pipeline to discover mutation-specific immune cells by integrating genomic and transcriptome data. The operation modes include inference of the relative abundance matrix of tumor-infiltrating immune cells, detection of differential abundance immune cells with respect to the gene mutation status, conversion of the abundance matrix of significantly dysregulated cells into two binary matrices (one for upregulated and one for downregulated cells), identification of somatic mutation-driven immune cells by comparing the gene mutation status with each immune cell in the binary matrices across all samples, and visualization of immune cell abundance of samples in different mutation status for each gene. SMDIC provides a user-friendly tool to identify somatic mutation-specific immune cell response. SMDIC may contribute to understand the mechanisms underlying anticancer immune response and find targets for cancer immunotherapy. The SMDIC was implemented as an R-based tool which was freely available from the CRAN website https://CRAN.R-project.org/package=SMDIC.

RhoA Signaling in Immune Cell Response and Cardiac Disease

Chronic inflammation, the activation of immune cells and their cross-talk with cardiomyocytes in the pathogenesis and progression of heart diseases has long been overlooked. However, with the latest research developments, it is increasingly accepted that a vicious cycle exists where cardiomyocytes release cardiocrine signaling molecules that spiral down to immune cell activation and chronic state of low-level inflammation. For example, cardiocrine molecules released from injured or stressed cardiomyocytes can stimulate macrophages, dendritic cells, neutrophils and even T-cells, which then subsequently increase cardiac inflammation by co-stimulation and positive feedback loops. One of the key proteins involved in stress-mediated cardiomyocyte signal transduction is a small GTPase RhoA. Importantly, the regulation of RhoA activation is critical for effective immune cell response and is being considered as one of the potential therapeutic targets in many immune-cell-mediated inflammatory diseases. In this review we provide an update on the role of RhoA at the juncture of immune cell activation, inflammation and cardiac disease.

In vitro immunogenicity prediction: bridging between innate and adaptive immunity

Development of antidrug antibodies (ADAs) is an undesirable potential outcome of administration of biotherapeutics and involves the innate and adaptive immune systems. ADAs can have detrimental clinical consequences: they can reduce biotherapeutic efficacy or produce adverse events. Because animal models are considered poor predictors of immunogenicity in humans, in vitro assays with human innate and adaptive immune cells are commonly used alternatives that can reveal cell-mediated unwanted immune responses. Multiple methods have been developed to assess the immune cell response following exposure to biotherapeutics and estimate the potential immunogenicity of biotherapeutics. This review highlights the role of innate and adaptive immune cells as the drivers of immunogenicity and summarizes the use of these cells in assays to predict clinical ADA.

RhoA Signaling in Immune Cell Response and Cardiac Disease

Chronic inflammation, the activation of immune cells and their cross-talk with cardiomyocytes in the pathogenesis and progression of heart diseases has long been overlooked. However, with the latest research developments, it is increasingly accepted that a vicious cycle exists where cardiomyocytes release cardiocrine signaling molecules that spirals down to immune cell activation and chronic state of low‐level inflammation. For example, cardiocrine molecules released from injured or stressed cardiomyocytes can stimulate macrophages, dendritic cells, neutrophils and even T-cells, which then subsequently increase cardiac inflammation by co-stimulation and positive feedback-loops. One of the key proteins involved in stress-mediated cardiomyocyte signal transduction is a small GTPase RhoA. Importantly, the regulation of RhoA activation is critical for effective immune cell response and is being considered as one of the potential therapeutic targets in many immune-cell-mediated inflammatory diseases. In this review we provide an update on the role of RhoA at the juncture of immune cell activation, inflammation and cardiac disease.

PIWIL1 governs the crosstalk of cancer cell metabolism and immunosuppressive microenvironment in hepatocellular carcinoma

Altered energy metabolism of cancer cells shapes the immune cell response in the tumor microenvironment that facilitates tumor progression. Herein, we reported the novel of tumor cell-expressed Piwi Like RNA-Mediated Gene Silencing 1 (PIWIL1) in mediating the crosstalk of fatty acid metabolism and immune response of human hepatocellular carcinoma (HCC). PIWIL1 expression in HCC was increased compared to normal hepatic tissues and was positively correlated with the proliferation rate of HCC cell lines. PIWIL1 overexpression accelerated in vitro proliferation and in vivo growth of HCC tumors, while PIWIL1 knockdown showed opposite effects. PIWIL1 increased oxygen consumption and energy production via fatty acid metabolism without altering aerobic glycolysis. Inhibition of fatty acid metabolism abolished PIWIL1-induced HCC proliferation and growth. RNA-seq analysis revealed that immune system regulation might be involved, which was echoed by the experimental observation that PIWIL1-overexpressing HCC cells attracted myeloid-derived suppressor cells (MDSCs) into the tumor microenvironment. MDSCs depletion reduced the proliferation and growth of PIWIL1-overexpressing HCC tumors. Complement C3, whose secretion was induced by PIWIL1 in HCC cells, mediates the interaction of HCC cells with MDSCs by activated p38 MAPK signaling in MDSCs, which in turn initiated expression of immunosuppressive cytokine IL10. Neutralizing IL10 secretion reduced the immunosuppressive activity of MDSCs in the microenvironment of PIWIL1-overexpressing HCC. Taken together, our study unraveled the critical role of PIWIL1 in initiating the interaction of cancer cell metabolism and immune cell response in HCC. Tumor cells-expressed PIWIL1 may be a potential target for the development of novel HCC treatment.

Leflunomide Inhibits rat-to-Mouse Cardiac Xenograft Rejection by Suppressing Adaptive Immune Cell Response and NF-κB Signaling Activation

Xenotransplantation is a potential solution for the severe shortage of human donor organs and tissues. The generation of humanized animal models attenuates strong innate immune responses, such as complement-mediated hyperacute rejection. However, acute vascular rejection and cell mediated rejection remain primary barriers to xenotransplantation, which limits its clinical application. In this study, we systematically investigated the immunosuppressive effect of LEF using a rat-to-mouse heart xenotransplantation model. SD rat xenogeneic hearts were transplanted into C57BL/6 mice, and survived 34.5 days after LEF treatment. In contrast, BALB/c allogeneic hearts were transplanted into C57BL/6 mice, and survived 31 days after LEF treatment. Compared to normal saline treatment, LEF treatment decreased xenoreactive T cells and CD19+ B cells in recipient splenocytes. Most importantly, LEF treatment protected myocardial cells by decreasing xenoreactive T and B cell infiltration, inflammatory gene expression, and IgM deposition in grafts. In vivo assays revealed that LEF treatment eliminated xenoreactive and alloreactive T and B lymphocytes by suppressing the activation of the NF-κB signaling pathway. Taken together, these observations complement the evidence supporting the potential use of LEF in xenotransplantation.

Detailed Immunophenotypic Profiling of Peripheral Blood Immune Cells in Patients with Hematologic Malignancies after Sars-Cov-2 Infection

Introduction:The spread of SARS-CoV-2 virus continues to pose a major public health threat. Patients with cancer are thought to be at increased risk from SARS-COV-2 infection due to the immunodeficiency that results from the underlying neoplasm and treatment. The immune response to this infection has been the subject of great interest, with an extreme variation in clinical severity between infected individuals. Variation in the immune cell response (B, T, and NK lymphocytes, monocytes, and myeloid derived suppressor cells (MDSCs), among others) and their function have been hypothesized to be responsible for this range of presentation. Methods:Two patients with a history of hematologic malignancies were matched with three non-cancer patients with similar baseline clinical characteristics and severity of COVID related illness. The critical group (CG) was defined as those requiring mechanical ventilation (MV) due to COVID related respiratory failure and the non-critical group (NCG) were hospitalized but did not require MV. All samples studied were obtained from peripheral blood and processed within 4-hours of collection. Peripheral blood mononuclear cell (PBMC) were isolated using ficoll density gradient separation. Flowcytometric analysis using CytekTM Aurora was done on fresh PBMC samples. Thirty antibody-based flow markers were used to identify 54 distinct immune cell populations. IRB approval was obtained. Results: Critical Group (CG):The CG included case 1, a 47 year-old (y.o.) female (F) with a history (hx) of acute myeloid leukemia and had an matched related donor allogeneic hematopoietic stem cell transplant (alloHSCT) 10-years prior remaining in remission, with hematologic recovery, and off immunosuppressants treated with remdesivir and coritcosteroids for COIVD directed therapy; and case 2, a 55 y.o. F with a hx of HIV treated with corticosteroids for COIVD directed therapy (see Figure 1a). Non-Critical Group (NCG):The NCG included case 3, a 73 y.o male (M) with hx of relapsed/refractory Philadelphia chromosome negative Acute Lymphoblastic Leukemia with loss of CD19 and CD22 expression following treatment with blinatumumab and inotuzumab, and most recently treated with decitabine/venetoclax; case 4, a 66 y.o. M with hx of cardiomyopathy; and case 6, a 54 y.o. M with hx of obesity. None of the NCG cases were treated with COVID directed therapy. See Table 1 for further clinical information. Immunophenotypic expression:Flow cytometry gating strategy done as outlined in Fig 1a. Case 1 had a high proportion of B-cells, CD8+ T-cells, and cells with exhaustion markers (CD8+CD94+ T-cells, CD4+PD1+ T-cells, CD4+PD1+CD94+ T-cells, PD1-CD94+ NK T-cells, Lag3+Cd11b- non-TB leukocytes) and MDSC immunophenotypes compared with matched case 2. Case 3 also had a high proportion of exhaustion markers (Lag3+CD39 low B-cells, CD8+PD1+ T-cells, CD8+CD94+PD1+ T-cells, CD4+PD1+CD94+ T-cells, PD1+CD94+ NK T-cells, PD1-CD94+ NK T-cells, Lag3+CD11b+, Lag3+CD11b- non-TB leukocytes) and high expression of immunosuppressive Treg and all MDSC; although high expression of granulocytic MDSC. Case 2 had a significant number of exhaustion and immunosuppressive cells as well. Cases 4 and 5 had a higher predominance of all T-cell subtypes and also had variable expression of exhaustion and immunosuppressive immunophenotypes (See Fib 1b). Conclusion:In our study of one critical and one non-critical patient with a history of hematologic malignancy matched with three non-cancer patients we demonstrate the high predominance of exhaustion markers (Lag3,PD1,CD94) and immunosuppressive cell types (Treg, granulocytic and monocytic MDSC). These findings are consistent with the fact that both CG and NCG, as hospitalized patients, represent the most severely ill COVID patient cohort. Of notable interest to the cancer population, cases 1 and 3 had a significant number of exhaustion and immunosuppressive immunophenotypes, suggestive of baseline exhaustion following alloHSCT even years after engraftment in case 1 and attenuated functional immunity in a patient undergoing active treatment in case 3. Interestingly, case 3 had lower expression of all MDSC, a known treatment effect of decitabine. Paired cytokine measurement and its effect on immunophenotype is underway. Additionally, we plan to present an atlas of the peripheral immune cell response on fifteen additional non-cancer COVID patients. Disclosures No relevant conflicts of interest to declare.