Beetler, Danielle J.

Background: Viral myocarditis presents a significant burden of disease, particularly among children and young adults. However, clinical guidelines and treatment strategies for pediatric patients are derived from those for adult patients due to a lack of pediatric data. Current animal models of viral myocarditis use adult mice, so conclusions from these models cannot necessarily be extrapolated to the pediatric population. We sought to develop a juvenile mouse model of myocarditis to examine differences between these two distinct clinical populations. Methods: Male and female BALB/c 3-4-week-old 'juvenile' and 8-week-old 'adult' mice were infected intraperitoneally with 103 PFU of heart-passaged coxsackievirus B3. Sera was used to evaluate testosterone and estradiol levels. Cardiac histological evaluations included overall inflammation, fibrosis, and specific cell-type infiltration. RNA was extracted from cardiac tissue and evaluated for changes in gene expression of cell-type markers, complement components, and NLRP3 inflammasome components. Results: Juvenile mice exhibited more severe inflammation than adult mice but no sex differences in overall inflammation. Juvenile mice demonstrated increased infiltration of CD11b+ cells, F4/80+ cells, and CD3+ T-cells vs. adults. Inflammasome genes NLRP3 and caspase-1 were significantly increased in juvenile compared with adult myocarditis. Conclusions: This paper is the first to describe a juvenile mouse model of coxsackievirus B3 myocarditis and provides a direct comparison to a translational adult mouse model. Juvenile mice had greater cardiac inflammation than adults. This model replicates clinical populations and provides a valuable tool to study age as a factor in the pathogenesis of myocarditis.

Stem cell-derived β (SC-β) cells are a promising therapy for type 1 diabetes (T1D), but their long-term efficacy is limited by immune-mediated graft rejection. Platelet-derived exosome product (PEP) has emerged as a novel immunomodulatory agent, although its effect on SC-β cell xenograft rejection remains unclear. Here, we evaluated PEP in stimulated human peripheral blood mononuclear cells (PBMCs) and in an immunocompetent mouse model of SC-β cell transplantation under the kidney capsule. Immune-related gene expression was assessed by quantitative PCR, immune cell infiltration by immunofluorescence, and graft function by circulating human insulin. In vitro, PEP reduced NK-cell-associated gene expression, including NK1.1, EOMES, and KLRK1, and increased IL-10 expression. In vivo, untreated SC-β cell xenografts showed progressive immune infiltration and loss of detectable human insulin by day 14, whereas PEP-treated grafts retained detectable insulin through day 14 across co-transplantation, pretreatment, and systemic administration strategies. PEP co-transplantation delayed, but did not prevent, xenograft rejection, with graft loss observed by day 21. This delay was associated with reduced NK1.1+ cell infiltration and lower expression of selected inflammatory and rejection-associated markers, including NK1.1, Nos2, and Nlrp3. These findings suggest that PEP modulates graft-associated immune responses and may serve as an adjunct immunomodulatory strategy for stem cell-based therapies for type 1 diabetes. Further studies are needed to evaluate sustained graft durability, safety, and translational efficacy.

Abstract Background Radiation-induced heart disease (RIHD) may develop months to years following radiation therapy for cancers near the heart such as lung or breast cancer. Several mouse models of RIHD exist to study the pathogenesis of disease, but to our knowledge no studies have examined whether sex differences exist in RIHD in mice. Methods Female and male BALB/cJ mice received one whole heart dose of 22 Gray (Gy) radiation via a C5 collimator vs. sham controls that received 0 Gy, anesthesia and were placed in the X-ray machine for the same length of time. Mechanisms of cardiotoxicity were examined at 24 h (innate cardiotoxicity), day 10 (acute cardiotoxicity) and day 35 (chronic cardiotoxicity) post irradiation. Results Females and males developed dsDNA breaks from radiation at day 10 but females had more damage than males ( p < 0.001). Vessel area and cardiac hypertrophy were increased by radiation at day 10 and 35 but were higher in males than females at day 35 ( p < 0.001, p = 0.001, respectively). Bulk RNA sequencing of the heart at day 10 revealed that females and males developed similar gene pathway responses to radiation by upregulating cellular repair pathways and downregulated mitochondrial function. However, females upregulated the master regulator of mitochondrial function, Pgc1α ( p = 0.0001), suggesting improved mitochondrial repair in females. By day 35, males developed heart failure with reduced ejection fraction (HFrEF), inflammatory dilated cardiomyopathy (iDCM) and fibrosis while females developed heart failure with preserved ejection fraction (HFpEF) without inflammation or fibrosis. Conclusions We observed sex differences in nearly every cardiac parameter that we examined following radiation exposure. BALB/c males exposed to 22 Gy radiation developed HFrEF and iDCM by day 35 while females developed HFpEF. Further research is needed to better understand mechanisms driving sex differences in RIHD using translational animal models.

Autoimmune diseases are a leading cause of disability worldwide. Most autoimmune diseases occur more often in women than men, with rheumatic autoimmune diseases being among those most highly expressed in women. Several key factors, identified mainly in animal models and cell culture experiments, are important in increasing autoimmune disease in females. These include sex hormones, immune genes including those found on the X chromosome, sex-specific epigenetic effects on genes by estrogen and the environment, and regulation of genes and messenger RNA by microRNAs found in extracellular vesicles. Evidence is also emerging that viruses as well as drugs or toxins that damage mitochondria may contribute to increased levels of autoantibodies against nuclear and mitochondrial antigens, which are common in many autoimmune diseases. The purpose of this Review is to summarize our current understanding of mechanisms that may determine sex differences in autoimmune disease.

Viral myocarditis is an inflammatory cardiomyopathy with no pharmacologic agent currently able to quell the immune mechanisms that damage the heart in their inflammatory crossfire to quell infections, without risk of immunocompromise. Sex differences are well known in inflammatory and/or infectious diseases. Females display more efficient, anti-inflammatory responses to external threats like viruses, while males launch a pro-inflammatory defensive response. Myocarditis is a classic case of an infectious, inflammatory disease being more frequent and more severe in men, while pre-menopausal women are most protected. Extracellular vesicles are biocompatible, highly abundant, stable messaging carriers with no transformative risk, and hold great promise as therapeutics due to their functional cargo. As EVs are soluble mediators of the conversations between cells, we seek to redirect protective anti-inflammatory messaging from pre-menopausal women to male mice, using a highly translational model of murine myocarditis. We derive EVs from omental fat, repurposing this source to derive a purified, therapeutic component. Our results show robust immune system control, with EV treatments effectively attenuating pro-inflammatory inflammasome and complement markers, while leaving regulatory components like M2b macrophages and complement component CR1 unchanged. Treated mice are healthier during acute disease and demonstrate better heart function than control mice, despite being a strain susceptible to myocarditis and subsequent dilated cardiomyopathy. Tests using EVs from similarly aged men do not demonstrate this anti-inflammatory capacity. Proteomic and RNA-sequencing content comparisons of these EVs from these two donor groups reveal important proteins and regulatory micro RNAs. Our data suggest that EVs from fat may be a novel anti-inflammatory treatment for viral myocarditis, however individual donors show varying therapeutic capacity, with EV content heavily influenced by sex and age. This study not only addresses a major therapeutic gap in the myocarditis field, but also provides functional and -omic based comparisons of EVs from donors differing only by sex, to test if female cardioprotection from myocarditis is transferrable.

AbstractPatients with viral myocarditis are at risk of sudden death and may progress to dilated cardiomyopathy (DCM). Currently, no disease‐specific therapies exist to treat viral myocarditis. Here it is examined whether reconstituted, lyophilized extracellular vesicles (EVs) from platelets from healthy men and women reduce acute or chronic myocarditis in male mice. Human‐platelet‐derived EVs (PEV) do not cause toxicity, damage, or inflammation in naïve mice. PEV administered during the innate immune response significantly reduces myocarditis with fewer epidermal growth factor (EGF)‐like module‐containing mucin‐like hormone receptor‐like 1 (F4/80) macrophages, T cells (cluster of differentiation molecules 4 and 8, CD4 and CD8), and mast cells, and improved cardiac function. Innate immune mediators known to increase myocarditis are decreased by innate PEV treatment including Toll‐like receptor (TLR)4 and complement. PEV also significantly reduces perivascular fibrosis and remodeling including interleukin 1 beta (IL‐1β), transforming growth factor‐beta 1, matrix metalloproteinase, collagen genes, and mast cell degranulation. PEV given at days 7–9 after infection reduces myocarditis and improves cardiac function. MicroRNA (miR) sequencing reveals that PEV contains miRs that decrease viral replication, TLR4 signaling, and T‐cell activation. These data show that EVs from the platelets of healthy individuals can significantly reduce myocarditis and improve cardiac function.

Viral infections are a leading cause of myocarditis and pericarditis worldwide, conditions that frequently coexist. Myocarditis and pericarditis were some of the early comorbidities associated with SARS-CoV-2 infection and COVID-19. Many epidemiologic studies have been conducted since that time concluding that SARS-CoV-2 increased the incidence of myocarditis/pericarditis at least 15× over pre-COVID levels although the condition remains rare. The incidence of myocarditis pre-COVID was reported at 1 to 10 cases/100 000 individuals and with COVID ranging from 150 to 4000 cases/100 000 individuals. Before COVID-19, some vaccines were reported to cause myocarditis and pericarditis in rare cases, but the use of novel mRNA platforms led to a higher number of reported cases than with previous platforms providing new insight into potential pathogenic mechanisms. The incidence of COVID-19 vaccine-associated myocarditis/pericarditis covers a large range depending on the vaccine platform, age, and sex examined. Importantly, the findings highlight that myocarditis occurs predominantly in male patients aged 12 to 40 years regardless of whether the cause was due to a virus-like SARS-CoV-2 or associated with a vaccine—a demographic that has been reported before COVID-19. This review discusses findings from COVID-19 and COVID-19 vaccine-associated myocarditis and pericarditis considering the known symptoms, diagnosis, management, treatment, and pathogenesis of disease that has been gleaned from clinical research and animal models. Sex differences in the immune response to COVID-19 are discussed, and theories for how mRNA vaccines could lead to myocarditis/pericarditis are proposed. Additionally, gaps in our understanding that need further research are raised.

Cancer stem cells (CSC) within non–small cell lung carcinoma (NSCLC) tumors drive NSCLC progression, metastasis, relapse, and intrinsic chemoresistance. Understanding the mechanisms that support the malignant phenotypes of NSCLC CSCs may provide insights for improved NSCLC therapeutic interventions. Here, we report that expression of RAB27B, a small GTPase, is significantly upregulated in NSCLC CSCs when compared with bulk cancer cells (BCC). Short hairpin RNA–mediated knockdown of RAB27B leads to a loss of stem cell marker gene expression and reduced NSCLC spheroid growth, clonal expansion, transformed growth, invasion, and tumorigenicity. We find that NSCLC CSCs secrete significantly more extracellular vesicles (EV) than BCCs, and that this is RAB27B-dependent. Furthermore, CSC-derived EVs, but not BCC-derived EVs, induce spheroid growth, clonal expansion, and invasion in BCCs. Finally, RAB27B is required for CSC-derived EV-induced stemness in BCCs. Taken together, our results indicate that RAB27B is required for maintenance of a highly tumorigenic, cancer-initiating, invasive stem-like cell population in NSCLC and RAB27B is involved in propagating EV-mediated communication from NSCLC CSCs to BCCs. Our findings further suggest that inhibition of RAB27B-dependent EV secretion may be a potential therapeutic strategy for NSCLC. Significance: Expression of RAB27B in CSCs leads to elevated levels of EVs that mediate communication between CSCs and BCCs that maintains a stem-like phenotype in NSCLC cells.

Abstract Non-small cell lung cancer (NSCLC), a major subtype of lung cancer, accounts for ~85% of all lung cancer diagnoses. A sub-population of cells within human NSCLC tumors called cancer stem cells (CSCs) have been found to exhibit distinct properties that contribute to factors associated with poor outcomes in NSCLC patients including metastasis, relapse, and therapeutic resistance. We previously performed RNAseq analysis on bulk cancer cells (BCCs) and CSCs from a panel of primary NSCLC tumors and cell lines to identify signaling mechanisms involved in the tumorigenic behavior of NSCLC CSCs. Our analysis revealed that expression of RAB27B, a small GTPase that plays a critical role in extracellular vesicle (EV) formation and secretion, was significantly upregulated in CSCs when compared to BCCs. Interestingly, RAB27B is overexpressed and predicts patient survival in NSCLC. The goal of this study was to assess the role of RAB27B in the tumorigenic behavior of NSCLC CSCs and determine the mechanism by which RAB27B promotes an aggressive stem-cell like phenotype in NSCLC cells an aspect of RAB27B function that has not been investigated in NSCLC. QPCR and immunoblot analysis was used to compare RAB27B mRNA and protein expression in a panel of NSCLC BCCs and CSCs. Two independent shRNA constructs were used to knockdown (KD) RAB27B in NSCLC CSCs. The effects of RAB27B KD on CSC expansion, transformed growth and invasion were analyzed by clonal expansion, soft agar colony formation and Boyden chamber assays, respectively. Xenograft and tail vein mouse models were used to assess the effects of RAB27B KD on CSC tumor growth and lung colonization. EVs were isolated from the conditioned media of BCCs, CSCs and RAB27B KD CSCs using differential ultracentrifugation, and characterized by nanoparticle tracking analysis, transmission electron microscopy and immunoblotting according to MISEV guidelines. Our analysis revealed that RAB27B is significantly upregulated in NSCLC CSCs compared to BCCs. RAB27B KD cells exhibited a significant decrease in clonal expansion, transformed growth and invasion when compared to non-target shRNA control CSCs. In vivo studies demonstrated that RAB27B is important for the tumorigenicity of CSCs and promotes tumor cell proliferation, and angiogenesis. EVs were positive for EV-specific markers and negative for an intracellular vesicle marker. CSCs secrete significantly more EVs when compared to BCCs and KD of RAB27B in CSCs led to a decrease in the levels of EVs secreted. Furthermore, delivery of CSC-derived, but not BCC-derived EVs to BCCs induced spheroid growth, clonal expansion, and invasion in BCCs. Taken together, our results indicate that RAB27B is required for maintenance of a highly tumorigenic, cancer-initiating and invasive stem-like cell population in NSCLC and RAB27B is involved in propagating EV-mediated communication from NSCLC CSCs to BCCs. Citation Format: Kayleah M. Meneses, Jennifer A. Lindemann, Prita Pandya, Dania S. Al-Qasrawi, Ryan Argo, Celeste Weems, Danielle J. Beetler, Irene K. Yan, Joy Wolfram, Tushar C. Patel, Verline Justilien. RAB27B drives a cancer stem cell phenotype in non-small cell lung cancer cells through enhanced extracellular vesicle secretion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2444.