Plasmacytoid Dendritic Cells Mediate CpG-ODN Induced Increase in Survival in a Mouse Model of Lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a devastating disease primarily found in women of reproductive age that leads to cystic destruction of the lungs. Recent work has shown that LAM causes immunosuppression and that checkpoint inhibitors can be used as LAM treatment. Toll-like receptor (TLR) agonists can also re-activate immunity and the TLR9 agonist, CpG-ODN, has been effective in treating lung cancer in animal models. Here we investigate the use of TLR9 agonist CpG-ODN as LAM immunotherapy in combination with checkpoint inhibitor, anti-PD1, standard of care rapamycin and determine the immune mechanisms underlying therapeutic efficacy. We used survival studies, flow cytometry, ELISA, and histology to assess immune response and survival after intranasal treatment with CpG-ODN in combination with rapamycin or anti-PD1 therapy in a mouse model of metastatic LAM. We found that local administration of CpG-ODN enhances survival in a mouse model of LAM. We found that a lower dose led to longer survival likely due to fewer local side effects but increased LAM nodule count and size compared to the higher dose. CpG-ODN treatment also reduced regulatory T cells and increased the number of Th17 helper T cells as well as cytotoxic T cells. These effects appear to be mediated in part by plasmacytoid dendritic cells (pDCs), as depletion of pDCs reduces survival and abrogates Th17 T cell response. Finally, we found that CpG-ODN treatment is effective in early stage and progressive disease and is additive with anti-PD1 therapy and rapamycin. In summary, we have found that TLR9 agonist CpG-ODN can be used as LAM immunotherapy and effectively synergizes with rapamycin and anti-PD1 therapy in LAM.

Microorganism-derived extracellular vesicles: emerging contributors to female reproductive health.

Extracellular vesicles (EVs) are cell-derived nanoparticles that carry small molecules, nucleic acids, and proteins long distances in the body facilitating cell-cell communication. Microorganism-derived EVs mediate communication between parent cells and host cells, with recent evidence supporting their role in biofilm formation, horizontal gene transfer, and suppression of the host immune system. As lipid-bound bacterial byproducts, EVs demonstrate improved cellular uptake and distribution in vivo compared to cell-free nucleic acids, proteins, or small molecules, allowing these biological nanoparticles to recapitulate the effects of parent cells and contribute to a range of human health outcomes. Here, we focus on how EVs derived from vaginal microorganisms contribute to gynecologic and obstetric outcomes. As the composition of the vaginal microbiome significantly impacts women's health, we discuss bacterial EVs from both healthy and dysbiotic vaginal microbiota. We also examine recent work done to evaluate the role of EVs from common vaginal bacterial, fungal, and parasitic pathogens in pathogenesis of female reproductive tract disease. We highlight evidence for the role of EVs in women's health, gaps in current knowledge, and opportunities for future work. Finally, we discuss how leveraging the innate interactions between microorganisms and mammalian cells may establish EVs as a novel therapeutic modality for gynecologic and obstetric indications.

Mechanical Dissociation of Tissues for Single Cell Analysis Using a Motorized Device.

Being able to isolate and prepare single cells for the analysis of tissue samples has rapidly become crucial for new biomedical discoveries and research. Manual protocols for single-cell isolations are highly time-consuming and prone to user variability. Automated mechanical protocols are able to reduce processing time and sample variability but aren't easily accessible or cost-effective in lower-resourced research settings. The device described here was designed for semi-automated tissue dissociation using commercially available materials as a low-cost alternative for academic laboratories. Instructions to fabricate, assemble, and operate the device design have been provided. The dissociation protocol reliably produces single-cell suspensions with comparable cell yields and sample viability to manual preparations across multiple mouse tissues. The protocol provides the ability to process up to 12 tissue samples simultaneously per device, making studies requiring large sample sizes more manageable. The accompanying software also allows for customization of the device protocol to accommodate varying tissues and experimental constraints.

Inhaled CpG increases survival and synergizes with checkpoint inhibition in lymphangioleiomyomatosis.

Rationale: Lymphangioleiomyomatosis (LAM) is a devastating disease primarily found in women of reproductive age that leads to cancer-like cystic destruction of the lungs. Recent work has shown that LAM causes immunosuppression and that checkpoint inhibitors can be used as LAM treatment. IN lung cancer, TLR agonist, in particular TLR9 agonist CpG has been shown to be effective. Objectives: Here we investigate the use of TLR9 agonist CpG as LAM immunotherapy in combination with checkpoint inhibitor, anti-PD1 and assess induced changes in anti-LAM immunity. Methods: We used a murine model of metastatic LAM to determine survival after intranasal treatment with TLR9 agonist CpG at two doses and in combination the checkpoint inhibitor immunotherapy, anti-PD-1. We used histology and flow cytometry to assess overall inflammation as well as changes in the immune response upon treatment. Measurements and Main Results: We found that local administration of CpG enhances survival in a murine model of LAM and that a lower dose more effectively balanced the inflammation induced by CpG with the anti-LAM therapeutic benefits. We also found that CpG reduces regulatory T cell infiltration in LAM lungs and that CD4 helper T cells are skewed toward pro-inflammatory phenotypes. We also found that CpG treatment is effective in both early stage and progressive disease and that CpG is synergistic with previously tested anti-PD1 therapy. Conclusions: We have found that TLR9 agonist CpG can be used as LAM immunotherapy and effectively synergizes with anti-PD1 therapy in LAM.

A Proteomic Strategy Identifies Lysine Methylation of Splicing Factor snRNP70 by the SETMAR Enzyme.

The lysine methyltransferase (KMT) SETMAR is implicated in the response to and repair of DNA damage, but its molecular function is not clear. SETMAR has been associated with dimethylation of histone H3 lysine 36 (H3K36) at sites of DNA damage. However, SETMAR does not methylate H3K36 in vitro. This and the observation that SETMAR is not active on nucleosomes suggest that H3K36 methylation is not a physiologically relevant activity. To identify potential non-histone substrates, we utilized a strategy on the basis of quantitative proteomic analysis of methylated lysine. Our approach identified lysine 130 of the mRNA splicing factor snRNP70 as a SETMAR substrate in vitro, and we show that the enzyme primarily generates monomethylation at this position. Furthermore, we show that SETMAR methylates snRNP70 Lys-130 in cells. Because snRNP70 is a key early regulator of 5' splice site selection, our results suggest a model in which methylation of snRNP70 by SETMAR regulates constitutive and/or alternative splicing. In addition, the proteomic strategy described here is broadly applicable and is a promising route for large-scale mapping of KMT substrates.

An unexpected journey: lysine methylation across the proteome.

The dynamic modification of histone proteins by lysine methylation has emerged over the last decade as a key regulator of chromatin functions. In contrast, our understanding of the biological roles for lysine methylation of non-histone proteins has progressed more slowly. Though recently it has attracted less attention, ε-methyl-lysine in non-histone proteins was first observed over 50 years ago. In that time, it has become clear that, like the case for histones, non-histone methylation represents a key and common signaling process within the cell. Recent work suggests that non-histone methylation occurs on hundreds of proteins found in both the nucleus and the cytoplasm, and with important biomedical implications. Technological advances that allow us to identify lysine methylation on a proteomic scale are opening new avenues in the non-histone methylation field, which is poised for dramatic growth. Here, we review historical and recent findings in non-histone lysine methylation signaling, highlight new methods that are expanding opportunities in the field, and discuss outstanding questions and future challenges about the role of this fundamental post-translational modification (PTM).

Proteome-wide enrichment of proteins modified by lysine methylation.

We present a protocol for using the triple malignant brain tumor domains of L3MBTL1 (3xMBT), which bind to mono- and di-methylated lysine with minimal sequence specificity, in order to enrich for such methylated lysine from cell lysates. Cells in culture are grown with amino acids containing light or heavy stable isotopic labels. Methylated proteins are enriched by incubating cell lysates with 3xMBT, or with the binding-null D355N mutant as a negative control. Quantitative liquid chromatography and tandem mass spectrometry (LC-MS/MS) are then used to identify proteins that are specifically enriched by 3xMBT pull-down. The addition of a third isotopic label allows the comparison of protein lysine methylation between different biological conditions. Unlike most approaches, our strategy does not require a prior hypothesis of candidate methylated proteins, and it recognizes a wider range of methylated proteins than any available method using antibodies. Cells are prepared by growing in isotopic labeling medium for about 7 d; the process of enriching methylated proteins takes 3 d and analysis by LC-MS/MS takes another 1-2 d.

A general molecular affinity strategy for global detection and proteomic analysis of lysine methylation.

Lysine methylation of histone proteins regulates chromatin dynamics and plays important roles in diverse physiological and pathological processes. However, beyond histone proteins, the proteome-wide extent of lysine methylation remains largely unknown. We have engineered the naturally occurring MBT domain repeats of L3MBTL1 to serve as a universal affinity reagent for detecting, enriching, and identifying proteins carrying a mono- or dimethylated lysine. The domain is broadly specific for methylated lysine ("pan-specific") and can be applied to any biological system. We have used our approach to demonstrate that SIRT1 is a substrate of the methyltransferase G9a both in vitro and in cells, to perform proteome-wide detection and enrichment of methylated proteins, and to identify candidate in-cell substrates of G9a and the related methyltransferase GLP. Together, our results demonstrate a powerful new approach for global and quantitative analysis of methylated lysine, and they represent the first systems biology understanding of lysine methylation.