Expression In Myeloid Cells Research Articles

Myeloid Cell Expression of LACC1 Is Required for Bacterial Clearance and Control of Intestinal Inflammation

Loss-of-function variants in the laccase domain containing 1 (LACC1) gene are associated with immune-mediated diseases, including inflammatory bowel disease. It is not clear how LACC1 balances defenses against intestinal bacteria vs intestinal inflammation or what cells are responsible for this balance in humans or mice. Lacc1-/- mice and mice with myeloid-specific disruption of Lacc1 (Lacc1Δmye) were given oral Salmonella Typhimurium or dextran sodium sulfate. CD45RBhiCD4+T cells were transferred to Lacc1-/-Rag2-/- mice to induce colitis. Organs were collected and analyzed by histology and protein expression. Bone marrow-derived macrophages and dendritic cells, lamina propria macrophages, and mesenteric lymph node dendritic cells were examined. We performed assays to measure intestinal permeability, cell subsets, bacterial uptake and clearance, reactive oxygen species, nitrite production, autophagy, signaling, messenger RNA, and cytokine levels. Lacc1-/- mice developed more severe T-cell transfer colitis than wild-type mice and had an increased burden of bacteria in intestinal lymphoid organs, which expressed lower levels of T helper (Th) 1 and Th17 cytokines and higher levels of Th2 cytokines. Intestinal lymphoid organs from mice with deletion of LACC1 had an increased burden of bacteria after oral administration of S Typhimurium and after administration of dextran sodium sulfate compared with wild-type mice. In macrophages, expression of LACC1 was required for toll-like receptor-induced uptake of bacteria, which required PDK1, and of mitogen-activated protein kinase (MAPK)- and nuclear factor κB-dependent induction of reactive oxygen species, reactive nitrogen species, and autophagy. Expression of LACC1 by dendritic cells was required for increasing expression of Th1 and Th17 cytokines and reducing expression of Th2 cytokines upon coculture with CD4+ T cells. Mice with LACC1-deficient myeloid cells had an increased burden of bacteria and altered T-cell cytokines in intestinal lymphoid organs, similar to Lacc1-/- mice. Complementation of cytokines produced by myeloid cells to cocultures of LACC1-deficient myeloid cells and wild-type CD4+ T cells restored T-cell cytokine regulation. When S Typhimurium-infected Lacc1Δmye mice were injected with these myeloid cell-derived cytokines, intestinal tissues increased production of Th1 and Th17 cytokines, and bacteria were reduced. Disruption of Lacc1 in mice increases the burden of bacteria in intestinal lymphoid organs and intestinal inflammation after induction of chronic colitis. LACC1 expression by myeloid cells in mice is required to clear bacteria and to regulate adaptive T-cell responses against microbes.

Folate Receptor β (FRβ) Expression in Tissue-Resident and Tumor-Associated Macrophages Associates with and Depends on the Expression of PU.1.

As macrophages exhibit a huge functional plasticity under homeostasis and pathological conditions, they have become a therapeutic target for chronic inflammatory diseases. Hence, the identification of macrophage subset-specific markers is a requisite for the development of macrophage-directed therapeutic interventions. In this regard, the macrophage-specific Folate Receptor β (FRβ, encoded by the FOLR2 gene) has been already validated as a target for molecular delivery in cancer as well as in macrophage-targeting therapeutic strategies for chronic inflammatory pathologies. We now show that the transcriptome of human macrophages from healthy and inflamed tissues (tumor; rheumatoid arthritis, RA) share a significant over-representation of the “anti-inflammatory gene set”, which defines the gene profile of M-CSF-dependent IL-10-producing human macrophages (M-MØ). More specifically, FOLR2 expression has been found to strongly correlate with the expression of M-MØ-specific genes in tissue-resident macrophages, tumor-associated macrophages (TAM) and macrophages from inflamed synovium, and also correlates with the presence of the PU.1 transcription factor. In fact, PU.1-binding elements are found upstream of the first exon of FOLR2 and most M-MØ-specific- and TAM-specific genes. The functional relevance of PU.1 binding was demonstrated through analysis of the proximal regulatory region of the FOLR2 gene, whose activity was dependent on a cluster of PU.1-binding sequences. Further, siRNA-mediated knockdown established the importance of PU.1 for FOLR2 gene expression in myeloid cells. Therefore, we provide evidence that FRβ marks tissue-resident macrophages as well as macrophages within inflamed tissues, and its expression is dependent on PU.1.

CCDC88B is required for mobility and inflammatory functions of dendritic cells.

The Coiled Coil Domain Containing Protein 88B (CCDC88B) gene is associated with susceptibility to several inflammatory diseases in humans and its inactivation in mice protects against acute neuroinflammation and models of intestinal colitis. We report that mice lacking functional CCDC88B (Ccdc88bMut ) are defective in several dendritic cells (DCs)-dependent inflammatory and immune reactions in vivo. In these mice, an inflammatory stimulus (LPS) fails to induce the recruitment of DCs into the draining lymph nodes (LNs). In addition, OVA-pulsed Ccdc88bMut DCs injected in the footpad do not induce recruitment and activation of antigen-specific CD4+ and CD8+ T cells in their draining LN. Experiments in vitro indicate that this defect is independent of the ability of mutant DCs to capture and present peptide antigen to T cells. Rather, kinetic analyses in vivo of wild-type and Ccdc88bMut DCs indicate a reduced migration capacity in the absence of the CCDC88B protein expression. Moreover, using time-lapse light microscopy imaging, we show that Ccdc88bMut DCs have an intrinsic motility defect. Furthermore, in vivo studies reveal that these reduced migratory properties lead to dampened contact hypersensitivity reactions in Ccdc88b mutant mice. These findings establish a critical role of CCDC88B in regulating movement and migration of DCs. Thus, regulatory variants impacting Ccdc88b expression in myeloid cells may cause variable degrees of DC-dependent inflammatory response in situ, providing a rationale for the genetic association of CCDC88B with several inflammatory and autoimmune diseases in humans.

LILRB1 Intron 1 Has a Polymorphic Regulatory Region That Enhances Transcription in NK Cells and Recruits YY1.

LILRB1 is a highly polymorphic receptor expressed by subsets of innate and adaptive immune cells associated with viral and autoimmune diseases and targeted by pathogens for immune evasion. LILRB1 expression on human NK cells is variegated, and the frequency of LILRB1+ cells differs among people. However, little is known about the processes and factors mediating LILRB1 transcription in NK cells. LILRB1 gene expression in lymphoid and myeloid cells arises from two distinct promoters that are separated by the first exon and intron. In this study, we identified a polymorphic 3-kb region within LILRB1 intron 1 that is epigenetically marked as an active enhancer in human lymphoid cells and not monocytes. This region possesses multiple YY1 sites, and complexes of the promoter/enhancer combination were isolated using anti-YY1 in chromatin immunoprecipitation-loop. CRISPR-mediated deletion of the 3-kb region lowers LILRB1 expression in human NKL cells. Together, these results indicate the enhancer in intron 1 binds YY1 and suggest YY1 provides a scaffold function enabling enhancer function in regulating LILRB1 gene transcription in human NK cells.

Disruption of CCR1-mediated myeloid cell accumulation suppresses colorectal cancer progression in mice

Tumor-stromal interaction is implicated in tumor progression. Although CCR1 expression in myeloid cells could be associated with pro-tumor activity, it remains elusive whether disruption of CCR1-mediated myeloid cell accumulation can suppress tumor progression. Here, we investigated the role of CCR1 depletion in myeloid cells in two syngeneic colorectal cancer mouse models: MC38, a transplanted tumor model and CMT93, a liver metastasis model. Both cells induced tumor accumulation of CCR1+ myeloid cells that express MMP2, MMP9, iNOS, and VEGF. Lack of the Ccr1 gene in host mice dramatically reduced MC38 tumor growth as well as CMT93 liver metastasis. To delineate the contribution of CCR1+ myeloid cells, we performed bone marrow (BM) transfer experiments in which sub-lethally irradiated wild-type mice were reconstituted with BM from either wild-type or Ccr1-/- mice. Mice reconstituted with Ccr1-/- BM exhibited marked suppression of MC38 tumor growth and CMT93 liver metastasis, compared with control mice. Consistent with these results, administration of a neutralizing anti-CCR1 monoclonal antibody, KM5908, significantly suppressed MC38 tumor growth and CMT93 liver metastases. Our findings highlight the importance of the application of CCR1 blockade as a therapeutic strategy.

Allograft inflammatory factor-1 in myeloid cells drives autoimmunity in type 1 diabetes.

Allograft inflammatory factor-1 (AIF1) is a calcium-responsive cytoplasmic scaffold protein that directs hematopoiesis and immune responses within dendritic cells (DC) and macrophages. Although the role of AIF1 in transplant rejection and rheumatoid arthritis has been explored, little is known about its role in type 1 diabetes. Here, we show that in vivo silencing of AIF1 in NOD mice restrained infiltration of immune cells into the pancreas and inhibited diabetes incidence. Analyses of FACS-sorted CD45neg nonleukocyte populations from resected pancreatic islets showed markedly higher expression of insulin in the AIF1-silenced groups. Evaluation of CD45+ leukocytes revealed diminished infiltration of effector T cells and DC in the absence of AIF1. Transcriptional profiling further revealed a marked decrease in cDC1 DC-associated genes CD103, BATF3, and IRF8, which are required for orchestrating polarized type 1 immunity. Reduced T cell numbers within the islets were observed, with concomitant lower levels of IFN-γ and T-bet in AIF1-silenced cohorts. In turn, there was a reciprocal increase in functionally suppressive pancreas-resident CD25+Foxp3+CD4+ Tregs. Taken together, results show that AIF1 expression in myeloid cells plays a pivotal role in promoting type 1 diabetes and that its suppression restrains insulitis by shifting the immune microenvironment toward tolerance.

Different regulation of PARP1, PARP2, PARP3 and TRPM2 genes expression in acute myeloid leukemia cells

BackgroundAcute myeloid leukemia (AML) is a heterogenic lethal disorder characterized by the accumulation of abnormal myeloid progenitor cells in the bone marrow which results in hematopoietic failure. Despite various efforts in detection and treatment, many patients with AML die of this cancer. That is why it is important to develop novel therapeutic options, employing strategic target genes involved in apoptosis and tumor progression.MethodsThe aim of the study was to evaluate PARP1, PARP2, PARP3, and TRPM2 gene expression at mRNA level using qPCR method in the cells of hematopoietic system of the bone marrow in patients with acute myeloid leukemia, bone marrow collected from healthy patients, peripheral blood of healthy individuals, and hematopoietic stem cells from the peripheral blood after mobilization.ResultsThe results found that the bone marrow cells of the patients with acute myeloid leukemia (AML) show overexpression of PARP1 and PARP2 genes and decreased TRPM2 gene expression. In the hematopoietic stem cells derived from the normal marrow and peripheral blood after mobilization, the opposite situation was observed, i.e. TRPM2 gene showed increased expression while PARP1 and PARP2 gene expression was reduced. We observed positive correlations between PARP1, PARP2, PARP3, and TRPM2 genes expression in the group of mature mononuclear cells derived from the peripheral blood and in the group of bone marrow-derived cells. In AML cells significant correlations were not observed between the expression of the examined genes. In addition, we observed that the reduced expression of TRPM2 and overexpression of PARP1 are associated with a shorter overall survival of patients, indicating the prognostic significance of these genes expression in AML.ConclusionsOur research suggests that in physiological conditions in the cells of the hematopoietic system there is mutual positive regulation of PARP1, PARP2, PARP3, and TRPM2 genes expression. PARP1, PARP2, and TRPM2 genes at mRNA level deregulate in acute myeloid leukemia cells.

OR04-04 Identification of a Novel Transcriptional Regulator of Metabolic Disease in Circulating and Central Myeloid Cells

Derangement in systemic metabolic homeostasis is tightly associated with widespread activation of resident and circulating immune cells, a phenomenon known as ‘metaflammation’. Numerous studies have explored the role of tissue resident and circulating macrophages in contributing to metaflammation, obesity, and their sequelae; however, there is a dearth of information regarding targetable transcriptional regulators of the genesis and persistence of metabolic disease. Here, we identify myeloid Krüppel-like factor 2 (KLF2) as a novel regulator of metabolic disease. Previous reports demonstrate that KLF2 serves as a critical regulator of myeloid cell quiescence and is downregulated in numerous acute and chronic inflammatory states. Specifically in the context of chronic metaflammation, we note that KLF2 expression is decreased in circulating immune cells of obese patients and in adipose tissue macrophages of high fat diet (HFD) fed mice, which is consistent with the hypothesis that KLF2 regulates metaflammation. To explore this further, we utilized mice with myeloid cell-specific deletion of KLF2 (K2KO) which exhibit accelerated obesity and insulin resistance. K2KO mice have widespread central (i.e. CNS) and peripheral metaflammation both in the basal and HFD-stimulated states. To discern whether the effect of myeloid deletion of KLF2 on metabolism is due to deletion in microglia in the feeding centers of the hypothalamus or in peripheral immune cells, bone marrow chimeras with head shielding were created. 50% reconstitution of circulating immune cells with K2KO cells in wildtype (WT) mice was sufficient to maintain the metabolic disease phenotype, while mice with K2KO microglia + WT circulating cells had only slightly improved outcomes compared to K2KO mice. Conversely, ablation of microglia in K2KO mice using PLX5622 formulated in HFD also successfully attenuated the aberrant feeding behavior, weight gain, and glucose dyshomeostasis seen in K2KO mice. Together, these data demonstrate a role for loss of KLF2 in hematopoietic and CNS resident cells in causing metabolic disease. Given that myeloid KLF2 expression decreases under metabolic stress in WT mice and humans, we sought to explore whether maintenance of KLF2 expression in these cells would be protective against diet-induced metabolic disease. Indeed, mice with myeloid-specific overexpression of KLF2 demonstrated a markedly improved metabolic phenotype when challenged with HFD, providing evidence that targeting KLF2 expression in myeloid cells may prove to be a therapeutic option against metaflammation.

OR05-01 Small Heterodimer Partner Modulates Antigen Presenting Myeloid Cells to Impair Regulatory T Cell Expansion, Promoting Anti-Tumor Immunity in Models of Breast Cancer

Immune checkpoint blockade has had underwhelming responses in breast cancer, in part due to the highly immune suppressive microenvironment. As a result, breast cancer continues to be the second most common cancer-related mortality amongst women, providing strong rationale for the development of new therapeutic approaches. Elevated circulating cholesterol is a poor prognostic, while breast cancer patients taking cholesterol-lowering drugs display increased time to recurrence. We and others have previously demonstrated that cholesterol metabolites mediate these effects by promoting breast cancer growth and metastasis, in part by suppressing the immune system. Therefore, given the demonstrated importance of cholesterol and its metabolites in breast cancer pathophysiology and immunology, we hypothesized that proteins involved in the regulation of cholesterol homeostasis would influence cancer progression. Through informatics analysis of breast tumors, we found that elevated expression of Small Heterodimer Partner (SHP; NR0B2) was a favorable prognostic. Antigen presenting cells such as macrophages and dendritic cells were found to express SHP, and manipulation of SHP altered the expression of genes involved in cross-talk with T cells. Intriguingly, when activated T cells were co-cultured with macrophages overexpressing SHP, there was a decrease in the expansion of regulatory T cells (Tregs) and vice versa in the absence of SHP. Adoptive transfer studies confirmed that loss of SHP resulted in immune suppressive Tregs. We hypothesized that myeloid cell-expressed SHP would promote immune surveillance and tumor clearance. In support of this hypothesis, tumors in the MMTV-PyMT model of mammary cancer grew at an accelerated rate in SHP-knockout mice. Tumors from these mice had significantly more Tregs and fewer effector T cells. Furthermore, orthotopic mammary tumor grafts grew at an increased rate in mice lacking SHP expression in myeloid cells (SHPfl/fl;LysMCre), compared to controls. A small molecule agonist of SHP impaired primary and metastatic tumor growth, and significantly enhanced the efficacy of immune checkpoint blockade in murine models of mammary cancer. Therefore, SHP represents a potential target to decrease suppressive Tregs, thereby allowing for immune-clearance of tumors.

Oridonin induces the apoptosis of mucoepidermoid carcinoma cell lines in a myeloid cell leukemia‑1‑dependent manner.

Oridonin, an active diterpenoid isolated from Rabdosia rubescens, has been reported to exhibit anticancer activities in several tumors. The aim of the present study was to investigate the anticancer effects and molecular mechanisms of oridonin in mucoepidermoid carcinoma (MEC). Treatment with oridonin induced the apoptosis of MC‑3 and YD‑15 cell and inhibited the expression of myeloid cell leukemia‑1 (MCL‑1) through the regulation of the protein level through post‑translational regulation in these cell lines. Oridonin significantly increased the expression level of truncated Bid (t‑Bid) as a downstream target of MCL‑1 and subsequently decreased the mitochondrial membrane potential. The ectopic expression of MCL‑1 protein was sufficient to reverse the induction of apoptosis and the increased t‑Bid expression induced by oridonin in both cell lines. Taken together, these results suggest that oridonin exerts an apoptotic effect through the modulation of MCL‑1 and t‑Bid in human MEC cell lines and may thus be a potential anticancer drug candidate for the treatment of human MEC.

Myeloid PTEN promotes chemotherapy-induced NLRP3-inflammasome activation and antitumour immunity.

PTEN is a dual-specificity phosphatase that is frequently mutated in human cancer, and its deficiency in cancer has been associated with therapy resistance and poor survival. Although the intrinsic tumour-suppressor function of PTEN has been well established, evidence of its role in the tumour immune microenvironment is lacking. Here, we show that chemotherapy-induced antitumour immune responses and tumour suppression rely on myeloid-cell PTEN, which is essential for chemotherapy-induced activation of the NLRP3 inflammasome and antitumour immunity. PTEN directly interacts with and dephosphorylates NLRP3 to enable NLRP3-ASC interaction, inflammasome assembly and activation. Importantly, supplementation of IL-1β restores chemotherapy sensitivity in mouse myeloid cells with a PTEN deficiency. Clinically, chemotherapy-induced IL-1β production and antitumour immunity in patients with cancer is correlated with PTEN expression in myeloid cells, but not tumour cells. Our results demonstrate that myeloid PTEN can determine chemotherapy responsiveness by promoting NLRP3-dependent antitumour immunity and suggest that myeloid PTEN might be a potential biomarker to predict chemotherapy responses.

The role of myeloid cell-specific ten eleven translocase 2 (TET2) in regulation of neuroinflammation

Abstract Multiple Sclerosis (MS), is an inflammatory demyelinating disease of the central nervous system (CNS) believed to be autoimmune in etiology. Analysis of legions from MS patients, as well as studies in Experimental Autoimmune Encephalomyelitis (EAE), a murine model of MS, have indicated that myeloid cells play a critical role in the pathogenesis of CNS autoimmunity. Recently, Ten-Eleven Translocation 2 (TET2) was identified as a susceptibility locus for MS. TET2 is a Fe(III), α-ketoglutarate dependent enzyme that catalyzes the oxidation of methylated cytosine to 5′-hydroxymethyl cytosine (5hmC), a stable epigenetic mark. There is growing evidence that TET2 acts as a negative regulator of myeloid cell activation. Further, TET2 and 5hmC are reduced in peripheral blood mononuclear cells of MS patients compared with healthy volunteers. The objective of the current study is to investigate the hypothesis that reductions of TET2 expression and activity in myeloid cells is a critical step in the development of pro-inflammatory myeloid cells during CNS autoimmunity. Our laboratory has found that Tet2 transcripts levels and 5hmC content are reduced in CNS-infiltrating myeloid cells at the peak of EAE compared with the onset or late stages. Adoptive transfer of encephalitogenic CD4+ Th17 cells into Tet2−/+ mice lead to an exacerbated clinical course compared with their Tet2+/+ counterparts. Our data suggests that TET2 expression and activity is dysregulated in myeloid cells and limits neuroinflammation and chronic neurological disability. Interventions that promote Tet2 expression in myeloid cells may ameliorate EAE and, by extension, MS.

Interferon regulatory factor 5 expression in myeloid cells and T cells negatively impacts the outcome of visceral leishmaniasis

Abstract Visceral leishmaniasis (VL) is a potentially fatal disease caused by Leishmania donovani and/or L. infantum. VL is characterized by hepatosplenomegaly, immune suppression, and chronic inflammation. We have previously reported that the transcription factor IRF-5 largely contributes to the induction of inflammation and the development of splenomegaly in mice infected with L. donovani. Moreover, Irf5−/− mice only develop limited IFNγ+ CD4 T responses and are more susceptible to infection. However, the cellular source of IRF-5 responsible for these effects is yet unknown. IRF-5 is constitutively expressed in several cells, such as macrophages, B cells, and dendritic cells. We have recently reported that CD4 T cells also express IRF-5 during chronic VL. Here, we investigate IRF-5 function in myeloid and CD4 T cells during VL, using cell-specific knockout mice. First, we show that IRF-5 expression in CD11c+ cells is required for the generation of splenomegaly and leads to increased disease susceptibility. We also demonstrate that IRF-5 expression in CD11c+ cells is not crucial for the development of Th1 responses. Moreover, we demonstrate that IRF-5 activation in Th1 cells results in cell death during chronic L. donovani infection. Triggering of TLR7 by apoptotic cell material promotes IRF-5 activation in Th1 cells. This results in the upregulation of death receptor 5 and caspase 8, making IFNγ+ CD4 T cells more prone to cell death. Because chronic inflammation and tissue disruption are common characteristics of persistent infections, the TLR7- IRF-5 pathway in CD4 T cells may represent a novel mechanism aimed at protecting tissues against sustained inflammation.

Genetic mechanisms contributing to IFNβ regulation of Lyme Arthritis: involvement of the Borrelia burgdorferi arthritis associated locus 1 (Bbaa1)

Abstract Lyme disease shows a spectrum of clinical disorders in patients that are infected with the tick borne spirochete Borrelia burgdorferi. Studies have shown that C3H mice develop severe arthritis while C57BL/6 (B6) mice display mild arthritis when infected with the same isolate of B. burgdorferi. In order to study the role of the host genetics in regulating the severity of Lyme arthritis development, we used forward genetics and identified a genetic region, Bbaa1, that includes the Type I IFN cluster on Chr4. We further developed congenic mice isolating the C3H allele of Bbaa1 onto the background of B6 mice and determined that Bbaa1 regulates arthritis severity by upregulating Type I IFN. Antibody blocking revealed IFNβ, but not IFNα, as the key effector that drives severe Lyme arthritis. Because there are no SNPs between the C3H and B6 IFNβ genes, we hypothesized that other genetic factors in the Bbaa1 locus regulate the differential expression of IFNβ and Lyme arthritis severity. RNA silencing was used to identify candidate genes within Bbaa1 that regulate IFNβ expression in myeloid cells. Cdkn2a was identified as a strong regulator of IFNβ expression. Although Cdkn2a encodes two proteins, p16 and ARF, only the silencing of Arf resulted in the suppression of IFNβ expression. Additionally, the overexpression of ARF led to enhancement of IFNβ. We propose that ARF regulates IFNβ by two pathways involving the proteins p53 and BCL6. Indeed, blocking of BCL6 by a small molecule inhibitor induced IFNβ activation in the joint tissue of B6 mice and enhanced the severity of Lyme arthritis. Future work will focus on the impact of the two pathways on IFNβ expression and arthritis severity in mice.

LncRNA HOTAIRM1 promotes the immunosuppressive functions of MDSCs through upregulating HOXA1 expression during chronic HCV infection

Abstract HOXA transcript antisense RNA myeloid-specific 1 (HOTAIRM1) is a long non-coding RNA (lncRNA) that is critical for the myeloid cell development by targeting HOXA1. We have previously shown that myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that have immunosuppressive functions. However, the role and mechanisms of HOTAIRM1 in the differentiation and function of MDSCs in the setting of viral infection remain elusive. In this study, we demonstrated that HOTAIRM1 and its target gene HOXA1 were upregulated and closely associated with the expression of immunosuppressive molecules, such as arginase 1 (Arg1), inducible nitric oxide synthase (iNOS), reactive oxygen species (ROS), and signal transducer and activator of transcription 3 (STAT3) in myeloid cells derived from individuals with chronic hepatitis C virus (HCV) infection. We further revealed that HCV-associated exosomes could induce HATAIRM1 and HOXA1 expression via the miRNA-mediated signaling pathway. Importantly, overexpression of HOTAIRM1 or HOXA1 in healthy myeloid cells promoted MDSC differentiation and suppressive functions, evidenced by an increased frequency of MDSCs, expression of immunosuppressive molecules, and inhibition of co-cultured CD4 T cells to produce IFN-γ. Conversely, silencing HOTAIRM1 or HOXA1 expression in HCV myeloid cells partially restored the frequencies of MDSCs, the expressions of their suppressive molecule, and the IFN-γ production in co-cultured T cells. Taken together, this study reveals that HOTAIRM1-HOXA1 enhances the immunosuppressive function of MDSCs and may be a potential target for immunomodulation in conjunction with antiviral therapy during chronic viral infection.

Gas Plasma-Treated Prostate Cancer Cells Augment Myeloid Cell Activity and Cytotoxicity.

Despite recent improvements in cancer treatment, with many of them being related to foster antitumor immunity, tumor-related deaths continue to be high. Novel avenues are needed to complement existing therapeutic strategies in oncology. Medical gas plasma technology recently gained attention due to its antitumor activity. Gas plasmas act via the local deposition of a plethora of reactive oxygen species (ROS) that promote the oxidative cancer cell death. The immunological consequences of plasma-mediated tumor cell death are only poorly understood, however. To this end, we exposed two prostate cancer cell lines (LNCaP, PC3) to gas plasma in vitro, and investigated the immunomodulatory effects of the supernatants in as well as of direct co-culturing with two human myeloid cell lines (THP-1, HL-60). After identifying the cytotoxic action of the kINPen plasma jet, the supernatants of plasma-treated prostate cancer cells modulated myeloid cell-related mitochondrial ROS production and their metabolic activity, proliferation, surface marker expression, and cytokine release. Direct co-culture amplified differentiation-like surface marker expression in myeloid cells and promoted their antitumor-toxicity in the gas plasma over the untreated control conditions. The results suggest that gas plasma-derived ROS not only promote prostate cancer cell death but also augment myeloid cell activity and cytotoxicity.

Survival of Mature Dendritic Cells Dependent on Clusterin Expression

Clusterin is a multifunctional glycoprotein found in various tissues and body fluids. It is involved in a number of physiological and pathological processes such as apoptosis, protein homeostasis, cancer and Alzheimer’s disease. Although clusterin expression in myeloid cells has been reported, its influence in dendritic cell (DC) function has yet to be analyzed. Therefore, the purpose of our experiment was to determine the role of clusterin in DC survival. First, we analyzed whether activation of human monocyte‐derived DCs resulted in clusterin production. This was done by culturing cells for 48 hours with or without LPS (10 ng/ml). Clusterin was analyzed by western blot and quantified by ELISA. A basal expression level of clusterin was observed in unstimulated DCs, while an increase in production was noted upon activation of these cells by LPS: clusterin concentration in cell supernatant was 0.04 ± 0.1 vs 7.47 ± 2.14 ng/ml (control vs LPS, mean ± ES, n=5, p<0.01 vs unstimulated cells). In order to understand the dynamics of production after activation, we constructed a production curve measuring the level of clusterin post stimulation over a period of four days. A peak in the production of intracellular and secreted clusterin was noted 48 – 72 hours post stimulus. Finally, a knockdown (KD) strategy using a lentiviral gene silencing protocol that delivered a clusterin and scramble (SCR) shRNA was used to determine the role of clusterin in DC function. Two lentiviral combinations were used due to their ability to knockdown >85% of clusterin expression, as evaluated by western blot (in HeLa cells) and rtPCR (DCs). Puromycin was used as a selection drug and a Ficoll gradient was used to separate live and dead cells. We found that silencing clusterin expression (CLU‐KD) resulted in a massive death of DCs upon stimulation with LPS, as evaluated by anexin‐V/propidium straining: 63.7 ± 11.8, 19.7 ± 4.9, and 25.2 ± 6.5% of dead cells, for LPS‐stimulated CLU‐KD DCs, LPS‐stimulated scramble DCs, and unstimulated DCs, respectively (mean ± ES, n=4, p<0.01 CLU‐KD vs scramble or unstimulated DCs). These observations uncover a novel function of clusterin as a key player in the control of the adaptive immune response.Support or Funding InformationRoy Mendoza was supported by the grant LA Basin Minority Health and Health Disparities Research Training Program (MHRT) T37MD001368 from the National Institute on Minority Health and Health Disparities, National Institute of Health.

MP17-05 MYELOID CELLS IN BLADDER CANCER MICROENVIRONMENT DEGRADE HYALURONIC ACID INTO INFLAMMATORY LOW MOLECULAR WEIGHT HYALURON

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology II (MP17)1 Apr 2020MP17-05 MYELOID CELLS IN BLADDER CANCER MICROENVIRONMENT DEGRADE HYALURONIC ACID INTO INFLAMMATORY LOW MOLECULAR WEIGHT HYALURON Paul Dominguez-Gutierrez*, Elizabeth Kwenda, Willim Donelan, Padraic O'Malley, Paul Crispen, and Sergei Kusmartsev Paul Dominguez-Gutierrez*Paul Dominguez-Gutierrez* More articles by this author , Elizabeth KwendaElizabeth Kwenda More articles by this author , Willim DonelanWillim Donelan More articles by this author , Padraic O'MalleyPadraic O'Malley More articles by this author , Paul CrispenPaul Crispen More articles by this author , and Sergei KusmartsevSergei Kusmartsev More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000842.05AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Hyaluronic acid (HA) is a multifunctional glycosaminoglycan that is involved in tissue regeneration, inflammation response, and angiogenesis. HA is degraded by hyaluronidases (HYAL) 1-3 that cleave HA into specific low molecular weights (LMW) HA (<20KDa) that act as ligands for CD44, RHAMM, and ICAM-1. Multiple cancers have increased levels of HA, suggesting abnormal HA metabolism. This study sought to elucidate the role and factors associated with increased HA accumulation within the bladder cancer tumor microenvironment. METHODS: IRB approval and patient informed consent were given before collection of normal and tumor bladder tissue during cystectomy. 300µm precision-cut tissue slice were cultured for 3-14 days. Polyacrylamide gel electrophoresis was used for analysis of HA size using. To test the effect of the tumor microenvironment on normal cells, naïve myeloid cells were isolated from mouse bone marrow and cultured in plates precoated with high molecular weight (HMW) HA (>200KDa) in the presence or absence of human tumor conditioned media and examined using fluorescent microscopy. Realtime PCR was used to measure HYAL 1-3 expression and WES capillary electrophoresis was used to validate protein levels of Hyal2. RESULTS: Cultured slices from 25 bladder cancer patients were characterized by LMW-HA (Fig 1A). Normal bladder tissue was characterized by long structured linear pericellular HA, low inflammatory infiltrates, and > 200KDa HMW-HA (Fig1B). LMW-HA levels were undetectable in normal bladder samples. Furthermore, tumor bladder tissue was infiltrated with Hyal2 expressing myeloid cells. Hyal-2 expression in myeloid cells was upregulated by exposure to tumor-conditioned medium. CONCLUSIONS: Experimental and clinical samples of bladder cancer demonstrate high infiltration of Hyal-2 expressing myeloid cells. Increased Hyal-2 expression promotes degradation of HMW-HA into highly fragmented LMW-HA. This LMW-HA triggers inflammation via increased production of cytokines, chemokines, and growth factors within the tumor microenvironment. Future studies should focus on Hyal-2 as a potential target for bladder cancer therapy and LMW-HA as a prognostic marker. Source of Funding: J&E King Florida Biomedical Research Program1923 Fund © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e227-e227 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Paul Dominguez-Gutierrez* More articles by this author Elizabeth Kwenda More articles by this author Willim Donelan More articles by this author Padraic O'Malley More articles by this author Paul Crispen More articles by this author Sergei Kusmartsev More articles by this author Expand All Advertisement PDF downloadLoading ...

Selective killing of leukemia cells: Yamanaka factors' new trick.

The four transcription factors of the Yamanaka cocktail (Oct4, Sox2, Klf4, and Myc, termed OSKM) are famously capable of reprogramming somatic cells into induced pluripotent stem cells (iPSCs). In an article recently published in Nature Communications, Wang et al describe the unexpected discovery that short-term activation of OSKM expression in acute myeloid leukemia cells in vivo induces apoptosis while negligibly affecting normal hematopoietic stem and progenitor cells (Nat Commun 2019;10:5594). These findings have potential implications for novel anticancer strategies.

Poly I:C stimulation in-vitro as a marker for an antiviral response in different cell types generated from Buffalo (Bubalus bubalis)

The innate immune system is activated upon virus invasion of a host cell by recognizing viral component, such as dsRNA through specific receptors, resulting in the production of type- I IFNs, which confer an antiviral state within the invaded as well as surrounding cells. In the present study, fibroblast, monocyte and macrophage cells derived from water Buffalo (Bubalus bubalis) were exposed to a synthetic dsRNA analogue, poly I:C to mimic viral invasion in each cell type. Recognition of poly I:C through cytosolic helicase receptors RIG-I and MDA5 molecule lead to the activation of the RLR pathway, subsequently activating the MAVS-IRF3/7 cascade and the production of antiviral effector molecule like IFNβ and ISGs. Within the different cell types, we identified variability in RLR receptor and IFNβ expression after poly I:C administration. Fibroblasts responded quickly and strongly with IFNβ production, followed by macrophages and monocytes. Despite absolute expression variability among different cell types the expression trend of RLRs pathway genes were similar. Length of poly I:C molecule also influence IFNβ expression in response of RLR pathway. Short (LMW) poly I:C induce stronger IFN-β expression in myeloid (macrophage and monocyte) cells. In contrast long (HMW) poly I:C preferably elicit higher IFNβ expression in non-myeloid (fibroblast) cell. Therefore, MDA5 and RIG-1 plays an indispensable role in eliciting antiviral response in non- immune (fibroblast) host cell. Thus, stimulation of RLR pathway with suitable and potentially cell-type specific agonist molecules successfully elicit antiviral state in the host animal, with fibroblasts conferring a stronger antiviral state compared with the monocytes and macrophages.