Breaking Entry-and Species Barriers: LentiBOOST® Plus Polybrene Enhances Transduction Efficacy of Dendritic Cells and Monocytes by Adenovirus 5

Due to their ability to trigger strong immune responses, adenoviruses (HAdVs) in general and the serotype5 (HAdV-5) in particular are amongst the most popular viral vectors in research and clinical application. However, efficient transduction using HAdV-5 is predominantly achieved in coxsackie and adenovirus receptor (CAR)-positive cells. In the present study, we used the transduction enhancer LentiBOOST® comprising the polycationic Polybrene to overcome these limitations. Using LentiBOOST®/Polybrene, we yielded transduction rates higher than 50% in murine bone marrow-derived dendritic cells (BMDCs), while maintaining their cytokine expression profile and their capability to induce T-cell proliferation. In human dendritic cells (DCs), we increased the transduction rate from 22% in immature (i)DCs or 43% in mature (m)DCs to more than 80%, without inducing cytotoxicity. While expression of specific maturation markers was slightly upregulated using LentiBOOST®/Polybrene on iDCs, no effect on mDC phenotype or function was observed. Moreover, we achieved efficient HAdV5 transduction also in human monocytes and were able to subsequently differentiate them into proper iDCs and functional mDCs. In summary, we introduce LentiBOOST® comprising Polybrene as a highly potent adenoviral transduction agent for new in-vitro applications in a set of different immune cells in both mice and humans.

Targeting the ERβ/HER Oncogenic Network in KRAS Mutant Lung Cancer Modulates the Tumor Microenvironment and Is Synergistic with Sequential Immunotherapy

High ERβ/HER oncogenic signaling defines lung tumors with an aggressive biology. We previously showed that combining the anti-estrogen fulvestrant with the pan-HER inhibitor dacomitinib reduced ER/HER crosstalk and produced synergistic anti-tumor effects in immunocompromised lung cancer models, including KRAS mutant adenocarcinoma. How this combination affects the tumor microenvironment (TME) is not known. We evaluated the effects of fulvestrant and dacomitinib on murine bone marrow-derived macrophages (BMDMs) and CD8+ T cells, and tested the efficacy of the combination in vivo, using the KRAS mutant syngeneic lung adenocarcinoma model, FVBW-17. While this combination synergistically inhibited proliferation of FVBW-17 cells, it had unwanted effects on immune cells, by reducing CD8+ T cell activity and phagocytosis in BMDMs and inducing PD-1. The effects were largely attributed to dacomitinib, which caused downregulation of Src family kinases and Syk in immune cells. In a subcutaneous flank model, the combination induced an inflamed TME with increased myeloid cells and CD8+ T cells and enhanced PD-1 expression in the splenic compartment. Concomitant administration of anti-PD-1 antibody with fulvestrant and dacomitinib was more efficacious than fulvestrant plus dacomitinib alone. Administering anti-PD-1 sequentially after fulvestrant plus dacomitinib was synergistic, with a two-fold greater tumor inhibitory effect compared to concomitant therapy, in both the flank model and in a lung metastasis model. Sequential triple therapy has potential for treating lung cancer that shows limited response to current therapies, such as KRAS mutant lung adenocarcinoma.

Carbon Monoxide Regulates Macrophage Differentiation and Polarization toward the M2 Phenotype through Upregulation of Heme Oxygenase 1

Carbon monoxide (CO) is generated by heme oxygenase (HO), and HO-1 is highly induced in monocytes and macrophages upon stimulation. Monocytes differentiate into macrophages, including pro-inflammatory (M1) and anti-inflammatory (M2) cells, in response to environmental signals. The present study investigated whether CO modulates macrophage differentiation and polarization, by applying the CO-releasing molecule-3 (CORM-3). Results showed that murine bone marrow cells are differentiated into macrophages by CORM-3 in the presence of macrophage colony-stimulating factor. CORM-3 increases expressions of macrophage markers, including F4/80 and CD11b, and alters the cell morphology into elongated spindle-shaped cells, which is a typical morphology of M2 cells. CORM-3 upregulates the expressions of genes and molecules involved in M2 polarization and M2 phenotype markers, such as STAT6, PPARγ, Ym1, Fizz1, arginase-1, and IL-10. However, exposure to CORM-3 inhibits the iNOS expression, suggesting that CO enhances macrophage differentiation and polarization toward M2. Increased HO-1 expression is observed in differentiated macrophages, and CORM-3 further increases this expression. Hemin, an HO-1 inducer, results in increased macrophage differentiation, whereas the HO-1 inhibitor zinc protoporphyrin IX inhibits differentiation. In addition, CORM-3 increases the proportion of macrophages in peritoneal exudate cells and enhances the expression of HO-1 and arginase-1 but inhibits iNOS. Taken together, these results suggest that the abundantly produced CO in activated macrophages enhances proliferation, differentiation, and polarization toward M2. It will probably help clear apoptotic cells, resolve inflammation, and promote wound healing and tissue remodeling.

Gram-Positive Staphylococcus aureus LTA Promotes Distinct Memory-like Effects in Murine Bone Marrow Neutrophils

Neutrophils as innate immune cells primarily act as first responders in acute infection and directly maintain inflammatory responses. However, a growing body of evidence suggests that neutrophils also bear the potential to mediate chronic inflammation by exhibiting memory-like features. We recently showed that priming by serial doses of lipopolysaccharide (LPS) from gram-negative bacteria can trigger opposing memory-like responses (exaggerated inflammation, i.e. trained sensitivity or suppression of inflammation, i.e. tolerance) depending on the LPS-dose. We now asked whether this observation could also hold true for lipoteichoic acid (LTA) from gram-positive S. aureus. We found comparable effects of LTA on neutrophil priming as seen for LPS. Low-dose (1 ng/mL) LTA-priming promoted increased production of pro-inflammatory mediators (i.e., TNF-α, IL-6, ROS), whereas high-dose (10 µg/mL) results in contrary reactions supporting anti-inflammatory responses by increased IL-10 and declined pro-inflammatory capacity. In vitro neutrophil recruitment was similarly regulated by LTA -priming. Investigation of signalling patterns revealed TLR2/MyD88-mediated regulation of NFκB-p65 through intermediate PI3Ks/MAPK. Collectively, our data suggest a previously unknown capacity of neutrophils to be differentially primed by varying doses of LTA, endorsing memory-like features in neutrophils.

Arhgap21 Deficiency Results in Increase of Osteoblastic Lineage Cells in the Murine Bone Marrow Microenvironment

ARHGAP21 is a member of the RhoGAP family of proteins involved in cell growth, differentiation, and adhesion. We have previously shown that the heterozygous Arhgap21 knockout mouse model (Arhgap21+/−) presents several alterations in the hematopoietic compartment, including increased frequency of hematopoietic stem and progenitor cells (HSPC) with impaired adhesion in vitro, increased mobilization to peripheral blood, and decreased engraftment after bone marrow transplantation. Although these HSPC functions strongly depend on their interactions with the components of the bone marrow (BM) niche, the role of ARHGAP21 in the marrow microenvironment has not yet been explored. In this study, we investigated the composition and function of the BM microenvironment in Arhgap21+/− mice. The BM of Arhgap21+/− mice presented a significant increase in the frequency of phenotypic osteoblastic lineage cells, with no differences in the frequencies of multipotent stromal cells or endothelial cells when compared to the BM of wild type mice. Arhgap21+/− BM cells had increased capacity of generating osteogenic colony-forming units (CFU-OB) in vitro and higher levels of osteocalcin were detected in the Arhgap21+/− BM supernatant. Increased expression of Col1a1, Ocn and decreased expression of Trap1 were observed after osteogenic differentiation of Arhgap21+/− BM cells. In addition, Arhgap21+/− mice recipients of normal BM cells showed decreased leucocyte numbers during transplantation recovery. Our data suggest participation of ARHGAP21 in the balanced composition of the BM microenvironment through the regulation of osteogenic differentiation.

The Antidiabetic Agent Metformin Inhibits IL-23 Production in Murine Bone-Marrow-Derived Dendritic Cells

Psoriasis is a chronic inflammatory skin disease, and its immune mechanism has been profoundly elucidated. Biologics targeting interleukin (IL)-23 have prevented the development of psoriasis. As major sources of IL-23, dendritic cells (DCs) play a pivotal role in psoriasis; however, the regulatory mechanism of IL-23 in DCs remains unclear. IL-36γ was reported to reflect the disease activity of psoriasis. Therefore, we hypothesized that IL-36γ may affect IL-23 production in DCs. To reveal the mechanism by which IL-36γ controls IL-23 production in DCs, we analyzed murine bone marrow-derived DCs (BMDCs) stimulated with IL-36γ. IL-36γ stimulation upregulated the mRNA and protein expression of Nfkbiz in BMDCs. Nfkbiz knockdown using siRNA transfection partially inhibited the upregulation of IL-23 mRNA expression induced by IL-36γ stimulation. Since NF-κB signaling regulates Nfkbiz expression and the anti-diabetic agent metformin reportedly modulates NF-κB signaling, we examined the effect of metformin treatment on IL-36γ-induced IL-23 production. Metformin treatment impaired the phosphorylation of NF-κB induced by IL-36γ stimulation with the subsequent downregulation of Nfkbiz, resulting in the inhibition of IL-23 production in BMDCs. These data provided evidence that metformin treatment can inhibit IL-36γ-mediated IL-23 production in BMDCs, which might contribute to the prevention of psoriasis.

Male Macrophages and Fibroblasts from C57/BL6J Mice Are More Susceptible to Inflammatory Stimuli

Mounting evidence argues for the significant impact of sex in numerous cardiac pathologies, including myocarditis. Macrophage polarization and activation of cardiac fibroblasts play a key role in myocardial inflammation and remodeling. However, the role of sex in these processes is still poorly understood. In this study, we investigated sex-specific alterations in the polarization of murine bone marrow-derived macrophages (BMMs) and the polarization-related changes in fibroblast activation. Cultured male and female murine BMMs from C57/BL6J mice were polarized into M1 (LPS) and M2 (IL-4/IL-13) macrophages. Furthermore, male and female cardiac fibroblasts from C57/BL6J mice were activated with TNF-α, TGF-β, or conditioned medium from M1 BMMs. We found a significant overexpression of M1 markers (c-fos, NFκB, TNF-α, and IL-1β) and M2 markers (MCP-1 and YM1) in male but not female activated macrophages. In addition, the ROS levels were higher in M1 male BMMs, indicating a stronger polarization. Similarly, the pro-fibrotic markers TGF-β and IL-1β were expressed in activated cardiac male fibroblasts at a significantly higher level than in female fibroblasts. In conclusion, the present study provides strong evidence for the male-specific polarization of BMMs and activation of cardiac fibroblasts in an inflammatory environment. The data show an increased inflammatory response and tissue remodeling in male mice.

Comprehensive Transcriptomic Profiling of Murine Osteoclast Differentiation Reveals Novel Differentially Expressed Genes and LncRNAs

Osteoclasts are the sole bone resorbing cells, which undertake opposing roles to osteoblasts to affect skeletal mass and structure. However, unraveling the comprehensive molecular mechanisms behind osteoclast differentiation is necessitated to overcome limitations and scarcity of available data, particularly in relation with the emerging roles of long non-coding RNAs (LncRNAs) in gene expression. In this study, we performed comprehensive and progressive analyses of the dynamic transcriptomes of murine osteoclasts, generated in vitro. We compared the total RNA-based transcriptomes of murine bone marrow derived cells with differentiated osteoclasts, while focusing on potentially novel genes and LncRNAs, to uncover critical genes and their associated pathways, which are differentially regulated during osteoclast differentiation. We found 4,214 differentially regulated genes during osteoclast differentiation, which included various types of LncRNAs. Among the upregulated protein coding genes not previously associated with osteoclast are Pheta1, Hagh, Gfpt1 and Nol4, while downregulated genes included Plau, Ltf, Sell and Zfp831. Notably, we report Nol4 as a novel gene related to osteoclast activity since Nol4 knockout mice Nol4em1(International Mouse Phenotyping Consortium)J exhibit increased bone mineral density. Moreover, the differentially expressed LncRNAs included antisense and long intergenic non-coding RNAs, among others. Overall, immune-related and metabolism-related genes were downregulated, while anatomical morphogenesis and remodeling-related genes were upregulated in early-differentiated osteoclasts with sustained downregulation of immune-related genes in mature osteoclasts. The gene signatures and the comprehensive transcriptome of osteoclast differentiation provided herein can serve as an invaluable resource for deciphering gene dysregulation in osteoclast-related pathologic conditions.