Mouse Dendritic Cells Research Articles

Ebola Virus Infection of Flt3-Dependent, Conventional Dendritic Cells and Antigen Cross-presentation Leads to High Levels of T-Cell Activation.

Previous studies have described that Ebola virus (EBOV) infection of human monocyte-derived dendritic cells (moDCs) inhibits dendritic cell (DC) maturation, resulting in poor T-cell activation. However, it is unknown how other DC subsets distinct from moDCs respond to EBOV infection. To better understand how DCs initiate T-cell activation during EBOV infection, we assessed the response of conventional mouse DCs (cDCs) to EBOV infection utilizing a recombinant EBOV expressing the model antigen ovalbumin. In contrast to moDCs, mouse cDC2s and cDC1s were poorly infected with EBOV but were highly activated. DCs were able to prime CD8 T cells via cross-presentation of antigens obtained from cell debris of EBOV-infected cells. EBOV infection further enhanced DC cross-presentation. Our findings indicate that EBOV infection of cDCs results in activation rather than inhibition, leading to high levels of T-cell activation. With that we propose a mechanistic explanation for the excess T-cell activation observed in human Ebola virus disease.

Approbation of a Homologous Model of the Antitumor Vaccine Based on Mature Mouse Dendritic Cells to Study the Biodistribution of the Cell Product.

Homologous animal cell product was obtained in protocol developed for female BALB/c mice. Dendritic cell (DC) migration from the injection site into the draining lymph nodes was evaluated. The number of DC labeled with carboxyfluorescein succinimidyl ester (CFSE) in draining lymph nodes increased from 5.3% (16 h) to 13.3% (48 h) (p=0.028) with a maximum at 72 h (15.4%, p=0.003). The immunophenotype of CFSE-DC detected in murine lymph nodes corresponded to the immunophenotype of mature vaccine DCs: they expressed differentiation markers CD11c, CD80, CD83, and CD86 (p>0.05 vs initial DC).

Lymphoid origin of intrinsically activated plasmacytoid dendritic cells in mice.

We identified a novel mouse plasmacytoid dendritic cell (pDC) lineage derived from the common lymphoid progenitors (CLPs) that is dependent on expression of Bcl11a. These CLP-derived pDCs, which we refer to as 'B-pDCs', have a unique gene expression profile that includes hallmark B cell genes, normally not expressed in conventional pDCs. Despite expressing most classical pDC markers such as SIGLEC-H and PDCA1, B-pDCs lack IFN-α secretion, exhibiting a distinct inflammatory profile. Functionally, B-pDCs induce T cell proliferation more robustly than canonical pDCs following Toll-like receptor 9 (TLR9) engagement. B-pDCs, along with another homogeneous subpopulation of myeloid-derived pDCs, display elevated levels of the cell surface receptor tyrosine kinase AXL, mirroring human AXL+ transitional DCs in function and transcriptional profile. Murine B-pDCs therefore represent a phenotypically and functionally distinct CLP-derived DC lineage specialized in T cell activation and previously not described in mice.

Immunogenicity risk assessment of empty capsids present in adeno-associated viral vectors using predictive innate immune responses

Immunogenicity of gene therapy and the impacts on safety and efficacy are of increasing interest in the pharmaceutical industry. Unique structural aspects of gene therapy delivery vectors, such as adeno-associated viral (AAV) vectors, are expected to activate the innate immune system. The risk of innate immune activation is critical to understand due to the potential impacts on safety and on subsequent adaptive immune responses. In this study, we investigated the responses of key innate immune players—dendritic cells, natural killer (NK) cells, and the complement system—to AAV8 capsids. Immunogenicity risk was also predicted in the presence empty AAV capsids for AAV gene therapy. Compared to genome-containing "full" AAV8 capsids, empty AAV8 capsids more strongly induced proinflammatory cytokine production and migration by human and mouse dendritic cells, but the “full” capsid increased expression of co-stimulatory markers. Furthermore, in an NK cell degranulation assay, we found mixtures of empty and full AAV8 capsids to activate expression of TNF-α, IFN-γ, and CD107a more strongly in multiple NK cell populations compared to either capsid type alone. Serum complement C3a was also induced more strongly in the presence of mixed empty and full AAV8 capsid formulations. Risk for innate immune activation suggests the importance to determine acceptable limits of empty capsids. Immunogenicity risk assessment of novel biological modalities will benefit from the aforementioned in vitro innate immune activation assays providing valuable mechanistic information.

Immune Responses Induced by a Recombinant Lactiplantibacillus plantarum Surface-Displaying the gD Protein of Pseudorabies Virus.

Pseudorabies virus (PRV) is one of the herpes viruses that can infect a wide range of animals including pigs, cattle, sheep, mice, and wild animals. PRV is a neurotropic alphaherpesvirus capable of infecting a variety of mammals. There is a rising interest in the targeted application of probiotic bacteria to prevent viral diseases, including PRV. In this study, the surface expression of enhanced green fluorescent protein (EGFP) on recombinant Lactiplantibacillus plantarum NC8 (rNC8) through the LP3065 LPxTG motif of Lactobacillus plantarum WCFS1 was generated. The surface expression was observed through confocal microscopy. Dendritic cell targeting peptides (DCpep) were also fused with LPxTG that help to bind with mouse DCs. The PRV-gD was cloned in LP3065 LPxTG, resulting in the generation of rNC8-LP3065-gD. Inactivated rNC8-LP3065-gD was administered intravenously in mice on days 1 and 7 at a dose of 200 µL (109 CFU/mouse) for monitoring immunogenicity. Subsequently, a challenge dose of PRV TJ (104 TCID50) was administered intramuscularly at 14 days post-immunization. The survival rate of the immunized mice reached 80% (4/5) with no significant signs of illness. A significant rise in anti-gD antibodies was detected in the immunized mice by ELISA. Quantitative PCR (qPCR) results showed decreased viral loading in different body tissues. Flow cytometry of lymphocytes derived from mice spleen indicated an increase in CD3+CD4+ T cells, but CD3+CD8+ T cells were not detected. Moreover, it offers a model to delineate immune correlates with rNC8-induced immunity against swine viral diseases.

TLR9-dependent dendritic cell maturation promotes IL-6-mediated upregulation of cathepsin X.

Cysteine cathepsins are lysosomal proteases subject to dynamic regulation within antigen-presenting cells during the immune response and associated diseases. To investigate the regulation of cathepsin X, a carboxy-mono-exopeptidase, during maturation of dendritic cells (DCs), we exposed immortalized mouse DCs to various Toll-like receptor agonists. Using a cathepsin X-selective activity-based probe, sCy5-Nle-SY, we observed a significant increase in cathepsin X activation upon TLR-9 agonism with CpG, and to a lesser extent with Pam3 (TLR1/2), FSL-1 (TLR2/6) and LPS (TLR4). Despite clear maturation of DCs in response to Poly I:C (TLR3), cathepsin X activity was only slightly increased by this agonist, suggesting differential regulation of cathepsin X downstream of TLR activation. We demonstrated that cathepsin X was upregulated at the transcriptional level in response to CpG. This occurred at late time points and was not dampened by NF-κB inhibition. Factors secreted from CpG-treated cells were able to provoke cathepsin X upregulation when applied to naïve cells. Among these factors was IL-6, which on its own was sufficient to induce transcriptional upregulation and activation of cathepsin X. IL-6 is highly secreted by DCs in response to CpG but much less so in response to poly I:C, and inhibition of the IL-6 receptor subunit glycoprotein 130 prevented CpG-mediated cathepsin X upregulation. Collectively, these results demonstrate that cathepsin X is differentially transcribed during DC maturation in response to diverse stimuli, and that secreted IL-6 is critical for its dynamic regulation.

Phalloidin-PAINT: Enhanced quantitative nanoscale imaging of F-actin

We present phalloidin-based points accumulation for imaging in nanoscale topography (phalloidin-PAINT), enabling quantitative superresolution imaging of filamentous actin (F-actin) in the cell body and delicate membrane protrusions. We demonstrate that the intrinsic phalloidin dissociation enables PAINT superresolution microscopy in an imaging buffer containing low concentrations of dye-conjugated phalloidin. We further show enhanced single-molecule labeling by chemically promoting phalloidin dissociation. Two benefits of phalloidin-PAINT are its ability to consistently quantify F-actin at the nanoscale throughout the entire cell and its enhanced preservation of fragile cellular structures. In a proof-of-concept study, we employed phalloidin-PAINT to superresolve F-actin structures in U2OS and dendritic cells (DCs). We demonstrate more consistent F-actin quantification in the cell body and structurally delicate membrane protrusions of DCs compared with direct stochastic optical reconstruction microscopy (dSTORM). Using DC2.4 mouse DCs as the model system, we show F-actin redistribution from podosomes to actin filaments and altered prevalence of F-actin-associated membrane protrusions on the culture glass surface after lipopolysaccharide exposure. The concept of our work opens new possibilities for quantitative protein-specific PAINT using commercially available reagents.

CCL22 Induces the Polarization of Immature Dendritic Cells into Tolerogenic Dendritic Cells in Radiation-Induced Lung Injury through the CCR4-Dectin2-PLC-γ2-NFATC2-Nr4a2-PD-L1 Signaling Pathway.

Recruitment of immune cells to the injury site plays a pivotal role in the pathology of radiation-associated diseases. In this study, we investigated the impact of the chemokine CCL22 released from alveolar type II epithelial (AT2) cells after irradiation on the recruitment and functional changes of dendritic cells (DCs) in the development of radiation-induced lung injury (RILI). By examining changes in CCL22 protein levels in lung tissue of C57BL/6N mice with RILI, we discovered that ionizing radiation increased CCL22 expression in irradiated alveolar AT2 cells, as did MLE-12 cells after irradiation. A transwell migration assay revealed that CCL22 promoted the migration of CCR4-positive DCs to the injury site, which explained the migration of pulmonary CCR4-positive DCs in RILI mice invivo. Coculture experiments demonstrated that, consistent with the response of regulatory T cells in the lung tissue of RILI mice, exogenous CCL22-induced DCs promoted regulatory T cell proliferation. Mechanistically, we demonstrated that Dectin2 and Nr4a2 are key targets in the CCL22 signaling pathway, which was confirmed in pulmonary DCs of RILI mice. As a result, CCL22 upregulated the expression of PD-L1, IL-6, and IL-10 in DCs. Consequently, we identified a mechanism in which CCL22 induced DC tolerance through the CCR4-Dectin2-PLC-γ2-NFATC2-Nr4a2-PD-L1 pathway. Collectively, these findings demonstrated that ionizing radiation stimulates the expression of CCL22 in AT2 cells to recruit DCs to the injury site and further polarizes them into a tolerant subgroup of CCL22 DCs to regulate lung immunity, ultimately providing potential therapeutic targets for DC-mediated RILI.

Modulation of Mouse Dendritic Cells In Vitro by Lactobacillus gasseri Postbiotic Proteins.

Different lactobacilli are probiotics for their beneficial effects that confer to the host. Recently, some of these effects were associated with released metabolic products/constituents (postbiotics). In the present study, the potential immunomodulatory capacity of the probiotic Lactobacillus gasseri OLL2809 cell-free supernatant (sup) was investigated in murine bone marrow-derived dendritic cells (DCs). Bacteria induced significantly higher expression of all examined cytokines than those induced by the stimulatory lipopolysaccharide (LPS) itself. On the contrary, sup only induced the anti-inflammatory IL-10 similarly to LPS, whereas IL-12 and IL-6 secretions were stimulated at a lower level. Moreover, sup reduced the surface expression of the analyzed co-stimulatory markers CD40, CD80, and CD86. Treatments of sup with different digestive enzymes indicated the proteinaceous nature of these immunomodulatory metabolites. Western blot and immunoadsorption analyzes revealed cross-reactivity of sup with the surface-layer proteins (SLPs) isolated from OLL2809. Therefore, we directly tested the ability of OLL2809 SLPs to stimulate specifically cytokine expression in iDCs. Interestingly, we found that all tested cytokines were induced by SLPs and in a dose-dependent manner. In conclusion, our results highlighted distinct immune properties between L. gasseri OLL2809 and its metabolites, supporting the concept that bacterial viability is not an essential prerequisite to exert immunomodulatory effects.

Activation of dendritic cells by a synthesized small molecule for breast cancer vaccination.

e13149 Background: Dendritic cell-based vaccination is a promising immunotherapy for treating various types of cancer. However, tumor-loaded dendritic cells alone have limited treatment efficacy. We have designed a small molecule mimicking the action of damage-associated molecular pattern molecules as an adjuvant for activating dendritic cells in vitro, which demonstrated augmented treatment efficacy. Methods: We aimed at exploring the roles of the synthesized small molecule in augmenting the efficacy of dendritic cell-based cancer vaccination to target triple negative breast cancer. Breast cancer mouse model is constructed using a 4T1 tumor cell line. Data were analyzed using a two-tailed unpaired t-test. Results: Mouse dendritic cells could be activated in vitro by the small molecule as an adjuvant evidenced by increased expression of MHC-I, MHC-II and CD40. Augmented motility of dendritic cells was also observed after treatment with the adjuvant. The signaling of NFkB is remarkably activated in dendritic cells by the adjuvant, which facilitates the maturation of dendritic cells. Engraftment of adjuvant-primed dendritic cells loaded with 4T1 tumor antigen into 4T1 tumor-bearing mice efficiently reduced the tumor size and improved survival, compared with the group treated with dendritic cells only. After injection of adjuvant-activated dendritic cells, both CD4 T cell and CD8 T cell populations were expanded in the spleen and lymph nodes draining the tumor sites. Furthermore, the adjuvant could promote the production of IFN-γ and IL-2, cytokines required for T cell activation and expansion. Next, splenocytes were obtained from individual mice after dendritic cell-based treatment. Upon re-encounter with the tumor antigen in vitro, remarkable expansion of T cell populations with a memory cell feature was confirmed in splenocytes derived from mice that received adjuvant-treated dendritic cells. Consistently, production of TNF-α, IFN-γ and granzyme B, was profoundly increased in these T cells associated with the adjuvant treatment. Conclusions: Our work suggests a novel dendritic cell-based cancer immunotherapy by using a small molecule mimicking the action of damage-associated molecular pattern molecules, which have documented immune potentiation activities.

Constitutive Flt3 signaling impacts conventional dendritic cell function.

The development of dendritic cells (DCs) depends on signaling via the FMS-like tyrosine kinase 3 (Flt3) receptor. How Flt3 signaling impacts terminally differentiated DC function is unknown. This is important given the increasing interest in exploiting Flt3 for vaccination and tumor immunotherapy. Here, we examined DCs in mice harboring constitutively activated Flt3 (Flt3-ITD). Flt3ITD/ITD mice possessed expanded splenic DC subsets including plasmacytoid DC, conventional DC (cDC)1, cDC2, double positive (DP) cDC1 (CD11c+ CD8+ CD11b- CD103+ CD86+), noncanonical (NC) cDC1 (CD11c+ CD8+ CD11b- CD103- CD86-) and single positive (SP) cDC1 (CD11c+ CD8+ CD11b- CD103- CD86+). Outcomes of constitutive Flt3 signaling differed depending on the cDC subset examined. In comparison with wild type (WT) DCs, all Flt3ITD/ITD cDCs displayed an altered surface phenotype with changes in costimulatory molecules, major histocompatibility complex class I (MHC I) and II (MHC II). Cytokine secretion patterns, antigen uptake, antigen proteolysis and antigen presenting function differed between WT and Flt3ITD/ITD subsets, particularly cDC2. In summary, Flt3 signaling impacts the function of terminally differentiated cDCs with important consequences for antigen presentation.

Mast cell-derived interleukin-4 mediates activation of dendritic cell during toll-like receptor 2-mediated inflammation.

During an innate inflammation, immune cells form distinct pro- and anti-inflammatory regions around pathogen-containing core-regions. Mast cells are localized in an anti-inflammatory microenvironment during the resolution of an innate inflammation, suggesting antiinflammatory roles of these cells. High-content imaging was used to investigated mast cell-dependent changes in the regional distribution of immune cells during an inflammation, induced by the toll-like receptor (TLR)-2 agonist zymosan. The distance between the zymosan-containing core-region and the anti-inflammatory region, described by M2-like macrophages, increased in mast cell-deficient mice. Absence of mast cells abolished dendritic cell (DC) activation, as determined by CD86-expression and localized the DCs in greater distance to zymosan particles. The CD86- DCs had a higher expression of the pro-inflammatory interleukins (IL)-1β and IL-12/23p40 as compared to activated CD86+ DCs. IL-4 administration restored CD86 expression, cytokine expression profile and localization of the DCs in mast cell-deficient mice. The IL-4 effects were mast cell-specific, since IL-4 reduction by eosinophil depletion did not affect activation of DCs. We found that mast cells induce DC activation selectively at the site of inflammation and thereby determine their localization within the inflammation. Overall, mast cells have antiinflammatory functions in this inflammation model and limit the size of the pro-inflammatory region surrounding the zymosan-containing core region.

Stat5b/Ezh2 axis governs high PD-L1 expressing tolerogenic dendritic cell subset in autoimmune diabetes

Dendritic cells (DCs) are specialized antigen-presenting cells that play an important role in inducing and maintaining immune tolerance. The altered distribution and/or function of DCs contributes to defective tolerance in autoimmune diseases such as type 1 diabetes (T1D). In human T1D and in NOD mouse models, DCs share some defects and are often described as less tolerogenic and excessively immunogenic. In the NOD mouse model, the autoimmune response is associated with a defect in the Stat5b signaling pathway. We have reported that expressing a constitutively active form of Stat5b in DCs of transgenic NOD mice (NOD.Stat5b-CA), re-established their tolerogenic function, restored autoimmune tolerance and conferred protection from diabetes. However, the role and molecular mechanisms of Stat5b signaling in regulating splenic conventional DCs tolerogenic signature remained unclear. In this study, we reported that, compared to immunogenic splenic DCs of NOD, splenic DCs of NOD.Stat5b-CA mice exhibited a tolerogenic profile marked by elevated PD-L1 and PD-L2 expression, reduced pro-inflammatory cytokine production, increased frequency of the cDC2 subset and decreased frequency of the cDC1 subset. This tolerogenic profile was associated with increased Ezh2 and IRF4 but decreased IRF8 expression. We also found an upregulation of PD-L1 in the cDC1 subset and high PD-L1 and PD-L2 expression in cDC2 of NOD.Stat5b-CA mice. Mechanistically, we demonstrated that Ezh2 plays an important role in the maintenance of high PD-L1 expression in cDC1 and cDC2 subsets and that Ezh2 inhibition resulted in PD-L1 but not PD-L2 downregulation which was more drastic in the cDC2 subset. Additionally, Ezh2 inhibition severely reduced the cDC2 subset and increased the cDC1 subset and Stat5b-CA.DC pro-inflammatory cytokine production. Together our data suggest that the Stat5b-Ezh2 axis is critical for the maintenance of tolerogenic high PD-L1-expressing cDC2 and autoimmune tolerance in NOD.Stat5b-CA mice.

TFEB regulates dendritic cell antigen presentation to modulate immune balance in asthma

ObjectiveAsthma stands as one of the most prevalent chronic respiratory conditions in children, with its pathogenesis tied to the actived antigen presentation by dendritic cells (DCs) and the imbalance within T cell subgroups. This study seeks to investigate the role of the transcription factor EB (TFEB) in modulating the antigen presentation process of DCs and its impact on the differentiation of T cell subgroups.MethodsBone marrow dendritic cells (BMDCs) were activated using house dust mites (HDM) and underwent RNA sequencing (RNA-seq) to pinpoint differentially expressed genes. TFEB mRNA expression levels were assessed in the peripheral blood mononuclear cells (PBMCs) of both healthy children and those diagnosed with asthma. In an asthma mouse model induced by HDM, the TFEB expression in lung tissue DCs was evaluated. Further experiments involved LV-shTFEB BMDCs co-cultured with T cells to explore the influence of TFEB on DCs’ antigen presentation, T cell subset differentiation, and cytokine production.ResultsTranscriptomic sequencing identified TFEB as a significantly differentially expressed gene associated with immune system pathways and antigen presentation. Notably, TFEB expression showed a significant increase in the PBMCs of children diagnosed with asthma compared to healthy counterparts. Moreover, TFEB exhibited heightened expression in lung tissue DCs of HDM-induced asthmatic mice and HDM-stimulated BMDCs. Silencing TFEB resulted in the downregulation of MHC II, CD80, CD86, and CD40 on DCs. This action reinstated the equilibrium among Th1/Th2 and Th17/Treg cell subgroups, suppressed the expression of pro-inflammatory cytokines like IL-4, IL-5, IL-13, and IL-17, while augmenting the expression of the anti-inflammatory cytokine IL-10.ConclusionTFEB might have a vital role in asthma’s development by impacting the antigen presentation of DCs, regulating T cell subgroup differentiation, and influencing cytokine secretion. Its involvement could be pivotal in rebalancing the immune system in asthma. These research findings could potentially unveil novel therapeutic avenues for treating asthma.

Sub-acute oral exposure to lowest observed adverse effect level of nivalenol exacerbates atopic dermatitis in mice via direct activation of mitogen-activated protein kinase signal in antigen-presenting cells.

This study investigated the immunotoxic effects of the mycotoxin nivalenol (NIV) using antigen-presenting cells and a mouse model of atopic dermatitis (AD). In vitro experiments were conducted using a mouse macrophage cell line (RAW 264.7) and mouse dendritic cell line (DC 2.4). After cells were exposed to NIV (0.19-5µmol) for 24h, the production of pro-inflammatory cytokines (IL-1β, IL-6, and TNFα) was quantified. To further investigate the inflammatory cytokine production pathway, the possible involvement of mitogen-activated protein kinase (MAPK) pathways, such as ERK1/2, p-38, and JNK, in NIV exposure was analyzed using MAPK inhibitors and phosphorylation analyses. In addition, the pro-inflammatory effects of oral exposure to NIV at low concentrations (1 or 5ppm) were evaluated in an NC/Nga mouse model of hapten-induced AD. In vitro experiments demonstrated that exposure to NIV significantly enhanced the production of TNFα. In addition, it also directly induced the phosphorylation of MAPK, indicated by the inhibition of TNFα production following pretreatment with MAPK inhibitors. Oral exposure to NIV significantly exacerbated the symptoms of AD, including a significant increase in helper T cells and IgE-produced B cells in auricular lymph nodes and secretion of pro-inflammatory cytokines, such as IL-4, IL-5, and IL-13, compared with the vehicle control group. Our findings indicate that exposure to NIV directly enhanced the phosphorylation of ERK1/2, p-38, and JNK, resulting in a significant increase in TNFα production in antigen-presenting cells, which is closely related to the development of atopic dermatitis.

Effects of stress-induced protein Sestrin2 on necroptosis of dendritic cells induced by lipopolysaccharide

To investigate the effect of stress-induced protein Sestrin2 (SESN2) on necroptosis of mouse dendritic cell (DC) induced by lipopolysaccharide (LPS) combined with zVAD, a panaspartate-specific cysteine protease (caspase) inhibitor. The DC2.4 cell line derived from the bone marrow of mouse in the 3rd to 10th generations was cultured. The cells were stimulated with LPS for 0 hour, 6 hours, 12 hours, and 24 hours, and grouped according to the stimulation time points. Western blotting was performed to determine the protein expression of SESN2 in each group. Overexpression empty lentivirus (NC), SESN2 gene overexpression RNA sequence lentivirus (SESN2 LV-RNA), small interfering empty lentivirus (NS), and SESN2 gene small interfering RNA sequence lentivirus (SESN2 siRNA) were transfected into DC2.4 cells. After 72 hours of transfection, cell fluorescence expression was observed under the inverted fluorescence microscope. Cells in each transfection group were stimulated with LPS for 24 hours. The blank control groups were set up and cultured with phosphate buffered saline (PBS) for 24 hours. Western blotting was performed to measure SESN2 protein expression. In the same groups as above, cells were stimulated with LPS+zVAD for 24 hours. The blank control groups were set up and cultured with PBS for 24 hours. Western blotting was used to determine the expression of mixed lineage kinase domain-like protein (MLKL) and phosphorylated-MLKL (p-MLKL). The p-MLKL levels and the number of positive cells were observed using laser scanning confocal microscopy. The necroptotic cell ratios were assessed by both flow cytometry and Hoechst staining. Compared to the LPS 0 hour group, the expression of SESN2 in the LPS 24 hours group showed a significant increase. Therefore, 24 hours was chosen as the subsequent stimulation time point. After successful lentivirus transduction and 24 hours of cultivation, the MLKL phosphorylation level in the SESN2 siRNA+LPS+zVAD group was significantly higher than that in the NS+LPS+zVAD group. The MLKL phosphorylation in the SESN2 LV-RNA+LPS+zVAD group was significantly lower than that in the NC+LPS+zVAD group. The MLKL phosphorylation levels in both the NS+LPS+zVAD group and the NC+LPS+zVAD group were obviously higher than those in the NS+PBS group and the NC+PBS group, respectively. Laser scanning confocal microscopy showed that the trends in quantity and fluorescence intensity of p-MLKL protein expressions were consistent with the above results. The results from flow cytometry analysis and Hoechst staining showed that the rates of cell necrotic apoptosis in SESN2 siRNA+LPS+zVAD group were significantly higher than those in NS+LPS+zVAD group [flow cytometry analysis: (30.800±1.153)% vs. (20.800±1.114)%, Hoechst staining: (75.267±0.451)% vs. (46.267±3.371)%, both P < 0.05], indicating that knocking down SESN2 further exacerbated the occurrence of necroptosis. The necrotic apoptosis rates in SESN2 LV-RNA+LPS+zVAD group were significantly lower than those in NC+LPS+zVAD group [flow cytometry analysis: (7.160±0.669)% vs. (19.240±2.322)%, Hoechst staining: (32.433±3.113)% vs. (48.567±4.128)%, both P < 0.05], indicating that overexpressing SESN2 reversed such response and markedly reduced the proportion of necroptotic cells compared to the corresponding empty vector group. SESN2 exhibits an inhibitory effect on necroptosis of DC in sepsis. Targeted SESN2 expression may regulate the process of DC-mediated immune response in sepsis.

Murine leukemia virus infection of non-dividing dendritic cells is dependent on nucleoporins.

Retroviral reverse transcription starts within the capsid and uncoating and reverse transcription are mutually dependent. There is still debate regarding the timing and cellular location of HIV's uncoating and reverse transcription and whether it occurs solely in the cytoplasm, nucleus or both. HIV can infect non-dividing cells because there is active transport of the preintegration complex (PIC) across the nuclear membrane, but Murine Leukemia Virus (MLV) is thought to depend on cell division for replication and whether MLV uncoating and reverse transcription is solely cytoplasmic has not been studied. Here, we used NIH3T3 and primary mouse dendritic cells to determine where the different stages of reverse transcription occur and whether cell division is needed for nuclear entry. Our data strongly suggest that in both NIH3T3 cells and dendritic cells (DCs), the initial step of reverse transcription occurs in the cytoplasm. However, we detected MLV RNA/DNA hybrid intermediates in the nucleus of dividing NIH3T3 cells and non-dividing DCs, suggesting that reverse transcription can continue after nuclear entry. We also confirmed that the MLV PIC requires cell division to enter the nucleus of NIH3T3 cells. In contrast, we show that MLV can infect non-dividing primary DCs, although integration of MLV DNA in DCs still required the viral p12 protein. Knockdown of several nuclear pore proteins dramatically reduced the appearance of integrated MLV DNA in DCs but not NIH3T3 cells. Additionally, MLV capsid associated with the nuclear pore proteins NUP358 and NUP62 during infection. These findings suggest that simple retroviruses, like the complex retrovirus HIV, gain nuclear entry by traversing the nuclear pore complex in non-mitotic cells.

CCDC88B interacts with RASAL3 and ARHGEF2 and regulates dendritic cell function in neuroinflammation and colitis

CCDC88B is a risk factor for several chronic inflammatory diseases in humans and its inactivation causes a migratory defect in DCs in mice. CCDC88B belongs to a family of cytoskeleton-associated scaffold proteins that feature protein:protein interaction domains. Here, we identified the Rho/Rac Guanine Nucleotide Exchange Factor 2 (ARHGEF2) and the RAS Protein Activator Like 3 (RASAL3) as CCDC88B physical and functional interactors. Mice defective in Arhgef2 or Rasal3 show dampened neuroinflammation, and display altered cellular response and susceptibility to colitis; ARHGEF2 maps to a human Chromosome 1 locus associated with susceptibility to IBD. Arhgef2 and Rasal3 mutant DCs show altered migration and motility in vitro, causing either reduced (Arhgef2) or enhanced (Rasal3) migratory properties. The CCDC88B/RASAL3/ARHGEF2 complex appears to regulate DCs migration by modulating activation of RHOA, with ARHGEF2 and RASAL3 acting in opposite regulatory fashions, providing a molecular mechanism for the involvement of these proteins in DCs immune functions.

Human and mouse peritoneal macrophages and dendritic cells compared.

Human and mouse peritoneal macrophages and dendritic cells compared.

Irisflorentin promotes bacterial phagocytosis and inhibits inflammatory responses in macrophages during bacterial infection

Bacterial infection remains a big concern in the patients of ICU, which is the main cause of life-threatening organ dysfunction, or even sepsis. The poor control of bacterial infection caused by antibiotic resistance, etc. or the overwhelming immune response are the most important patho genic factors in intensive care unit (ICU) patients. As main pathogens, antibiotic-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), impose serious challenges during sepsis and require alternative therapeutic options. Irisflorentin (IFL) is one of the major bioactive compounds isolated from the roots of Belamcanda chinensis (Shegan). In this study, IFL could suppress inflammatory response induced by MRSA or a synthetic mimic of bacterial lipoprotein (Pam3CSK4). IFL treatment enhanced the ability of macrophages to phagocytose bacteria likely through up-regulating the expression of phagocytic receptors SR-A1 and FcγR2a. Furthermore, IFL inhibited Pam3CSK4-induced production of pro-inflammatory cytokines, including IL-6 and TNF-α in Raw 264.7 cells, mouse primary macrophages or dendritic cells. IFL treatment also inhibited heat-killed MRSA-induced secretion of IL-6 and TNF-α in mouse bone marrow-derived macrophages. Moreover, IFL attenuated M1 polarization of macrophages as indicated by the down-regulated expression of its polarization markers CD86 and iNOS. Mechanistically, IFL markedly decreased the Pam3CSK4-induced activation of ERK, JNK or p38 MAPK pathways in macrophages. Taken together, IFL may serve as a promising compound for the therapy of bacterial infection, particularly those caused by antibiotic-resistant bacteria, such as MRSA.