The effects of dietary vitamin D supplementation and in vitro 1,25 dihydroxyvitamin D3 treatment on autophagy in bone marrow-derived dendritic cells from high-fat diet-induced obese mice.

Obesity is associated with the dysregulation of vitamin D metabolism and altered immune responses in bone marrow-derived dendritic cells (BMDCs). Vitamin D can affect the differentiation, maturation, and activation of dendritic cells (DCs) and regulate autophagy via vitamin D receptor signaling. Autophagy was shown to be involved in the functions of DCs. We investigated the effects of dietary vitamin D supplementation and in vitro 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) treatment on autophagy in BMDCs from control diet (CON)-fed lean and high-fat diet (HFD)-induced obese mice. C57BL/6 male mice were fed CON or HFD with 10% or 45% kcal fat, respectively, supplemented with 1,000 or 10,000 IU vitamin D/kg diet (vDC or vDS) for 12 weeks. BMDCs were generated by culturing bone marrow cells from the mice with 20 ng/mL rmGM-CSF and treated with 1 nM 1,25(OH)2D3. Maturation of BMDCs was induced by lipopolysaccharide (50 ng/mL) stimulation. Treatment with 1,25(OH)2D3 inhibited the expression of phenotypes related to DC function (MHC class Ⅱ, CD86, CD80) and production of IL-12p70 by BMDCs from control and obese mice, regardless of dietary vitamin D supplementation. LC3Ⅱ/Ⅰ and VPS34 protein levels increased, and p62 expression decreased, after 1,25(OH)2D3 treatment of the BMDCs in CON-vDC only. Vdr mRNA levels decreased following 1,25(OH)2D3 treatment of BMDCs in the HFD-vDC. In conclusion, autophagy flux was increased by 1,25(OH)2D3 treatment of the BMDCs in CON-vDC but not in the HFD-vDC group. This suggests that the decreased expression of Vdr following 1,25(OH)2D3 treatment might have affected autophagy flux in BMDCs from obese mice.

Immunosuppressive effect of Columbianadin on maturation, migration, allogenic T cell stimulation and phagocytosis capacity of TNF-α induced dendritic cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Angelicae pubescentis radix (APR) has a long history in the treatment of rheumatoid arthritis (RA) in China. It has the effects of dispelling wind to eliminate dampness, removing arthralgia and stopping pain in the Chinese Pharmacopeia, but its mechanisms was unclear. Columbianadin (CBN) was one of the main bioactive compounds of APR, and has many pharmacological effects. But the immunosuppressive effect of CBN on DCs and the potential mechanism needed to be explored. AIM OF THE STUDY: The study was aimed to clarify the immunosuppressive effect of CBN on maturation, migration, allogenic T cell stimulation and phagocytosis capacity of TNF-α induced DCs. MATERIALS AND METHODS: Bone marrow-derived DCs were obtained and cultured from C57BL/6 mice in accordance with protocol. The phenotypic study (CD11c, CD40, CD80, CD86 and MHC Ⅱ) were measured by flow cytometry. FITC-dextran were uptaked by DCs and the change of endocytosis activity were mediated by acquired mannose receptor. Transwell chambers were used to detect the migration ability of DCs. Mixed leukocyte reaction (MLR) assay was used to detect the allostimulatory ability of CBN on TNF-α stimulated DCs. The secretion of cytokines and chemokines was measured by ELISA Kit. TLRs gene and MAPKs/NF-κB protein expression were checked by qRT-PCR and Western blot. RESULTS: CBN inhibited the maturation of TNF-α-induced DCs while maintaining phagocytosis capabilities. Additionally, CBN inhibited the migration of TNF-α stimulated DCs, which related to reduce the production of chemokines (MCP-1, MIP-1α). Notably, CBN could suppress the proliferation of CD4+T cells by inhibiting DCs maturation, and decrease the proinflammatory cytokines IL-6 production. Furthermore, CBN inhibited mRNA expression of TLR2, TLR7 and TLR9 in TNF-α-activated DCs. Meanwhile, the phosphorylation of p38, JNK1/2 and NF-κB protein were significantly inhibited in CBN treated DCs. CONCLUSIONS: These findings provided novel insights into the pharmacological activity of CBN. They also indicated that inhibition DCs maturation owning to the immunosuppressive effect of CBN. CBN was expected as a potential immunosuppressant and TLRs/MAPKs/NF-κB pathway may be an important mechanism for CBN's immunosuppressive activity.

Photoactivation of mitochondrial reactive oxygen species-mediated Src and protein kinase C pathway enhances MHC class II-restricted T cell immunity to tumours.

High fluence low-level laser (HF-LLL), a mitochondria-targeted tumour phototherapy, results in oxidative damage and apoptosis of tumour cells, as well as damage to normal tissue. To circumvent this, the therapeutic effect of low fluence LLL (LFL), a non-invasive and drug-free therapeutic strategy, was identified for tumours and the underlying molecular mechanisms were investigated. We observed that LFL enhanced antigen-specific immune response of macrophages and dendritic cells by upregulating MHC class II, which was induced by mitochondrial reactive oxygen species (ROS)-activated signalling, suppressing tumour growth in both CD11c-DTR and C57BL/6 mice. Mechanistically, LFL upregulated MHC class II in an MHC class II transactivator (CIITA)-dependent manner. LFL-activated protein kinase C (PKC) promoted the nuclear translocation of CIITA, as inhibition of PKC attenuated the DNA-binding efficiency of CIITA to MHC class II promoter. CIITA mRNA and protein expression also improved after LFL treatment, characterised by direct binding of Src and STAT1, and subsequent activation of STAT1. Notably, scavenging of ROS downregulated LFL-induced Src and PKC activation and antagonised the effects of LFL treatment. Thus, LFL treatment altered the adaptive immune response via the mitochondrial ROS-activated signalling pathway to control the progress of neoplastic disease.

Ginsenoside-Rg1 acts as an IDO1 inhibitor, protects against liver fibrosis via alleviating IDO1-mediated the inhibition of DCs maturation.

BACKGROUND: Indoleamine 2,3-dioxygenase 1 (IDO1) has been reported as a hallmark of hepatic fibrosis. Ginseng Rg1(G-Rg1) is a characterized bioactive component isolated from a traditional Chinese medicinal herb Panax ginseng C. A. Meyer (Ginseng) that used in China widely. However, the anti-hepatic fibrosis property of G-Rg1 and the underlying mechanisms of action are poorly reported. PURPOSE: Here, we researched the effect of G-Rg1 on experimental liver fibrosis in vivo and in vitro. STUDY DESIGN AND METHODS: We applied a CCL4-induced liver fibrosis in mice (wild-type and those overexpressing IDO1 by in vivo AAV9 vector) and HSC-T6 cells to detect the anti-hepatic fibrosis effect of G-Rg1 in vivo and in vitro. RESULTS: We found that G-Rg1 reduced serum levels of AST and ALT markedly. Histologic examination indicated that G-Rg1 dramatically improved the extent of liver fibrosis and suppressed the hepatic levels of fibrotic marker α-SMA in vivo and in vitro. The proliferation of HSC-T6 was significantly inhibited by G-Rg1 in vitro. Both TUNEL staining and flow cytometry demonstrated that G-Rg1 attenuated the levels of hepatocyte apoptosis in fibrotic mice. Additionally, G-Rg1 up-regulated the maturation of hepatic DCs via reducing the expression level of hepatic IDO1, which played an inverse role in the maturation of DCs. Furthermore, oral administration of G-Rg1 ameliorated IDO1 overexpression-induced worsen liver fibrosis as well as IDO1 overexpression-mediated more apparent inhibition of maturation of DCs. CONCLUSION: These results suggest that G-Rg1, which exerts its antifibrotic properties via alleviating IDO1-mediated the inhibition of DCs maturation, may be a potential therapeutic drug in treating liver fibrosis.

Bombyx batryticatus Protein-Rich Extract Induces Maturation of Dendritic Cells and Th1 Polarization: A Potential Immunological Adjuvant for Cancer Vaccine.

Bombyx batryticatus, a protein-rich edible insect, is widely used as a traditional medicine in China. Several pharmacological studies have reported the anticancer activity of B. batryticatus extracts; however, the capacity of B. batryticatus extracts as immune potentiators for increasing the efficacy of cancer immunotherapy is still unverified. In the present study, we investigated the immunomodulatory role of B. batryticatus protein-rich extract (BBPE) in bone marrow-derived dendritic cells (BMDCs) and DC vaccine-immunized mice. BBPE-treated BMDCs displayed characteristics of mature immune status, including high expression of surface molecules (CD80, CD86, major histocompatibility complex (MHC)-I, and MHC-II), increased production of proinflammatory cytokines (tumor necrosis factor-α and interleukin-12p70), enhanced antigen-presenting ability, and reduced endocytosis. BBPE-treated BMDCs promoted naive CD4+ and CD8+ T-cell proliferation and activation. Furthermore, BBPE/ovalbumin (OVA)-pulsed DC-immunized mice showed a stronger OVA-specific multifunctional T-cell response in CD4+ and CD8+ T cells and a stronger Th1 antibody response than mice receiving differently treated DCs, which showed the enhanced protective effect against tumor growth in E.G7 tumor-bearing mice. Our data demonstrate that BBPE can be a novel immune potentiator for a DC-based vaccine in anticancer therapy.

Endo-1,3(4)-ß-Glucanase-Treatment of Oat ß-Glucan Enhances Fermentability by Infant Fecal Microbiota, Stimulates Dectin-1 Activation and Attenuates Inflammatory Responses in Immature Dendritic Cells.

Background: Non-digestible carbohydrates are added to infant formula to mimic the effects of human milk oligosaccharide by acting as prebiotics and stimulating the immune system. Although not yet used in infant formulas, ß-glucans are known to have beneficial health effects, and are therefore of potential interest for supplementation. Methods and results: We investigated the in vitro fermentation of native and endo-1,3(4)-ß-glucanase-treated oat ß-glucan using pooled fecal inocula of 2- and 8-week-old infants. While native oat ß-glucan was not utilized, both inocula specifically utilized oat ß-glucan oligomers containing ß(1→4)-linkages formed upon enzyme treatment. The fermentation rate was highest in the fecal microbiota of 2-week-old infants, and correlated with a high lactate production. Fermentation of media supplemented with native and enzyme-treated oat ß-glucans increased the relative abundance of Enterococcus and attenuated pro-inflammatory cytokine production (IL-1ß, IL-6, TNFα) in immature dendritic cells. This attenuating effect was more pronounced after enzyme treatment. This attenuation might result from the enhanced ability of fermented oat ß-glucan to stimulate Dectin-1 receptors. Conclusion: Our findings demonstrate that endo-1,3(4)-ß-glucanase treatment enhances the fermentability of oat ß-glucan and attenuates pro-inflammatory responses. Hence, this study shows that especially enzyme-treated oat ß-glucans have a high potential for supplementation of infant formula.

Myrothecine A modulates the proliferation of HCC cells and the maturation of dendritic cells through downregulating miR-221.

Myrothecine A, characterized from the extracts of myrothecium roridum strain IFB-E012, isolated as endophytic fungi found in the traditional Chinese medicinal plant Artemisia annua. Here we investigated its roles on anti-tumor and immune regulation in vitro. Dendritic cells (DCs) are the most potent antigen presenting cells in immune responses. Recent studies have indicated that miRNAs are indispensable in regulating the development, differentiation, maturation and function of DC. MiR-221, acted as an oncogene, is an important regulator in cancer development by binding to 3' untranslated regions (3' UTR) of target mRNA. Here, we investigated whether myrothecine A could inhibit cell proliferation in hepatocellular carcinoma (HCC) cell line SMMC-7721 by regulating miR-221. The HCC cells were treated with myrothecine A at different concentration, and the cell growth ability was measured by MTT assay. Then we observed whether myrothecine A could affect the maturation of DC by regulating miR-221. The HCC cell line was co-cultured with immature DC from mice bone marrow, and the levels of CD86 and CD40 was detected by FCM. Our results showed that myrothecine A could rescue miR-221-induced cell proliferation and influence the protein level of p27 by inhibiting the expression of miR-221. In addition, myrothecine A could enhance the expression of CD86 and CD40 by reversing the function of miR-221. Therefore, myrothecine A may be acted as an anti-tumor drug to promote the maturation of DC in the microenvironment of hepatocellular carcinoma.

Maturation of dendritic cells by maitake α-glucan enhances anti-cancer effect of dendritic cell vaccination.

Dendritic cells (DCs) play a primary role in antigen presentation to CD4+ and CD8+ T cells and induce acquired immune response against cancer cells. Therefore, determining positive modulators of DC activation to improve therapeutic approaches for cancer immunotherapy is greatly needed. In this study, we investigated the effect of maitake α-glucan YM-2A, isolated from Grifola frondosa, on the maturation and function of DCs and its adjuvant effect on a tumor-associated antigen (TAA)-loaded DC vaccine against murine tumor. We showed that YM-2A induced morphological changes and increased cell-surface maturation markers and cytokine production in DCs. In a mixed lymphocyte reactions assay, YM-2A-treated DCs increased proliferation and production of IFN-γ by allogeneic CD4+ and CD8+ T cells. These results indicate that YM-2A phenotypically and functionally activates DCs. Furthermore, YM-2A-treated TAA-loaded DC vaccine significantly reduced tumor growth and improved survival in two murine tumor models, CT-26 tumor-bearing BALB/c mice and B16 melanoma-bearing C57BL/6 mice. YM-2A-treated TAA-loaded DC vaccine increased splenic IFN-γ producing CD4+ and CD8+ T cells in CT-26 tumor-bearing BALB/c mice. Antibody neutralization studies indicated that YM-2A-induced DC maturation is mediated, in part, by the Dectin-1-dependent pathway. Overall, YM-2A-treatment with a TAA-loaded DC vaccine could be an excellent candidate for immunotherapy against cancer.

Skewing effect of sulprostone on dendritic cell maturation compared with dinoprostone.

BACKGROUND: Dendritic cells (DCs) are the most efficient antigen-presenting cells and act at the center of the immune system owing to their ability to control both immune tolerance and immunity. In cancer immunotherapy, DCs play a key role in the regulation of the immune response against tumors and can be generated ex vivo with different cytokine cocktails. METHODS: We evaluated the feasibility of dinoprostone (PGE2) replacement with the molecular analog sulprostone, in our good manufacturing practice (GMP) protocol for the generation of DC-based cancer vaccine. We characterized the phenotype and the function of DCs matured in the presence of sulprostone as a potential substitute of dinoprostone in the pro-inflammatory maturation cocktail consisting of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1ß) and IL-6. RESULTS: We found that sulprostone invariably reduces the recovery, but does not significantly modify the viability and the purity of DCs. The presence of sulprostone in the maturation cocktail increases the adhesion of single cells and of clusters of DCs to the flask, making them more similar to their immature counterpart in terms of adhesion and spreading proprieties. Moreover, we observed that sulprostone impairs the expression of co-stimulatory molecules and the spontaneous as well as the directed migration capacity of DCs. DISCUSSION: These findings underscore that the synthetic analog sulprostone strongly reduces the functional quality of DCs, thus cannot replace dinoprostone in the maturation cocktail of monocyte-derived DCs.

Isorhamnetin, the active constituent of a Chinese herb Hippophae rhamnoides L, is a potent suppressor of dendritic-cell maturation and trafficking.

Dendritic cells (DCs) have been recognized as major targets of immunosuppressive therapies for their significant roles in connecting innate and adaptive immunity. Isorhamnetin (Iso), one of the most common flavonoid compounds extracted from the Chinese herb Hippophae rhamnoides L, has been proved to have anti-inflammatory, anticarcinogenic, and antioxidant activities in many chronic inflammatory conditions, but the effects of Iso on DCs have rarely been reported before. Here we investigated the functions and the mechanisms of Iso on bone marrow-derived DCs (BMDCs) including maturation, phagocytosis, and trafficking. Our data showed that Iso effectively inhibited the maturation of lipopolysaccharide (LPS)-treated BMDCs by down regulation of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1ß and IL-12p70, up regulation of IL-10, and depression of costimulatory molecules CD40, CD80, and CD86, while had no effects on phagocytosis. Furthermore, Iso inhibited the migration of LPS-treated BMDCs, which may be due to its inhibition on chemokine receptor 7 (CCR7) expression. These findings strongly suggest that Iso is a potent immunosuppressive agent by inhibiting DC activation and trafficking, and may be used to prevent or treat chronic inflammation, autoimmune diseases, and graft rejections.

1,25-Dihydroxyvitamin D3 modulates the phenotype and function of Monocyte derived dendritic cells in cattle.

BACKGROUND: The active form of the vitamin D3, 1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) has been shown to have major effects not only on physiological processes but also on the regulation of the immune system of vertebrates. Dendritic cells are specialised antigen presenting cells which are in charge of the initiation of T-cell dependant immune responses and as such are key regulators of responses towards pathogens. In this study we set out to evaluate the effects of 1,25-(OH)2D3 on the phenotype of cattle monocyte-derived dendritic cells (MoDCs) and how the conditioning with this vitamin affects the function of these myeloid cells. RESULTS: MoDCs were generated from CD14+ monocytes with bovine IL-4 and GM-CSF with or without 1,25-(OH)2D3 supplementation for 10 days. Vitamin D conditioned MoDCs showed a reduced expression of co-stimulatory and antigen presenting molecules, as well as a reduced capability of endocytose ovalbumin. Furthermore, the capacity of MoDCs to induce proliferation in an allogeneic mixed leukocyte reaction was abolished when MoDCs were generated in presence of 1,25-(OH)2D3. LPS induced maturation of 1,25-(OH)2D3conditioned MoDCs resulted in lower secretion of IL-12 and higher IL-10 than that observed in MoDCs. CONCLUSIONS: The typical immunotolerant phenotype observed in cattle DCs after exposure to 1,25-(OH)2D3 has a significant effect on the functionality of these immune cells, inhibiting the T-cell stimulatory capacity of MoDCs. This could have profound implications on how the bovine immune system deals with pathogens, particularly in diseases such as tuberculosis or paratuberculosis.

ATP promotes the fast migration of dendritic cells through the activity of pannexin 1 channels and P2X7 receptors.

Upon its release from injured cells, such as infected, transformed, inflamed, or necrotic cells, extracellular adenosine-5'-triphosphate (ATP) acts as a danger signal that recruits phagocytes, such as neutrophils, macrophages, and dendritic cells (DCs), to the site of injury. The sensing of extracellular ATP occurs through purinergic (P2) receptors. We investigated the cellular mechanisms linking purinergic signaling to DC motility. We found that ATP stimulated fast DC motility through an autocrine signaling loop, which was initiated by the activation of P2X7 receptors and further amplified by pannexin 1 (Panx1) channels. Upon stimulation of the P2X7 receptor by ATP, Panx1 contributed to fast DC motility by increasing the permeability of the plasma membrane, which resulted in supplementary ATP release. In the absence of Panx1, DCs failed to increase their speed of migration in response to ATP, despite exhibiting a normal P2X7 receptor-mediated Ca2+ response. In addition to DC migration, Panx1 channel- and P2X7 receptor-dependent signaling was further required to stimulate the reorganization of the actin cytoskeleton. In vivo, functional Panx1 channels were required for the homing of DCs to lymph nodes, although they were dispensable for DC maturation. These data suggest that P2X7 receptors and Panx1 channels are crucial players in the regulation of DC migration to endogenous danger signals.

Dietary grape seed proanthocyanidins inactivate regulatory T cells by promoting NER-dependent DNA repair in dendritic cells in UVB-exposed skin.

Ultraviolet B (UVB) radiation induces regulatory T cells (Treg cells) and depletion of these Treg cells alleviates immunosuppression and inhibits photocarcinogenesis in mice. Here, we determined the effects of dietary grape seed proanthocyanidins (GSPs) on the development and activity of UVB-induced Treg cells. C3H/HeN mice fed a GSPs (0.5%, w/w)-supplemented or control diet were exposed to UVB (150 mJ/cm2) radiation, sensitized to 2,4-dinitrofluorobenzene (DNFB) and sacrificed 5 days later. FACS analysis indicated that dietary GSPs decrease the numbers of UVB-induced Treg cells. ELISA analysis of cultured sorted Treg cells indicated that secretion of immunosuppressive cytokines (interleukin-10, TGF-ß) was significantly lower in Treg cells from GSPs-fed mice. Dietary GSPs also enhanced the ability of Treg cells from wild-type mice to stimulate production of IFNγ by T cells. These effects of dietary GSPs on Treg cell function were not found in XPA-deficient mice, which are incapable of repairing UVB-induced DNA damage. Adoptive transfer experiments revealed that naïve recipients that received Treg cells from GSPs-fed UVB-irradiated wild-type donors that had been sensitized to DNFB exhibited a significantly higher contact hypersensitivity (CHS) response to DNFB than mice that received Treg cells from UVB-exposed mice fed the control diet. There was no significant difference in the CHS response between mice that received Treg cells from UVB-irradiated XPA-deficient donors fed GSPs or the control diet. Furthermore, dietary GSPs significantly inhibited UVB-induced skin tumor development in wild-type mice but not in XPA-deficient mice. These results suggest that GSPs inactivate Treg cells by promoting DNA repair in dendritic cells in UVB-exposed skin.

Paeonol ameliorates imiquimod-induced psoriasis-like skin lesions in BALB/c mice by inhibiting the maturation and activation of dendritic cells.

Paeonol, an active component derived from the traditional Chinese medicine Cortex Moutan, possesses anti-inflammatory, analgesic, antioxidant and anti-allergic properties. Psoriasis is a chronic, recurrent, inflammatory dermatosis accompanied by excessive activation of Toll­like receptors (TLRs) in dendritic cells (DCs), which are primarily responsible for initiating an immune response. We investigated the effect of paeonol on inflammation in an imiquimod (IMQ)-induced psoriasis-like mouse model and murine bone marrow-derived dendritic cells (BMDCs) stimulated by R848. Mice were intragastrically administered 100 mg/kg (high), 50 mg/kg (medium) and 25 mg/kg (low) paeonol, respectively. We evaluated inflammation of psori-asis­like lesions based on histological changes, protein levels of myeloid differentiation factor 88 (MyD88) and TLR8 in skin lesions by western blotting, and levels of CD11c+ DCs in skin by immunoassay and in spleens by flow cytometry. Inflammatory cytokines [interleukin (IL)-23, IL-12 and IL-1ß] in skin lesions and BMDCs were also assessed by RT-PCR and ELISA. Application of paeonol decreased IMQ-induced keratinocyte proliferation, and infiltration of CD3+ cells, while the treatment ameliorated CD11c+ cells in the spleen and skin, and reduced MyD88 and TLR8 proteins in skin lesions. Paeonol inhibited IMQ-induced mRNA expression of IL-23, but not IL-12 and IL-1ß in BMDCs, along with significantly lower levels of DCs expressing MHCⅡ, CD80 and CD86 in vitro. These results indicate that paeonol suppresses the maturation and activation of DCs by decreasing MyD88 and TLR8 proteins in the TLR7/8 signaling pathway which finally alleviates psoriasis­like skin lesions. The TLR7/8 signaling pathway in DCs provides an important insight into the mechanism of psoriasis, and paeonol may be a potent therapeutic drug for psoriasis.

Small-molecule inhibitor sorafenib regulates immunoreactions by inducing survival and differentiation of bone marrow cells.

Sorafenib has been used for the treatment of liver cancer. However, its clinical impact on human immunity system remains poorly known. Our previous study has shown that sorafenib modulates immunosuppressive cell populations in murine liver cancer models. Here, we continue to report that low doses of sorafenib promotes the survival of murine bone marrow cells (BMCs) in a dose-dependent manner by up-regulating the anti-apoptotic protein survivin. Sorafenib induces differentiation of BMCs into suppressive dendritic cells that inhibit autologous T-cell proliferation and stimulate CD4(+) T cells to express increased IL-1ß, IL-2, IL-4, IL-10, IFN-γ and TNF-α, and reduced levels of IL-6 and CD25, which indicates that sorafenib-induced dendritic cells represent a distinct cellular subset with unique properties. Taken together, our findings suggest that in addition to its anticancer effects, sorafenib has an immunoregulatory property that is apparent at low doses.

Biogenic selenium nanoparticles: characterization, antimicrobial activity and effects on human dendritic cells and fibroblasts.

Tailored nanoparticles offer a novel approach to fight antibiotic-resistant microorganisms. We analysed biogenic selenium nanoparticles (SeNPs) of bacterial origin to determine their antimicrobial activity against selected pathogens in their planktonic and biofilm states. SeNPs synthesized by Gram-negative Stenotrophomonas maltophilia [Sm-SeNPs(-)] and Gram-positive Bacillus mycoides [Bm-SeNPs(+)] were active at low minimum inhibitory concentrations against a number of clinical isolates of Pseudomonas aeruginosa but did not inhibit clinical isolates of the yeast species Candida albicans and C. parapsilosis. However, the SeNPs were able to inhibit biofilm formation and also to disaggregate the mature glycocalyx in both P. aeruginosa and Candida spp. The Sm-SeNPs(-) and Bm-SeNPs(+) both achieved much stronger antimicrobial effects than synthetic selenium nanoparticles (Ch-SeNPs). Dendritic cells and fibroblasts exposed to Sm-SeNPs(-), Bm-SeNPs(+) and Ch-SeNPs did not show any loss of cell viability, any increase in the release of reactive oxygen species or any significant increase in the secretion of pro-inflammatory and immunostimulatory cytokines. Biogenic SeNPs therefore appear to be reliable candidates for safe medical applications, alone or in association with traditional antibiotics, to inhibit the growth of clinical isolates of P. aeruginosa or to facilitate the penetration of P. aeruginosa and Candida spp. biofilms by antimicrobial agents.

Thymus: the next (re)generation.

As the primary site of T-cell development, the thymus plays a key role in the generation of a strong yet self-tolerant adaptive immune response, essential in the face of the potential threat from pathogens or neoplasia. As the importance of the role of the thymus has grown, so too has the understanding that it is extremely sensitive to both acute and chronic injury. The thymus undergoes rapid degeneration following a range of toxic insults, and also involutes as part of the aging process, albeit at a faster rate than many other tissues. The thymus is, however, capable of regenerating, restoring its function to a degree. Potential mechanisms for this endogenous thymic regeneration include keratinocyte growth factor (KGF) signaling, and a more recently described pathway in which innate lymphoid cells produce interleukin-22 (IL-22) in response to loss of double positive thymocytes and upregulation of IL-23 by dendritic cells. Endogenous repair is unable to fully restore the thymus, particularly in the aged population, and this paves the way toward the need for exogenous strategies to help regenerate or even replace thymic function. Therapies currently in clinical trials include KGF, use of the cytokines IL-7 and IL-22, and hormonal modulation including growth hormone administration and sex steroid inhibition. Further novel strategies are emerging in the preclinical setting, including the use of precursor T cells and thymus bioengineering. The use of such strategies offers hope that for many patients, the next regeneration of their thymus is a step closer.

Impaired signaling through the Fms-like tyrosine kinase 3 receptor increases osteoclast formation and bone damage in arthritis.

Osteoclasts are bone-resorbing cells that accumulate in the joints of patients with rheumatoid arthritis causing severe bone damage. Fms-like tyrosine kinase 3 ligand is enriched in the synovial fluid of patients with rheumatoid arthritis, and local exposure to Fms-like tyrosine kinase 3 ligand aggravates arthritis in mice. Because Fms-like tyrosine kinase 3 ligand has been suggested to facilitate osteoclast differentiation, we asked whether Fms-like tyrosine kinase 3 ligand affects bone remodeling in arthritis. The effect of Fms-like tyrosine kinase 3 signaling on osteoclast development was studied by immunohistochemistry in methylated bovine serum albumin-induced arthritis using mice that lack the gene for Flt3l (Flt3L(-/-)) and by an in vitro assay. Bone and joint changes were studied morphologically and by microcomputer tomography. We found that Flt3L(-/-) mice had increased accumulations of osteoclasts in the periarticular area of the arthritic joint. This triggered bone destruction and trabecular bone loss. The increased number of osteoclasts in Flt3L(-/-) mice may be a consequence of insufficient expression of interferon regulatory factor 8. Treatment of Flt3L(-/-) mice with Fms-like tyrosine kinase 3 ligand increased expression of interferon regulatory factor 8, reduced the number of osteoclasts in arthritic mice, and promoted trabecular bone formation. Finally, the reduced number of regulatory T cells in the bone marrow of Flt3L(-/-) mice could further contribute to the increased osteoclastogenesis by reducing the ratio of regulatory T cells to T helper 17 cells. This study shows that Fms-like tyrosine kinase 3 ligand may serve as a negative regulator of osteoclast development by promoting transcription of interferon regulatory factor 8 and sustaining a balance between protective regulatory T cells and pathogenic T helper 17 cells in the pathogenesis of arthritis.

Near-infrared photoactivatable control of Ca(2+) signaling and optogenetic immunomodulation.

The application of current channelrhodopsin-based optogenetic tools is limited by the lack of strict ion selectivity and the inability to extend the spectra sensitivity into the near-infrared (NIR) tissue transmissible range. Here we present an NIR-stimulable optogenetic platform (termed 'Opto-CRAC') that selectively and remotely controls Ca(2+) oscillations and Ca(2+)-responsive gene expression to regulate the function of non-excitable cells, including T lymphocytes, macrophages and dendritic cells. When coupled to upconversion nanoparticles, the optogenetic operation window is shifted from the visible range to NIR wavelengths to enable wireless photoactivation of Ca(2+)-dependent signaling and optogenetic modulation of immunoinflammatory responses. In a mouse model of melanoma by using ovalbumin as surrogate tumor antigen, Opto-CRAC has been shown to act as a genetically-encoded 'photoactivatable adjuvant' to improve antigen-specific immune responses to specifically destruct tumor cells. Our study represents a solid step forward towards the goal of achieving remote and wireless control of Ca(2+)-modulated activities with tailored function.

Deficiency of autophagy in dendritic cells protects against experimental autoimmune encephalomyelitis.

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) in the immune system. DCs present antigens to CD8 and CD4 T cells in the context of class I or II MHC. Recent evidence suggests that autophagy, a conserved intracellular degradation pathway, regulates class II antigen presentation. In vitro studies have shown that deletion of autophagy-related genes reduced antigen presentation by APCs to CD4 T cells. In vivo studies confirmed these findings in the context of infectious diseases. However, the relevance of autophagy-mediated antigen presentation in autoimmunity remains to be elucidated. Here, we report that loss of autophagy-related gene 7 (Atg7) in DCs ameliorated experimental autoimmune encephalomyelitis (EAE), a CD4 T cell-mediated mouse model of multiple sclerosis, by reducing in vivo priming of T cells. In contrast, severity of hapten-induced contact hypersensitivity, in which CD8 T cells and NK cells play major roles, was unaffected. Administration of the autophagy-lysosomal inhibitor chloroquine, before EAE onset, delayed disease progression and, when administered after the onset, reduced disease severity. Our data show that autophagy is required in DCs for induction of EAE and suggest that autophagy might be a potential target for treating CD4 T cell-mediated autoimmune conditions.