Tiliroside,a dietary glycosidic flavonoid,inhibits TRAF-6/NF-κB/p38-mediated neuroinflammation in activated BV2 microglia

Background

Tiliroside is a dietary glycosidic flavonoid which has shown in vivo anti-inflammatory activity. This study is aimed at evaluating the effect of tiliroside on neuroinflammation in BV2 microglia, and to identify its molecular targets of anti-neuroinflammatory action.

Methods

BV2 cells were stimulated with LPS + IFNγ in the presence or absence of tiliroside. TNFα, IL-6, nitrite and PGE₂ production was determined with ELISA, Griess assay and enzyme immunoassay, respectively. iNOS, COX-2, phospho-p65, phospho-IκBα, phospho-IKKα, phospho-p38, phospho-MK2, phosopho-MKK3/6 and TRAF-6 were determined by western blot analysis. NF-κB activity was also investigated using a reporter gene assay in HEK293 cells. LPS-induced microglia ROS production was tested using the DCFDA method, while HO-1 and Nrf2 activation was determined with western blot.

Results

Tiliroside significantly suppressed TNFα, IL-6, nitrite and PGE₂ production, as well as iNOS and COX-2 protein expression from LPS + IFNγ-activated BV2 microglia. Further mechanistic studies showed that tiliroside inhibited neuroinflammation by targeting important steps in the NF-κB and p38 signalling in LPS + IFNγ-activated BV2 cells. This compound also inhibited LPS-induced TRAF-6 protein expression in BV2 cells. Antioxidant activity of tiliroside in BV2 cells was demonstrated through attenuation of LPS + IFNγ-induced ROS production and activation of HO-1/Nrf2 antioxidant system.

Conclusions

Tiliroside inhibits neuroinflammation in BV2 microglia through a mechanism involving TRAF-6-mediated activation of NF-κB and p38 MAPK signalling pathways. These activities are possibly due, in part, to the antioxidant property of this compound.

General Significance

Tiliroside is a potential novel natural compound for inhibiting neuroinflammation in neurodegenerative disorders.     

The stimulation of arginine transport by TNFα in human endothelial cells depends on NF-κB activation

In human saphenous vein endothelial cells (HSVECs), tumor necrosis factor-α (TNFα) and bacterial lipopolysaccharide (LPS), but neither interferon γ (IFNγ) nor interleukin 1β (IL-1β), stimulate arginine transport. The effects of TNFα and LPS are due solely to the enhancement of system y⁺ activity, whereas system y⁺L is substantially unaffected. TNFα  causes an increased expression of SLC7A2/CAT-2B gene while SLC7A1/CAT-1 expression is not altered by the cytokine. The suppression of PKC-dependent transduction pathways, obtained with the inhibitor chelerytrhine, the inhibitor peptide of PKCζ isoform, or chronic exposure to phorbol esters, does not prevent TNFα effect on arginine transport. Likewise, ERK, JNK, and p38 MAP kinases are not involved in the cytokine effect, since arginine transport stimulation is unaffected by their specific inhibitors. On the contrary, inhibitors of NF-κB pathway hinder the increase in CAT2B mRNA and the stimulation of arginine uptake. These results indicate that in human endothelial cells the activation of NF-κB pathway mediates the TNFα effects on arginine transport.     

CD8+ T cell/adipocyte inflammatory cross talk and ensuing M1 macrophage polarization are reduced by fish-oil-derived n-3 polyunsaturated fatty acids,in part by a TNF-α-dependent mechanism

Obese visceral adipose tissue (AT) inflammation is driven by adipokine-mediated cross talk between CD8⁺ T cells and adipocytes, a process mitigated by long-chain (LC) n-3 polyunsaturated fatty acids (PUFA) but underlying mechanisms and ensuing effects on macrophage polarization status are unknown. Using an in vitro co-culture model that recapitulates the degree of CD8⁺ T cell infiltration reported in obese AT, 3T3-L1 adipocytes were co-cultured for 24 h with purified splenic CD8⁺ T cells from C57Bl/6 mice consuming either a 10% w/w safflower oil (control, CON) or 7% w/w safflower oil + 3% w/w fish oil (FO) diet for 4 weeks (n=8–10/diet). Co-cultured cells were in direct contact or in a contact-independent condition separated by a Transwell permeable membrane and stimulated with lipopolysaccharide (10 ng/ml) to mimic in vivo obese endotoxin levels. In contact-dependent co-cultures, FO reduced inflammatory (IL-6, TNFα, IFN-γ) and macrophage chemotactic (CCL2, CCL7, CCL3) mRNA expression and/or secreted protein, NF-κB p65 activation, ROS accumulation, NLRP3 inflammasome priming (Nlrp3, Il1β mRNA) and activation (caspase-1 activity) compared to CON (P<.05). The anti-inflammatory action of FO was reproduced by the addition of a TNF-α neutralizing antibody (1 μg/ml) to CON co-cultures (CON/anti-TNF-α), albeit to a lesser degree. Conditioned media from FO and CON/anti-TNF-α co-cultures, in turn, reduced RAW 264.7 macrophage mRNA expression of M1 polarization markers (iNos, Cd11c, Ccr2) and associated inflammatory cytokines (Il6, Tnfα, Il1β) compared to CON. These data suggest that inflammatory CD8⁺ T cell/adipocyte cross talk is partially attributable to TNF-α signaling, which can be mitigated by LC n-3 PUFA.     

Dietary long-chain n-3 PUFAs mitigate CD4+ T cell/adipocyte inflammatory interactions in co-culture models of obese adipose tissue

Obese adipose tissue (AT) inflammation is partly driven by accumulation of CD4⁺ T helper (Th)1 cells and reduced Th2 and T regulatory subsets, which promotes macrophage chemotaxis and ensuing AT metabolic dysfunction. This study investigated CD4⁺ T cell/adipocyte cytokine-mediated paracrine interactions (cross talk) as a target for dietary intervention to mitigate obese AT inflammation. Using an in vitro co-culture model designed to recapitulate CD4⁺ T cell accumulation in obese AT (5% of stromal vascular cellular fraction), 3T3-L1 adipocytes were co-cultured with purified splenic CD4⁺ T cells from C57Bl/6 mice consuming one of two isocaloric diets containing either 10% w/w safflower oil (control, CON) or 7% w/w safflower oil+3% w/w fish oil (FO) for 4 weeks (n=8–11/diet). The FO diet provided 1.9% kcal from the long-chain (LC) n-3 polyunsaturated fatty acids (PUFAs) eicosapentaenoic acid and docosahexaenoic acid, a dose that can be achieved by supplementation. Co-cultures were stimulated for 48 h with lipopolysaccharide (LPS) to mimic in vivo obese endotoxin levels or with conditioned media collected from LPS-stimulated visceral AT isolated from CON-fed mice. In both stimulation conditions, FO reduced mRNA expression and/or secreted protein levels of Th1 markers (T-bet, IFN-γ) and increased Th2 markers (GATA3, IL-4), concomitant with reduced inflammatory cytokines (IL-1β, IL-6, IL-12p70, TNF-α), macrophage chemokines (MCP-1, MCP-3, MIP-1α, MIP-2) and levels of activated central regulators of inflammatory signaling (NF-κB, STAT-1, STAT-3) (P<.05). Therefore, CD4⁺ T cell/adipocyte cross talk represents a potential target for LC n-3 PUFAs to mitigate obese AT inflammation.     

Human adipose CD34+CD90+ stem cells and collagen scaffold constructs grafted in vivo fabricate loose connective and adipose tissues

Stem cell based therapies for the repair and regeneration of various tissues are of great interest for a high number of diseases. Adult stem cells, instead, are more available, abundant and harvested with minimally invasive procedures. In particular, mesenchymal stem cells (MSCs) are multi‐potent progenitors, able to differentiate into bone, cartilage, and adipose tissues. Human adult adipose tissue seems to be the most abundant source of MSCs and, due to its easy accessibility; it is able to give a considerable amount of stem cells. In this study, we selected MSCs co‐expressing CD34 and CD90 from adipose tissue. This stem cell population displayed higher proliferative capacity than CD34^?CD90^? cells and was able to differentiate in vitro into adipocytes (PPARγ⁺ and adiponectin⁺) and endothelial cells (CD31⁺VEGF⁺Flk1⁺). In addition, in methylcellulose without VEGF, it formed a vascular network. The aim of this study was to investigate differentiation potential of human adipose CD34⁺/CD90⁺ stem cells loaded onto commercial collagen sponges already used in clinical practice (Gingistat) both in vitro and in vivo. The results of this study clearly demonstrate that human adult adipose and loose connective tissues can be obtained in vivo, highlighting that CD34⁺/CD90 ASCs are extremely useful for regenerative medicine. J. Cell. Biochem. 114: 1039–1049, 2013. © 2012 Wiley Periodicals, Inc.     

In vitro activation and differentiation of naïve CD4 and CD8 T cells into HCV Core- and NS3-specific armed effector cells: A new role for CD4 T cells

Viral clearance in hepatitis C virus (HCV) infection has been correlated with strong, multi-specific and sustained T cell responses. The number of functionally active effector T cells determines the outcome of infection. Only a small number of antigen-specific naïve T cells are originally present. Upon infection, they undergo activation, clonal expansion and differentiation to become effector cells. In this study, we determined the ability of dendritic cells (DCs) to prime T cells in vitro to become effector cells upon stimulation with various TLR ligands or IFNα. T cell priming and activation was determined by proliferation and production of effector molecules, IFN-γ and Granzyme B (GrB). HCV Core-specific T cells showed significant increase in proliferation, and the number of HCV Core-specific CD4⁺ and CD8⁺ T cells producing IFN-γ and GrB was higher than control or NS3-specific T cells. These in vitro-primed CD4⁺ and CD8⁺ T cells exhibit the phenotype of just-activated and/or armed effector lymphocytes confirming the transition of naïve T cells to effector cells. This is the first study demonstrating the activation of GrB⁺CD4⁺ T cells against antigen(s) derived from HCV. Our study suggests a novel role of CD4⁺ T cells in immunity against HCV.     

A semi-synthetic derivative of artemisinin,artesunate inhibits prostaglandin E2 production in LPS/IFNγ-activated BV2 microglia

Artesunate is a semi-synthetic derivative of artemisinin used to treat malaria, and has been shown to possess anti-inflammatory activity. In this study, we have investigated the effect of artesunate on PGE₂ production/COX-2 protein expression in LPS + IFNγ-activated BV2 microglia. To further understand the mechanism of action of this compound, we investigated its interference with NF-κB and p38 MAPK signalling pathways. PGE₂ production was determined using EIA, while protein expressions of inflammatory targets like COX-2, mPGES-1, IκB, p38 and MAPKAPK2 were evaluated using western blot. An NF-κB-bearing luciferase reporter gene assay was used to test the effect of artesunate on NF-κB-mediated pro-inflammatory gene expression in HEK293 cells stimulated with TNFα (1 ng/ml). Artesunate (2 and 4 μM), significantly (p <0.01) suppressed PGE₂ production in LPS + IFNγ-activated BV2 microglia. This effect was found to be mediated via reduction in COX-2 and mPGES-1 proteins. Artesunate also produced significant inhibition of TNFα and IL-6 production in activated BV2 microglia. Further investigations showed that artesunate (0.5–4 μM) significantly (p <0.001) reduced NF-κB-driven luciferase expression, and inhibited IκB phosphorylation and degradation, through inhibition of IKK. Artesunate inhibited phosphorylation of p38 MAPK and its substrate MAPKAPK2 following stimulation of microglia with LPS + IFNγ. Taken together, we have shown that artesunate prevents neuroinflammation in BV2 microglia by interfering with NF-κB and p38 MAPK signalling.     

Identification of new cytokine combinations for antigen-specific T-cell therapy products via a high-throughput multi-parameter assay

Infusion of viral-specific T cells (VSTs) is an effective treatment for viral infection after stem cell transplant. Current manufacturing approaches are rapid, but growth conditions can still be further improved. To optimize VST cell products, the authors designed a high-throughput flow cytometry-based assay using 40 cytokine combinations in a 96-well plate to fully characterize T-cell viability, function, growth and differentiation. Peripheral blood mononuclear cells (PBMCs) from six consenting donors were seeded at 100 000 cells per well with pools of cytomegalovirus peptides from IE1 and pp65 and combinations of IL-15, IL-6, IL-21, interferon alpha, IL-12, IL-18, IL-4 and IL-7. Ten-day cultures were tested by 13-color flow cytometry to evaluate viable cell count, lymphocyte phenotype, memory markers and interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) expression. Combinations of IL-15/IL-6 and IL-4/IL-7 were optimal for the expansion of viral-specific CD3+ T cells, (18-fold and 14-fold, respectively, compared with unstimulated controls). CD8+ T cells expanded 24-fold in IL-15/IL-6 and 9-fold in IL-4/IL-7 cultures (P < 0.0001). CD4+ T cells expanded 27-fold in IL-4/IL-7 and 15-fold in IL-15/IL-6 (P < 0.0001). CD45RO+ CCR7– effector memory (CD45RO+ CCR7– CD3+), central memory (CD45RO+ CCR7+ CD3+), terminal effector (CD45RO– CCR7– CD3+), and naive (CD45RO– CCR7+ CD3+). T cells were the preponderant cells (76.8% and 72.3% in IL-15/IL-6 and IL-15/IL-7 cultures, respectively). Cells cultured in both cytokine conditions were potent, with 19.4% of CD3+ cells cultured in IL-15/IL-6 producing IFNγ (7.6% producing both TNFα and IFNγ) and 18.5% of CD3+ cells grown in IL-4/IL-7 producing IFNγ (9% producing both TNFα and IFNγ). This study shows the utility of this single-plate assay to rapidly identify optimal growth conditions for VST manufacture using only 10⁷ PBMCs.     

Semiquantitative analysis of Th1 and Th2 cytokine expression in CD3+, CD4+, and CD8+renal-cell-carcinoma-infiltrating lymphocytes

The mRNA expression of Th1 and Th2 cytokines was compared in freshly isolated CD3⁺ tumor-infiltrating lymphocytes (CD3⁺ TIL) and in autologous CD3⁺ peripheral blood lymphocytes (CD3⁺ PBL) obtained simultaneously from 20 patients with renal cell carcinomas (RCC). In addition cytokine expression was compared in CD4⁺ TIL and CD8⁺ TIL from another group of 20 patients with RCC. TIL were isolated from mechanically disaggregated tumor material and PBL from peripheral blood by gradient centrifugation and subsequent selection with anti-CD3, anti-CD4 or anti-CD8 magnetic beads. In these pure lymphocyte preparations the constitutive expression of interleukin-1 (IL-1), IL-2, IL-10, interferon γ (IFN), and tumor necrosis factor α (TNF) was determined by using a polymerase-chain-reaction-assisted mRNA amplification assay. In the CD3⁺ TIL, levels of mRNA for IFN, IL-10, IL-1 and TNF were significantly higher than in the autologous CD3⁺ PBL whereas IL-2 expression was rather low and did not differ in the two populations. Comparison of cytokine mRNA expression in CD4⁺ TIL and simultaneously obtained CD8⁺ TIL revealed a significantly higher expression of IFN in the CD8⁺ cells. These data reflect an in vivo activation of RCC-infiltrating lymphocytes at the mRNA level with respect to the Th1 as well as the Th2 immune response. Th1 activation seems to be most evident in the CD8⁺ TIL. Received: 14 January 1999 / Accepted: 30 April 1999     

Increased intratumoral IL-22-producing CD4+ T cells and Th22 cells correlate with gastric cancer progression and predict poor patient survival

IL-22-producing CD4⁺ T cells (IL-22⁺CD4⁺ T cells) and Th22 cells (IL-22⁺IL-17^?IFN-γ^?CD4⁺ T cells) represent newly discovered T-cell subsets, but their nature, regulation, and clinical relevance in gastric cancer (GC) are presently unknown. In our study, the frequency of IL-22⁺CD4⁺ T cells in tumor tissues from 76 GC patients was significantly higher than that in tumor-draining lymph nodes, non-tumor, and peritumoral tissues. Most intratumoral IL-22⁺CD4⁺ T cells co-expressed IL-17 and IFN-γ and showed a memory phenotype. Locally enriched IL-22⁺CD4⁺ T cells positively correlated with increased CD14⁺ monocytes and IL-6 and IL-23 detection ex vivo, and in vitro IL-6 and IL-23 induced the polarization of IL-22⁺CD4⁺ T cells in a dose-dependent manner and the polarized IL-22⁺CD4⁺ T cells co-expressed of IL-17 and IFN-γ. Moreover, IL-22⁺CD4⁺ T-cell subsets (IL-22⁺IL-17⁺CD4⁺, IL-22⁺IL-17^?CD4⁺, IL-22⁺IFN-γ⁺CD4⁺, IL-22⁺IFN-γ^?CD4⁺, and IL-22⁺IL-17⁺IFN-γ⁺CD4⁺ T cells), and Th22 cells were also increased in tumors. Furthermore, higher intratumoral IL-22⁺CD4⁺ T-cell percentage and Th22-cell percentage were found in patients with tumor-node-metastasis stage advanced and predicted reduced overall survival. In conclusion, our data indicate that IL-22⁺CD4⁺ T cells and Th22 cells are likely important in establishing the tumor microenvironment for GC; increased intratumoral IL-22⁺CD4⁺ T cells and Th22 cells are associated with tumor progression and predict poorer patient survival, suggesting that tumor-infiltrating IL-22⁺CD4⁺ T cells and Th22 cells may be suitable therapeutic targets in patients with GC.     

Extracellular purine metabolism and signaling of CD73-derived adenosine in murine Treg and Teff cells

CD73-derived adenosine acts as potent inhibitor of inflammation, and regulatory T cells (Treg) have been shown to express CD73 as a novel marker. This study explored the role of endogenously formed adenosine in modulating NF-κB activity and cytokine/chemokine release from murine Treg and effector T cells (Teff) including key enzymes/purinergic receptors of extracellular ATP catabolism. Stimulating murine splenocytes and CD4(+) T cells with anti-CD3/anti-CD28 significantly upregulated activated NF-κB in CD73(-/-) T cells (wild type: 4.36 ± 0.21; CD73(-/-): 6.58 ± 0.75; n = 4; P = 0.029). This was associated with an augmented release of proinflammatory cytokines IL-2, TNF-α, and IFN-γ. Similar changes were observed with the CD73 inhibitor APCP (50 μM) on NF-κB and IFN-γ in wild-type CD4(+) T-cells. Treatment of stimulated CD4(+) T-cells with adenosine (25 μM) potently reduced IFN-γ release which is mediated by adenosine A2a receptors (A2aR). AMP (50 μM) also reduced cytokine release which was not inhibited by APCP. In Teff, A2aR activation (CGS21680) potently inhibited the release of IL-1, IL-2, IL-3, IL-4, IL-12, IL-13, IFN-γ, TNF-α, granulocyte-macrophage colony-stimulating factor (GM-CSF), CCL3, and CCL4. However, in Treg, CGS21680 did not alter cytokine/chemokine release. In summary, CD73-derived adenosine tonically inhibits active NF-κB in CD4(+) T-cells, thereby modulating the release of a broad spectrum of proinflammatory cytokines and chemokines. Downregulation of P2X7 and upregulation of CD73 in Treg after antigenic stimulation may be an important mechanism to maintain the ability of Treg to generate immunosuppressive adenosine.     

A DNA vaccine encoding multiple HIV CD4 epitopes elicits vigorous polyfunctional, long-lived CD4+ and CD8+ T cell responses

T-cell based vaccines against HIV have the goal of limiting both transmission and disease progression by inducing broad and functionally relevant T cell responses. Moreover, polyfunctional and long-lived specific memory T cells have been associated to vaccine-induced protection. CD4(+) T cells are important for the generation and maintenance of functional CD8(+) cytotoxic T cells. We have recently developed a DNA vaccine encoding 18 conserved multiple HLA-DR-binding HIV-1 CD4 epitopes (HIVBr18), capable of eliciting broad CD4(+) T cell responses in multiple HLA class II transgenic mice. Here, we evaluated the breadth and functional profile of HIVBr18-induced immune responses in BALB/c mice. Immunized mice displayed high-magnitude, broad CD4(+)/CD8(+) T cell responses, and 8/18 vaccine-encoded peptides were recognized. In addition, HIVBr18 immunization was able to induce polyfunctional CD4(+) and CD8(+) T cells that proliferate and produce any two cytokines (IFNγ/TNFα, IFNγ/IL-2 or TNFα/IL-2) simultaneously in response to HIV-1 peptides. For CD4(+) T cells exclusively, we also detected cells that proliferate and produce all three tested cytokines simultaneously (IFNγ/TNFα/IL-2). The vaccine also generated long-lived central and effector memory CD4(+) T cells, a desirable feature for T-cell based vaccines. By virtue of inducing broad, polyfunctional and long-lived T cell responses against conserved CD4(+) T cell epitopes, combined administration of this vaccine concept may provide sustained help for CD8(+) T cells and antibody responses- elicited by other HIV immunogens.     

Human CD8+ T cells display a differential ability to undergo cytokine-driven bystander activation

A subset of CD44^hiCD8⁺ T cells in some, but not all mice, can be induced to rapidly secrete IFNγ during infection with Listeria monocytogenes. This response is dependent on the presence of both IL-12 and IL-18 and does not require engagement of the T cell receptor. In this study, we demonstrate that human CD8⁺ T cells also vary widely in their ability to secrete IFNγ within 15 h of either Listeria infection or cytokine stimulation. The magnitude of the rapid IFNγ response correlated more closely with the intrinsic responsiveness of the T cells to cytokine stimulation rather than the amount of IL-12 produced. CD8⁺ T cells from 2 out of 16 blood donors (12.5%) failed to generate a significant IFNγ response. These results demonstrate that bystander activation of CD8⁺ T cells varies among individuals and validate further study of the differential responses observed using BALB/c vs. C57BL/6 mice.     

Differential regulation of IFNα, IFNβ and IFNε gene expression in human cervical epithelial cells

Interferonε (IFNε) is a unique type I IFN that has distinct functions from IFNα/β. IFNε is constitutively expressed at mucosal tissues, including the female genital mucosa, and is reported to be modulated by estrogen and seminal plasma. However, its regulation by cytokines, including TNFα, IL-1β, IL-6, IL-8, IL-17, IL-22 and IFNα, which are commonly present in the female genital mucosa, is not well documented in freshly isolated primary cervical cells from tissues. We determined the effect of these cytokines on gene expression of type I IFNs in an immortalized endocervical epithelial cell line (A2EN) and in primary cervical epithelial cells. Several pro-inflammatory cytokines were found to induce IFNε, and TNFα induced the strongest response in both cell types. Pretreatment of cells with the IκB inhibitor, which blocks the NF-κB pathway, suppressed TNFα-mediated IFNε gene induction and promoter activation. Expression of IFNα, IFNβ, and IFNε was differentially regulated in response to various cytokines. Taken together, our results show that regulation of these IFNs depends on cell type, cytokine concentration, and incubation time, highlighting the complexity of the cytokine network in the cervical epithelium.     

TLR8 stimulation enhances cetuximab-mediated natural killer cell lysis of head and neck cancer cells and dendritic cell cross-priming of EGFR-specific CD8+ T cells

Background

Cetuximab is an anti-epidermal growth factor receptor (EGFR) monoclonal antibody that prolongs survival in the treatment for head and neck cancer (HNC), but only in 10–20 % of patients. An immunological mechanism of action such as natural killer (NK) cell–mediated antibody-dependent cellular cytotoxicity (ADCC) has been suggested. We investigated the effects of activating toll-like receptor (TLR)-8 to enhance activity of cetuximab-stimulated, FcγR-bearing cells.

Objective

To determine the capability of TLR8-stimulation to enhance the activation and function of NK cells and dendritic cells (DC) in the presence of cetuximab-coated HNC cells.

Methods

Peripheral blood mononuclear cells (PBMC), NK, DC, and CD8⁺ T cells were isolated and analyzed using ⁵¹Cr release ADCC, flow cytometry analysis, cytokine ELISA, and EGFR_853-861 tetramer staining.

Results

TLR8 stimulation of unfractionated PBMC led to enhanced cetuximab-mediated ADCC in healthy donors (p < 0.01) and HNC patients (p < 0.001), which was dependent on NK cells. Secretion of Th1 cytokines TNFα (p < 0.0001), IFNγ (p < 0.0001), and IL-12p40 (p < 0.005) was increased. TLR8 stimulation of PBMC augmented cetuximab-enhanced NK cell degranulation (p < 0.001). TLR8-stimulated NK cells enhanced DC maturation markers CD80, CD83, and CD86 in co-culture with cetuximab-treated HNC cells. TLR8 stimulation of NK-DC co-cultures significantly increased DC priming of EGFR-specific CD8⁺ T cells in the presence of cetuximab.

Discussion

VTX-2337 and cetuximab combination therapy can activate innate and adaptive anti-cancer immune responses. Further investigation in human trials will be important for determining the clinical benefit of this combination and for determining biomarkers of response.     

IgE production in CD40/CD40L cross-talk of B and mast cells and mediator release via TGase 2 in mouse allergic asthma

TGase 2 is over-expressed in a variety of inflammatory diseases including allergic asthma. This study aimed to investigate the role of TGase 2 on IgE production and signaling pathways in mast cell activation related to OVA-induced allergic asthma. Bone marrow-derived mast cells (BMMCs) isolated from WT or TGase 2^?/? mice were activated with Ag/Ab (refer to act-WT-BMMCs and act-KO-BMMCs, respectively). B cells isolated from splenocytes were activated with anti-mouse IgM (act-B cells), and B cells were co-cultured with BMMCs. WT and TGase 2^?/? mice were sensitized and challenged with OVA adsorbed in alum hydroxide. Intracellular Ca^2 + ([Ca^2 +]_i) levels were determined by fluorescence intensity; IgE, mediators and TGase 2 activity by ELISA; the CD138 expression by FACS analyzer; cell surface markers and signal molecules by Western blot; NF-κB by EMSA; co-localization of mast cells and B cells by immunohistochemistry; Fcε RI-mediated mast cell activation by PCA test; expression of cytokines, MMPs, TIMPs, TLR2 and Fc?RI by RT-PCR. In vitro, act-KO-BMMCs reduced the [Ca^2 +]i levels, NF-κB activity, expression of CD40/CD40L, plasma cells, total IgE levels and TGase 2 activity in act-B cells co-cultured with act-BMMCs, expression of inflammatory cytokines and MMPs2/9, release of mediators (TNF-α, LTs and cytokines), and activities of signal molecules (PKCs, MAP kinases, I-κB and PLA2), which were all increased in act-WT-BMMCs. TGase 2 siRNA transfected/activated-BMMCs reduced all responses as same as those in act-KO-BMMCs. In allergic asthma model, TGase 2^?/? mice protected against PCA reaction, OVA-specific IgE production and AHR, and they reduced co-localization of mast cells and B cells or IgE in lung tissues, expression and co-localization of surface molecules in mast cells (c-kit and CD40L) and B cells (CD23 and CD40), inflammatory cells including mast cells, goblet cells, amounts of collagen and mediator release in BAL fluid and/or lung tissues, which were all increased in WT mice. TLR expression in TGase 2^?/? mice did not differ from those in WT mice. Our data suggest that TGase 2 expression and Ca^2 + influx required by bidirectional events in mast cell activation facilitate IgE production in B cells via up-regulating mast cell CD40L expression, and induce the expression of numerous signaling molecules associated with airway inflammation and remodeling in allergic asthma.     

BCG Skin Infection Triggers IL-1R-MyD88-Dependent Migration of EpCAMlow CD11bhigh Skin Dendritic cells to Draining Lymph Node During CD4+ T-Cell Priming

The transport of antigen from the periphery to the draining lymph node (DLN) is critical for T-cell priming but remains poorly studied during infection with Mycobacterium bovis Bacille Calmette-Guérin (BCG). To address this we employed a mouse model to track the traffic of Dendritic cells (DCs) and mycobacteria from the BCG inoculation site in the skin to the DLN. Detection of BCG in the DLN was concomitant with the priming of antigen-specific CD4⁺ T cells at that site. We found EpCAM^low CD11b^high migratory skin DCs to be mobilized during the transport of BCG to the DLN. Migratory skin DCs distributed to the T-cell area of the LN, co-localized with BCG and were found in close apposition to antigen-specific CD4⁺ T cells. Consequently, blockade of skin DC traffic into DLN dramatically reduced mycobacterial entry into DLN and muted T-cell priming. Interestingly, DC and mycobacterial entry into the DLN was dependent on IL-1R-I, MyD88, TNFR-I and IL-12p40. In addition, we found using DC adoptive transfers that the requirement for MyD88 in BCG-triggered migration was not restricted to the migrating DC itself and that hematopoietic expression of MyD88 was needed in part for full-fledged migration. Our observations thus identify a population of DCs that contribute towards the priming of CD4⁺ T cells to BCG infection by transporting bacilli into the DLN in an IL-1R-MyD88-dependent manner and reveal both DC-intrinsic and -extrinsic requirements for MyD88 in DC migration.     

Cyclophilin a increases CD68<Superscript>+</Superscript> cell infiltration in rat experimental periodontitis

Cyclophilin A (CyPA) is a potent chemokine, which can directly induce leukocyte chemotaxis and contribute to the pathogenesis of inflammation-mediated diseases. This study is to observe the expression and distribution of CyPA and CD68⁺ cells in the histopathogenesis of rat ligation-induced experimental periodontitis, and assess the role of CyPA in CD68⁺ cell infiltration in rat experimental periodontitis. Experimental periodontitis was induced by ligation according to our previous method. CyPA expression in gingival tissues was detected by western blotting. Immunohistochemistry was applied for CyPA and CD68 distribution. For further certifying the role of CyPA in CD68⁺ cell infiltration, the right mandibular first molar received 0.1 μM CyPA locally by gingival injection every 2 days (L?+?C group), while the left mandibular first molar received saline as a control group (L group). The number of CD68⁺ cells in the experimental periodontitis was observed by immunohistochemistry. Alveolar bone destruction was assessed by micro-computerized tomography (micro-CT). Osteoclast was observed through TRAP staining. Nuclear factor (NF)-κB phospho-p65 (p p65) and phosphor-IκBα (p IκBα) expressions were detected to investigate NF-κB activation. CyPA showed an increasing trend at 1–6 weeks after ligation. CyPA and CD68⁺ cells were present in the gingival inflammatory infiltration, and participated in alveolar bone destruction. In the L?+?C group, the number of CD68⁺ cells was increased compared with the L group, and greater alveolar bone destruction was observed. NF-κB p p65 and p IκBα expressions were upregulated in the L?+?C group compared with the L group indicating NF-κB activation. CyPA increases CD68⁺ cell infiltration in rat experimental periodontitis, suggesting CyPA might be an anti-inflammatory therapeutic target.     

Priming of naive CD8+ T cells in the presence of IL-12 selectively enhances the survival of CD8<Superscript>+</Superscript>CD62L<Superscript>hi</Superscript> cells and results in superior anti-tumor activity in a tolerogenic murine model

During the antigen-dependant activation process several subsets CD8⁺ T cells appear with different phenotypic and functional characteristics. Recent studies indicate that the state of T cell differentiation radically affects their ability to effectively respond to tumor challenge, with early effector CD8⁺ T (CD62L^high) cells having better anti-tumor activity. Thus strategies aimed at optimizing the generation of such subpopulations could significantly enhance the effectiveness of adoptive cell therapy (ACT) for cancer. In this study, we show that priming of naïve CD8⁺ T cells in the presence of IL-12 selectively rescued early CD8⁺ CD62L^hi from activation induced cell death and resulted in the increased accumulation of this subset of CD8⁺ T cells. Furthermore, we demonstrated that IL-12 directly modulated the expression of CD62L on activated CD8⁺ T cells. When used for ACT, naïve CD8⁺ T cells primed in vitro in the presence of IL-12 showed superior anti-tumor activity toward B16 melanoma. Importantly, using the Pmel-1 model, priming pmel-1 cells in vitro with IL-12 reduced the state of functional tolerance associated with the non-mutated “self” tumor antigen gp100, as demonstrated by significant tumor responses in the absence of vaccination. Together, our results suggest that in vitro conditioning of naïve CD8⁺ T cells with IL-12 prior to ACT could significantly enhance their anti-tumor activity.