Dual Inhibition of the MEK/ERK and PI3K/AKT Pathways Prevents Pulmonary Graft-Versus-Host Disease through Suppression of Arteriovenous Inflammation and Bronchiolitis

Abstract Introduction Despite clinical development of novel immunosuppressants, the prognosis of pulmonary graft-versus-host disease (pGVHD) following allogeneic hematopoietic stem cell transplantation (allo-HSCT) is still poor. It is clinically classified into obstructive and constrictive subtypes, but its pathophysiology is not fully elucidated due to difficulty in collecting human lung specimens. We have shown that the MEK inhibitors that target the MAP kinase pathway suppress gastrointestinal and cutaneous GVHD in mice. Therefore, other kinase inhibitors may have potentials to suppress pGVHD. We here explored key molecules in pGVHD patients who underwent lung transplantation and verified the potency of dual inhibition of the MEK/ERK and PI3K/AKT pathways. Methods For human histopathological analysis, we reviewed 45 lung specimens from the allo-HSCT recipients who underwent lung transplantation at our institute for pGVHD from 2008 through 2018. Single-cell level imaging mass cytometry of human pGVHD specimens was performed to visualize interactions of lymphocytes and monocytes around bronchioles. As for a murine pGVHD model, eight-week-old B10.BR (H2K k) mice were pretreated with 240 mg/kg cyclophosphamide and 7 Gy total body irradiation, followed by infusion of C57BL/6J (H2K b) bone marrow cells and splenocytes. Vehicles, the MEK inhibitors trametinib (tra) 0.1 mg/kg/day, cobimetinib (cobi) 0.1 mg/kg/day, the PI3K inhibitor taselisib (tase) 5 mg/kg/day and tacrolimus (tac) 1 mg/kg were given from day 0 through 28. Evaluation was made at day 42 following transplant: primary endpoint was survival rate and secondary endpoints included development of bronchiolitis and values of airway resistance and lung compliance. Immunosuppressive effects of those reagents for human cells were evaluated in vitro: CSFE dilution of T cells stimulated with allogeneic dendritic cells; expression of CD23/CD69 on B cells activated with IL-4/CD40L; TNF-α production of monocytes stimulated with lipopolysaccharide. Results Three pathological findings characterize human pGVHD: bronchiolitis obliterans, lymphocyte infiltration and wall thickening of bronchioles; pleuroparenchymal fibroelastosis (PPFE), proliferation of subpleural elastic fibers; peribronchitis, macrophage aggregation around the bronchi. Most cases with PPFE had subpleural arteriovenous inflammation, and immunostaining revealed that B cells and macrophages were abundantly infiltrated around the bronchioles. These results indicated that suppressing B cells and macrophages may be crucial to prevent pGVHD through avoiding vascular inflammation and bronchiolitis. In the murine pGVHD, cobi but not tra/tac improved survival rate (vehicle 31%; cobi 62%, tra 7%, tac 10% at day 42; p < 0.01). Cobi attenuated development of bronchiolitis, venulitis and peribronchial macrophage aggregation. Cobi improved respiratory functions, such as airway resistance (vehicle 1.65 ± 0.19, cobi 2.86 ± 0.75 cmH 2O.s/mL, p < 0.001) and lung compliance (vehicle 13.14 ± 0.71 cobi 8.99 ± 1.08 μL/cmH 2O, p < 0.05). Notably, T cell infiltration around bronchioles was inhibited by cobi and tra, but B cell infiltration was inhibited only by cobi. Whereas cobi inhibited phosphorylation of AKT, tra compensatory upregulated it. Cobi inhibited B cell activation more than tra (%CD23 +CD69 + B cells: 32.8% with cobi 1μM, 53.0% with tra 1μM, p < 0.05). In addition, cobi suppressed TNF-α production by monocytes more strongly than tra (%TNF-α + monocytes: 53.1% with cobi 1μM vs 71.6% with tra 1μM, p < 0.05). Dual inhibition of the MEK/ERK and PI3K/AKT pathways with combination of tra and tase strongly suppressed B cells in vitro, and improved survival rate compared with vehicle and monotherapy of tra or tase in mice (Figure). Finally, imaging mass cytometry of human pGVHD specimens revealed that lymphocytes around bronchioles and venules were positive for phospho-ERK1/2 and phospho-AKT. Conclusions pGVHD is associated with vascular inflammation and bronchiolitis in humans and mice. Furthermore, phosphorylation of ERK1/2 and AKT is upregulated in human pGVHD. Given that dual inhibition of MEK/ERK and PI3K/AKT signaling suppresses vascular inflammation and bronchiolitis thorough inhibition of B cells and monocytes in mice, this strategy may provide a novel treatment option against human pGVHD. Figure 1 Figure 1. Disclosures Takaori-Kondo: ONO PHARMACEUTICAL CO., LTD.: Research Funding; Bristol-Myers K.K.: Honoraria; Celgene: Research Funding.

Download Full-text

In vitro regulation of immunoglobulin synthesis after marrow transplantation. I. T-cell and B-cell deficiencies in patients with and without chronic graft-versus-host disease

Blood ◽

10.1182/blood.v58.3.431.431 ◽

1981 ◽

Vol 58 (3) ◽

pp. 431-439 ◽

Cited By ~ 65

Author(s):

LG Lum ◽

MC Seigneuret ◽

RF Storb ◽

RP Witherspoon ◽

ED Thomas

Keyword(s):

T Cell ◽

B Cells ◽

B Cell ◽

Graft Versus Host Disease ◽

Marrow Transplantation ◽

Cell Function ◽

Cell Activity ◽

Graft Versus Host ◽

B Cell Function

Abstract Twenty-four patients with aplastic anemia or acute leukemia were treated by marrow grafts from HLA-identical donors after conditioning with high doses of cyclophosphamide and/or today body irradiation. They were studied between 4 and 63 mo (median 14.2) after transplantation. Seventeen patients had chronic graft-versus-host disease (C-GVHD) and 7 were healthy. They were studied for defects in their T- and B-cell function using and indirect hemolytic plaque assay for Ig production after 6 days of culture in the presence of pokeweek mitogen. T or B cells from the patients with or without C-GVHD were cocultured with T or B cells from their HLA-identical marrow donors or unrelated normal controls. Intrinsic B-cell defects, lack of helper T-cell activity, and suppressor T-cell activity were more frequently found in patients with C-GVHD than in healthy patients. Fifteen of the 17 patients with C-GVHD showed on or more defects in their T-and B-cell function compared to only 3 of the 7 patients without C-GVHD. None of the healthy controls, including the marrow donors, showed defects in their T- and B-cell functions. These in vitro findings may be helpful in assessing the process of immune reconstitution and the immunologic aberration found after human marrow transplantation.

Download Full-text

IL-35 and Rapamycin Reduce Acute Graft-Versus-Host Disease Associated with Decreased Platelet Aggregation in a Mouse Model

Blood ◽

10.1182/blood.v124.21.3814.3814 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3814-3814 ◽

Cited By ~ 1

Author(s):

Xiao-Hui Zhang ◽

Yi Zhou ◽

Shi-yuan Zhou ◽

Fei-er Feng ◽

Qian-ming Wang ◽

...

Keyword(s):

T Cells ◽

Survival Rate ◽

Platelet Aggregation ◽

Inflammatory Cytokine ◽

Control Group ◽

Graft Versus Host ◽

Tnf Α ◽

Thrombotic Complications ◽

Ifn Γ ◽

Acute Graft

Abstract Introduction: Acute graft-versus-host disease (aGVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT) caused by the activation of donor T lymphocytes by host antigen-presenting cells and the immune-mediated inflammatory response. Epithelial cells of the skin and mucous membranes, biliary ducts, and intestinal tract crypts are the primary tissue systems damaged during the pathobiological course of GVHD. IL-35, a member of the IL-12 family of cytokines, comprising an IL-12 p35 subunit and an IL-12 p40-related protein subunit, EBV-induced gene 3 (EBI3). It is an anti-inflammatory cytokine that suppresses the immune response through the expansion of regulatory T cells and suppression of Th17 cell development (Niedbala W, et al. European journal of immunology 2007). Rapamycin (Sirolimus; RAPA), a macrolide antibiotic produced by Streptomyces hygroscopicus, has been used for the prophylaxis and treatment of several immune reactions including GVHD and solid organ rejection (Ho-Jin Shin, et al. Blood 2011). We hypothesized that IL-35 has a protective effect in aGVHD, and that its function may be increased by RAPA. Methods: We used C57BL/6 (B6, H-2b) mice as donors and (B6×DBA/2)F1 (BDF1, H-2b×d) mice as recipients to create an aGVHD model (Kuroiwa T, et al. The Journal of clinical investigation 2001). Mice were divided into five groups, including a BMT control group, aGVHD control group, aGVHD treated with IL-35 group, aGVHD treated with RAPA group and aGVHD treated with IL-35 and RAPA group. Morbidity and mortality related to aGVHD were observed, and 2 weeks after BMT, tissues from the intestine and liver were stained with hematoxylin and eosin and examined by light microscopy. To detect apoptosis in intestinal sections, a modified terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) method was applied. CD4+CD25+Foxp3+ regulatory T cells were measured by flow cytometry. Quantitative RT-PCR was used to measure the production of IFN-γ, TNF-α and IL-17A in the spleen and intestine of each group of mice. We also measured platelet aggregation using a turbidimetric aggregation-monitoring device. Finally, western blotting was conducted to test the signaling pathways of IL-35. Results: Mice receivingIL-35 exhibited a higher survival rate compared with GVHD mice as well as those mice receiving RAPA. When the two drugs were given together, the survival rate was much higher than that in the other groups. The aGVHD control group had the highest morbidity rate of aGVHD, and IL-35 plus RAPA could prevent the occurrence of aGVHD. Additionally, this treatment inhibited apoptosis of intestinal epithelial cells as well as donor T-cell infiltration into the liver, thereby ameliorating the enteropathy and liver injury caused by aGVHD. The importance of the inflammatory cytokine cascade in the pathogenesis of both clinical and experimental GVHD is now well accepted. We found that IL-35 and RAPA also markedly suppressed IFN-γ, TNF-α and IL-17A expression in the intestine and liver. Because studies by other have showed that Tregs have the ability to inhibit aGVHD, we measured Tregs in serum and found that IL-35 and RAPA treatment expanded serum Tregs. We further explored the relationship between IL-35 and platelet aggregation. Platelet aggregation was high in aGVHD mice, and the ratio of platelet aggregation was inhibited by IL-35 and RAPA. Finally, we found that the phosphorylation of STAT1 and STAT4 were inhibited in GVHD mice, and thatSTAT1 and STAT4 were phosphorylated when mice were treated with IL-35. Conclusions: IL-35 may be useful for controlling aGVHD after allo-HSCT. IL-35 suppresses inflammatory cytokines and expands anti-inflammatory cells in aGVHD. IL-35 also prevents platelet aggregation in aGVHD mice, which could be helpful in treating thrombotic complications after HSCT. These results are readily translatable to the clinic in future clinical trials. IL-35 and RAPA may have potential clinical use for the prevention or treatment of aGVHD and thrombotic complications after HSCT. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Systems Immune Monitoring with Mass Cytometry Characterizes Altered Peripheral Immune Cell Environments in Patients with Chronic Graft Versus Host Disease

Blood ◽

10.1182/blood.v128.22.4572.4572 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4572-4572 ◽

Cited By ~ 2

Author(s):

Hannah G. Polikowsky ◽

Michael T. Byrne ◽

Caroline E. Maier ◽

Allison R. Greenplate ◽

Madan H. Jagasia ◽

...

Keyword(s):

B Cells ◽

Clinical Research ◽

Graft Versus Host Disease ◽

Peripheral Blood ◽

Research Funding ◽

Immune Monitoring ◽

Mass Cytometry ◽

Computational Tools ◽

Graft Versus Host ◽

Treatment Responses

Abstract Introduction: Allogeneic stem cell transplant (SCT) fundamentally alters the immune milieu of an individual and provides a curative treatment for patients with blood cancer. However, a common side effect of SCT is the development of chronic graft versus host disease (cGVHD). Given the growth of new experimental therapeutics for GVHD, a systems immune monitoring approach based on mass cytometry analysis of peripheral blood may provide a robust and minimally invasive way to characterize the post-SCT cGVHD immune environment and systematically track cellular biomarkers of immune status, disease severity, or ongoing treatment responses (Greenplate et al., Euro J Cancer 2016, Kordasti et al., Blood 2016). It may also be possible to identify shifts in the immune milieu or cell signaling that predict treatment responses or stratify risks (Greenplate et al., Cancer Immunol Res 2016). This study tested the feasibility of using mass cytometry peripheral blood analysis in clinical research with the goals of 1) better understanding the post-SCT and cGVHD immune environment and 2) developing a knowledge base for monitoring treatment responses. Methods: Peripheral blood mononuclear cells (PBMC) were collected with informed consent from cGVHD patients or healthy donors with approval of the local Institutional Review Board (IRB) and in accordance with the Declaration of Helsinki. A total of 88 viably cryopreserved PBMC samples were analyzed from 11 individual patients undergoing extracorporeal photopheresis (ECP) therapy. For each patient, PBMC from 4 clinical timepoints representing pre-ECP and 2, 4, and 6 months post-ECP were characterized with 2 systems immune monitoring antibody panels emphasizing B cells and T cells (Table 1). Staining, data collection using a CyTOF mass cytometer, bead based normalization, and analysis with Cytobank software followed established protocols (Leelatian et al., Methods Mol Bio 2016, Polikowsky et al., J Immunol 2015). Next, cell subsets were algorithmically revealed and characterized by computational tools (Diggins et al., Methods 2015) and patterns in the abundance and phenotype of T, B, NK, and myeloid cell subsets systematically compared to patient clinical features. Results: Single cell tools revealed profound, abnormal shifts in the peripheral immune environment in individual patients that contrasted with relatively stable immunophenotypes observed in healthy individuals and prior studies. Expected immune changes, such a profound loss of B cells post-treatment with Rituximab, were readily detected by computational tools and expert review. Expression of signaling, activation, and subset identity markers in B and T cell subsets displayed striking variation between patients and was effectively quantified on at least 5 major cell subsets and numerous minor cell subsets (Figure 1). These data highlighted how computational tools paired with high-dimensional mass cytometry can systematically characterize key protein features and cell subsets in complex and heterogeneous diseases including cancer and cGVHD and provided a rationale for ongoing association analyses between cellular subsets and clinical outcomes. Conclusion: Systems immune monitoring successfully revealed shifts in cell abundance and immunophenotype pre- and post-ECP for patients with cGVHD. These shifts were detected by computational tools and revealed alterations to the immune system following treatment. This study indicates that single cell quantitative analysis is feasible as a clinical research tool to characterize immunotherapy treatment responses, and reveal cellular biomarkers. Integration of such tools in prospective clinical trials is essential to establish and once validated, may augment physician decision making. Disclosures Polikowsky: Cytobank: Employment. Greenplate:Cytobank: Consultancy. Jagasia:Therakos: Consultancy. Irish:Cytobank, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Janssen: Research Funding; Incyte: Research Funding.

Download Full-text

In vitro regulation of immunoglobulin synthesis after marrow transplantation. I. T-cell and B-cell deficiencies in patients with and without chronic graft-versus-host disease

Blood ◽

10.1182/blood.v58.3.431.bloodjournal583431 ◽

1981 ◽

Vol 58 (3) ◽

pp. 431-439 ◽

Cited By ~ 1

Author(s):

LG Lum ◽

MC Seigneuret ◽

RF Storb ◽

RP Witherspoon ◽

ED Thomas

Keyword(s):

T Cell ◽

B Cells ◽

B Cell ◽

Graft Versus Host Disease ◽

Marrow Transplantation ◽

Cell Function ◽

Cell Activity ◽

Graft Versus Host ◽

B Cell Function

Twenty-four patients with aplastic anemia or acute leukemia were treated by marrow grafts from HLA-identical donors after conditioning with high doses of cyclophosphamide and/or today body irradiation. They were studied between 4 and 63 mo (median 14.2) after transplantation. Seventeen patients had chronic graft-versus-host disease (C-GVHD) and 7 were healthy. They were studied for defects in their T- and B-cell function using and indirect hemolytic plaque assay for Ig production after 6 days of culture in the presence of pokeweek mitogen. T or B cells from the patients with or without C-GVHD were cocultured with T or B cells from their HLA-identical marrow donors or unrelated normal controls. Intrinsic B-cell defects, lack of helper T-cell activity, and suppressor T-cell activity were more frequently found in patients with C-GVHD than in healthy patients. Fifteen of the 17 patients with C-GVHD showed on or more defects in their T-and B-cell function compared to only 3 of the 7 patients without C-GVHD. None of the healthy controls, including the marrow donors, showed defects in their T- and B-cell functions. These in vitro findings may be helpful in assessing the process of immune reconstitution and the immunologic aberration found after human marrow transplantation.

Download Full-text

The Immune Microenvironment of Hashimoto’s Thyroiditis Regulates the Glycosylation Modification of IgG

Journal of the Endocrine Society ◽

10.1210/jendso/bvab048.1724 ◽

2021 ◽

Vol 5 (Supplement_1) ◽

pp. A845-A845

Author(s):

Yedi Cao ◽

Zhijing Song ◽

Yan Gong ◽

Keli Zhao ◽

Xue Zhao ◽

...

Keyword(s):

Sialic Acid ◽

B Cells ◽

Hashimoto’S Thyroiditis ◽

Culture System ◽

Hashimoto's Thyroiditis ◽

Differentiation Process ◽

Immune Microenvironment ◽

Tnf Α ◽

Ifn Γ

Abstract Objectives: Elevation of anti-thyroglobulin antibodies that are primarily IgG isotype is a hallmark of Hashimoto’s thyroiditis (HT). As for IgG,it bears two conserved repertoire of N-linked glycans attached to its crystallizable fragment (Fc) at the 297 asparagine residue (Asn297). In our previous study, we found that serum TgAb IgG from HT patients exhibits higher glycosylation levels than those observed from healthy controls. Previous studies confirmed that imbalance of Th1/Th2 and Th17/Treg leading to altered immune microenvironment with elevation of certain cytokines was found in the thyroid tissue of HT, including IFN-γ, TNF-α, IL-21, IL-17A, IL-6, BAFF, APRIL. Thus, the aim of our study was to investigate the influence of the elevated cytokines on the differentiation process of B cells and the glycosylation levels of IgG. Methods: We formed a two-phase culture system in vitro to promote B cells to differentiate to antibody-secreting cells (ASCs). In the process of cell culture, B cells were co-cultured with cytokines as followed: IFN-γ, TNF-α, IL-21, IL-17A, IL-6, BAFF and APRIL. Flow cytometry was performed to identify the percentage of plasmablasts (CD38+CD27high) and plasma cells (CD20-CD138+). ELISA was used to measure the yield of IgG in culture supernatants. The glycosylation levels of secreted IgG under different stimulation conditions were detected by lectin microarray. Results: We found that IL-21, TNF-α and BAFF can significantly promote the differentiation of B cells into ASCs in vitro culture system, and augment the production of IgG to over 4-fold. In addition, cytokines affected the glycosylation modification profile of IgG diversely: 1) IL-21, IL-17A, TNF-α, BAFF significantly increased the glycosylation level of sialic acid of total IgG; 2) IFN-γ significantly increased the level of galactose; 3) IL-21, IL-17A, IFN-γ, BAFF, and APRIL significantly increased the level of mannose; 4) IL-6 significantly decreased the level of sialic acid, galactose and mannose; 5) IL-17A, IFN-γ, TNF-α, BAFF significantly increased the level of GalNAc that was a component of O-Glycan,which only exists in the hinge region of IgG3 subclass. Conclusions: The abnormally elevated cytokines in microenvironment participated in the regulation of B cell terminal differentiation process and glycosylation level of IgG, thereby involving in the pathogenesis of AITD.

Download Full-text

Inhibition of Graft-Versus-Host Disease (GVHD) by Histone Deacetylase Inhibitor (HDAC) SAHA Leads to Downregulation of STAT1 Activation.

Blood ◽

10.1182/blood.v106.11.1311.1311 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1311-1311

Author(s):

Corinna Leng ◽

Cuiling Li ◽

Judy Ziegler ◽

Anna Lokshin ◽

Suzanne Lentzsch ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Histone Deacetylase ◽

Graft Versus Host Disease ◽

Host Disease ◽

Graft Versus Host ◽

Tnf Α ◽

Ifn Γ

Abstract Histone deacetylase (HDAC) inhibitors have been shown to reduce development of graft versus host disease [GVHD] following allogeneic bone marrow transplantation [BMT]. Administration of the HDAC inhibitor suberonylanilide hydroxamic acid [SAHA] resulted in a significantly reduced GVHD-dependent mortality following fully MHC-mismatched allogeneic BMT. Median Survival Time (MST) for vehicle and SAHA-treated mice were 7.5 days and 38 days respectively. However, SAHA treatment did not affect T cell activation nor T cell expansion in vitro and in vivo as determined by MLR assays, phenotypic analysis of donor T cells with regard to expression of the CD25 activation antigen and calculation of donor CD4+ and CD8+ T cell numbers on days +3 and +6 post-BMT. Thus, SAHA treatment was not able to inhibit the strong upregulation of CD25 antigen on CD8+ T cells observed during induction of GVHD on days +3 and +6 post-BMT. We therefore focused on the effects of SAHA treatment on efferent immune effects including cytokine secretion and intracellular signaling events in vitro and in vivo following GVHD induction. SAHA treatment broadly inhibited lipopolysaccharide [LPS] and allo-antigen-induced cytokine/chemokine secretion in vitro like MIP-1-α, IP-10, IFN-γ, TNF-α and IL-6 and led also to a significant decrease in IFN-γ and TNF-α levels in vivo following induction of GVHD. Concomitantly, SAHA treatment inhibited phosphorylation of STAT1 and STAT3 in response to LPS and allo-activation in vitro. Furthermore, analysis of liver tissue and spleens from SAHA-treated animals with GVHD showed a significant decrease in phosphorylated STAT1. In contrast SAHA treatment had only moderate effects on p38 or ERK1,2 Mitogen-activated Protein Kinase (MAPK) pathway underscoring the relevance of the inhibition of the STAT1 pathway. In conclusion, GVHD is associated with a strong induction of phosphorylation of STAT1 in the liver and spleen and SAHA-dependent reduction of GVHD is associated with systemic and local inhibition of pSTAT1 and modulation of the inflammatory cytokine milieu during the efferent immune response.

Download Full-text

Efficient Treatment of Murine Acute Graft-Versus-Host Disease with In Vitro Expanded CD4+CD25+ Regulatory T Cells

Blood ◽

10.1182/blood.v118.21.2987.2987 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2987-2987

Author(s):

Tina J Boeld ◽

Kristina Doser ◽

Corinna Lang-Schwarz ◽

Elisabeth Huber ◽

Reinhard Andreesen ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

Regulatory T Cells ◽

Graft Versus Host Disease ◽

Acute Gvhd ◽

Treg Cells ◽

Host Disease ◽

Graft Versus Host ◽

Acute Graft

Abstract Abstract 2987 Acute graft-versus-host disease (GVHD) is a frequent complication after allogeneic bone marrow transplantation (BMT). We previously showed that the adoptive transfer of donor-type CD4+CD25+ regulatory T cells (Treg) at the time of BMT prevents acute GVHD in murine models. However, the therapeutic potential of donor-derived Treg cells for the treatment of established acute GVHD has not yet been examined in detail. In analogy to potential clinical applications we now tested the capacity of in vitro expanded Treg cells to ameliorate acute GVHD after haploidentical BMT (BALB/c→CB6F1). CD4+CD25highCD62L+ Treg cells were purified by FACS and stimulated polyclonally using anti-CD3/CD28-coated beads. Cells expanded on average 130±19-fold (n=7) within 2 wks and maintained high levels of FoxP3 expression (96, 8±0, 8% FoxP3+ cells; n=7) as well as potent immunosuppressive activity in vitro. For the induction of acute GVHD CB6F1 recipients were lethally irradiated and transplanted with 2.5×106 BM cells in combination with 5×106 splenocytes. All animals developed severe GVHD by d11, as revealed by an increase of the GVHD severity score (2.3±0.4 in GVHD animals vs 0±0 in BM controls, p<0.001, n=1–11) and by histological analyses of the gut (score: 7.8±0.4 for the GVHD group vs 0.2±0.2 for BM controls, p =0.046, n=3). When animals with acute GVHD were treated with 5×106 expanded CD4+CD25highCD62L+ Treg cells on d11 after BMT, they initially developed progressive GVHD comparable to non-treated GVHD animals, as indicated by weight loss and an increase of the GVHD score. However from d44 post BMT onwards, Treg-treated GVHD animals regained body weight (d44: 75±3% vs 67±2% of initial weight; p <0.05; n=9–10) and their clinical GVHD score (d44: 6±0 vs 4.3±0.4; p <0.05; n=9–10) decreased. While all non-treated GVHD animals succumbed to disease by d67 after transplantation, 50% of Treg-treated GVHD animals survived for at least 100d (p =0, 002; n=16–21). As immune reconstitution and in particular reconstitution of the lymphocyte compartment is impaired in animals with GVHD, we analyzed the effect of Treg therapy on the reconstitution of the lymphoid and myeloid compartment. At d21 after BMT spleen and BM of non-treated as well as Treg-treated GVHD animals were completely lymphopenic as compared to control mice and both organs contained exceptionally high numbers of granulocytes. Unlike non-treated GVHD animals, however, Treg-treated recipients by d60 showed a recovery of the lymphocyte compartment in spleen (10±2.6×106 T cells and 23.5±12.5×106 B cells in Treg-treated vs 3.0±0.6×106 T cells and 1.5±0.4×106 B cells in non-treated GVHD animals vs 26.25±2.6×106 T cells and 63.9±9.1×106 B cells in BM controls) and BM (0.7±0.1×106 T cells and 8.6±4×106 B cells in Treg-treated vs 0.3±0.01×106 T cells and 0.7±0.4 ×106 B cells in non-treated GVHD animals vs 0.4±0.03×106 T cells and 11.2±0.6×106 B cells in BM controls), while the number of granulocytes decreased constantly. Successful treatment with Treg cells was finally accompanied by a reconstitution of the lymphatic system comparable to control mice. Furthermore, successfully treated mice showed only mild histological signs of gut GVHD at d100 that was significantly lower then those in non-treated GVHD animals with end-stage disease (score: 4.2±1 vs 9.9±1.5 in treated vs non-treated animals, p =0.006, n=4–6). Taken together, these results indicate that in vitro expanded natural Treg cells may not only be effective for the prevention, but also for the treatment of acute GVHD after allogeneic BMT. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Identification of Previously Uncharacterized Delta 4-Positive Inflammatory Plasmacytoid Dendritic Cells That Play Important Roles in Eliciting Graft-Versus-Host Disease in Mice

Blood ◽

10.1182/blood.v120.21.337.337 ◽

2012 ◽

Vol 120 (21) ◽

pp. 337-337 ◽

Cited By ~ 1

Author(s):

Kazuhiro Mochizuki ◽

Fang Xie ◽

Shan He ◽

Qing Tong ◽

Yongnian Liu ◽

...

Keyword(s):

T Cells ◽

Effector T Cells ◽

Activated T Cells ◽

Graft Versus Host ◽

Notch Receptors ◽

Tnf Α ◽

Donor T Cells ◽

Ifn Γ ◽

Antigen Presenting

Abstract Abstract 337 Graft-versus-host disease (GVHD) remains a major barrier to the success of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Host antigen-presenting cells (APCs) are known to be essential for presenting alloantigens to activate donor T cells to become effector cells mediating GVHD after allo-HSCT. However, APCs are heterogeneous populations. The identity of APC subset(s) that directs effector differentiation of alloantigen-activated T cells and by which mechanism this effect may be achieved remain largely unknown. The Notch signaling pathway controls cell proliferation, differentiation and survival. Upon interaction with Notch ligands of the δ-like family (Dll1, Dll3 and Dll4) and Jagged family (J1, J2), Notch receptors (Notch 1, 2, 3, and 4) are cleaved by γ-secretase and translocate into the nucleus to modify gene transcription. We have recently demonstrated that activation of Notch receptors in donor T cells is critical to the production of alloreactive effector T cells producing multiple inflammatory cytokines (e.g., IFN-γ, TNF-α and IL-17) during GVH reaction (Blood 2011). Building on these findings, we hypothesized that: 1) Notch ligand(s) derived from APCs may be important for directing effector differentiation of alloantigen-activated T cells, and 2) the expression of Notch ligand(s) may differentiate the capability of APCs to prime GVH responses. Using mouse models of GVHD, here we report the identification of previously uncharacterized Dll4-positive (Dll4+) inflammatory plasmacytoid dendritic cells (i-pDCs) and their roles in eliciting allogeneic T-cell responses. Host-derived Dll4+ i-pDCs occurred in the spleen of allo-HSCT recipients one day after transplantation, peaked by three days and declined by seven days. In contrast, host-derived inflammatory conventional DCs (i-cDCs) were Dll4-negative (Dll4−) and rapidly diminished by three days after transplantation. Notably, donor-derived DCs which occurred seven days after HSCT did not express Dll4. In vitro mixed lymphocyte-reaction (MLR) assay showed that these host-derived Dll4+ i-pDCs induced approximately 2.5-fold and 7-fold more IFN-γ- and IL-17-producing effector T cells than Dll4− i-cDCs, respectively. Addition of neutralizing antibody specific to Dll4 to the MLR cultures markedly reduced the production of IFN-γ and IL-17 in donor T cells stimulated by host Dll4+ i-pDCs, but had minimal impact on donor T cells cultured in the presence of Dll4− i-cDCs. These results suggest that Dll4+ i-pDCs may play important roles in directing effector differentiation of alloantigen-activated T cells. Further characterization of biological properties of Dll4+ i-pDCs revealed that as compared to unstimulated host pDCs at steady state conditions, Dll4+ i-pDCs expressed higher levels of antigen-presenting and costimulatory molecules, upregulated other Notch ligands (e.g.,J1 and J2) on their surface and produced more Ifnb and Il23. Notably, Dll4+ i-pDCs were mainly located in the spleen and intestine of mice receiving allogeneic HSCT. In vivo administration of Dll4 antibody reduced donor alloreactive effector T cell producing IFN-γ, IL-17 and TNF-α in GVHD target organs (in particular of the intestine), leading to reduction of GVHD and significantly improved survival of mice after allogeneic HSCT. Furthermore, adoptive transfer of in vitro generated Dll4+ i-pDCs caused severe GVHD in MHC-II-deficient mice (in which host DCs are incapable to elicit GVHD). Our findings identify that Dll4+ i-pDCs may represent a previously uncharacterized inflammatory APC population developed during GVH reaction. These Dll4+ i-pDCs and their-derived Dll4 are critical for directing differentiation of alloreactive effector T cells and may be beneficial therapeutic targets for modulating GVHD. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Protective effects of 4, 5-O-dicaffeoylquinic acid against mouse sepsis via down-regulation of TNF-α, IL-6 and IL-8

Tropical Journal of Pharmaceutical Research ◽

10.4314/tjpr.v19i4.6 ◽

2020 ◽

Vol 19 (4) ◽

pp. 715-720

Author(s):

XiaoBo Wang ◽

JianHua Wu ◽

BuKao Ni

Keyword(s):

Survival Rate ◽

Mrna Expression ◽

Molecular Mechanisms ◽

Cecal Ligation ◽

Enzyme Linked Immunosorbent Assay ◽

Protective Effects ◽

Dicaffeoylquinic Acid ◽

Xanthium Sibiricum ◽

Tnf Α

Purpose: To investigate the protective effects of 4, 5-O-dicaffeoylquinic acid (DCQA) isolated from Xanthium sibiricum Patr. against mouse sepsis caused by cecal ligation/puncture (CLP) in vivo, as well as the molecular mechanisms of action involved.Methods: DCQA (7.5, 15, and 30 mg/kg/day) were administered to the mice with sepsis and the survival rate was obtained. Tumor necrosis factor (TNF)-α and interleukin (IL)-6 were examined by enzyme-linked immunosorbent assay (ELISA). Subsequently, the lipopolysaccharide (LPS) neutralizing ability of DCQA (2, 4, and 8 μg/mL) was measured using limulus amebocyte lysate (LAL) test in vitro. Furthermore, the effect of DCQA (10, 20, and 40 μg/mL) on mRNA expression of TNF-α, IL-6, and IL-8 in LPS (100 ng/mL)-treated RAW 264.7 cells was assessed using quantitative real time-polymerase chain reaction (RT-qPCR) assay.Results: DCQA significantly improved the survival rate of mice with sepsis caused by CLP (35, 50, and 65 %, respectively vs. 15 % for control, p < 0.05). LPS levels fell on co-incubation with DCQA in vitro. Moreover, ELISA and RT-qPCR results revealed that DCQA treatment lowered tendency in the mRNA expression of TNF-α, IL-6, and IL-8 (p < 0.01).Conclusion: DCQA exhibits protective effects against sepsis in mice mediated by downregulating TNF-α, IL-6, and IL-8. Further studies, in animals and humans are requied to determine the safety and efficacy of DCQA in both animal and clinical management of sepsis. Keywords: Xanthium sibiricum, 4,5-O-Dicaffeoylquinic acid, Sepsis, Cecal ligation and puncture, Lipopolysaccharide, TNF-α, IL-6, IL-8

Download Full-text