Tolerogenic Vaccination Reduced Effector Memory CD4 T Cells and Induced Effector Memory Treg Cells for Type I Diabetes Treatment

Background

Vaccination could induce immune tolerance and protected NOD mice from the development of type I diabetes (T1D). We previously demonstrated that insulin peptide (B9-23) combined with dexamethasone (DEX) stimulated the expansion of antigen specific regulatory T (Treg) cells which in turn effectively prevented T1D in NOD mice. Here, we aimed to investigate the therapeutic effect of tolerogenic vaccination for T1D treatment.

Methodology/Principal Findings

The diabetic NOD mice (Blood glucose level ≧250 mg/dl) were treated with B9-23 and DEX twice. The tolerance was restored by blocking maturation of dendritic cells (DCs) and inducing Treg cells in treated NOD mice. Remarkably, the reduction of autoreactive effector memory CD4 T (Tm) cells and the induction of functional effector memory Treg (mTreg) cells contributed to the improvement of T1D in treated NOD mice.

Conclusions/Significance

Tolerogenic vaccination restored tolerance and ameliorated T1D by suppressing effector CD4 Tm cells and inducing effector mTreg cells. Our findings implicate the potential of tolerogenic vaccination for T1D treatment.     

MSC Therapy Attenuates Obliterative Bronchiolitis after Murine Bone Marrow Transplant

Rationale

Obliterative bronchiolitis (OB) is a significant cause of morbidity and mortality after lung transplant and hematopoietic cell transplant. Mesenchymal stromal cells (MSCs) have been shown to possess immunomodulatory properties in chronic inflammatory disease.

Objective

Administration of MSCs was evaluated for the ability to ameliorate OB in mice using our established allogeneic bone marrow transplant (BMT) model.

Methods

Mice were lethally conditioned and received allogeneic bone marrow without (BM) or with spleen cells (BMS), as a source of OB-causing T-cells. Cell therapy was started at 2 weeks post-transplant, or delayed to 4 weeks when mice developed airway injury, defined as increased airway resistance measured by pulmonary function test (PFT). BM-derived MSC or control cells [mouse pulmonary vein endothelial cells (PVECs) or lung fibroblasts (LFs)] were administered. Route of administration [intratracheally (IT) and IV] and frequency (every 1, 2 or 3 weeks) were compared. Mice were evaluated at 3 months post-BMT.

Measurements and Main Results

No ectopic tissue formation was identified in any mice. When compared to BMS mice receiving control cells or no cells, those receiving MSCs showed improved resistance, compliance and inspiratory capacity. Interim PFT analysis showed no difference in route of administration. Improvements in PFTs were found regardless of dose frequency; but once per week worked best even when administration began late. Mice given MSC also had decreased peribronchiolar inflammation, lower levels of hydroxyproline (collagen) and higher frequencies of macrophages staining for the alternatively activated macrophage (AAM) marker CD206.

Conclusions

These results warrant study of MSCs as a potential management option for OB in lung transplant and BMT recipients.     

Antigen-sensitized CD4+CD62Llow memory/effector T helper 2 cells can induce airway hyperresponsiveness in an antigen free setting

Background

Airway hyperresponsiveness (AHR) is one of the most prominent features of asthma, however, precise mechanisms for its induction have not been fully elucidated. We previously reported that systemic antigen sensitization alone directly induces AHR before development of eosinophilic airway inflammation in a mouse model of allergic airway inflammation, which suggests a critical role of antigen-specific systemic immune response itself in the induction of AHR. In the present study, we examined this possibility by cell transfer experiment, and then analyzed which cell source was essential for this process.

Methods

BALB/c mice were immunized with ovalbumin (OVA) twice. Spleen cells were obtained from the mice and were transferred in naive mice. Four days later, AHR was assessed. We carried out bronchoalveolar lavage (BAL) to analyze inflammation and cytokine production in the lung. Fluorescence and immunohistochemical studies were performed to identify T cells recruiting and proliferating in the lung or in the gut of the recipient. To determine the essential phenotype, spleen cells were column purified by antibody-coated microbeads with negative or positive selection, and transferred. Then, AHR was assessed.

Results

Transfer of spleen cells obtained from OVA-sensitized mice induced a moderate, but significant, AHR without airway antigen challenge in naive mice without airway eosinophilia. Immunization with T helper (Th) 1 elicited antigen (OVA with complete Freund''s adjuvant) did not induce the AHR. Transferred cells distributed among organs, and the cells proliferated in an antigen free setting for at least three days in the lung. This transfer-induced AHR persisted for one week. Interleukin-4 and 5 in the BAL fluid increased in the transferred mice. Immunoglobulin E was not involved in this transfer-induced AHR. Transfer of in vitro polarized CD4⁺ Th2 cells, but not Th1 cells, induced AHR. We finally clarified that CD4⁺CD62L^low memory/effector T cells recruited in the lung and proliferated, thus induced AHR.

Conclusion

These results suggest that antigen-sensitized memory/effector Th2 cells themselves play an important role for induction of basal AHR in an antigen free, eosinophil-independent setting. Therefore, regulation of CD4⁺ T cell-mediated immune response itself could be a critical therapeutic target for allergic asthma.     

A Comparison of the Inflammatory and Proteolytic Effects of Dung Biomass and Cigarette Smoke Exposure in the Lung

Rationale

Biomass is the energy source for cooking and heating for billions of people worldwide. Despite their prevalent use and their potential impact on global health, the effects of these fuels on lung biology and function remain poorly understood.

Methods

We exposed human small airway epithelial cells and C57BL/6 mice to dung biomass smoke or cigarette smoke to compare how these exposures impacted lung signaling and inflammatory and proteolytic responses that have been linked with disease pathogenesis.

Results

The in vitro exposure and siRNA studies demonstrated that biomass and cigarette smoke activated ERK to up regulate IL-8 and MMP-1 expression in human airway epithelial cells. In contrast to cigarette smoke, biomass also activated p38 and JNK within these lung cells and lowered the expression of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1). Similarly, in the lungs of mice, both biomass and cigarette smoke exposure increased macrophages, activated ERK and p38 and up regulated MMP-9 and MMP-12 expression. The main differences seen in the exposure studies was that mice exposed to biomass exhibited more perivascular inflammation and had higher G-CSF and GM-CSF lavage fluid levels than mice exposed identically to cigarette smoke.

Conclusion

Biomass activates similar pathogenic processes seen in cigarette smoke exposure that are known to result in the disruption of lung structure. These findings provide biological evidence that public health interventions are needed to address the harm associated with the use of this fuel source.     

Intra-Tumoral Heterogeneity in Metastatic Potential and Survival Signaling between Iso-Clonal HCT116 and HCT116b Human Colon Carcinoma Cell Lines

Background

Colorectal cancer (CRC) metastasis is a leading cause of cancer-related deaths in the United States. The molecular mechanisms underlying this complex, multi-step pathway are yet to be completely elucidated. Recent reports have stressed the importance of intra-tumoral heterogeneity in the development of a metastatic phenotype. The purpose of this study was to characterize the intra-tumoral phenotypic heterogeneity between two iso-clonal human colon cancer sublines HCT116 and HCT116b on their ability to undergo metastatic colonization and survive under growth factor deprivation stress (GFDS).

Materials and Methods

HCT116 and HCT116b cells were transfected with green fluorescence protein and subcutaneously injected into BALB/c nude male mice. Once xenografts were established, they were excised and orthotopically implanted into other male BALB/c nude mice using microsurgical techniques. Animal tissues were studied for metastases using histochemical techniques. Microarray analysis was performed to generate gene signatures associated with each subline. In vitro assessment of growth factor signaling pathway was performed under GFDS for 3 and 5 days.

Results

Both HCT116 and HCT116b iso-clonal variants demonstrated 100% primary tumor growth, invasion and peritoneal spread. However, HCT116 was highly metastatic with 68% metastasis observed in liver and/or lungs compared to 4% in HCT116b. Microarray analysis revealed an upregulation of survival and metastatic genes in HCT116 cells compared to HCT116b cells. In vitro analysis showed that HCT116 upregulated survival and migratory signaling proteins and downregulated apoptotic agents under GFDS. However, HCT116b cells effectively showed the opposite response under stress inducing cell death.

Conclusions

We demonstrate the importance of clonal variation in determining metastatic potential of colorectal cancer cells using the HCT116/HCT116b iso-clonal variants in an orthotopic metastatic mouse model. Determination of clonal heterogeneity in patient tumors can serve as useful tools to identify clinically relevant biomarkers for diagnostic and therapeutic assessment of metastatic colorectal cancer.     

Attenuation of antigen-induced airway hyperresponsiveness and inflammation in CXCR3 knockout mice

Background

CD8+ T cells participate in airway hyperresponsiveness (AHR) and allergic pulmonary inflammation that are characteristics of asthma. CXCL10 by binding to CXCR3 expressed preferentially on activated CD8+ T cells, attracts T cells homing to the lung. We studied the contribution and limitation of CXCR3 to AHR and airway inflammation induced by ovalbumin (OVA) using CXCR3 knockout (KO) mice.

Methods

Mice were sensitized and challenged with OVA. Lung histopathological changes, AHR, cellular composition and levels of inflammatory mediators in bronchoalveolar lavage (BAL) fluid, and lungs at mRNA and protein levels, were compared between CXCR3 KO mice and wild type (WT) mice.

Results

Compared with the WT controls, CXCR3 KO mice showed less OVA-induced infiltration of inflammatory cells around airways and vessels, and less mucus production. CXCR3 KO mice failed to develop significant AHR. They also demonstrated significantly fewer CD8+ T and CD4+ T cells in BAL fluid, lower levels of TNFα and IL-4 in lung tissue measured by real-time RT-PCR and in BAL fluid by ELISA, with significant elevation of IFNγ mRNA and protein expression levels.

Conclusions

We conclude that CXCR3 is crucial for AHR and airway inflammation by promoting recruitment of more CD8+ T cells, as well as CD4+ T cells, and initiating release of proinflammatory mediators following OVA sensitization and challenge. CXCR3 may represent a novel therapeutic target for asthma.     

CD4+ T cells in aged or thymectomized recipients of allogeneic stem cell transplantations

Background

CD4+CD25highFOXP3+ regulatory T (Treg) cells, which include thymus-derived and peripherally induced cells, play a central role in immune regulation, and are therefore crucial to prevent graft-versus-host disease (GVHD). The increasing use of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for elderly patients with thymus regression, and our case of allo-HSCT shortly after total thymectomy, raised questions about the activity of thymus-derived Treg cells and peripherally induced Treg cells, which are otherwise indistinguishable.

Results

We found that despite pre-transplant thymectomy or older age, both naïve and effector Treg cells, as well as naïve and effector conventional T cells, proliferated in allo-HSCT recipients. Higher proportions of total Treg cells 1 month post allo-HSCT, and naïve Treg cells 1 year post allo-HSCT, appeared in patients achieving complete chimera without developing significant chronic GVHD, including our thymectomized patient, compared with patients who developed chronic GVHD.

Conclusions

Treg cells that modulate human allogeneic immunity may arise peripherally as well as in the thymus of allo-HSCT recipients.     

Toll-Like Receptor 4 Stimulation before or after Streptococcus pneumoniae Induced Sepsis Improves Survival and Is Dependent on T-Cells

Introduction

Endotoxin tolerance improves outcomes from gram negative sepsis but the underlying mechanism is not known. We determined if endotoxin tolerance before or after pneumococcal sepsis improved survival and the role of lymphocytes in this protection.

Methods

Mice received lipopolysaccharide (LPS) or vehicle before or after a lethal dose of Streptococcus pneumoniae. Survival, quantitative bacteriology, liver function, and cytokine concentrations were measured. We confirmed the necessity of Toll-like receptor 4 (TLR4) for endotoxin tolerance using C3H/HeN (TLR4 replete) and C3H/HeJ (TLR4 deficient) mice. The role of complement was investigated through A/J mice deficient in C5 complement. CBA/CaHN-Btk^xid//J mice with dysfunctional B cells and Rag-1 knockout (KO) mice deficient in T and B cells delineated the role of lymphocytes.

Results

Endotoxin tolerance improved survival from pneumococcal sepsis in mice with TLR4 that received LPS pretreatment or posttreatment. Survival was associated with reduced bacterial burden and serum cytokine concentrations. Death was associated with abnormal liver function and blood glucose concentrations. Endotoxin tolerance improved survival in A/J and CBA/CaHN-Btk^xid//J mice but not Rag-1 KO mice.

Conclusions

TLR4 stimulation before or after S. pneumoniae infection improved survival and was dependent on T-cells but did not require an intact complement cascade or functional B cells.     

Oroxylin-A Rescues LPS-Induced Acute Lung Injury via Regulation of NF-κB Signaling Pathway in Rodents

Background and Purpose

Successful drug treatment for sepsis-related acute lung injury (ALI) remains a major clinical problem. This study was designed to assess the beneficial effects of post-treatment of oroxylin A (OroA), a flavonoid, in ameliorating lipopolysaccharides (LPS)-induced lung inflammation and fatality.

Experimental Approach

Rats were injected with LPS (10 mg/kg, iv) to induce ALI, and OroA was given (15 mg/kg, iv) 1 hr or 6 hrs after LPS challenge. Twenty four hrs after LPS challenge, biochemical changes in the blood and lung tissues, and morphological/histological alterations in the lung associated with inflammation and injury were examined. Therapeutic effect of OroA was assessed by measuring the survival rate in endotoxemic mice.

Key Results

LPS (10 mg/kg, iv) significantly altered WBC counts, elevated plasma tumor necrosis factor (TNF)-α and nitric oxide (NO), increased pulmonary edema, thickened alveolar septa, and decreased survival rate. These changes were ameliorated by OroA (15 mg/kg, iv) administered 1 hr or 6 hrs after LPS challenge. This post-treatment also significantly attenuated LPS-induced activation of nuclear factor-κB (NF-κB) and the release of high mobility group box 1 (HMGB1) in lung tissues. Furthermore, post-treatment with OroA (60 mg/kg, ip) administered 1 hr or 6 hrs after LPS challenge in mice significantly increased survival rate.

Conclusion and Implication

OroA administered after induction of ALI by LPS significantly prevent and revere lung tissues injuries with increased survival rate. Positive post-treatment effects of OroA suggest that OroA is a potentially useful candidate for managing lung inflammation in LPS-induced endotoxemia and septic shock.     

CD103 deficiency prevents graft-versus-host disease but spares graft-versus-tumor effects mediated by alloreactive CD8 T cells

Background

Graft-versus-host disease (GVHD) remains the main barrier to broader application of allogeneic hematopoietic stem cell transplantation (alloSCT) as a curative therapy for host malignancy. GVHD is mediated by allogeneic T cells directed against histocompatibility antigens expressed by host tissues. Based on previous studies, we postulated that the integrin CD103 is required for CD8-mediated GVHD, but not for graft-versus-tumor effects (GVT).

Methodology/Principal Findings

We herein provide evidence in support of this hypothesis. To circumvent the potentially confounding influence of donor CD4 T cells, we developed an alloSCT model in which GVHD mortality is mediated by purified CD8 T cells. In this model, host-reactive CD8 T cells receive CD4 T cell help at the time of initial activation but not in the effector phase in which mature CD8 T effectors migrate into host tissues. We show that donor CD8 T cells from wild-type BALB/c mice primed to host alloantigens induce GVHD pathology and eliminate tumors of host origin in the absence of host CD4 T cells. Importantly, CD103 deficiency dramatically attenuated GVHD mortality, but had no detectable impact on the capacity to eliminate a tumor line of host origin. We provide evidence that CD103 is required for accumulation of donor CD8 T cells in the host intestinal epithelium but not in the tumor or host lymphoid compartments. Consistent with these data, CD103 was preferentially expressed by CD8 T cells infiltrating the host intestinal epithelium but not by those infiltrating the tumor, lamina propria, or lymphoid compartments. We further demonstrate that CD103 expression is not required for classic CD8 effector activities including cytokine production and cytotoxicity.

Conclusions/Significance

These data indicate that CD103 deficiency inhibits GVHD pathology while sparing anti-tumor effects mediated by CD8 T cells, identifying CD103 blockade as an improved strategy for GVHD prophylaxis.     

Effector and naturally occurring regulatory T cells display no abnormalities in activation induced cell death in NOD mice

Background

Disturbed peripheral negative regulation might contribute to evolution of autoimmune insulitis in type 1 diabetes. This study evaluates the sensitivity of naïve/effector (Teff) and regulatory T cells (Treg) to activation-induced cell death mediated by Fas cross-linking in NOD and wild-type mice.

Principal Findings

Both effector (CD25⁻, FoxP3⁻) and suppressor (CD25⁺, FoxP3⁺) CD4⁺ T cells are negatively regulated by Fas cross-linking in mixed splenocyte populations of NOD, wild type mice and FoxP3-GFP tranegenes. Proliferation rates and sensitivity to Fas cross-linking are dissociated in Treg cells: fast cycling induced by IL-2 and CD3/CD28 stimulation improve Treg resistance to Fas-ligand (FasL) in both strains. The effector and suppressor CD4⁺ subsets display balanced sensitivity to negative regulation under baseline conditions, IL-2 and CD3/CD28 stimulation, indicating that stimulation does not perturb immune homeostasis in NOD mice. Effective autocrine apoptosis of diabetogenic cells was evident from delayed onset and reduced incidence of adoptive disease transfer into NOD.SCID by CD4⁺CD25⁻ T cells decorated with FasL protein. Treg resistant to Fas-mediated apoptosis retain suppressive activity in vitro. The only detectable differential response was reduced Teff proliferation and upregulation of CD25 following CD3-activation in NOD mice.

Conclusion

These data document negative regulation of effector and suppressor cells by Fas cross-linking and dissociation between sensitivity to apoptosis and proliferation in stimulated Treg. There is no evidence that perturbed AICD in NOD mice initiates or promotes autoimmune insulitis.     

Effect of attenuation of Treg during BCG immunization on anti-mycobacterial Th1 responses and protection against Mycobacterium tuberculosis

Background

The functional equilibrium between natural regulatory T cells (Treg) and effector T cells can affect the issue of numerous infections. In unvaccinated mice, the influence of Treg in the control of primary infection with mycobacteria remains controversial.

Methodology

Here, we evaluated the role of Treg during prophylactic vaccination with Mycobacterium bovis BCG (Bacillus Calmette-Guérin) on the induction of T cell responses and on the protective effect against subsequent M. tuberculosis challenge in mice.

Principal Findings

We demonstrated that, subsequent to BCG injection, Treg were recruited to the draining lymph nodes and negatively control anti-mycobacterial CD4⁺ — but not CD8⁺ — T-cell responses. Treatment of BCG-immunized mice with an anti-CD25 mAb (PC61) induced an increase IFN-γ response against both subdominant and immunodominant regions of the protective immunogen TB10.4. In Treg-attenuated, BCG-immunized mice, which were then infected with M. tuberculosis, the lung mycobacterial load was significantly, albeit moderately, reduced compared to the control mice.

Conclusions

Our results provide the first demonstration that attenuation of Treg subset concomitant to BCG vaccination has a positive, yet limited, impact on the protective capacity of this vaccine against infection with M. tuberculosis. Thus, for rational design of improved BCG, it should be considered that, although the action of Treg does not represent the major cause of the limited efficiency of BCG, the impact of this cell population on the subsequent control of M. tuberculosis growth is significant and measurable.     

Protective effects of intratracheally administered quercetin on lipopolysaccharide-induced acute lung injury

Background

Acute respiratory distress syndrome (ARDS) can result in a life-threatening form of respiratory failure, and established, effective pharmacotherapies are therefore urgently required. Quercetin is one of the most common flavonoids found in fruits and vegetables, and has potent anti-inflammatory and anti-oxidant activities. Quercetin has been demonstrated to exhibit cytoprotective effects through the induction of heme oxygenase (HO)-1. Here, we investigated whether the intratracheal administration of quercetin could suppress lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice as well as the involvement of HO-1 in quercetin’s suppressive effects.

Methods

Mouse model of ALI were established by challenging intratracheally LPS. The wet lung-to-body weight ratio, matrix metalloproteinase (MMP)-9 activities, and pro-inflammatory cytokine productions, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 in bronchoalveolar lavage fluid (BALF) were examined in ALI mice with or without quercetin pretreatment. We also examined the effects of quercetin on LPS stimulation in the mouse alveolar macrophage cell line, AMJ2-C11 cells.

Results

Intratracheal administration of quercetin decreased the wet lung-to-body weight ratio. Moreover, quercetin decreased MMP-9 activity and the production of pro-inflammatory cytokines in BALF cells activated by LPS in advance. We determined the expression of quercetin-induced HO-1 in mouse lung, e.g., alveolar macrophages (AMs), alveolar and bronchial epithelial cells. When AMJ2-C11 cells were cultured with quercetin, a marked suppression of LPS-induced pro-inflammatory cytokine production was observed. The cytoprotective effects were attenuated by the addition of the HO-1 inhibitor SnPP. These results indicated that quercetin suppressed LPS-induced lung inflammation, and that an HO-1-dependent pathway mediated these cytoprotective effects.

Conclusions

Our findings indicated that quercetin suppressed LPS-induced lung inflammation, and that an HO-1-dependent pathway mediated these cytoprotective effects. Intratracheal administration of quercetin will lead to new supportive strategies for cytoprotection in these serious lung conditions.     

Small interfering RNA against CD86 during allergen challenge blocks experimental allergic asthma

Background

CD86-CD28 interaction has been suggested as the principal costimulatory pathway for the activation and differentiation of naïve T cells in allergic inflammation. However, it remains uncertain whether this pathway also has an essential role in the effector phase. We sought to determine the contribution of CD86 on dendritic cells in the reactivation of allergen-specific Th2 cells.

Methods

We investigated the effects of the downregulation of CD86 by short interfering RNAs (siRNAs) on Th2 cytokine production in the effector phase in vitro and on asthma phenotypes in ovalbumin (OVA)-sensitized and -challenged mice.

Results

Treatment of bone marrow-derived dendritic cells (BMDCs) with CD86 siRNA attenuated LPS-induced upregulation of CD86. CD86 siRNA treatment impaired BMDCs’ ability to activate OVA-specific Th2 cells. Intratracheal administration of CD86 siRNA during OVA challenge downregulated CD86 expression in the airway mucosa. CD86 siRNA treatment ameliorated OVA-induced airway eosinophilia, airway hyperresponsiveness, and the elevations of OVA-specific IgE in the sera and IL-5, IL-13, and CCL17 in the bronchoalveolar lavage fluid, but not the goblet cell hyperplasia.

Conclusion

These results suggest that local administration of CD86 siRNA during the effector phase ameliorates lines of asthma phenotypes. Targeting airway dendritic cells with siRNA suppresses airway inflammation and hyperresponsiveness in an experimental model of allergic asthma.     

FcεR(1)-Mediated Mast Cell Reactivity Is Amplified through Prolonged Toll-Like Receptor-Ligand Treatment

Background

Mast cell-derived mediators mediate several of the pathological features of asthma. Microbial infections induce asthma exacerbations in which the contribution of mast cells remains incomprehensible.

Principal Findings

In this study we have investigated the characteristic expression pattern of Toll-like receptors (TLRs) 1–9 and the effect of TLR ligand treatment on IgE-receptor mediated mast cell reactivity. For the studies we employed in vitro differentiated connective tissue like mast cells (CTLMC) and mucosal like mast cells (MLMC) from mice. Both phenotypes were treated for 24 h or 96 h with ligands for TLR1/2, TLR2/6, TLR3 and TLR4, before activation with IgE and antigen. Prolonged exposure (96 h) with TLR-ligands promoted mast cell reactivity following IgE-receptor activation. TLR4 activation with LPS generated the most pronounced effect, with an enhanced degranulation and secretion of leukotrienes, cytokines and chemokines, in both CTLMC and MLMC. The effect of LPS was mediated through a Myd88-dependent pathway and the increased effect involved JNK-dependent pathway.

Conclusion

We find that prolonged exposure of mast cells to pathogens/TLR-ligands modulates their effector responses by priming them for increased release of several inflammatory mediators when subsequently activated by IgE-receptors. These data suggest that infections might exaggerate the severity of allergic reactions such as in asthma, by enhancing mediator release from mast cells.     

Molecular targeting of hepatocyte growth factor by an antagonist,NK4, in the treatment of rheumatoid arthritis

Introduction

Hepatocyte growth factor (HGF) is a potent proangiogenic molecule that induces neovascularization. The HGF antagonist, NK4, competitively antagonizes HGF binding to its receptor. In the present study, we determined the inhibitory effect of NK4 in a rheumatoid arthritis (RA) model using SKG mice.

Methods

Arthritis was induced in SKG mice by a single intraperitoneal injection of β-glucan. Recombinant adenovirus containing NK4 cDNA (AdCMV.NK4) was also injected intravenously at the time of or 1 month after β-glucan injection. Ankle bone destruction was examined radiographically. The histopathologic features of joints were examined using hematoxylin and eosin and immunohistochemical staining. Enzyme-linked immunosorbent assays were used to determine the serum levels of HGF, interferon γ (IFN-γ, interleukin 4 (IL-4) and IL-17 production by CD4⁺ T cells stimulated with allogeneic spleen cells.

Results

The intravenous injection of AdCMV.NK4 into SKG mice suppressed the progression of β-glucan-induced arthritis. Bone destruction was also inhibited by NK4 treatment. The histopathologic findings of the ankles revealed that angiogenesis, inflammatory cytokines and RANKL expression in synovial tissues were significantly inhibited by NK4 treatment. Recombinant NK4 (rNK4) proteins inhibited IFN-γ, IL-4 and IL-17 production by CD4⁺ T cells stimulated with allogeneic spleen cells.

Conclusions

These results indicate that NK4 inhibits arthritis by inhibition of angiogenesis and inflammatory cytokine production by CD4⁺ T cells. Therefore, molecular targeting of angiogenic inducers by NK4 can potentially be used as a novel therapeutic approach for the treatment of RA.     

Houttuynia cordata Thunb extract modulates G0/G1 arrest and Fas/CD95-mediated death receptor apoptotic cell death in human lung cancer A549 cells

Background

Houttuynia cordata Thunb (HCT) is commonly used in Taiwan and other Asian countries as an anti-inflammatory, antibacterial and antiviral herbal medicine. In this study, we investigated the anti-human lung cancer activity and growth inhibition mechanisms of HCT in human lung cancer A549 cells.

Results

In order to investigate effects of HCT on A549 cells, MTT assay was used to evaluate cell viability. Flow cytometry was employed for cell cycle analysis, DAPI staining, and the Comet assay was used for DNA fragmentation and DNA condensation. Western blot analysis was used to analyze cell cycle and apoptotic related protein levels. HCT induced morphological changes including cell shrinkage and rounding. HCT increased the G₀/G₁ and Sub-G₁ cell (apoptosis) populations and HCT increased DNA fragmentation and DNA condensation as revealed by DAPI staining and the Comet assay. HCT induced activation of caspase-8 and caspase-3. Fas/CD95 protein levels were increased in HCT-treated A549 cells. The G₀/G₁ phase and apoptotic related protein levels of cyclin D1, cyclin A, CDK 4 and CDK 2 were decreased, and p27, caspase-8 and caspase-3 were increased in A549 cells after HCT treatment.

Conclusions

The results demonstrated that HCT-induced G₀/G₁ phase arrest and Fas/CD95-dependent apoptotic cell death in A549 cells