Zhi Yao

IL-17/Th17 Promotes Type 1 T Cell Immunity against Pulmonary Intracellular Bacterial Infection through Modulating Dendritic Cell Function

Although their contribution to host defense against extracellular infections has been well defined, IL-17 and Th17 are generally thought to have limited impact on intracellular infections. In this study, we investigated the role and mechanisms of IL-17/Th17 in host defense against Chlamydia muridarum, an obligate intracellular bacterium, lung infection. Our data showed rapid increase in IL-17 production and expansion of Th17 cells following C. muridarum infection and significant detrimental impact of in vivo IL-17 neutralization by anti-IL-17 mAb on disease course, immune response, and dendritic cell (DC) function. Specifically, IL-17-neutralized mice exhibited significantly greater body weight loss, higher organism growth, and much more severe pathological changes in the lung compared with sham-treated control mice. Immunological analysis showed that IL-17 neutralization significantly reduced Chlamydia-specific Th1 responses, but increased Th2 responses. Interestingly, the DC isolated from IL-17-neutralized mice showed lower CD40 and MHC II expression and IL-12 production, but higher IL-10 production compared with those from sham-treated mice. In two DC-T cell coculture systems, DC isolated from IL-17-neutralized mice induced higher IL-4, but lower IFN-γ production by Ag-specific T cells than those from sham-treated mice in cell priming and reaction settings. Adoptive transfer of DC isolated from IL-17-neutralized mice, unlike those from sham-treated mice, failed to protect the recipients against challenge infection. These findings provide in vivo evidence that IL-17/Th17 plays an important role in host defense against intracellular bacterial infection, and suggest that IL-17/Th17 can promote type 1 T cell immunity through modulating DC function.

IL-17A synergizes with IFN-γ to upregulate iNOS and NO production and inhibit chlamydial growth.

IFN-γ-mediated inducible nitric oxide synthase (iNOS) expression is critical for controlling chlamydial infection through microbicidal nitric oxide (NO) production. Interleukin-17A (IL-17A), as a new proinflammatory cytokine, has been shown to play a protective role in host defense against Chlamydia muridarum (Cm) infection. To define the related mechanism, we investigated, in the present study, the effect of IL-17A on IFN-γ induced iNOS expression and NO production during Cm infection in vitro and in vivo. Our data showed that IL-17A significantly enhanced IFN-γ-induced iNOS expression and NO production and inhibited Cm growth in Cm-infected murine lung epithelial (TC-1) cells. The synergistic effect of IL-17A and IFN-γ on Chlamydia clearance from TC-1 cells correlated with iNOS induction. Since one of the main antimicrobial mechanisms of activated macrophages is the release of NO, we also examined the inhibitory effect of IL-17A and IFN-γ on Cm growth in peritoneal macrophages. IL-17A (10 ng/ml) synergizes with IFN-γ (200 U/ml) in macrophages to inhibit Cm growth. This effect was largely reversed by aminoguanidine (AG), an iNOS inhibitor. Finally, neutralization of IL-17A in Cm infected mice resulted in reduced iNOS expression in the lung and higher Cm growth. Taken together, the results indicate that IL-17A and IFN-γ play a synergistic role in inhibiting chlamydial lung infection, at least partially through enhancing iNOS expression and NO production in epithelial cells and macrophages.

Glypican-3 expression and its relationship with recurrence of HCC after liver transplantation

AIMnTo investigate the diagnostic value of glypican-3 (GPC3) and its relationship with hepatocellular carcinoma (HCC) recurrence after liver transplantation.nnnMETHODSnHCC tissue samples (n = 31) obtained from patients who had undergone liver transplantation were analyzed. GPC3 mRNA and protein expression were analyzed by TaqMan real-time reverse transcription-polymerase chain reaction and immunohistochemistry. Correlation between the GPC3 expression and clinicopathological features was analyzed. The potential prognostic value of GPC3 was investigated by comparing recurrence-free survival between HCC patients with and without GPC3 expression.nnnRESULTSnUsing a cutoff value of 3.5 × 10⁻², 20 of 31 cancerous tissues had expression values of > 3.5 × 10⁻², whereas 3 of 31 adjacent non-neoplastic parenchyma and 0 of 20 control liver tissues had expression values of > 3.5 × 10⁻² (P < 0.001). GPC3 protein was immunoexpressed in 68% of cancerous tissues, but not in adjacent non-neoplastic parenchyma and control liver tissues. Vascular invasion was significantly related to GPC3 expression (P < 0.05). Recurrence-free survival was significantly longer for patients without GPC3 mRNA overexpression (> 3.5 × 10⁻²) and those without vascular invasion (P < 0.05 for both).nnnCONCLUSIONnGPC3 expression may serve as a valuable diagnostic marker for HCC. GPC3 mRNA overexpression may be an adverse indicator for HCC patients after liver transplantation.

Reciprocal regulation of 17β-estradiol, interleukin-6 and interleukin-8 during growth and progression of epithelial ovarian cancer

Estrogens have been associated with risk for epithelial ovarian cancer (OVCA). Both IL-6 and IL-8 are also likely involved in the progression of OVCA. In order to discover the underline molecular mechanism, we investigated the modulation of estrogen and two cytokines in the growth and progression of epithelial OVCA. In these studies, the effect of 17beta-estradiol (E(2)) on the expression levels of IL-6, IL-8 and their receptors was investigated. The effect of IL-6 and IL-8 on activation of estrogen-responsive promoter as well as estrogen receptor (ER)alpha and ER beta expression was also analyzed. Gene expression profile analysis revealed that CAOV-3 and OVCAR-3 cells, which express ER, IL-6 and IL-8 receptors, are suitable model for this study. We found that E(2) not only enhanced IL-6 and IL-8 production via NF-kappaB signaling pathway, but also modulated their respective receptor expression. Tamoxifen (Txf), an ER antagonist, completely abolished E(2)-stimulated cell growth and the expression of IL-6 and IL-8. IL-6/IL-8-induced cell proliferation was completely blocked by their specific neutralizing antibodies, which partially inhibited E(2)-induced cell growth. In the absence of estrogen, both cytokines activated estrogen-responsive promoter, which was completely blocked by Txf, and caused a dose-dependent ER alpha increase and ER beta decrease. Pretreatment of OVCAR-3 with p38 MAPK, MEK1/2 or ErbB2 MAPK inhibitors, respectively, blocked IL-6-mediated induction of estrogen-responsive promoter while Src inhibitor blocked IL-8-induced activation of estrogen-responsive promoter. These results provide a novel mechanism that estrogens, IL-6 and IL-8 may form a common amplifying signaling cascade to modulate OVCA growth and progression. Estrogen-induced OVCA proliferation is partially occurring via enhanced IL-6 and IL-8 production and modulated their receptors, and IL-6/IL-8 could also promote OVCA growth through an ER alpha pathway.

Fibulin-3 suppresses Wnt/β-catenin signaling and lung cancer invasion

The 5 year survival rate of lung cancer is <20%, with most patients dying from distant metastasis. However, the molecular mechanisms underlying lung cancer invasion and metastasis have not been fully characterized. In this study, we found that fibulin-3, a fibulin family extracellular matrix protein, functions as a suppressor of lung cancer invasion and metastasis. Fibulin-3 was downregulated in large fractions of lung tumors and cell lines, and inhibited lung cancer cell invasion and the expression of matrix metalloproteinase-7 (MMP-7), a promoter of lung cancer invasion. The expression levels of fibulin-3 and MMP-7 were inversely correlated in lung tumors. Fibulin-3 inhibited extracellular signal-regulated kinase (ERK) to activate glycogen synthase kinase 3β and suppress Wnt/β-catenin signaling, which induces MMP-7 expression in lung cancer cells. Furthermore, fibulin-3 expression impeded the growth and metastasis of lung tumors in mice. Collectively, these results suggest that downregulation of fibulin-3 contributes to lung cancer invasion and metastasis by activating Wnt/β-catenin signaling and MMP-7 expression.

Roles of full-length and truncated neurokinin-1 receptors on tumor progression and distant metastasis in human breast cancer

Substance P (SP) regulates various physiologic and pathophysiologic responses predominantly by acting through its primary receptor, the neurokinin-1 receptor (NK1R). There are two naturally occurring forms of NK1R: full-length NK1R-FL and truncated NK1R-Tr. SP-coupled NK1R can directly or indirectly regulate the proliferation and metastatic progression of many types of human cancer cells. However, the exact roles played by the two isoforms of NK1R in breast carcinogenesis still remain largely unclear. In the present study, we first examined the expression profile of total NK1Rs, NK1R-FL and NK1R-Tr in multiple breast cancer cell lines as well as in breast tumor samples. We found that total NK1Rs are present in normal, benign and breast tumor tissues; while, NK1R-FL expression are significantly decreased in tumor specimens, particularly in metastatic carcinomas. More interestingly, NK1R-FL is highly expressed in nontumorigenic HBL-100 breast cells, whereas MDA-MB-231, MCF-7 and T47D breast cancer cells express only NK1R-Tr. To further investigate potential implications of NK1R-FL and NK1R-Tr in the malignant phenotypes of breast cancer, we studied the impacts of ectopically overexpressed NK1R-FL and NK1R-Tr in MDA-MB-231 and HBL-100 cells, respectively. Our in vitro and in vivo data showed that NK1R-FL expression was inversely associated with proliferation, invasiveness and metastasis of MDA-MB-231 cells, but overexpression of NK1R-Tr was able to promote malignant transformation of HBL-100 cells and NK1R-Tr may contribute to tumor progression and promote distant metastasis in human breast cancer. A long-term treatment of NK1R antagonist ASN-1377642 exerted antitumor action in breast cancer cells with NK1R-Tr high expression.

Monitoring of CD95 and CD38 expression in peripheral blood T lymphocytes during active human cytomegalovirus infection after orthotopic liver transplantation

Aim:u2002 The aim of the present study was to quantitatively monitor the response of CD95 molecules expressed on CD3+ T cells (CD95+CD3+ cells) and CD38 molecules expressed on CD8+ T cells (CD38+CD8+ cells) to ganciclovir treatment after orthotopic liver transplant (OLT) in recipients with active human cytomegalovirus (HCMV) infection.

Effect of truncated neurokinin‐1 receptor expression changes on the interaction between human breast cancer and bone marrow‐derived mesenchymal stem cells

Previous studies in breast cancer cell lines showed that truncated neurokinin receptor‐1 (NK1R‐Tr) was able to promote malignant transformation of breast cells, and NK1R‐Tr may contribute to tumor progression and promote distant metastasis in human breast cancer. A co‐culture model of breast cancer and bone marrow‐derived human mesenchymal stem (HMSC‐bm) cells showed that HMSC‐bm inhibited the growth of breast cancer cells and entered the bone marrow at early stages. Down‐regulation of NK1R‐Tr may be a key factor in maintaining the quiescent phenotype of breast cancer cells among bone marrow stroma. Stromal‐derived factor (SDF)‐1α expression was negatively correlated with NK1R‐Tr expression in breast cancer cells. Secretion of SDF‐1α by HMSC‐bm may maintain the quiescent phenotype of breast cancer cells among bone marrow stroma by down‐regulating NK1R‐Tr expression. Transforming growth factor (TGF)‐β1 expression was positively associated with NK1R‐Tr expression in breast cancer cells. In a co‐culture system, MDA‐MB‐231‐TGF‐β1I (TGF‐β genes were suppressed using specific shRNA) cells were able to attach to HMSC‐bm quickly, indicating that TGF‐β1 was also a key factor for maintaining the quiescent phenotype of breast cancer cells in bone marrow stroma. However, the detailed mechanism still remained unclear and could involve other molecules, in addition to NK1R‐Tr.

Temporal evolution of soluble Fas and Fas ligand in patients with orthotopic liver transplantation.

AIMnThe aim of this study was to analyze the expression levels of plasma soluble Fas (sFas) and soluble Fas ligand (sFasL) in patients with orthotopic liver transplantation (OLT) procedures routinely performed without venovenous bypass.nnnMETHODSnThe sFas and sFasL were analyzed in the blood of 20 consecutive patients who underwent transplantation. Blood samples were drawn from the radial artery at serial time points before, during, and after surgery. Plasma levels of sFas and sFasL were detected by Enzyme Linked-Immuno-Sorbent Assay. Plasma aspartate transaminase (AST) and alanine transaminase (ALT) were assayed by routine clinical chemistry testing.nnnRESULTSnMarked elevation of plasma AST and ALT were detected at the reperfusion and postoperation time points (P<0.001), with a peak on the first postoperative day. The mean plasma concentration of sFas and sFasL remained unchanged from preoperative to anhepatic phase (T1 to T3) (P> or =0.268). The sFas and sFasL concentrations were significantly higher at 15 and 60 min after reperfusion compared to the preoperative value (P< or =0.048). Postoperatively, sFas and sFasL concentration were decreased to preoperative levels on the first postoperative day (P> or =0.127).nnnCONCLUSIONnThe sFas and sFasL seem to be involved in reperfusion injury during OLT. The understanding of Fas may provide new insights into the mechanisms of ischemia/reperfusion injury during OLT.

Effects of age on biological and functional characterization of adipose‑derived stem cells from patients with end‑stage liver disease

Adipose‑derived mesenchymal stromal cells (ADSCs) possess a multilineage potential and immunoregulatory properties, and may have great potential in autologous cell‑based technologies. The aim of the present study was to investigate how the age of patients with benign end‑stage liver disease affected the biological and functional characteristics of ADSCs, which is important for increasing the potential effectiveness of autologous cell therapy. ADSCs were obtained and cultured from three distinct age groups: Infant, adult and elderly. Cell immunophenotypic characteristics and antiapoptotic capacity were determined by flow cytometry, and cell proliferation and migration were monitored with a Real‑Time Cell Analyzer. Multilineage differentiation potential was investigated by evaluating the induction response and by reverse transcription‑quantitative polymerase chain reaction. Suppression of T cell proliferation was assessed in a co‑culture system by MTT assay. The regulatory T cells (Tregs) were analyzed by flow cytometry, and ELISAs were performed to detect the cytokine profile in culture supernatants. All ADSC sample phenotypes were characterized as CD90+/CD73+/CD105+/CD45‑/CD34‑, and the apoptotic rate was not statistically different among all ages. However, the proliferation and migratory capacity were significantly increased in infant‑derived ADSCs. In addition, ADSCs derived from infant patients demonstrated a relatively high proclivity for osteogenic differentiation compared with cells derived from either adult or elderly patients. Furthermore, ADSCs co‑cultured with mitogen‑activated T cells significantly suppressed T‑cell proliferation, downregulated the secretion of interferon‑γ and increased the percentage of Tregs, with infant‑derived ADSCs being most effective. Results from the present study indicated that ADSCs derived from infant patients may have biological advantages compared with older cell sources, and may provide an effective reference for the clinical application of ADSCs.