Jie Zhao

Toll-Like Receptors on Tumor Cells Facilitate Evasion of Immune Surveillance

The signal pathways that trigger tumor cell escape from immune surveillance are incompletely understood. Toll-like receptors (TLRs), which activate innate and adaptive immune responses, are thought to be restricted to immune cells. We show here that TLRs, including TLR4, are expressed on tumor cells from a wide variety of tissues, suggesting that TLR activation may be an important event in tumor cell immune evasion. Activation of TLR4 signaling in tumor cells by lipopolysaccharide induces the synthesis of various soluble factors and proteins including interleukin-6, inducible nitric oxide synthase, interleukin-12, B7-H1, and B7-H2, and results in resistance of tumor cells to CTL attack. In addition, lipopolysaccharide-stimulated tumor cell supernatants inhibit both T cell proliferation and natural killer cell activity. Blockade of the TLR4 pathway by either TLR4 short interfering RNA or a cell-permeable TLR4 inhibitory peptide reverses tumor-mediated suppression of T cell proliferation and natural killer cell activity in vitro, and in vivo, delays tumor growth and thus prolongs the survival of tumor-bearing mice. These findings indicate that TLR signaling results in a cascade leading to tumor evasion from immune surveillance. These novel functions of TLRs in tumor biology suggest a new class of therapeutic targets for cancer therapy.

TLR signaling by tumor and immune cells: a double-edged sword

The tumor cell signaling pathways that trigger the uncontrolled proliferation, resistance to apoptosis, metastasis and escape from immune surveillance are partially understood. Toll-like receptors (TLRs), which recognize a variety of pathogen-associated molecular patterns, are centrally involved in the initiation of the innate and adaptive immune responses. However, recent evidence shows that functional TLRs are also expressed on a wide variety of tumors suggesting that TLRs may play important roles in tumor biology. Activation of tumor cell TLRs not only promotes tumor cell proliferation and resistance to apoptosis, but also enhances tumor cell invasion and metastasis by regulating metalloproteinases and integrins. Moreover, the activation of TLR signaling in tumor cells induces the synthesis of proinflammatory factors and immunosuppressive molecules, which enhance the resistance of tumor cells to cytotoxic lymphocyte attack and lead to immune evasion. Thus, the neoplastic process may usurp TLR signaling pathways to advance cancer progression, which suggests that targeting tumor TLR signaling pathways may open novel therapeutic avenues.

miR‐142‐3p restricts cAMP production in CD4+CD25− T cells and CD4+CD25+ TREG cells by targeting AC9 mRNA

Cyclic AMP (cAMP) is a ubiquitous second messenger that regulates diverse cellular functions. It has been found that CD4+CD25+ regulatory T (TREG) cells exert their suppressor function by transferring cAMP to responder T cells. Here, we show that miR‐142‐3p regulates the production of cAMP by targeting adenylyl cyclase (AC) 9 messenger RNA in CD4+CD25− T cells and CD4+CD25+ TREG cells. miR‐142‐3p limits the level of cAMP in CD4+CD25− T cells by inhibiting AC9 production, whereas forkhead box P3 (FOXP3) downregulates miR‐142‐3p to keep the AC9/cAMP pathway active in CD4+CD25+ TREG cells. These findings reveal a new molecular mechanism through which CD4+CD25+ TREG cells contain a high level of cAMP for their suppressor function, and also suggest that the microRNA controlling AC expression might restrict the final level of cAMP in various types of cells.

Listeria monocytogenes Promotes Tumor Growth via Tumor Cell Toll-Like Receptor 2 Signaling

The contribution of bacterial infection to tumorigenesis is usually ascribed to infection-associated inflammation. An alternate view is that direct interaction of bacteria with tumor cells promotes tumor progression. Here, we show that the microenvironment of large tumors favors bacterial survival, which in turn directly accelerates tumor growth by activating tumor cell Toll-like receptors (TLR). Listeria monocytogenes (Lm) survives in the microenvironment of large but not small tumors, resulting in the promotion of tumor growth. Lm did not affect the percentage of regulatory T cells or myeloid suppressor cells in the tumor. Through TLR2 signaling, Lm activated mitogen-activated protein kinases and nuclear factor-kappaB in tumor cells, resulting in the increased production of nitric oxide and interleukin-6 and increased proliferation of tumor cells. All of these effects were abrogated by silencing expression of TLR2, but not TLR4. The interaction of Helicobacter pylori with tumor cells from gastric carcinoma patients resulted in similar effects. These findings provide a new insight into infection-associated tumorigenesis and illustrate the importance of antibiotic therapy to treat tumors with bacterial infiltration.

Selective Depletion of CD4+CD25+Foxp3+ Regulatory T Cells by Low-Dose Cyclophosphamide Is Explained by Reduced Intracellular ATP Levels

CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells have been shown to play important roles in mediating cancer development. Although cyclophosphamide (CY) has shown promise as a drug to selectively target Treg cells with low-dose in vivo, the underlying molecular mechanism remains unclear. In this report, we provide evidence that ATP, the energy molecule and signal element, accounts for the selective depletion of Treg cells by low-dose CY. Relative to conventional T cells or other cell types, ATP levels were much lower in Treg cells. This was due to Treg cells that downregulate one microRNA, miR-142-3p, and upregulate ecto-nucleoside triphosphate diphosphohydrolase CD39. The transfection of miR-142-3p or the blockade of CD39 could increase intracellular ATP levels of Treg cells, consequently decreasing the sensitivity of Treg cells to low-dose CY. On the other hand, the transfection of miR-142-3p inhibitor or the addition of soluble CD39 to the cultured CD4(+)CD25(-) T cells resulted in the decrease of intracellular ATP levels and increase of sensitivity of conventional T cells to low-dose CY. Furthermore, we found that the low levels of ATP attenuated the synthesis of glutathione, leading to the decrease of CY detoxification, thus increasing the sensitivity of Treg cells to low-dose CY. Therefore, we here identify a molecular pathway through which low-dose CY selectively ablates Treg cells. Our findings also imply that low levels of ATP are probably related to Treg cell function.

IRF-8/Interferon (IFN) Consensus Sequence-binding Protein Is Involved in Toll-like Receptor (TLR) Signaling and Contributes to the Cross-talk between TLR and IFN-γ Signaling Pathways

Toll-like receptor (TLR) and interferon-γ (IFN-γ) signaling pathways are important for both innate and adaptive immune responses. However, the cross-talk between these two signaling pathways is incompletely understood. Here we show that IFN-γ and LPS synergistically induce the expression of proinflammatory factors, including interleukin-1 (IL-1), IL-6, IL-12, NO, and tumor necrosis factor-α (TNF-α). Comparable synergism was observed between IFN-γ and peptidoglycan (PGN; a TLR2 ligand) and poly(I:C) (a TLR3 ligand) in the induction of IL-12 promoter activity. IFN-γ enhanced lipopolysaccharide (LPS)-induced ERK and JNK phosphorylation but had no effect on LPS-induced NF-κB activation. Interestingly, we found that IRF-8–/– macrophages were impaired in the activation of LPS-induced ERK and JNK and the production of proinflammatory cytokines induced by LPS or IFN-γ plus LPS. Retroviral transduction of IRF-8 into IRF-8–/– macrophages rescued ERK and JNK activation. Furthermore, co-immunoprecipitation experiments show that IRF-8 physically interacts with TRAF6 at a binding site between amino acid residues 356 and 305 of IRF-8. Transfection of IRF-8 enhanced TRAF6 ubiquitination, which is consistent with a physical interaction of IRF-8 with TRAF6. Taken together, the results suggest that the interaction of IRF-8 with TRAF6 modulates TLR signaling and may contribute to the cross-talk between IFN-γ and TLR signal pathways.

Human Pregnancy Up-Regulates Tim-3 in Innate Immune Cells for Systemic Immunity

Pregnant women have both the local immune tolerance at the maternal-fetal interface and the systemic immune defense against pathogens. To date, regardless of the extensive investigation on the maternal-fetal immune tolerance, the maintenance of systemic immune defense in pregnant women still remains poorly understood. In the present study, we demonstrate that the immunoregulatory molecule T cell Ig and mucin domain (Tim)-3 plays important roles in innate and adaptive immunity of human pregnancy. During pregnancy, Tim-3 is strikingly up-regulated in peripheral blood of pregnant women, most by monocytes but not by T or B cells. The increased IL-4/STAT6 signaling may contribute to such up-regulation of Tim-3. In turn, the increased Tim-3 enhances not only innate immunity but also Th1-associated immune responses of pregnant women against pathogens. In contrast, our clinical data show that abnormal Tim-3 expression level might be connected to the pregnancy loss. In conclusion, our data show in this study that an immune regulatory molecule Tim-3, by virtue of its up-regulation in innate immune cells in pregnant women, enhances both innate and adaptive immune responses. Nevertheless, the abnormality of Tim-3 in pregnant woman may be deleterious to normal pregnancy.

Local Accumulation of FOXP3+ Regulatory T Cells: Evidence for an Immune Evasion Mechanism in Patients with Large Condylomata Acuminata

Condylomata acuminata derived from the infection of human papillomavirus is a common sexually transmitted disease. Although T cell-mediated cellular immunity is considered as the main arm against such infection, the regulation of T cell immune responses in genital condylomata is unclear to date. In this study, we analyzed FOXP3+ regulatory T cells in genital condylomata of patients. The results show that FOXP3+ regulatory T cells with suppressive function accumulated in large warts. Consistently, the immunosuppressive milieu in large warts was characterized by high expression of IL-10 and TGF-β1 and low expression of IL-2 and IFN-γ. The responsiveness of wart-infiltrating T cells both in vitro and in vivo can be increased by depleting FOXP3+ T cells. The accumulation of FOXP3+ regulatory T cells in large warts can be partly ascribed to the chemotaxis of CCL17 and CCL22, derived from Langerhans cells and macrophages in wart. Although such accumulation favors the local immunosuppression, it seems not to influence the systemic immunity. In conclusion, these findings demonstrate that FOXP3+ regulatory T cells play an important role in genital condylomata, which has multiple implications in the comprehensive treatment of condylomata acuminata.

Mast cell: insight into remodeling a tumor microenvironment

Mast cells are of paramount importance to allergies, pathogen immune responses during infections, and angiogenesis, as well as innate and adaptive immune regulations. Beyond all these roles, mast cells are now more and more being recognized as modulators of tumor microenvironment. Notwithstanding mounting evidences of mast cell accumulation in tumors, their exact role in tumor microenvironment is still incompletely understood. In this review, we discuss the significant role of mast cells in the remodeling of tumor microenvironment by either releasing various factors after activation or interacting with other cells within tumor and, as a result, the possible role of mast cell in cancer invasion and metastasis. We also discuss recent findings that mast cells actively release microparticles, which account for the transfer of membrane-type receptor signal and regulatory molecules such as microRNAs to tumor cells and immune cells. These findings on mast cells provide further insights into the complexity of tumor microenvironment remodeling.

Spontaneous Miscarriages Are Explained by the Stress/Glucocorticoid/Lipoxin A4 Axis

Despite various suspected causes, ranging from endocrine and genetic to infectious and immunological aspects, the molecular mechanisms of miscarriage still remain enigmatic. This work provides evidence that downregulation of 11β-hydroxysteroid dehydrogenase (HSD) type 2, the key enzyme inactivating glucocorticoid activities, insults the pregnant inflammatory milieu by inhibiting the biosynthesis of lipoxin A4 (LXA4), a metabolite of arachidonic acid, leading to an early loss of the pregnancy. Both LXA4 and its biosynthetic enzymes were found to be decreased in women with spontaneous miscarriages and in the murine miscarriage model. Replenishing LXA4 reversed LPS-induced miscarriages in mouse models, whereas blocking LXA4 signaling resulted in miscarriages in the pregnant mice. The protective effect of LXA4 might be explained by LXA4’s role in regulating uterine and placental inflammatory factors and mast cells. The underlying molecular mechanism involved miscarriage-inducing infections or stresses that downregulate the expression of 11β-HSD2, but not 11β-HSD1, resulting in increases in glucocorticoid activity and decreases in LXA4. Together, these findings suggest that the stress/glucocorticoid/LXA4 axis might be a common pathway through which miscarriages occur.