Yuan-Ji Day

Tumor-Derived Chemokine CCL5 Enhances TGF-β–Mediated Killing of CD8+ T Cells in Colon Cancer by T-Regulatory Cells

Chemokine CCL5/RANTES is highly expressed in cancer where it contributes to inflammation and malignant progression. In this study, we show that CCL5 plays a critical role in immune escape in colorectal cancer. We found that higher levels of CCL5 expression in human and murine colon tumor cells correlated with higher levels of apoptosis of CD8+ T cells and infiltration of T-regulatory cells (T(reg)). In mouse cells, RNA interference (RNAi)-mediated knockdown of CCL5 delayed tumor growth in immunocompetent syngeneic hosts but had no effect on tumor growth in immunodeficient hosts. Reduced tumor growth was correlated with a reduction in T(reg) infiltration and CD8(+) T-cell apoptosis in tumors. Notably, we found that CCL5 enhanced the cytotoxicity of T(reg) against CD8(+) T cells. We also found tumor growth to be diminished in mice lacking CCR5, a CCL5 receptor, where a similar decrease in both T(reg) cell infiltration and CD8(+) T-cell apoptosis was noted. TGF-β signaling blockade diminished apoptosis of CD8(+) T cells, implicating TGF-β as an effector of CCL5 action. In support of this concept, CCL5 failed to enhance the production of TGF-β by CCR5-deficient T(reg) or to enhance their cytotoxic effects against CD8(+) T cells. CCR5 signaling blockade also diminished the in vivo suppressive capacity of T(reg) in inhibiting the antitumor responses of CD8(+) T cells, in the same way as CCL5 signaling blockade. Together, our findings establish that CCL5/CCR5 signaling recruits T(reg) to tumors and enhances their ability to kill antitumor CD8(+) T cells, thereby defining a novel mechanism of immune escape in colorectal cancer.

Myeloid Expression of Adenosine A2A Receptor Suppresses T and NK Cell Responses in the Solid Tumor Microenvironment

High concentrations of adenosine in tumor microenvironments inhibit antitumor cytotoxic lymphocyte responses. Although T cells express inhibitory adenosine A2A receptors (A2AR) that suppress their activation and inhibit immune killing of tumors, a role for myeloid cell A2ARs in suppressing the immune response to tumors has yet to be investigated. In this study, we show that the growth of transplanted syngeneic B16F10 melanoma or Lewis lung carcinoma cells is slowed in Adora2a(f/f)-LysMCre(+/-) mice, which selectively lack myeloid A2ARs. Reduced melanoma growth is associated with significant increases in MHCII and IL12 expression in tumor-associated macrophages and with >90% reductions in IL10 expression in tumor-associated macrophages, dendritic cells (DC), and Ly6C(+) or Ly6G(+) myeloid-derived suppressor cells (MDSC). Myeloid deletion of A2ARs significantly increases CD44 expression on tumor-associated T cells and natural killer (NK) cells. Depletion of CD8(+) T cells or NK cells in tumor-bearing mice indicates that both cell types initially contribute to slowing melanoma growth in mice lacking myeloid A2A receptors, but tumor suppression mediated by CD8(+) T cells is more persistent. Myeloid-selective A2AR deletion significantly reduces lung metastasis of melanomas that express luciferase (for in vivo tracking) and ovalbumin (as a model antigen). Reduced metastasis is associated with increased numbers and activation of NK cells and antigen-specific CD8(+) T cells in lung infiltrates. Overall, the findings indicate that myeloid cell A2ARs have direct myelosuppressive effects that indirectly contribute to the suppression of T cells and NK cells in primary and metastatic tumor microenvironments. The results indicate that tumor-associated myeloid cells, including macrophages, DCs, and MDSCs all express immunosuppressive A2ARs that are potential targets of adenosine receptor blockers to enhance immune killing of tumors.

Insulin and resveratrol act synergistically, preventing cardiac dysfunction in diabetes, but the advantage of resveratrol in diabetics with acute heart attack is antagonized by insulin

Resveratrol (RSV), a natural phenolic compound, has been found to display cardiovascular protective and insulin-sensitizing properties. In this study, the effects of RSV and its combination with insulin on mortality, hemodynamics, insulin signaling, and nitrosative stress were compared in streptozotocin (STZ)-induced diabetic rats with or without acute myocardial ischemia/reperfusion (I/R) injury. Under normoxic conditions, cardiac systolic and diastolic functions and insulin-mediated Akt/GLUT4 (glucose transporter 4) activation were impaired in STZ-diabetic rats. The combination of RSV and insulin significantly prevented the above diabetes-associated abnormalities. Notwithstanding that, the diabetic state rendered the animals more susceptible to myocardial I/R injury, and the mortality rate and inducible nitric oxide synthase (iNOS)/nitrotyrosine protein expression and superoxide anion production were also further increased in I/R-injured diabetic hearts. In contrast, RSV treatment alone resulted in a lower mortality rate (from 62.5 to 18%) and better cardiac systolic function than its combination with insulin. RSV also inhibited iNOS/nitrotyrosine protein overexpression and superoxide anion overproduction in I/R-injured diabetic myocardium. Hyperglycemia, impairment of insulin signaling, overexpression of iNOS/nitrotyrosine, and superoxide anion overproduction were markedly rescued by the combination treatment, which did not show an improvement in mortality rate (30%) or cardiac performance over RSV treatment alone. These results indicate that insulin and RSV synergistically prevented cardiac dysfunction in diabetes and this may be in parallel with activation of the insulin-mediated Akt/GLUT4 signaling pathway. Although activation of the protective signal (Akt/GLUT4) and suppression of the adverse markers (iNOS, nitrotyrosine, and superoxide anion) were simultaneously observed in insulin and RSV combination treatment, insulin counteracted the advantage of RSV in diabetics with acute heart attack.

Inflammation Confers Dual Effects on Nociceptive Processing in Chronic Neuropathic Pain Model

Background:Although inflammation induces pain, immune cells also produce mediators that can effectively counteract it. To further elucidate the role of the immune response, we analyzed the relationship of pain behavior, several inflammatory signals, and opioid peptides using partial sciatic nerve ligation in mice at different levels of immunocompromise. Methods:Sciatic nerves of C57BL/6C, nonobese diabetic (NOD), or nonobese diabetic–severe combined immune deficiency (NOD-SCID) mice were partially ligated. Responses to mechanical and radiant heat stimuli were observed. Inflammation was detected by immunohistochemistry and flow cytometry. Inflammatory cytokines and opioid peptides were analyzed using real-time polymerase chain reaction and enzyme-linked immunosorbent assay or immunostaining. Results:Inflammation in immunocompromised mice was subordinate when compared with that seen in C57BL/6C mice. In addition, immunocompromised mice had less pain hypersensitivity at early stages. Whereas proinflammatory tumor necrosis factor-&agr; (TNF-&agr;), interleukin 1&bgr; (IL-1&bgr;), interleukin 6 (IL-6), and interferon-&ggr; (IFN-&ggr;), as well as antiinflammatory interleukin 1 receptor antagonist (IL-1Ra), interleukin 4 (IL-4), interleukin 10 (IL-10), and interleukin 13 (IL-13) cytokine expression and protein were increased in C57BL/6C mice, they were lower in immunocompromised mice. Although enkephalin, dynorphin, and &bgr;-endorphin messenger RNA expression also increased in C57BL/6C mice, peaking on day 14, this result was not observed in immunocompromised mice. Conclusion:The contribution of inflammation to nerve injury is complex with biphasic modulation. During the early phase, a wide range of proinflammatory cytokines are released, leading to enhanced pain. In contrast, the analgesic effect of opioid peptides and antiinflammatory cytokines was more predominate in the later phases of injury, leading to attenuated pain responses.

Extracellular adenosine regulates naive T cell development and peripheral maintenance

Adenosine receptor signaling inhibits TCR-induced activation of PI3K–Akt to reduce IL-7Rα expression on T cells, thereby regulating development and maintenance of naive T cells in the periphery.

Peritoneal Administration of Met-RANTES Attenuates Inflammatory and Nociceptive Responses in a Murine Neuropathic Pain Model

UNLABELLED The C-C motif chemokine ligand 5 (CCL5; also known as regulated on activation, normal T expressed and secreted, or RANTES) is a member of the CC family of chemokines that specifically attract and activate leukocytes to sites of inflammation. Although CCL5 has been implicated in the processing of pain, its detailed mechanisms of action are still unknown. In this study, we investigated the potential of the Met-RANTES, a selective CCL5 receptor antagonist, via peritoneal administration to modulate the recruitment of inflammatory cells in injured sites and attenuate nociceptive responses in a neuropathic pain model in mice. Nociceptive sensitization, immune cell infiltration, multiple cytokine secretion, and opioid peptide expression in damaged nerves were studied. Our results indicated that Met-RANTES-treated mice had less behavioral hypersensitivity after partial sciatic nerve ligation. Macrophage infiltration, pro-inflammatory cytokine (TNFα, IL-1β, IL-6, and IFNγ) protein secretion, and enkephalin, β-endorphin, and dynorphin mRNA expression in damaged nerves following partial sciatic nerve ligation were significantly decreased, and anti-inflammatory cytokine (IL-10) protein was significantly increased in Met-RANTES-treated mice. These results suggest that CCL5 is capable of regulating the microenvironment that controls behavioral hypersensitivity at the level of the peripheral injured site in a murine chronic neuropathic pain model. PERSPECTIVE The present study identifies the potent pro-inflammatory potential of CCL5 and verifies the possible role of selective CCL5 receptor inhibitor in a murine neuropathic pain model.

Hydroxyethyl starch interferes with human blood ex vivo coagulation, platelet function and sedimentation.

BACKGROUND Hydroxyethyl starch (HES) solutions are widely used for intravascular volume expansion. In Taiwan, the medium molecular weight of HES 200/0.5 and HES 130/0.4 solutions are most commonly used. It has been demonstrated that HES may affect coagulation and platelet function significantly. However, the differential effects of each medium molecular weight HES on platelets remain poorly reported. Therefore, we studied the influence of the two HES solutions on platelet function in vitro by mixing whole blood with different proportions of HES 130 kD, HES 200 kD, and saline to determine the differences. METHODS Human blood samples for platelet function analyzer (PFA), aggregometry and blood/HES mixed test were drawn from the antecubital vein and put into test tubes containing 3.2% trisodium citrate (blood:citrate, 9:1). The specimens were divided into four groups, designated as whole blood, 10%, 20%, and 30% dilution with normal saline (N/S), HES130 or HES200 solution. The platelet function of each sample was measured by both PFA and platelet aggregometry. RESULTS The results showed that the PFA-100 closure times CEPI-CT and CADP-CT were significantly prolonged in the samples diluted with normal saline, HES130 and HES200 than in the controls. The ADP triggered whole blood aggregometry showed that attenuated impedance was observed in samples of 20% diluted with HES130 and HES200 groups. The blood/HES mixed sedimentation test showed significantly increased proportion of the upper liquid layer in the HES200 group than in other groups. CONCLUSION Our data demonstrated that HES200 and HES130 possess noticeably inhibitory effects on platelet function, especially when the HES replaced proportion was more than 20%. HES200 has a greater effect on blood cells and plasma separation than does HES130.

Exogenous granulocyte colony-stimulating factor exacerbate pain-related behaviors after peripheral nerve injury

Previous studies have demonstrated that inflammatory cells produce several mediators that can effectively counteract pain. This study was designed to test the hypothesis that exogenous administration of recombinant mouse granulocyte-colony-stimulating factor (rmG-CSF) to enhance the recruitment of inflammatory cells to painful inflamed sites could attenuate pain in a chronic neuropathic pain model in mice. Our results indicate that treatment with rmG-CSF increased several cytokines and opioid peptides content; however, it did not attenuate but exacerbate neuropathic pain. Our study highlights the potent pro-inflammatory potential of G-CSF and suggests they may be targets for therapeutic intervention in chronic neuropathic pain.

lack of interleukin-17 leads to a modulated micro-environment and amelioration of mechanical hypersensitivity after peripheral nerve injury in mice

Summary Lack of interleukin‐17 leads to a modulated micro‐environment and amelioration of mechanical hypersensitivity after peripheral nerve injury in mice. ABSTRACT Interleukin‐17 (IL‐17) is involved in a wide range of inflammatory disorders and in recruitment of inflammatory cells to injury sites. A recent study of IL‐17 knock‐out mice revealed that IL‐17 contributes to neuroinflammation and neuropathic pain after peripheral nerve injury. Surprisingly, little is known of micro‐environment modulation by IL‐17 in injured sites and in pathologically related neuroinflammation and chronic neuropathic pain. Therefore, we investigated nociceptive sensitization, immune cell infiltration, myeloperoxidase (MPO) activity, and expression of multiple cytokines and opioid peptides in damaged nerves of wild‐type (IL‐17+/+) and IL‐17 knock‐out (IL‐17−/−) mice after partial sciatic nerve ligation. Our results demonstrated that the IL‐17−/− mice had less behavioral hypersensitivity after partial sciatic nerve ligation, and inflammatory cell infiltration and pro‐inflammatory cytokine (tumor necrosis factor–&agr;, IL‐6, and interferon‐&ggr;) levels in damaged nerves were significantly decreased, with the levels of anti‐inflammatory cytokines IL‐10 and IL‐13, and expressions of enkephalin, &bgr;‐endorphin, and dynorphin were also decreased compared to those in wild‐type control mice. In conclusion, we provided evidence that IL‐17 modulates the micro‐environment at the level of the peripheral injured nerve site and regulates progression of behavioral hypersensitivity in a murine chronic neuropathic pain model. The attenuated behavioral hypersensitivity in IL‐17−/− mice could be a result of decreased inflammatory cell infiltration to the injured site, resulting in modulation of the pro‐ and anti‐inflammatory cytokine milieu within the injured nerve. Therefore, IL‐17 may be a critical component for neuropathic pain pathogenesis and a novel target for therapeutic intervention for this and other chronic pain states.

P-selectin is required for neutrophils and macrophage infiltration into injured site and contributes to generation of behavioral hypersensitivity following peripheral nerve injury in mice.

Summary P‐selectin is required for neutrophils and macrophage infiltration into injured site and contributes to generation of behavioral hypersensitivity in a murine model of neuropathic pain. Abstract Growing evidence suggests that leukocyte extravasation is initiated by the interaction of selectins with their ligands; as well as an essential role for P‐selectin in the initial recruitment of inflammatory cells to sites of inflammation. In this study, P‐selectin‐deficient (P‐sel−/−) mice were used to test the hypothesis that lack of P‐selectin would attenuate the recruitment of inflammatory cells to the site of inflammation, thereby modulating pain in a murine chronic neuropathic pain model. Nociceptive sensitization and the microenvironment of the peripheral injury site were studied in wild‐type (P‐sel+/+) and P‐selectin‐deficient (P‐sel−/−) mice after partial sciatic nerve ligation (PSNL). Variables measured included myeloperoxidase (MPO) activity, several inflammatory cell infiltration profiles, cytokines, and endogenous opioid peptide expression in damaged nerves. Results indicate that behavioral hypersensitivity, MPO activity, and infiltration of neutrophils and macrophages were attenuated in P‐sel−/− mice after PSNL. Proinflammatory cytokines, tumor necrosis factor &agr;, and interleukin (IL)‐6, were reduced in damaged nerves following PSNL; however, several antiinflammatory cytokines – IL‐1Ra, IL‐4, and IL‐10 – were significantly increased in P‐sel−/− mice. In addition, endogenous opioid peptides mRNA was significantly lower in P‐sel−/− mice compared with P‐sel +/+ mice. The current results demonstrated that the absence of P‐selectin in mice leads to an altered microenvironment that attenuated behavioral hypersensitivity. The specific role of P‐selectin could have been a result of decreased neutrophils, as well as the accumulation of macrophages at the site of injury, which may subsequently modulate the inflammatory cytokine expression and impact behavioral hypersensitivity within the injured nerve.