CB2 Cannabinoid Receptors Contribute to Bacterial Invasion and Mortality in Polymicrobial Sepsis

BACKGROUND:Sepsis is a major healthcare problem and current estimates suggest that the incidence of sepsis is approximately 750,000 annually. Sepsis is caused by an inability of the immune system to eliminate invading pathogens. It was recently proposed that endogenous mediators produced during sepsis can contribute to the immune dysfunction that is observed in sepsis. Endocannabinoids that are produced excessively in sepsis are potential factors leading to immune dysfunction, because they suppress immune cell function by binding to G-protein-coupled CB(2) receptors on immune cells. Here we examined the role of CB(2) receptors in regulating the host's response to sepsis. METHODS AND FINDINGS:The role of CB(2) receptors was studied by subjecting CB(2) receptor wild-type and knockout mice to bacterial sepsis induced by cecal ligation and puncture. We report that CB(2) receptor inactivation by knockout decreases sepsis-induced mortality, and bacterial translocation into the bloodstream of septic animals. Furthermore, CB(2) receptor inactivation decreases kidney and muscle injury, suppresses splenic nuclear factor (NF)-kappaB activation, and diminishes the production of IL-10, IL-6 and MIP-2. Finally, CB(2) receptor deficiency prevents apoptosis in lymphoid organs and augments the number of CD11b(+) and CD19(+) cells during CLP. CONCLUSIONS:Taken together, our results establish for the first time that CB(2) receptors are important contributors to septic immune dysfunction and mortality, indicating that CB(2) receptors may be therapeutically targeted for the benefit of patients suffering from sepsis

A pilot clinical study of Δ9-tetrahydrocannabinol in patients with recurrent glioblastoma multiforme

Δ9-Tetrahydrocannabinol (THC) and other cannabinoids inhibit tumour growth and angiogenesis in animal models, so their potential application as antitumoral drugs has been suggested. However, the antitumoral effect of cannabinoids has never been tested in humans. Here we report the first clinical study aimed at assessing cannabinoid antitumoral action, specifically a pilot phase I trial in which nine patients with recurrent glioblastoma multiforme were administered THC intratumoraly. The patients had previously failed standard therapy (surgery and radiotherapy) and had clear evidence of tumour progression. The primary end point of the study was to determine the safety of intracranial THC administration. We also evaluated THC action on the length of survival and various tumour-cell parameters. A dose escalation regimen for THC administration was assessed. Cannabinoid delivery was safe and could be achieved without overt psychoactive effects. Median survival of the cohort from the beginning of cannabinoid administration was 24 weeks (95% confidence interval: 15–33). Δ9-Tetrahydrocannabinol inhibited tumour-cell proliferation in vitro and decreased tumour-cell Ki67 immunostaining when administered to two patients. The fair safety profile of THC, together with its possible antiproliferative action on tumour cells reported here and in other studies, may set the basis for future trials aimed at evaluating the potential antitumoral activity of cannabinoids

Amelioration of Experimental Autoimmune Encephalomyelitis by Plumbagin through Down-Regulation of JAK-STAT and NF-κB Signaling Pathways

Plumbagin(PL), a herbal compound derived from roots of the medicinal plant Plumbago zeylanica, has been shown to have immunosuppressive properties. Present report describes that PL is a potent novel agent in control of encephalitogenic T cell responses and amelioration of mouse experimental autoimmune encephalomyelitis (EAE), through down-regulation of JAK-STAT pathway. PL was found to selectively inhibit IFN-γ and IL-17 production by CD4+ T cells, which was mediated through abrogated phosphorylation of JAK1 and JAK2. Consistent with IFN-γ and IL-17 reduction was suppressed STAT1/STAT4/T-bet pathway which is critical for Th1 differentiation, as well as STAT3/ROR pathway which is essential for Th17 differentiation. In addition, PL suppressed pro-inflammatory molecules such as iNOS, IFN-γ and IL-6, accompanied by inhibition of IκB degradation as well as NF-κB phosphorylation. These data give new insight into the novel immune regulatory mechanism of PL and highlight the great value of this kind of herb compounds in probing the complex cytokine signaling network and novel therapeutic targets for autoimmune diseases

Resveratrol Prevents Endothelial Cells Injury in High-Dose Interleukin-2 Therapy against Melanoma

Immunotherapy with high-dose interleukin-2 (HDIL-2) is an effective treatment for patients with metastatic melanoma and renal cell carcinoma. However, it is accompanied by severe toxicity involving endothelial cell injury and induction of vascular leak syndrome (VLS). In this study, we found that resveratrol, a plant polyphenol with anti-inflammatory and anti-cancer properties, was able to prevent the endothelial cell injury and inhibit the development of VLS while improving the efficacy of HDIL-2 therapy in the killing of metastasized melanoma. Specifically, C57BL/6 mice were injected with B16F10 cells followed by resveratrol by gavage the next day and continued treatment with resveratrol once a day. On day 9, mice received HDIL-2. On day 12, mice were evaluated for VLS and tumor metastasis. We found that resveratrol significantly inhibited the development of VLS in lung and liver by protecting endothelial cell integrity and preventing endothelial cells from undergoing apoptosis. The metastasis and growth of the tumor in lung were significantly inhibited by HDIL-2 and HDIL-2 + resveratrol treatment. Notably, HDIL-2 + resveratrol co-treatment was more effective in inhibiting tumor metastasis and growth than HDIL-2 treatment alone. We also analyzed the immune status of Gr-1+CD11b+ myeloid-derived suppressor cells (MDSC) and FoxP3+CD4+ regulatory T cells (Treg). We found that resveratrol induced expansion and suppressive function of MDSC which inhibited the development of VLS after adoptive transfer. However, resveratrol suppressed the HDIL-2-induced expansion of Treg cells. We also found that resveratrol enhanced the susceptibility of melanoma to the cytotoxicity of IL-2-activated killer cells, and induced the expression of the tumor suppressor gene FoxO1. Our results suggested the potential use of resveratrol in HDIL-2 treatment against melanoma. We also demonstrated, for the first time, that MDSC is the dominant suppressor cell than regulatory T cell in the development of VLS

Activation through cannabinoid receptors 1 and 2 on dendritic cells triggers NF-kappa B-dependent apoptosis: Novel role for endogenous and exogenous cannabinoids in immunoregulation

The precise role of cannabinoid receptors (CB)1 and CB2, as well as endogenous ligands for these receptors, on immune cells remains unclear. In the current study, we examined the effect of endogenous and exogenous cannabinoids on murine bone marrow-derived dendritic cells (DCs). Addition of ?? 9-tetrahydrocannabinol (THC), a major psychoactive component found in marijuana or anandamide, an endogenous cannabinoid, to DC cultures induced apoptosis in DCs. DCs expressed CB1 and CB2 receptors and the engagement of both receptors was necessary to trigger apoptosis. Treatment with THC induced caspase-2, -8, and -9 activation, cleavage of Bid, decreased mitochondrial membrane potential, and cytochrome c release, suggesting involvement of death-receptor and mitochondrial pathways. DCs from Bid-knockout mice were sensitive to THC-induced apoptosis thereby suggesting that Bid was dispensable. There was no induction of p44/p42 MAPK, p38 MAPK, or stress-activated protein/JNK pathway in THC-treated DCs. However, THC treatment induced phosphorylation of I??B-??, and enhanced the transcription of several apoptotic genes regulated by NF-??B. Moreover, inhibition of NF-??B was able to block THC-induced apoptosis in DCs. Lastly, in vivo treatment of mice with THC caused depletion of splenic DCs. Together, our study demonstrates for the first time that endogenous and exogenous cannabinoids may suppress the immune response through their ability to induce apoptosis in DCs.close878

Combined deficiency in CD44 and Fas leads to exacerbation of lymphoproliferative and autoimmune disease

Patients with mutations in Fas develop autoimmune lymphoproliferative disease (ALPS), While their family members with similar mutations are often normal, thereby suggesting that additional factors may play a role in the development of ALPS. In the current study, we tested the role of CD44 in the development of lymphoproliferative disease by generating CD44 -/-/Fas -/-mice, which failed to express CD44 and Fas, and compared them to CD44 +/+/Fas -/- mice that expressed CD44, but not Fas. The results showed that CD44 -/-/Fas -/- mice developed a more severe lymphoproliferative and autoimmune disease when compared to CD44 +/+/Fas -/- mice. This was indicated by increased numbers of cells in their lymph nodes, and a greater proportion of B220 +CD4 -CD8 - (double-negative) T cells as well as antibodies against single-stranded DNA and chromatin. The heightened severity of lymphoproliferative disease seen in CD44 -/-/Fas -/- mice correlated with increased resistance of T cells, but not B cells, to undergo activation-induced cell death (AICD). The current study suggests that deficiency in CD44 in combination with a defect in one of the molecules involved in the death receptor family such as Fas can further down-regulate AICD, and exacerbate the lymphoproliferative and autoimmune disease.close7

Role of CD44 in activation-induced cell death: CD44-deficient mice exhibit enhanced T cell response to conventional and superantigens

T cells upon activation are known to up-regulate CD44 expression. However, the precise function of CD44 on activated T cells is not clear. In this report, we demonstrate that signaling through CD44 plays an important role in activation-induced cell death (AICD). CD44 knockout (KO) mice had an elevated in vivo primary and in vitro secondary response to challenge with conalbumin, anti-CD3 mAb and staphylococcal enterotoxin A (SEA), which correlated with reduced AICD when compared to CD44 wild-type mice. In addition, CD44 KO mice exhibited increased delayed-type hypersensitivity response to dinitrofluorobenzene. In a model examining in vitro AICD, splenocytes from CD44 KO mice showed resistance to TCR-mediated apoptosis when compared to splenocytes from CD44 wild-type mice. In addition, signaling through CD44 led to increased apoptosis in TCR-activated but not resting T cells from CD44 wild-type mice without affecting Fas expression. Injection of SEA into mice deficient in CD44 and Fas (CD44 KO/lpr) led to an increased primary response when compared to mice that expressed CD44 but not Fas (CD44 WT/lpr), suggesting that the enhanced response to SEA was dependent on CD44 but not Fas expression. Administration of anti-CD44 mAb into CD44 wild-type mice caused a significant decrease in antigen-specific T cell response. Together, these data implicate CD44 as an important regulator of AICD in T cells. Furthermore, targeting CD44 in vivo may constitute a novel approach to induce apoptosis in activated T cells, and therefore to treat autoimmune diseases, allograft rejection and graft versus host disease.close343

Evidence for estradiol-induced apoptosis and dysregulated T cell maturation in the thymus

In an attempt to delineate the immunological alterations that may occur following treatment with estrogen, groups of C57BL/6 mice were treated with 75 mg/kg body weight of ??-estradiol-17-valerate (E2) or the vehicle. The thymus from these mice were harvested on days 1, 4 and 7 following treatment. The thymocytes from E2-treated mice when cultured in vitro for 24 h, showed increased levels of apoptosis when compared to controls. The apoptosis was demonstrable by both TUNEL assay and Annexin V/propidium iodide (PI) staining. Also, thymic atrophy and increased apoptosis of thymocytes when cultured in vitro were seen when lower doses of E2 (5 mg/kg) were administered. The thymus from E2-treated mice on days 4 and 7 also showed a decrease in the percentage of CD4+CD8+ (DP) T cells and an increase in the percentage of CD4-CD8- (DN), CD4+ and CD8+ T cells. However, the total cellularity of all T cell subsets in the thymus was decreased following E2 treatment. Earlier studies from our laboratory and elsewhere have demonstrated that in thymocytes undergoing apoptosis, there is increased expression of surface markers including CD3, ????TCR and CD44 with a simultaneous decrease in the expression of J11d. Similar changes were observed in thymocytes from mice on days 4 and 7 following E2 treatment. These data therefore confirmed that the thymocytes were indeed undergoing apoptosis following E2 treatment. Together, our studies suggest for the first time that estrogen may induce thymic atrophy by triggering apoptosis.close697

Attenuation of myocardial fibrosis with curcumin is mediated by modulating expression of angiotensin II AT1/AT2 receptors and ACE2 in rats

Xue-Fen Pang,1 Li-Hui Zhang,2 Feng Bai,1 Ning-Ping Wang,3 Ron E Garner,3 Robert J McKallip,4 Zhi-Qing Zhao1,3 1Department of Physiology, Shanxi Medical University, Taiyuan, Shanxi, People’s Republic of China; 2Department of Cardiology, Shanxi Academy of Medical Sciences and Shanxi Dayi Hospital, Shanxi Medical University, Taiyuan, Shanxi, People’s Republic of China; 3Department of Basic Biomedical Sciences, Mercer University School of Medicine, Savannah, GA, USA; 4Division of Basic Biomedical Sciences, Mercer University School of Medicine, Macon, GA, USA Abstract: Curcumin is known to improve cardiac function by balancing degradation and synthesis of collagens after myocardial infarction. This study tested the hypothesis that inhibition of myocardial fibrosis by curcumin is associated with modulating expression of angiotensin II (Ang II) receptors and angiotensin-converting enzyme 2 (ACE2). Male Sprague Dawley rats were subjected to Ang II infusion (500 ng/kg/min) using osmotic minipumps for 2 and 4 weeks, respectively, and curcumin (150 mg/kg/day) was fed by gastric gavage during Ang II infusion. Compared to the animals with Ang II infusion, curcumin significantly decreased the mean arterial blood pressure during the course of the observation. The protein level of the Ang II type 1 (AT1) receptor was reduced, and the Ang II type 2 (AT2) receptor was up-regulated, evidenced by an increased ratio of the AT2 receptor over the AT1 receptor in the curcumin group (1.2±0.02%) vs in the Ang II group (0.7±0.03%, P<0.05). These changes were coincident with less locally expressed AT1 receptor and enhanced AT2 receptor in the intracardiac vessels and intermyocardium. Along with these modulations, curcumin significantly decreased the populations of macrophages and alpha smooth muscle actin-expressing myofibroblasts, which were accompanied by reduced expression of transforming growth factor beta 1 and phosphorylated-Smad2/3. Collagen I synthesis was inhibited, and tissue fibrosis was attenuated, as demonstrated by less extensive collagen-rich fibrosis. Furthermore, curcumin increased protein level of ACE2 and enhanced its expression in the intermyocardium relative to the Ang II group. These results suggest that curcumin could be considered as an add-on therapeutic agent in the treatment of fibrosis-derived heart failure patient who is intolerant of ACE inhibitor therapy. Keywords: angiotensin II receptors, angiotensin-converting enzyme 2, curcumin, collagen, myocardial fibrosi