Milada Mahic

FOXP3+CD4+CD25+ Adaptive Regulatory T Cells Express Cyclooxygenase-2 and Suppress Effector T Cells by a Prostaglandin E2-Dependent Mechanism

CD4+CD25+ regulatory T (TR) cells suppress effector T cells by partly unknown mechanisms. In this study, we describe a population of human suppressive CD4+CD25+ adaptive TR (TRadapt) cells induced in vitro that express cyclooxygenase 2 (COX-2) and the transcription factor FOXP3. TRadapt cells produce PGE2 and suppress effector T cell responses in a manner that is reversed by COX inhibitors and PGE2 receptor-specific antagonists. In resting CD4+CD25− T cells, treatment with PGE2 induced FOXP3 expression. Thus, autocrine and paracrine effects of PGE2 produced by COX-2-positive TRadapt cells may be responsible for both the FOXP3+ phenotype and the mechanism used by these cells to suppress effector T cells.

Generation of highly suppressive adaptive CD8+CD25+FOXP3+ regulatory T cells by continuous antigen stimulation

Continuous antigen stimulation of CD4+CD25– T cells leads to generation of adaptive CD4+CD25+FOXP3+ regulatory T (TR) cells. Here, we show that highly suppressive adaptive CD8+CD25+FOXP3+ T cells can be generated in the same manner by continuous antigen stimulation in the presence of CD14+ monocytes. During the course of stimulation, acquisition of immunosuppressive properties develops in parallel with up‐regulation and expression of cytotoxic molecules. The CD8+ TR cells inhibit CD4+ and CD8+ T cell proliferation and cytokine production, but do not alter the expression of granzyme A and granzyme B or perforin in CD8+ effector T cells. Although, the CD8+ TR cells express prostaglandin E2, IL‐10 and TGF‐β, the mechanism of suppression was independent of these soluble factors. In contrast to adaptive CD4+ TR cells, the CD8+ TR cells suppress mainly by a contact‐dependent mechanism as evident from transwell experiments. However, neither blocking antibodies to CTLA‐4, CD80 nor CD86 could reverse CD8+ TR‐mediated suppression, indicating that other mechanism(s) must be employed by these cells.

Regulatory T cells in colorectal cancer patients suppress anti-tumor immune activity in a COX-2 dependent manner

ObjectiveNaturally occurring regulatory T (TR) cells suppress autoreactive T cells whereas adaptive TR cells, induced in the periphery, play an important role in chronic viral diseases and cancer. Several studies indicate that cyclooxygenase (COX) inhibitors prevent cancer development of colon adenomas and delay disease progression in patients with colorectal cancer (CRC). We have shown that adaptive TR cells express COX-2 and produce PGE2 that suppress effector T cells in a manner that is reversed by COX-inhibitors.Methods and resultsHere we demonstrate that CRC patients have elevated levels of PGE2 in peripheral blood, and CRC tissue samples and draining lymph nodes display increased numbers of FOXP3+ TR cells. Depletion of TR cells from PBMC enhanced anti-tumor T-cell responses to peptides from carcinoembryonic antigen. Furthermore, the COX inhibitor indomethacin and the PKA type I antagonist Rp-8-Br-cAMPS significantly improved the anti-tumor immune activity.ConclusionWe suggest that adaptive TR cells contribute to an immunosuppressive microenvironment in CRC and inhibit effector T cells by a COX-2–PGE2-dependent mechanism and thereby facilitate tumor growth. Therapeutic strategies targeting TR cells and the PGE2–cAMP pathway may be interesting to pursue to enhance anti-tumor immune activity in CRC patients.

The Cyclic AMP-Epac1-Rap1 Pathway Is Dissociated from Regulation of Effector Functions in Monocytes but Acquires Immunoregulatory Function in Mature Macrophages

cAMP mediates its intracellular effects through activation of protein kinase A (PKA), nucleotide-gated ion channels, or exchange protein directly activated by cAMP (Epac). Although elevation of cAMP in lymphocytes leads to suppression of immune functions by a PKA-dependent mechanism, the effector mechanisms for cAMP regulation of immune functions in monocytes and macrophages are not fully understood. In this study, we demonstrate the presence of Epac1 in human peripheral blood monocytes and activation of Rap1 in response to cAMP. However, by using an Epac-specific cAMP analog (8-CPT-2′-O-Me-cAMP), we show that monocyte activation parameters such as synthesis and release of cytokines, stimulation of cell adhesion, chemotaxis, phagocytosis, and respiratory burst are not regulated by the Epac1-Rap1 pathway. In contrast, activation of PKA by a PKA-specific compound (6-Bnz-cAMP) or physiological cAMP-elevating stimuli like PGE2 inhibits monocyte immune functions. Furthermore, we show that the level of Epac1 increases 3-fold during differentiation of monocytes into macrophages, and in monocyte-derived macrophages cAMP inhibits FcR-mediated phagocytosis via both PKA and the Epac1-Rap1 pathway. However, LPS-induced TNF-α production is only inhibited through the PKA pathway in these cells. In conclusion, the Epac1-Rap1 pathway is present in both monocytes and macrophages, but only regulates specific immune effector functions in macrophages.

Differentiation of naive CD4+ T cells into CD4+CD25+FOXP3+ regulatory T cells by continuous antigen stimulation

Human CD4+CD25+ regulatory T (TR) cells express the transcription factor forkhead box p3 (FOXP3) and have potent immunosuppressive properties. While naturally occurring TR cells develop in the thymus, adaptive TR cells develop in the periphery from naive CD4+ T cells. Adaptive TR cells may express cyclooxygenase type 2 (COX‐2) and suppress effector T cells by a PGE2‐dependent mechanism, which is reversible with COX inhibitors. In this study we have characterized the differentiation of naive CD4+ T cells into adaptive TR cells in detail during 7 days of continuous antigen stimulation. After 2 days of stimulation of CD4+CD25– T cells, the cells expressed FOXP3 and COX‐2 and displayed potent immunosuppressive properties. The suppressive phenotype was present at all observed time‐points from Day 2, although suppression was merely present at Day 7. The adaptive TR cells expressed cell surface markers consistent with an activated phenotype and secreted high levels of TGF‐β, IL‐10, and PGE2. However, the suppressive phenotype was found exclusively in cells that proliferated upon activation. These data support the notion that activation of naive CD4+ T cells leads to concomitant acquisition of effector and suppressive properties.

Chronic pain and use of opioids: a population-based pharmacoepidemiological study from the Norwegian prescription database and the Nord-Trøndelag health study.

Summary Three quarters of patients using opioids persistently reported strong or very strong pain despite of the medication. ABSTRACT In previous studies on prescription patterns of opioids, accurate data on pain are missing, and previous epidemiological studies of pain lack accurate data on opioid use. The present linkage study, which investigates the relationship between pain and opioid use, is based on accurate individual data from the complete national Norwegian prescription database and the Nord‐Trøndelag health study 3, which includes about 46,000 people. Baseline data were collected in 2006 to 2008, and the cohort was followed up for 3 years. Of 14,477 people who reported chronic nonmalignant pain, 85% did not use opioids at all, 3% used opioids persistently, and 12% used opioids occasionally. Even in the group reporting severe or very severe chronic pain, the number not using opioids (2680) was far higher than the number who used opioids persistently (304). However, three quarters of people using opioids persistently reported strong or very strong pain in spite of the medication. Risk factors for the people with chronic pain who were not persistent opioid users at baseline to use opioids persistently 3 years later were occasional use of opioids, prescription of >100 defined daily doses per year of benzodiazepines, physical inactivity, reports of strong pain intensity, and prescription of drugs from 8 or more Anatomical Therapeutic Chemical groups. The study showed that most people having chronic nonmalignant pain are not using opioids, even if the pain is strong or very strong. However, the vast majority of patients with persistent opioid use report strong or very strong pain in spite of opioid treatment.

A pharmacoepidemiological cohort study of subjects starting strong opioids for nonmalignant pain: A study from the Norwegian Prescription Database

Summary In a complete national cohort of persons starting opioids for chronic nonmalignant pain, only 24% of the cohort received opioids 5 years later. Abstract Clinical studies of short duration have demonstrated that strong opioids improve pain control in selected patients with chronic nonmalignant pain. However, high discontinuation rates and dose escalation during long‐term treatment have been indicated. The aim of the present study was to determine discontinuation rates, dose escalation, and patterns of co‐medication with benzodiazepines. The Norwegian Prescription Database provides complete national data at an individual level on dispensed drugs. A complete national cohort of new users of strong opioids was followed up for 5 years after initiation of therapy with strong opioids. Of the 17,248 persons who were new users of strong opioids in 2005, 7229 were dispensed a second prescription within 70 days and were assumed to be intended long‐term users. A total of 1233 persons in the study cohort were still on opioid therapy 5 years later. This equals 24% of the study cohort who were still alive. Of the participants, 21% decreased their annual opioid dose by 25% or more, whereas 21% kept a stable dose (±24%) and 34% more than doubled their opioid dose from the first to the fifth year. High annual doses of opioids were associated with high annual doses of benzodiazepines at the end of follow‐up. It is an issue of major concern that large dose escalation is common during long‐term treatment, and that that high doses of opioids are associated with high doses of benzodiazepines. These findings make it necessary to question whether the appropriate patient population receives long‐term opioid treatment.

Short-Interfering RNA-Mediated Lck Knockdown Results in Augmented Downstream T Cell Responses

The Src family kinase Lck is essential for T cell Ag receptor-mediated signaling. In this study, we report the effects of acute elimination of Lck in Jurkat TAg and primary T cells using RNA interference mediated by short-interfering RNAs. In cells with Lck knockdown (kd), proximal TCR signaling was strongly suppressed as indicated by reduced ζ-chain phosphorylation and intracellular calcium mobilization. However, we observed sustained and elevated phosphorylation of ERK1/2 in Lck kd cells 30 min to 2 h after stimulation. Downstream effects on immune function as determined by activation of a NFAT-AP-1 reporter, and TCR/CD28-stimulated IL-2 secretion were strongly augmented in Jurkat and primary T cells, respectively. As expected, overexpression of SHP-1 in Jurkat cells inhibited TCR-induced NFAT-AP-1 activation, but this effect could be overcome by simultaneous kd of Lck. Furthermore, acute elimination of Lck also suppressed TCR-mediated activation of SHP-1, suggesting the possible role of SHP-1 in a negative feedback loop originating from Lck. This report underscores Lck as an important mediator of proximal TCR signaling, but also indicates a suppressive role on downstream immune function.

Aspirin As Secondary Prevention in Patients With Colorectal Cancer: An Unselected Population-Based Study

PURPOSE Regular use of aspirin (acetylsalicylic acid) is associated with reduced incidence and mortality of colorectal cancer (CRC). However, aspirin as primary prevention is debated because of the risk of hemorrhagic adverse effects. Aspirin as secondary prevention may be more justified from a risk-benefit perspective. We have examined the association between aspirin use after the diagnosis of CRC with CRC-specific survival (CSS) and overall survival (OS). MATERIALS AND METHODS An observational, population-based, retrospective cohort study was conducted by linking patients diagnosed with CRC from 2004 through 2011 (Cancer Registry of Norway) with data on their aspirin use (The Norwegian Prescription Database). These registries cover more than 99% of the Norwegian population and include all patients in an unselected and consecutive manner. Exposure to aspirin was defined as receipt of aspirin prescriptions for more than 6 months after the diagnosis of CRC. Multivariable Cox-proportional hazard analyses were used to model survival. The main outcome measures of the study were CSS and OS. RESULTS A total of 23,162 patients diagnosed with CRC were included, 6,102 of whom were exposed to aspirin after the diagnosis of CRC (26.3%). The median follow-up time was 3.0 years. A total of 2,071 deaths (32.9%, all causes) occurred among aspirin-exposed patients, of which 1,158 (19.0%) were CRC specific. Among unexposed patients (n = 17,060), there were 7,218 deaths (42.3%), of which 5,375 (31.5%) were CRC specific. In multivariable analysis, aspirin exposure after the diagnosis of CRC was independently associated with improved CSS (hazard ratio [HR], 0.85; 95% CI, 0.79 to 0.92) and OS (HR, 0.95; 95% CI, 0.90 to 1.01). CONCLUSION Aspirin use after the diagnosis of CRC is independently associated with improved CSS and OS.

Prevalence of use of non-prescription analgesics in the Norwegian HUNT3 population: Impact of gender, age, exercise and prescription of opioids

BackgroundThere are concerns about potential increasing use of over-the-counter (OTC) analgesics. The aims of this study were to examine 1) the prevalence of self-reported use of OTC analgesics; 2) the prevalence of combining prescription analgesics drugs with OTC analgesics and 3) whether lifestyle factors such as physical activity were associated with prevalence of daily OTC analgesic use.MethodsQuestionnaire data from the Nord–Trøndelag health study (HUNT3, 2006–08), which includes data from 40,000 adult respondents. The questionnaire included questions on use of OTC analgesics, socioeconomic conditions, health related behaviour, symptoms and diseases. Data were linked to individual data from the Norwegian Prescription Database. A logistic regression was used to investigate the association between different factors and daily use of paracetamol and/or non-steroid anti-inflammatory drugs (NSAIDs) in patients with and without chronic pain.ResultsThe prevalence of using OTC analgesics at least once per week in the last month was 47%. Prevalence of paracetamol use was almost 40%, compared to 19% and 8% for NSAIDs and acetylsalicylic acid (ASA), respectively. While the use of NSAIDs decreased and the use of ASA increased with age, paracetamol consumption was unaffected by age. Overall more women used OTC analgesics. About 3-5% of subjects using OTC analgesics appeared to combine these with the same analgesic on prescription. Among subjects reporting chronic pain the prevalence of OTC analgesic use was almost twice as high as among subjects without chronic pain. Subjects with little physical activity had 1.5-4 times greater risk of daily use of OTC compared to physically active subjects.ConclusionsUse of OTC analgesics is prevalent, related to chronic pain, female gender and physical inactivity.