IL-11 induces NLRP3 inflammasome activation in monocytes and inflammatory cell migration to the central nervous system.

The objective of this study is to examine IL-11-induced mechanisms of inflammatory cell migration to the central nervous system (CNS). We report that IL-11 is produced at highest frequency by myeloid cells among the peripheral blood mononuclear cell (PBMC) subsets. Patients with relapsing-remitting multiple sclerosis (RRMS) have an increased frequency of IL-11+ monocytes, IL-11+ and IL-11R+ CD4+ lymphocytes, and IL-11R+ neutrophils in comparison to matched healthy controls. IL-11+ and granulocyte-macrophage colony-stimulating factor (GM-CSF)+ monocytes, CD4+ lymphocytes, and neutrophils accumulate in the cerebrospinal fluid (CSF). The effect of IL-11 in-vitro stimulation, examined using single-cell RNA sequencing, revealed the highest number of differentially expressed genes in classical monocytes, including up-regulated NFKB1, NLRP3, and IL1B. All CD4+ cell subsets had increased expression of S100A8/9 alarmin genes involved in NLRP3 inflammasome activation. In IL-11R+-sorted cells from the CSF, classical and intermediate monocytes significantly up-regulated the expression of multiple NLRP3 inflammasome-related genes, including complement, IL18, and migratory genes (VEGFA/B) in comparison to blood-derived cells. Therapeutic targeting of this pathway with αIL-11 mAb in mice with RR experimental autoimmune encephalomyelitis (EAE) decreased clinical scores, CNS inflammatory infiltrates, and demyelination. αIL-11 mAb treatment decreased the numbers of NFκBp65+, NLRP3+, and IL-1ß+ monocytes in the CNS of mice with EAE. The results suggest that IL-11/IL-11R signaling in monocytes represents a therapeutic target in RRMS.

CSF-1 maintains pathogenic but not homeostatic myeloid cells in the central nervous system during autoimmune neuroinflammation.

The receptor for colony stimulating factor 1 (CSF-1R) is important for the survival and function of myeloid cells that mediate pathology during experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). CSF-1 and IL-34, the ligands of CSF-1R, have similar bioactivities but distinct tissue and context-dependent expression patterns, suggesting that they have different roles. This could be the case in EAE, given that CSF-1 expression is up-regulated in the CNS, while IL-34 remains constitutively expressed. We found that targeting CSF-1 with neutralizing antibody halted ongoing EAE, with efficacy superior to CSF-1R inhibitor BLZ945, whereas IL-34 neutralization had no effect, suggesting that pathogenic myeloid cells were maintained by CSF-1. Both anti­CSF-1 and BLZ945 treatment greatly reduced the number of monocyte-derived cells and microglia in the CNS. However, anti­CSF-1 selectively depleted inflammatory microglia and monocytes in inflamed CNS areas, whereas BLZ945 depleted virtually all myeloid cells, including quiescent microglia, throughout the CNS. Anti­CSF-1 treatment reduced the size of demyelinated lesions and microglial activation in the gray matter. Lastly, we found that bone marrow­derived immune cells were the major mediators of CSF-1R­dependent pathology, while microglia played a lesser role. Our findings suggest that targeting CSF-1 could be effective in ameliorating MS pathology, while preserving the homeostatic functions of myeloid cells, thereby minimizing risks associated with ablation of CSF-1R­dependent cells.

Anesthesia Choice and Its Potential Impact on Endoluminal Functional Lumen Imaging Probe Measurements in Esophageal Motility Disorders.

BACKGROUND AND AIMS: The widespread use of peroral endoscopic myotomy (POEM) has revolutionized the management of esophageal motility disorders (EMDs). The introduction of an endoluminal functional lumen imaging probe (EndoFLIP) can serve as a complimentary diagnostic tool to assess the mechanical properties (i.e., pressure, diameter, distensibility and topography) of the esophagus. During EndoFLIP measurements, different anesthesia techniques may induce variable degrees of neuromuscular blockade, potentially affecting esophageal motility and altering the results of EndoFLIP metrics. Our study aimed to compare the impact of using total intravenous anesthesia (TIVA) versus general anesthesia with inhalational anesthetics (GAIA) on diagnostic EndoFLIP measurements. METHODS: We conducted a retrospective study of all adult patients (≥18 years) undergoing EndoFlip during the POEM procedure at our institution between February 2017 and February 2022. We obtained the differences in pressure, diameter, and distensibility index using propofol-based TIVA vs sevoflurane-based GAIA with a 30ml and 60ml balloon. The differences were divided into terciles and compared between diagnoses using univariate comparisons and logistic regression models. RESULTS: 49 patients were included (39% Type 1 achalasia, 43% Type 2 or 3 achalasia, and 18% jackhammer esophagus (JE)). Compared to spastic disorders (Type 2, 3 and JE), Type 1 had lower values of pressure differences at 60 mL in univariate (3.75 vs 15.20 p=0.001) and multivariate (aOR 0.89 95%CI 0.82-0.978) analyses. Compared to Type 1, Type 2 and 3 had higher rates of pressure differences at 60 mL in univariate (9.85 vs 3.75 p=0.04); and nearly reached significance in multivariate analysis (1.09 95%CI 1-1.20). Compared to Type 1, JE demonstrated higher values in pressure differences at 60 mL (27.7 vs 3.75 p<0.001) CONCLUSION: Esophageal pressure, as measured by EndoFLIP, was significantly reduced when patients were sedated with sevoflurane-based GAIA. The use sevoflurane-based GAIA for diagnostic EndoFLIP may potentially lead to the misclassification of spastic disorders as Type I achalasia. Therefore, propofol-based TIVA should be considered over sevoflurane-based GAIA for sedation during the diagnostic test.

Reproducibility efforts as a teaching tool: A pilot study.

The "replication crisis" is a methodological problem in which many scientific research findings have been difficult or impossible to replicate. Because the reproducibility of empirical results is an essential aspect of the scientific method, such failures endanger the credibility of theories based on them and possibly significant portions of scientific knowledge. An instance of the replication crisis, analytic replication, pertains to reproducing published results through computational reanalysis of the authors' original data. However, direct replications are costly, time-consuming, and unrewarded in today's publishing standards. We propose that bioinformatics and computational biology students replicate recent discoveries as part of their curriculum. Considering the above, we performed a pilot study in one of the graduate-level courses we developed and taught at our University. The course is entitled Intro to R Programming and is meant for students in our Master's and PhD programs who have little to no programming skills. As the course emphasized real-world data analysis, we thought it would be an appropriate setting to carry out this study. The primary objective was to expose the students to real biological data analysis problems. These include locating and downloading the needed datasets, understanding any underlying conventions and annotations, understanding the analytical methods, and regenerating multiple graphs from their assigned article. The secondary goal was to determine whether the assigned articles contained sufficient information for a graduate-level student to replicate its figures. Overall, the students successfully reproduced 39% of the figures. The main obstacles were the need for more advanced programming skills and the incomplete documentation of the applied methods. Students were engaged, enthusiastic, and focused throughout the semester. We believe that this teaching approach will allow students to make fundamental scientific contributions under appropriate supervision. It will teach them about the scientific process, the importance of reporting standards, and the importance of openness.

Developing and Evaluating an AI-Based Computer-Aided Diagnosis System for Retinal Disease: Diagnostic Study for Central Serous Chorioretinopathy.

BACKGROUND: Although previous research has made substantial progress in developing high-performance artificial intelligence (AI)-based computer-aided diagnosis (AI-CAD) systems in various medical domains, little attention has been paid to developing and evaluating AI-CAD system in ophthalmology, particularly for diagnosing retinal diseases using optical coherence tomography (OCT) images. OBJECTIVE: This diagnostic study aimed to determine the usefulness of a proposed AI-CAD system in assisting ophthalmologists with the diagnosis of central serous chorioretinopathy (CSC), which is known to be difficult to diagnose, using OCT images. METHODS: For the training and evaluation of the proposed deep learning model, 1693 OCT images were collected and annotated. The data set included 929 and 764 cases of acute and chronic CSC, respectively. In total, 66 ophthalmologists (2 groups: 36 retina and 30 nonretina specialists) participated in the observer performance test. To evaluate the deep learning algorithm used in the proposed AI-CAD system, the training, validation, and test sets were split in an 8:1:1 ratio. Further, 100 randomly sampled OCT images from the test set were used for the observer performance test, and the participants were instructed to select a CSC subtype for each of these images. Each image was provided under different conditions: (1) without AI assistance, (2) with AI assistance with a probability score, and (3) with AI assistance with a probability score and visual evidence heatmap. The sensitivity, specificity, and area under the receiver operating characteristic curve were used to measure the diagnostic performance of the model and ophthalmologists. RESULTS: The proposed system achieved a high detection performance (99% of the area under the curve) for CSC, outperforming the 66 ophthalmologists who participated in the observer performance test. In both groups, ophthalmologists with the support of AI assistance with a probability score and visual evidence heatmap achieved the highest mean diagnostic performance compared with that of those subjected to other conditions (without AI assistance or with AI assistance with a probability score). Nonretina specialists achieved expert-level diagnostic performance with the support of the proposed AI-CAD system. CONCLUSIONS: Our proposed AI-CAD system improved the diagnosis of CSC by ophthalmologists, which may support decision-making regarding retinal disease detection and alleviate the workload of ophthalmologists.

IL-9 Controls Central Nervous System Autoimmunity by Suppressing GM-CSF Production.

Multiple sclerosis and experimental autoimmune encephalomyelitis (EAE) are inflammatory diseases of the CNS in which Th17 cells play a major role in the disease pathogenesis. Th17 cells that secrete GM-CSF are pathogenic and drive inflammation of the CNS. IL-9 is a cytokine with pleiotropic functions, and it has been suggested that it controls the pathogenic inflammation mediated by Th17 cells, and IL-9R-/- mice develop more severe EAE compared with wild-type counterparts. However, the underlying mechanism by which IL-9 suppresses EAE has not been clearly defined. In this study, we investigated how IL-9 modulates EAE development. By using mice knockout for IL-9R, we show that more severe EAE in IL-9R-/- mice correlates with increased numbers of GM-CSF+ CD4+ T cells and inflammatory dendritic cells (DCs) in the CNS. Furthermore, DCs from IL-9R-/- mice induced more GM-CSF production by T cells and exacerbated EAE upon adoptive transfer than did wild-type DCs. Our results suggest that IL-9 reduces autoimmune neuroinflammation by suppressing GM-CSF production by CD4+ T cells through the modulation of DCs.

Retinal Pigment Epithelium Tears after Anti-Vascular Endothelial Growth Factor Therapy for Neovascular Age-Related Macular Degeneration.

PURPOSE: The aim of this study was to assess the visual prognostic factors of retinal pigment epithelium (RPE) tears and describe their clinical features. METHODS: The medical records of treatment-naive neovascular age-related macular degeneration patients who received intravitreal anti-vascular endothelial growth factor (VEGF) injections were retrospectively reviewed. RESULTS: The incidence of RPE tears was 1.36% (10 out of 733 eyes). The type of anti-VEGF agent administered did not affect the incidence (p = 0.985). The median best-corrected visual acuity (BCVA) of 10 patients decreased after an RPE tear (0.4-0.6 logarithm of the minimum angle of resolution [logMAR]); however, subsequent injections restored the BCVA to a level similar to that before the RPE tear (0.4 logMAR, p = 0.436). Central macular thickness improved significantly during the study (794.4-491.9 µm, p = 0.013). The final BCVA was positively correlated with the BCVA before and immediately after the RPE tear (p = 0.025 and 0.002, respectively) and was weakly correlated with foveal involvement of the RPE tear (p = 0.061). CONCLUSION: The incidence of RPE tears did not differ according to the type of anti-VEGF agent. The final BCVA was proportional to the BCVA before and after RPE tears. Continuous treatment with anti-VEGF after the occurrence of RPE tears can benefit the final visual acuity and macular anatomy.

A Google Trends Approach to Identify Distinct Diurnal and Day-of-Week Web-Based Search Patterns Related to Conjunctivitis and Other Common Eye Conditions: Infodemiology Study.

BACKGROUND: Studies suggest diurnal patterns of occurrence of some eye conditions. Leveraging new information sources such as web-based search data to learn more about such patterns could improve the understanding of patients' eye-related conditions and well-being, better inform timing of clinical and remote eye care, and improve precision when targeting web-based public health campaigns toward underserved populations. OBJECTIVE: To investigate our hypothesis that the public is likely to consistently search about different ophthalmologic conditions at different hours of the day or days of week, we conducted an observational study using search data for terms related to ophthalmologic conditions such as conjunctivitis. We assessed whether search volumes reflected diurnal or day-of-week patterns and if those patterns were distinct from each other. METHODS: We designed a study to analyze and compare hourly search data for eye-related and control search terms, using time series regression models with trend and periodicity terms to remove outliers and then estimate diurnal effects. We planned a Google Trends setting, extracting data from 10 US states for the entire year of 2018. The exposure was internet search, and the participants were populations who searched through Google's search engine using our chosen study terms. Our main outcome measures included cyclical hourly and day-of-week web-based search patterns. For statistical analyses, we considered P<.001 to be statistically significant. RESULTS: Distinct diurnal (P<.001 for all search terms) and day-of-week search patterns for eye-related terms were observed but with differing peak time periods and cyclic strengths. Some diurnal patterns represented those reported from prior clinical studies. Of the eye-related terms, "pink eye" showed the largest diurnal amplitude-to-mean ratios. Stronger signal was restricted to and peaked in mornings, and amplitude was higher on weekdays. By contrast, "dry eyes" had a higher amplitude diurnal pattern on weekends, with stronger signal occurring over a broader evening-to-morning period and peaking in early morning. CONCLUSIONS: The frequency of web-based searches for various eye conditions can show cyclic patterns according to time of the day or week. Further studies to understand the reasons for these variations may help supplement the current clinical understanding of ophthalmologic symptom presentation and improve the timeliness of patient messaging and care interventions.

Endotoxin May Not Be the Major Cause of Postprandial Inflammation in Adults Who Consume a Single High-Fat or Moderately High-Fat Meal.

BACKGROUND: Metabolic endotoxemia is considered a cause for high-fat diet (HFD)-induced inflammation. However, convincing experimental evidence in humans is scant. OBJECTIVE: We determined whether a HFD or moderately HFD increases LPS and LPS-mediated cytokine production in the postprandial blood (PPB). METHODS: Ninety-eight volunteers (age: 37.3 ± 1.5 y) from the cross-sectional phenotyping study (PS) and 62 volunteers (age: 26.8 ± 1.2 y) from the intervention study (IS) consumed a breakfast containing 60% kcal fat (HF) and 36% kcal fat (moderately HF), respectively. For the IS, only the results from the placebo group are presented. Blood samples were probed for LPS-mediated cytokine production by incubating them with LPS inhibitor polymyxin B (PMB) for 24 h at 37°C besides the Limulus amebocyte lysate (LAL) assay. Repeated-measures ANOVA was used to compare the temporal changes of metabolic profiles and treatment outcomes. RESULTS: At least 87.5% of the plasma LPS measurements in 32 PS volunteers from each time point were below the LAL assay sensitivity (0.002 EU/mL). PMB suppressed IL-1ß (P = 0.035) and IL-6 (P = 0.0487) production in the 3 h PPB of the PS after 24 h incubation at 37°C compared to the vehicle control, suggesting the presence of LPS. However, the amount of LPS did not increase the cytokine concentrations in the 3 h PPB above the fasting concentrations. Such suppression was not detected in the PPB of the IS. Treating whole blood with lipoprotein lipase (LPL) significantly (P < 0.05) increased FFA and cytokine (IL-1ß, IL-6, TNF-α) concentrations in both studies. CONCLUSION: LPS may not be the major cause of postprandial inflammation in healthy adults consuming a moderately HF meal (36% kcal fat, similar to the typical American diet) or a HF meal (60% kcal fat). Plasma FFAs may modulate postprandial inflammation. The prevailing concept of HFD-induced metabolic endotoxemia requires careful re-evaluation. The PS was registered at clinicaltrials.gov as NCT02367287 and the IS as NCT02472171.

Resveratrol increases phagocytosis and lipopolysaccharide-induced interleukin-1ß production, but decreases surface expression of Toll-like receptor 2 in THP-1 monocytes.

THP-1 monocytes were used to evaluate the effects of physiological levels of resveratrol aglycone, resveratrol-3-O-glucuronide, resveratrol-4'-O-glucuronide, and resveratrol-3-O-sulfate on phagocytosis, IL-1ß, IL-1α, and IL-18 production, viability, and TLR2 and TLR4 expression. THP-1 cells were treated with 1, 5, 10, and 15µM resveratrol or metabolites. Resveratrol-3-O-glucuronide, resveratrol-4'-O-glucuronide, and resveratrol-3-O-sulfate had no effect on the functional parameters tested. Resveratrol aglycone increased phagocytosis at concentrations of 5, 10, and 15µM and LPS-induced IL-1ß production at concentrations of 10 and 15µM. Expression of TLR4 increased slightly after resveratrol treatment, but surface expression of TLR2 was reduced as resveratrol concentrations increased. Our data suggest that resveratrol may be effective in modulating monocyte function in an environment where there is direct exposure to the aglycone, such as at the gut epithelium.

Expression of GM-CSF in T Cells Is Increased in Multiple Sclerosis and Suppressed by IFN-ß Therapy.

Multiple sclerosis (MS) is an autoimmune disease of the CNS. Studies in animal models of MS have shown that GM-CSF produced by T cells is necessary for the development of autoimmune CNS inflammation. This suggests that GM-CSF may have a pathogenic role in MS as well, and a clinical trial testing its blockade is ongoing. However, there have been few reports on GM-CSF production by T cells in MS. The objective of this study was to characterize GM-CSF production by T cells of MS patients and to determine the effect of IFN-ß therapy on its production. GM-CSF production by peripheral blood (PB) T cells and the effects of IFN-ß were characterized in samples of untreated and IFN-ß-treated MS patients versus healthy subjects. GM-CSF production by T cells in MS brain lesions was analyzed by immunofluorescence. Untreated MS patients had significantly greater numbers of GM-CSF(+)CD4(+) and CD8(+) T cells in PB compared with healthy controls and IFN-ß-treated MS patients. IFN-ß significantly suppressed GM-CSF production by T cells in vitro. A number of CD4(+) and CD8(+) T cells in MS brain lesions expressed GM-CSF. Elevated GM-CSF production by PB T cells in MS is indicative of aberrant hyperactivation of the immune system. Given its essential role in animal models, abundant GM-CSF production at the sites of CNS inflammation suggests that GM-CSF contributes to MS pathogenesis. Our findings also reveal a potential mechanism of IFN-ß therapy, namely suppression of GM-CSF production.

Postprandial Inflammatory Responses and Free Fatty Acids in Plasma of Adults Who Consumed a Moderately High-Fat Breakfast with and without Blueberry Powder in a Randomized Placebo-Controlled Trial.

BACKGROUND: Saturated fatty acids (FAs) released from triglyceride-rich lipoproteins (TGRLs) activate Toll-like receptor 2 (TLR-2) and induce the expression of proinflammatory cytokines in monocytes. Certain plant polyphenols inhibit TLR-mediated signaling pathways. OBJECTIVE: We determined whether plasma free FAs (FFAs) after a moderately high-fat (MHF, 40% kcal from fat) breakfast modulate the inflammatory status of postprandial blood, and whether blueberry intake suppresses FFA-induced inflammatory responses in healthy humans. METHODS: Twenty-three volunteers with a mean ± SEM age and body mass index (in kg/m(2)) of 30 ± 3 y and 21.9 ± 0.4, respectively, consumed an MHF breakfast with either a placebo powder or 2 or 4 servings of blueberry powder in a randomized crossover design. The placebo powder was provided on the first test day and the blueberry powder doses were randomized with a 2-wk washout period. Plasma concentrations of lipids, glucose, and cytokines were determined. To determine whether FFAs derived from TGRL stimulate monocyte activation, and whether this is inhibited by blueberry intake, whole blood was treated with lipoprotein lipase (LPL). RESULTS: The median concentrations of FFAs and cytokines [tumor necrosis factor-α, interleukin (IL)-6 and IL-8] in postprandial plasma (3.5 h) decreased compared with fasting plasma regardless of the blueberry intake (P < 0.001 for FFAs and P < 0.05 for cytokines). However, concentrations of FFAs and cytokines including IL-1ß increased in LPL-treated whole blood compared with untreated blood samples from participants who consumed the placebo powder. Blueberry intake suppressed IL-1ß and IL-6 production in LPL-treated postprandial blood compared with the placebo control when fasting changes were used as a covariate. CONCLUSIONS: The plasma FFA concentration may be an important determinant affecting inflammatory cytokine production in blood. Supplementation with blueberry powder did not affect plasma FFA and cytokine concentrations; however, it attenuated the cytokine production induced by ex vivo treatment of whole blood with LPL. This trial was registered at clinicaltrials.gov as NCT01594008.

Acylcarnitines: potential implications for skeletal muscle insulin resistance.

Insulin resistance may be linked to incomplete fatty acid ß-oxidation and the subsequent increase in acylcarnitine species in different tissues including skeletal muscle. It is not known if acylcarnitines participate in muscle insulin resistance or simply reflect dysregulated metabolism. The aims of this study were to determine whether acylcarnitines can elicit muscle insulin resistance and to better understand the link between incomplete muscle fatty acid ß-oxidation, oxidative stress, inflammation, and insulin-resistance development. Differentiated C2C12, primary mouse, and human myotubes were treated with acylcarnitines (C4:0, C14:0, C16:0) or with palmitate with or without carnitine acyltransferase inhibition by mildronate. Treatment with C4:0, C14:0, and C16:0 acylcarnitines resulted in 20-30% decrease in insulin response at the level of Akt phosphorylation and/or glucose uptake. Mildronate reversed palmitate-induced insulin resistance concomitant with an ∼25% decrease in short-chain acylcarnitine and acetylcarnitine secretion. Although proinflammatory cytokines were not affected under these conditions, oxidative stress was increased by 2-3 times by short- or long-chain acylcarnitines. Acylcarnitine-induced oxidative stress and insulin resistance were reversed by treatment with antioxidants. Results are consistent with the conclusion that incomplete muscle fatty acid ß-oxidation causes acylcarnitine accumulation and associated oxidative stress, raising the possibility that these metabolites play a role in muscle insulin resistance.

Toll-like receptor 4-induced endoplasmic reticulum stress contributes to impairment of vasodilator action of insulin.

Impairment of vasodilator action of insulin is associated with endothelial dysfunction and insulin resistance. Activation of Toll-like receptor 4 (TLR4) induces proinflammatory response and endoplasmic reticulum (ER) stress. Saturated fatty acids (SFA) activate TLR4, which induces ER stress and endothelial dysfunction. Therefore, we determined whether TLR4-mediated ER stress is an obligatory step mediating SFA-induced endothelial dysfunction. Palmitate stimulated proinflammatory responses and ER stress, and this was suppressed by knockdown of TLR4 in primary human aortic endothelial cells (HAEC). Next, we examined the role of TLR4 in vasodilatory responses in intact vessels isolated from wild-type (WT, C57BL/6) and TLR4-KO mice after feeding high-fat (HFD) or normal chow diet (NCD) for 12 wk. Arterioles isolated from HFD WT mice exhibited impaired insulin-stimulated vasodilation compared with arterioles isolated from NCD WT mice. Deficiency of TLR4 was protective from HFD-induced impairment of insulin-stimulated vasodilation. There were no differences in acetylcholine (Ach)- or sodium nitroprusside (SNP)-stimulated vasodilation between the two groups. Furthermore, we examined whether ER stress is involved in SFA-induced impairment of vasodilator actions of insulin. Infusion of palmitate showed the impairment of vasodilatory response to insulin, which was ameliorated by coinfusion with tauroursodeoxycholic acid (TUDCA), an ER stress suppressor. Taken together, the results suggest that TLR4-induced ER stress may be an obligatory step mediating the SFA-mediated endothelial dysfunction.

Committed Tc17 cells are phenotypically and functionally resistant to the effects of IL-27.

IL-17-secreting CD8(+) T cells (Tc17 cells) have been implicated in immunity to infections, cancer, and autoimmune diseases. Thus far, studies on Tc17 cells have primarily investigated their development from naïve precursors, while the biology of committed Tc17 cells has been less characterized, in particular during the effector phase of immune responses. IL-27 is an important regulator of inflammation through the induction of regulatory Tr1 cells, as well as a suppressor of Th17-cell development. IL-27 suppresses the development of Tc17 cells, but its effects on committed Tc17 cells are unknown. Here we demonstrate that even though IL-27 completely inhibited the development of C57BL/6 mouse Tc17 cells, it had little effect on previously committed Tc17 cells. Although committed Tc17 cells were capable of responding to IL-27, it had no effect on expression of RORγt and RORα, or production of various cytokines. Committed Tc17 cells did not express granzyme B and lacked cytotoxicity in vitro, features that remained unaltered by IL-27 treatment. Nonetheless, they efficiently induced diabetes, irrespective of treatment with IL-27 prior to transfer into RIP-mOVA mice. These findings suggest that use of IL-27 to modulate autoimmune diseases might have limited therapeutic efficacy if autoaggressive Tc17 cells have already developed.

Inflammasome-mediated secretion of IL-1ß in human monocytes through TLR2 activation; modulation by dietary fatty acids.

Many studies have shown that TLR4- and TLR2-deficient mice are protected from high-fat diet-induced inflammation and insulin resistance, suggesting that saturated fatty acids derived from the high-fat diet activate TLR-mediated proinflammatory signaling pathways and induce insulin resistance. However, evidence that palmitic acid, the major dietary saturated fatty acid, can directly activate TLR has not been demonstrated. In this article, we present multiple lines of evidence showing that palmitic acid directly activates TLR2, a major TLR expressed on human monocytes, by inducing heterodimerization with TLR1 in an NADPH oxidase-dependent manner. Dimerization of TLR2 with TLR1 was inhibited by the n-3 fatty acid docosahexaenoic acid. Activation of TLR2 by palmitic acid leads to expression of pro-IL-1ß that is cleaved by caspase-1, which is constitutively present in monocytes, to release mature IL-1ß. Our results reveal mechanistic insight about how palmitic acid activates TLR2, upregulates NALP3 expression, and induces inflammasome-mediated IL-1ß production in human monocytes, which can trigger enhanced inflammation in peripheral tissues, and suggest that these processes are dynamically modulated by the types of dietary fat we consume.

Acylcarnitines activate proinflammatory signaling pathways.

Incomplete ß-oxidation of fatty acids in mitochondria is a feature of insulin resistance and type 2 diabetes mellitus (T2DM). Previous studies revealed that plasma concentrations of medium- and long-chain acylcarnitines (by-products of incomplete ß-oxidation) are elevated in T2DM and insulin resistance. In a previous study, we reported that mixed D,L isomers of C12- or C14-carnitine induced an NF-κB-luciferase reporter gene in RAW 264.7 cells, suggesting potential activation of proinflammatory pathways. Here, we determined whether the physiologically relevant L-acylcarnitines activate classical proinflammatory signaling pathways and if these outcomes involve pattern recognition receptor (PRR)-associated pathways. Acylcarnitines induced the expression of cyclooxygenase-2 in a chain length-dependent manner in RAW 264.7 cells. L-C14 carnitine (5-25 µM), used as a representative acylcarnitine, stimulated the expression and secretion of proinflammatory cytokines in a dose-dependent manner. Furthermore, L-C14 carnitine induced phosphorylation of JNK and ERK, common downstream components of many proinflammatory signaling pathways including PRRs. Knockdown of MyD88, a key cofactor in PRR signaling and inflammation, blunted the proinflammatory effects of acylcarnitine. While these results point to potential involvement of PRRs, L-C14 carnitine promoted IL-8 secretion from human epithelial cells (HCT-116) lacking Toll-like receptors (TLR)2 and -4, and did not activate reporter constructs in TLR overexpression cell models. Thus, acylcarnitines have the potential to activate inflammation, but the specific molecular and tissue target(s) involved remain to be identified.

PPARδ deficiency disrupts hypoxia-mediated tumorigenic potential of colon cancer cells.

Peroxisome proliferator-activated receptor (PPAR) δ is highly expressed in colon epithelial cells and closely linked to colon carcinogenesis. However, the role of PPARδ in colon cancer cells in a hypoxic tumor microenvironment is not fully understood. We found that expression of the tumor-promoting cytokines, IL-8 and VEGF, induced by hypoxia (<1% O2) and deferoxamine (a hypoxia mimetic) was significantly attenuated in PPARδ-deficient HCT116 colon cancer cells. Consequently, PPARδ-knockout colon cancer cells exposed to hypoxia and deferoxamine failed to stimulate endothelial cell vascularization and macrophage migration/proliferation, whereas wild-type cells were able to induce angiogenesis and macrophage activation in response to hypoxic stress. Hypoxic stress induced transcriptional activation of PPARδ, but not its protein expression, in HCT116 cells. Exogenous expression of p300 potentiated deferoxamine-induced PPARδ transactivation, while siRNA knockdown of p300 abolished hypoxia- and deferoxamine-induced PPARδ transactivation. PPARδ associated with p300 upon hypoxic stress as demonstrated by coimmunoprecipitation studies. PI3K inhibitors or siRNA knockdown of Akt suppressed the PPARδ transactivation induced by hypoxia and deferoxamine in HCT116 cells, leading to decreased expression of IL-8 and VEGF. Collectively, these results reveal that PPARδ is required for hypoxic stress-mediated cytokine expression in colon cancer cells, resulting in promotion of angiogenesis, macrophage recruitment, and macrophage proliferation in the tumor microenvironment. p300 and the PI3K/Akt pathway play a role in the regulation of PPARδ transactivation induced by hypoxic stress. Our results demonstrate the positive crosstalk between PPARδ in tumor cells and the hypoxic tumor microenvironment and provide potential therapeutic targets for colon cancer.