LPS Response Research Articles

COVID-19 extracellular vesicles display heterogeneity based on viral and host RNA expression: implications for host immune response.

Viral RNA and miRNAs released by immune cells contribute to inflammation in COVID-19 patients. Here, we investigated the role of SARS-CoV2 RNA and host miRNAs carried within extracellular vesicles (EVs) in modulating inflammation. EVs were classified as positive or negative depending on their viral RNA cargo. To assess the function of viral RNA, EVs, and LPS were used to stimulate whole blood samples from healthy subjects, and the secretion of 27 serum analytes was measured. EVs alone did not induce cytokines, chemokines, or growth factors. However, under LPS stimulation, (SARS-CoV2+) EVs increased IL-12 and decreased IL-13 secretion, while (SARS-CoV2-) EVs increased MIP-1α and IL-1β secretion. Host miR-19a-3p, -192-5p, -let-7c-5p, and -92b-3a were differentially expressed in association with viral RNA. EVs from COVID-19 patients exhibited differences in viral RNA and miRNA expression profiles that modulate LPS responses. This knowledge sheds light on the immunopathology of COVID-19.

Monocyte and macrophage profiles in patients with inherited long-chain fatty acid oxidation disorders

Patients with inherited disorders of the long-chain fatty acid oxidation (lcFAO) machinery present with a heterogeneous profile of disease manifestations and aggravation of symptoms is often triggered by inflammatory activation. Monocytes and macrophages are innate immune cells that play a major role in the onset and resolution of inflammation. These cells undergo metabolic rewiring upon activation including the regulation of the FAO rate. The rewiring of FAO and the effect of lcFAO disorders (lcFAOD) on human monocyte and macrophage phenotype and function remain largely unknown. Here, we performed extensive phenotyping of circulating monocytes and analyzed plasma cytokine levels in 11 lcFAOD patients and 11 matched control subjects. In patients with lcFAOD, we observed induced plasma levels of the inflammatory cytokines IL-1β and IL-6, and enhanced CD206 and CD62L surface marker expression in circulating monocyte subsets. To mimic the most common lcFAOD very-long-chain acyl-CoA dehydrogenase disorder (VLCADD), we used siRNA-mediated knockdown of the ACADVL gene (encoding VLCAD) in macrophages derived from healthy volunteers. Hereby, we found that siVLCAD affected IL-4-induced alternative macrophage activation while leaving LPS responses and cellular metabolism intact. In the same line, monocyte-derived macrophages from lcFAOD patients had elevated levels of the IL-4-induced alternative macrophage markers CD206 and CD200R. Still, they did not show major metabolic defects or changes in the LPS-induced inflammatory response. Our results indicate that monocytes and macrophages from lcFAOD patients present no major inflammatory or metabolic differences and show that IL-4-induced surface markers are intertwined with lcFAO in human macrophages.

Effects of Azithromycin on Blood Inflammatory Gene Expression and Cytokine Production in Sarcoidosis

IntroductionIn sarcoidosis granulomas, monocyte-derived macrophages are activated by pro-inflammatory cytokines including TNF and IL-6. Current drug treatment for sarcoidosis aims to suppress inflammation but disabling side effects can ensue. The macrolide azithromycin may be anti-inflammatory. We aimed to determine whether treatment with azithromycin affects blood inflammatory gene expression and monocyte functions in sarcoidosis.MethodsBlood samples were collected from patients with chronic pulmonary sarcoidosis enrolled in a single arm, open label clinical trial who received oral azithromycin 250 mg once daily for 3 months. Whole blood inflammatory gene expression with or without LPS stimulation was measured using a 770-mRNA panel. Phenotypic analysis and cytokine production were conducted by flow cytometry and ELISA after 24h stimulation with growth factors and TLR ligands. mTOR activity was assessed by measuring phosphorylated S6RP.ResultsDifferential gene expression analysis indicated a state of heightened myeloid cell activation in sarcoidosis. Compared with controls, sarcoidosis patients showed increased LPS responses for several cytokines and chemokines. Treatment with azithromycin had minimal effect on blood gene expression overall, but supervised clustering analysis identified several chemokine genes that were upregulated. At the protein level, azithromycin treatment increased LPS-stimulated TNF and unstimulated IL-8 production. No other cytokines showed significant changes following azithromycin. Blood neutrophil counts fell during azithromycin treatment whereas mononuclear cells remained stable. Azithromycin had no detectable effects on mTOR activity or activation markers.ConclusionBlood myeloid cells are activated in sarcoidosis, but azithromycin therapy did not suppress inflammatory gene expression or cytokine production in blood.Trial registration: EudraCT 2019-000580-24 (17 May 2019)

Impact of Extreme Prematurity, Chorioamnionitis, and Sepsis on Neonatal Monocyte Characteristics and Functions

Introduction: The innate branch of the immune system is important in early life, in particular for infants born preterm. Methods: We performed a longitudinal analysis of the peripheral monocyte compartment in extremely preterm children from a randomized, placebo-controlled study of probiotic supplementation. PBMCs and fecal samples were collected at several timepoints during the first months of life. Monocyte characteristics were analyzed by flow cytometry, and LPS-stimulated PBMC culture supernatants were analyzed by Luminex or ELISA. Plasma cytokines and gut microbiota composition were analyzed by ELISA and 16S rRNA-sequencing, respectively. Results: The extremely preterm infants had persistent alterations in their monocyte characteristics that were further aggravated in chorioamnionitis cases. They showed a markedly reduced TLR4 expression and hampered LPS-stimulated cytokine responses 14 days after birth. Notably, at later timepoints, TLR4 expression and LPS responses no longer correlated. Sepsis during the first weeks of life strongly associated with increased pro-inflammatory, and reduced IL-10, responses also at postmenstrual week 36. Further, we report a correlation between gut microbiota features and monocyte phenotype and responses, but also that probiotic supplementation associated with distinct monocyte phenotypic characteristics, without significantly influencing their responsiveness. Conclusion: Extremely preterm infants have monocyte characteristics and functional features that deviate from infants born full-term. Some of these differences persist until they reach an age corresponding to full-term, potentially making them more vulnerable to microbial exposures during the first months of life.

Comprehensive Analysis of scRNA-Seq and Bulk RNA-Seq Reveals Transcriptional Signatures of Macrophages in Intrahepatic Cholestasis of Pregnancy.

Intrahepatic cholestasis of pregnancy (ICP) is a disorder that characterized by maternal pruritus, abnormal liver function, and an elevation in total bile acid concentrations during pregnancy. Immune factors have been recognized as playing a vital role in the mechanism of ICP. However, the underlying mechanisms regulating dysfunctional immune cells and immune genes remain to be fully elucidated. Single-cell RNA sequencing and bulk RNA sequencing data of the placenta were downloaded from the SRA database. The AUCell package, Monocle package and SCENIC package were utilized to explored immune cell activity, cell trajectory and transcription factor, respectively. GO, KEGG, and GSEA were employed to explore potential biological mechanisms. Cell-cell communications were further investigated using the CellChat package. RT-PCR, and Western blot were used to verify the gene expression in placenta. In placenta cells, macrophages were found to be significantly increased in ICP. Additionally, macrophages exhibited the highest immune gene score and were divided into four subclusters (MF1-4). Our analysis revealed significant elevations in MF2, associated with LPS response and antigen presentation, and MF4, associated with TNF and cytokine production. MF3 displayed an anti-inflammatory phenotype. MF1, closely related to ribosomes and proteins, exhibited a sharp decrease. Although ICP maintained an anti-inflammatory state, macrophage trajectories showed a gradual progression toward inflammation. Subsequently, we confirmed that cytokine- and chemokine-related signaling pathways were emphasized in macrophages. Within the CXCL signaling pathway, the increased expression of CXCL1 in macrophages can interact with CXCR2 in neutrophils, potentially inducing macrophage infiltration, stimulating neutrophil chemotaxis, and leading to an inflammatory response and cellular damage. In conclusion, we firstly revealed the transcriptional signatures of macrophages in ICP and discovered a tendency toward an inflammatory state. This study also provides new evidence that the CXCL1-CXCR2 axis may play an important role in the pathogenesis of ICP.

Microglial responses to inflammatory challenge in adult rats altered by developmental exposure to polychlorinated biphenyls in a sex-specific manner

Polychlorinated biphenyls are ubiquitous environmental contaminants linkedc with peripheral immune and neural dysfunction. Neuroimmune signaling is critical to brain development and later health; however, effects of PCBs on neuroimmune processes are largely undescribed. This study extends our previous work in neonatal or adolescent rats by investigating longer-term effects of perinatal PCB exposure on later neuroimmune responses to an inflammatory challenge in adulthood. Male and female Sprague-Dawley rats were exposed to a low-dose, environmentally relevant, mixture of PCBs (Aroclors 1242, 1248, and 1254, 1:1:1, 20 μg / kg dam BW per gestational day) or oil control during gestation and via lactation. Upon reaching adulthood, rats were given a mild inflammatory challenge with lipopolysaccharide (LPS, 50 μg / kg BW, ip) or saline control and then euthanized 3 hours later for gene expression analysis or 24 hours later for immunohistochemical labeling of Iba1+ microglia. PCB exposure did not alter gene expression or microglial morphology independently, but instead interacted with the LPS challenge in brain region- and sex–specific ways. In the female hypothalamus, PCB exposure blunted LPS responses of neuroimmune and neuromodulatory genes without changing microglial morphology. In the female prefrontal cortex, PCBs shifted Iba1+ cells from reactive to hyperramified morphology in response to LPS. Conversely, in the male hypothalamus, PCBs shifted cell phenotypes from hyperramified to reactive morphologies in response to LPS. The results highlight the potential for long-lasting effects of environmental contaminants that are differentially revealed over a lifetime, sometimes only after a secondary challenge. These neuroimmune endpoints are possible mechanisms for PCB effects on a range of neural dysfunction in adulthood, including mental health and neurodegenerative disorders. The findings suggest possible interactions with other environmental challenges that also influence neuroimmune systems.

Effect of short-term turmeric supplementation on postprandial LPS AND APO-B48 response in IBS patients

Effect of short-term turmeric supplementation on postprandial LPS AND APO-B48 response in IBS patients

Platelet CD47 Maintains Circulating Monocyte Immune Homeostasis

In addition to their well-studied hemostatic functions, platelets are immune cells. Platelets circulate at the interface between the vascular wall and white blood cells, and transient platelet-leukocyte complexes are found in both healthy and disease states, positioning platelets to provide physiologic cues of both vascular health and injury. Roles for activated platelets in inducing and amplifying immune responses have received an increasing amount of research attention, but our past studies also showed that normal platelet counts are needed in healthy conditions to maintain immune homeostasis. Circulating monocytes are a part of the first line of host defenses that recognize and clear pathogens in part by releasing inflammatory cytokines, including IL-6, IL-8 and TNFα, that trigger a robust inflammatory reaction. However, this needs to be tightly regulated and in disease conditions like sepsis, dysregulated monocyte responses contribute to a “cytokine storm” that leads to exaggerated inflammation, tissue damage and potentially mortality. The cellular and molecular mechanisms that regulate monocyte immune phenotypes are complex and include roles for platelets. Considering the immune regulatory role of platelets and that thrombocytopenia is associated with worse sepsis outcomes, we hypothesize that resting platelets maintain monocyte immune homeostasis in healthy conditions and thrombocytopenia independently leads to monocyte dysregulation that exacerbates sepsis responses in a manner that is dependent on direct platelet-monocyte CD47 homotypic interactions that regulate monocyte immunometabolism and gene expression. Compared to monocytes from mice with normal platelet counts, circulating monocytes from thrombocytopenic mice had increased toll-like receptor (TLR) induced cytokine responses. Furthermore, ex vivo co-incubation of resting platelets with platelet naïve bone marrow monocytes, induced monocyte metabolic programming skewed toward glycolysis, consistent with our in vitro and in vivo RNA-Seq results. Enhanced glycolysis was mediated by AKT/mTOR pathway activation and resulted in durable changes in TLR agonist responses. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-Seq) on monocytes from thrombocytopenic mice showed open chromatin at LPS response genes, and resting platelet interactions with monocytes induced histone H3K9 and H3K27 trimethylation in a CD47 dependent manner. Using mouse models of thrombocytopenia and sepsis, we found that normal platelet numbers limited monocyte immune dysregulation and systemic inflammation. This was dependent on platelet CD47 as only WT platelets, not CD47 -/- platelet transfusion rescued the exaggerated inflammatory responses in thrombocytopenic mice (Fig 1). Both plasma IL-6 and IL-8 and circulating monocyte IL6 and IL8 gene expression from human patients with sepsis and non-septic conditions inversely correlated with patient platelet counts, further suggesting a resting platelet role in maintaining monocyte immune quiescence. Overall, our studies demonstrate that resting platelets maintain monocyte immune homeostasis by limiting TLR responses through regulating monocyte immunometabolic processes that lead to epigenetic changes in monocyte immune response genes (Fig 2). Thrombocytopenia therefore independently leads to immune dysregulation in sepsis, exacerbating systemic inflammatory processes. This is the first demonstration of sterile endogenous cell interactions that regulate innate immune-metabolism and monocyte pathogen responses.

Hyaluronan synthesis inhibition normalizes ethanol-enhanced hepatic stellate cell activation.

Chronic ethanol overconsumption promotes alcohol-associated liver disease (ALD), characterized by hepatocyte injury, inflammation, hepatic stellate cell (HSC) activation, and fibrosis. Hyaluronan (HA) concentration is greater in livers and blood from advanced ALD patients than patients with advanced non-ALD. In the liver, HSCs are the major HA producers. The relationship between ethanol, HA, and HSC activation is incompletely understood. Thus, here, we tested the hypothesis that ethanol enhances HSC activation in a HA-dependent manner. Liver tissue microarrays (TMAs) containing steatotic livers from donors with or without a history of alcohol consumption were used to measure HA and collagen content. Mice were fed a moderate (2%, v/v) ethanol-containing diet or pair-fed control diet for 2 days, after which they were given a single carbon tetrachloride (CCl4 ) injection. To inhibit HA synthesis, we provided 4-methylumbelliferone (4MU) daily. We used LX2 cells, a human HSC cell line, to determine the impact ethanol had on LPS responses, with or without concurrent 4MU exposure. CCl4 induced liver injury, but it did not differ between ethanol or control diet fed mice with or without 4MU treatment. Ethanol feeding enhanced CCl4 -induced hepatic HA content, which was paralleled by HA synthase (Has)2 transcript abundance; 4MU treatment normalized both. Consistently, HSC activation, assessed by measuring αSMA mRNA and protein, was induced by CCl4 exposure, enhanced by ethanol feeding, and normalized by 4MU. Hepatic transcripts, but not protein, for Ccl2 were enhanced by ethanol feeding and normalized by 4MU exposure. Finally, ethanol-exposed LX2 cells made more LPS-stimulated CCL2 mRNA and protein than cells not exposed to ethanol; 4MU prevented this. These data show that ethanol augments HSC activation through HA synthesis and enhances hepatic profibrogenic features. Therefore, targeting HSC HA production could potentially attenuate liver disease in ALD patients.

Abstract 163: Thrombocytopenia Independently Leads To Monocyte Immune Dysfunction

In addition to their well-studied hemostatic functions, platelets are immune cells. Platelets circulate at the interface between the vascular wall and leukocytes, and transient platelet-leukocyte complexes are found in both healthy and disease states, positioning platelets to provide physiologic cues of both vascular health and injury. Roles for activated platelets in inducing and amplifying immune responses have received an increasing amount of research attention, but our past studies also showed that normal platelet counts are needed in healthy conditions to maintain immune homeostasis. We have now found that thrombocytopenia (a low platelet count) leads to monocyte dysfunction, independent of the cause of thrombocytopenia, in a manner that is dependent on direct platelet-monocyte CD47 interactions that regulate monocyte immunometabolism and gene expression. Compared to monocytes from mice with normal platelet counts, monocytes from thrombocytopenic mice had increased toll-like receptor (TLR) responses, including increased IL-6 production. Furthermore, ex vivo co-incubation of resting platelets with platelet naïve bone marrow monocytes, induced monocyte metabolic programming and durable changes in TLR agonist responses. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-Seq) on monocytes from thrombocytopenic mice showed persistently open chromatin at LPS response genes. Using mouse models of thrombocytopenia and sepsis we found that normal platelet numbers are needed to limit immune dysregulation in sepsis. IL6 and TNF expression in monocytes from human patients with sepsis also inversely correlated with patient platelet counts. Our studies demonstrate that in healthy conditions resting platelets maintain monocyte immune tolerance by regulating monocyte immunometabolic processes that lead to epigenetic changes in TLR-related genes. Platelets therefore function as immune rheostats in both health and disease.

HuR modulation with tanshinone mimics impairs LPS response in murine macrophages.

Lipopolysaccharide exposure to macrophages induces an inflammatory response that is heavily regulated at the transcriptional and post-transcriptional levels. HuR (ELAVL1) is an RNA binding protein that binds and regulates the maturation and half-life of AU/U rich elements (ARE) containing cytokines and chemokines transcripts, mediating the LPS-induced response. Here we investigated how and to what extent small molecule tanshinone mimics (TMs) inhibiting HuR-RNA interaction counteract LPS stimulus in macrophages. We show TMs exist in solution in keto-enolic tautomerism and that, by molecular dynamic calculations, the orto quinone form is the bioactive species interacting with HuR and inhibiting its binding mode vs mRNA targets. A chemical blockage of the diphenolic, reduced form as a diacetate caused the loss of activity of TMs in vitro but resulted to prodrug-like activity in vivo. The murine macrophage cell line RAW264.7 was treated with LPS and TMs, and the modulation of cellular LPS-induced response was monitored by RNA and Ribonucleoprotein immunoprecipitation sequencing. Correlation analyses indicated that LPS induced a strong coupling between differentially expressed genes and HuR-bound genes, and that TMs reduced such interactions. Functional annotation addressed a specific set of genes involved in chemotaxis and immune response, such as Cxcl10, Il1b, Cd40, and Fas, with a decreased association with HuR, a reduction of their expression and protein secretion. The same effect was observed in primary murine bone marrow-derived macrophages, and in vivo in an LPS induced peritonitis model, in which the serum level of Cxcl10 and Il1b was strongly reduced, endowing TMs such as TM7nox with remarkable anti-inflammatory properties in vivo.

Increased Weight Gain and Insulin Resistance in HF-Fed PLTP Deficient Mice Is Related to Altered Inflammatory Response and Plasma Transport of Gut-Derived LPS

Bacterial lipopolysaccharides (LPS, endotoxins) are found in high amounts in the gut lumen. LPS can cross the gut barrier and pass into the blood (endotoxemia), leading to low-grade inflammation, a common scheme in metabolic diseases. Phospholipid transfer protein (PLTP) can transfer circulating LPS to plasma lipoproteins, thereby promoting its detoxification. However, the impact of PLTP on the metabolic fate and biological effects of gut-derived LPS is unknown. This study aimed to investigate the influence of PLTP on low-grade inflammation, obesity and insulin resistance in relationship with LPS intestinal translocation and metabolic endotoxemia. Wild-type (WT) mice were compared with Pltp-deficient mice (Pltp-KO) after a 4-month high-fat (HF) diet or oral administration of labeled LPS. On a HF diet, Pltp-KO mice showed increased weight gain, adiposity, insulin resistance, lipid abnormalities and inflammation, together with a higher exposure to endotoxemia compared to WT mice. After oral administration of LPS, PLTP deficiency led to increased intestinal translocation and decreased association of LPS to lipoproteins, together with an altered catabolism of triglyceride-rich lipoproteins (TRL). Our results show that PLTP, by modulating the intestinal translocation of LPS and plasma processing of TRL-bound LPS, has a major impact on low-grade inflammation and the onset of diet-induced metabolic disorders.

Lacticaseibacillus casei Strain Shirota Modulates Macrophage-Intestinal Epithelial Cell Co-Culture Barrier Integrity, Bacterial Sensing and Inflammatory Cytokines.

Probiotic bacteria modulate macrophage immune inflammatory responses, with functional cytokine responses determined by macrophage subset polarisation, stimulation and probiotic strain. Mucosal macrophages exhibit subset functional heterogeneity but are organised in a 3-dimensional tissue, over-laid by barrier epithelial cells. This study aimed to investigate the effects of the probiotic Lacticaseibacillus casei strain Shirota (LcS) on macrophage-epithelial cell cytokine responses, pattern recognition receptor (PRR) expression and LPS responses and the impacts on barrier integrity. THP-1-derived M1 and M2 subset macrophages were co-cultured in a transwell system with differentiated Caco-2 epithelial cells in the presence or absence of enteropathogenic LPS. Both Caco-2 cells in monoculture and macrophage co-culture were assayed for cytokines, PRR expression and barrier integrity (TEER and ZO-1) by RT-PCR, ELISA, IHC and electrical resistance. Caco-2 monocultures expressed distinct cytokine profiles (IL-6, IL-8, TNFα, endogenous IL-10), PRRs and barrier integrity, determined by inflammatory context (TNFα or IL-1β). In co-culture, LcS rescued ZO-1 and TEER in M2/Caco-2, but not M1/Caco-2. LcS suppressed TLR2, TLR4, MD2 expression in both co-cultures and differentially regulated NOD2, TLR9, Tollip and cytokine secretion. In conclusion, LcS selectively modulates epithelial barrier integrity, pathogen sensing and inflammatory cytokine profile; determined by macrophage subset and activation status.

Which ICU patients need stress ulcer prophylaxis?

Critically ill patients are at an increased risk for developing stress ulcers of the mucosa of the upper gastrointestinal (GI) tract. Bleeding from stress ulcers was previously associated with a longer stay in the intensive care unit and an increased risk of death. Thus, most patients admitted to the intensive care unit receive stress ulcer prophylaxis. However, there is a growing concern that acid-suppression drugs may be associated with increased frequency of nosocomial pneumonia and Clostridioides difficile infection. In this article, the authors address controversies regarding stress ulcer prophylaxis in critically ill patients and provide guidance for its appropriate use in this setting.

Supplementing Plant-Based Docosahexaenoic Acid From Bioactive Canola, with Arachidonic Acid, in Maternal Diet Is Beneficial for Immune System of the 3 Week BALB/c Offspring

ObjectivesTo understand the mechanism behind the programming effect of docosahexaenoic acid (DHA, derived from genetically modified Canola) supplementation (along with arachidonic acid, AA) during breastfeeding period on immune development of 3 wk pups using BALB/c mouse.MethodsPregnant mice, 5 days prior to parturition, were randomized to consume DHA diet (1% DHA, 1% AA w/w total fat, n = 10) or control diet (0% DHA and AA, n = 12) for 3-wk post-parturition (suckling period). At birth, pups were cross fostered to ensure equal pups per dam. Dams and 3-wk old pups were euthanized and spleen was collected for analysis. Immune cells were isolated to study ex-vivo cytokine production by innate (bacterial stimulant, lipopolysaccharide; LPS) and adaptive (lymphocyte stimulant phorbol myristate acetate + ionomycin; PMAi) immune cell. Cytokine were measured by Meso Scale (electrochemiluminescent assay) and splenocyte phenotyping by flow cytometry. Data was analyzed using Student's T test.ResultsFood intake, bodyweight, spleen weight and splenocyte count showed no diet effect in dams or pups. DHA diet in dams resulted in lower proportion of CD86+ (effector cells, P = 0.01) and higher proportion of CD138 + plasma B cells (P < 0.001) in splenocyte than control, however, total B cells (CD45R+) and total T cells (CD3+) showed no diet effect. Although, ex-vivo cytokine production (IL-1β, IL2, IL-4, IL-5, IL-6, IL-10, IL-12, CXCL-1, TNFα and IFN-γ) by splenocytes to PMAi in dams did not differ between diet groups. In pups, splenocytes proportion of effector T cells (CD3 + CD28+) was 20% lower, whereas mature B cells (CD19 + and IgG+) was higher in DHA diet group (P's < 0.05). Additionally, dendritic cell (CD103+) was 30% higher in DHA group with no difference in other innate cells. DHA diet significantly improved inflammatory response of innate cells to LPS (TNFα, IFNγ and IL-1β) and overall response of adaptive cells to PMAi (∼ 50% higher IL-10, IL-6, and CXCL-1) compared to control 3-wk pups.ConclusionsAdaptive immune cell response improved in 3-wk pups from dams supplemented with dietary DHA and AA, despite lower effector T cells, this may be due to higher antigen presentation function of mature B cells and dendritic cells.Funding SourcesAlberta Canola, NSERC, and product from Nuseed; ALES AGES (awarded to Patel, D.).

T cells dominate peripheral inflammation in obesity-associated diabetes

Abstract Many studies have examined obese/insulin-resistant mice to conclude that macrophages dominate pathogenic inflammation in metabolic disease, but few studies have ranked chronic sources of peripheral inflammation in obese people across a natural spectrum of metabolic health. We compared the relative contributions of myeloid and T cells to peripheral inflammation in samples from metabolically healthy and unhealthy obese people to address disappointing outcomes from clinical trials of anti-inflammatories in obesity-associated type 2 diabetes (T2D). We stimulated PBMCs from obese subjects with euglycemia, prediabetes, or T2D with T cell-targeting CD3/CD28 or myeloid-targeting LPS, and measured supernatant cytokines over time. Cytokines increased with time following CD3/CD28, but LPS responses were time-independent. A series of surface marker and intracellular staining assays taking into account these temporal differences in cytokine production unexpectedly showed that T cells contribute more TNFα to peripheral inflammation than do myeloid cells from all subject groups. Bioinformatic modeling combining cytokine outcomes from all subjects, stimuli, and time points, indicated IFNγ generated by CD3/CD28 stimulation was most important for differentiating peripheral inflammation in T2D compared to non-T2D samples. This result was consistent with the previously identified Th1/Th17 profile in T2D. We conclude that human T cells dominate peripheral inflammation in obesity-associated T2D, and thus targeting T cells may be an effective approach for prevention/management of obesity-associated metabolic diseases. Supported by NIH Training Grants T32 DK007778 and TL1 UL1TR001998, R01DK108056, the Shared Resource Facility of the University of Kentucky Markey Cancer Center P30 CA177558, University of Kentucky College of Medicine, Barnstable Brown Diabetes and Obesity Center, The Center for Clinical and Translational Research (UL1TR000117), and NIH National Center for Advancing Translational Sciences UL1TR001998.

LPS Response Is Impaired by Urban Fine Particulate Matter.

Fine particulate matter (PM2.5) is a complex mixture of components with diverse chemical and physical characteristics associated with increased respiratory and cardiovascular diseases mortality. Our study aimed to investigate the effects of exposure to concentrated PM2.5 on LPS-induced lung injury onset. BALB/c male mice were exposed to either filtered air or ambient fine PM2.5 in an ambient particle concentrator for 5 weeks. Then, an acute lung injury was induced with nebulized LPS. The animals were euthanized 24 h after the nebulization to either LPS or saline. Inflammatory cells and cytokines (IL-1β, IL-4, IL-5, IL-6, IL-10, IL-17, TNF) were assessed in the blood, bronchoalveolar lavage fluid (BALF), and lung tissue. In addition, lung morphology was assessed by stereological methods. Our results showed that the PM+LPS group showed histological evidence of injury, leukocytosis with increased neutrophils and macrophages, and a mixed inflammatory response profile, with increased KC, IL-6, IL-1β, IL-4, and IL-17. Our analysis shows that there is an interaction between the LPS nebulization and PM2.5 exposure, differently modulating the inflammatory response, with a distinct response pattern as compared to LPS or PM2.5 exposure alone. Further studies are required to explain the mechanism of immune modulation caused by PM2.5 exposure.

Unravelling the immunomodulatory role of apple phenolic rich extracts on human THP-1- derived macrophages using multiplatform metabolomics

Apples represent a significant source of dietary phenolic compounds with evidenced anti-inflammatory and immunomodulatory activities. Nevertheless, the effect of the whole apple matrix on human macrophages is unknown. In this context, our study attempts to evaluate the effect of apple-derived phenolic compounds-rich extracts (pulp, peel and leaf) on IL-1β production in THP-1-differentiated macrophages and derived metabolic alterations through untargeted metabolomics. Our results have showed that apple pulp treatment inhibited the release of the pro-inflammatory cytokine IL-1β induced by LPS in THP-1 macrophages by ELISA analysis. Metabolomics demonstrate that different proportions of phenolic compounds led to differential alterations in the metabolism of THP-1 macrophages. Indeed, apple extracts promoted alterations in lipid, carbohydrate, amino acid and vitamins as well as cofactors metabolism. Specifically, leaf extracts were characterized by alteration of galactose metabolism while the extracts derived from the fruit showed predominant alterations in lipids metabolism. All extracts mimicked the response observed under normal conditions in LPS-stimulated macrophages, inhibiting LPS response. Thus, the phenolic enriched extracts from apples will be a good source of natural compounds with a beneficial effect against inflammation, and they may be applied as a food supplement and/or functional ingredient for the treatment of inflammatory diseases.

Isoorientin Affects Markers of Alzheimer's Disease via Effects on the Oral and Gut Microbiota in APP/PS1 Mice

BackgroundThere is growing evidence of strong associations between the pathogenesis of Alzheimer's disease (AD) and dysbiotic oral and gut microbiota. Recent studies demonstrated that isoorientin (ISO) is anti-inflammatory and alleviates markers of AD, which were hypothesized to be mediated by the oral and gut microbiota. ObjectivesWe studied the effects of oral administration of ISO on AD-related markers and the oral and gut microbiota in mice. MethodsEight-month-old amyloid precursor protein/presenilin-1 (AP) transgenic male mice were randomly allocated to 3 groups of 15 mice each: vehicle (AP) alone or with a low dose of ISO (AP + ISO-L; 25 mg/kg) or a high dose of ISO (AP + ISO-H; 50 mg/kg). Age-matched wild-type (WT) C57BL/6 male littermates were used as controls. The 4 groups were treated intragastrically with ISO or sterilized ultrapure water for 2 months. AD-related markers in the brain, serum, colon, and liver were analyzed with immunohistochemical and histochemical staining, Western blotting, and ELISA. Oral and gut microbiotas were analyzed using 16S ribosomal RNA gene sequencing. ResultsThe high-dose ISO treatment significantly decreased amyloid beta 42–positive deposition by 38.1% and 45.2% in the cortex and hippocampus, respectively, of AP mice (P < 0.05). Compared with the AP group, both ISO treatments reduced brain phospho-Tau, phosphor-p65, phosphor–inhibitor of NF-κB, and brain and serum LPS and TNF-α by 17.9%–72.5% and increased brain and serum IL-4 and IL-10 by 130%–210% in the AP + ISO-L and AP + ISO-H groups (P < 0.05). Abundances of 26, 25, and 23 microbial taxa in oral, fecal and cecal samples, respectively, were increased in both the AP + ISO-L and AP + ISO-H groups relative to the AP group [linear discriminant analysis (LDA) >3.0; P < 0.05]. Gram-negative bacteria, Alteromonas, Campylobacterales, and uncultured Bacteroidales bacterium were positively correlated (rho = 0.28–0.59; P < 0.05) with the LPS levels and responses of inflammatory cytokines. ConclusionsThe microbiota-gut-brain axis is a potential mechanism by which ISO reduces AD-related markers in AP mice.

Lipopolysaccharide-Induced Cytokine Secretion from In Vitro Mouse Slow and Fast Limb Muscle.

Skeletal muscles play important roles in innate immunity. However, in vitro, their sensitivity to LPS is low. In other tissues, LPS sensing is facilitated by the presence of plasma, LPS binding protein (LBP), or soluble CD14 (sCD14). This study addressed whether these are critical for LPS sensitivity in skeletal muscle and whether LPS responsiveness is different between slow versus fast muscle. Soleus (SOL) or extensor digitorum longus (EDL) muscles from adult male C57bl/6 mice were mounted in 1 mL oxygenated baths containing: buffer only; buffer+1% mouse plasma; buffer+1 μg/mL LBP; or buffer+1% plasma from sCD14-/- mice. In each condition, muscles were exposed to LPS from 0 μg/mL to 1.0 μg/mL. Bath samples were collected at 0, 1, and 2 h, and analyzed using cytokine multiplex arrays. In both SOL and EDL the predominant responding cytokines/chemokines were KC(CXCL1), IL-6, and MCP-1(CCL2) and their average responses were amplified by ∼10-fold in the presence of 1% plasma. Overall, SOL and EDL exhibited similar secretory responses in the presence of 1% plasma, with a lower limit of sensitivity to LPS of 0.01 μg/mL. LBP supplementation did not augment secretion; however, 1% plasma from CD14-/- mice suppressed cytokine/chemokine secretion from EDL muscle. In conclusion, intact slow and fast mouse muscles have similar cytokine/chemokine responses to LPS but depend on the presence of low levels of plasma constituents. Though sCD14 plays some role in EDL muscle, neither sCD14 nor LBP can fully account for the strong effects of plasma on LPS sensitivity.