ß-Glucan Reverses the Epigenetic State of LPS-Induced Immunological Tolerance.

Innate immune memory is the phenomenon whereby innate immune cells such as monocytes or macrophages undergo functional reprogramming after exposure to microbial components such as lipopolysaccharide (LPS). We apply an integrated epigenomic approach to characterize the molecular events involved in LPS-induced tolerance in a time-dependent manner. Mechanistically, LPS-treated monocytes fail to accumulate active histone marks at promoter and enhancers of genes in the lipid metabolism and phagocytic pathways. Transcriptional inactivity in response to a second LPS exposure in tolerized macrophages is accompanied by failure to deposit active histone marks at promoters of tolerized genes. In contrast, ß-glucan partially reverses the LPS-induced tolerance in vitro. Importantly, ex vivo ß-glucan treatment of monocytes from volunteers with experimental endotoxemia re-instates their capacity for cytokine production. Tolerance is reversed at the level of distal element histone modification and transcriptional reactivation of otherwise unresponsive genes. VIDEO ABSTRACT.

The ATP-Binding Cassette Gene ABCF1 Functions as an E2 Ubiquitin-Conjugating Enzyme Controlling Macrophage Polarization to Dampen Lethal Septic Shock.

Sepsis is a bi-phasic inflammatory disease that threatens approximately 30 million lives and claims over 14 million annually, yet little is known regarding the molecular switches and pathways that regulate this disease. Here, we have described ABCF1, an ATP-Binding Cassette (ABC) family member protein, which possesses an E2 ubiquitin enzyme activity, through which it controls the Lipopolysaccharide (LPS)- Toll-like Receptor-4 (TLR4) mediated gram-negative insult by targeting key proteins for K63-polyubiquitination. Ubiquitination by ABCF1 shifts the inflammatory profile from an early phase MyD88-dependent to a late phase TRIF-dependent signaling pathway, thereby regulating TLR4 endocytosis and modulating macrophage polarization from M1 to M2 phase. Physiologically, ABCF1 regulates the shift from the inflammatory phase of sepsis to the endotoxin tolerance phase, and modulates cytokine storm and interferon-ß (IFN-ß)-dependent production by the immunotherapeutic mediator, SIRT1. Consequently, ABCF1 controls sepsis induced mortality by repressing hypotension-induced renal circulatory dysfunction.

Nitric oxide and tumor necrosis factor-⍺ levels are negatively correlated in endotoxin tolerance recovery in vitro.

Endotoxin tolerance (ET) is the hyporesponsiveness to lipopolysaccharide (LPS) after prior exposure. It is characterized by the downregulation of pro-inflammatory cytokine levels. Although ET protects against inflammation, its abolishment or recovery is critical for immunity. Nitric oxide (NO) plays various roles in the development of ET; however, its specific role in ET recovery remains unknown. To induce ET, RAW264.7 cells (a murine macrophage cell line) were pre-exposed to LPS (LPS1, 100 ng/mL for 24 h) and subsequently re-stimulated with LPS (LPS2, 100 ng/mL for 24 h). Expression of cytokines, NO, nitrite and inducible NO synthase (iNOS) were measured after 0, 12, 24, and 36 h of resting after LPS1 treatment with or without the iNOS-specific inhibitor, 1400W. LPS2-induced tumor necrosis factor-⍺ (TNF-⍺) and interleukin-6 (IL-6) were downregulated after LPS1 treatment, confirming the development of ET. Notably, TNF-⍺ and IL-6 levels spontaneously rebounded after 12-24 h of resting following LPS1 treatment. In contrast, levles of NO, nitrite and iNOS increased during ET development and decreased during ET recovery. Moreover, 1400W inhibited ET development and blocked the early production of NO (<12 h) during ET recovery. Our findings suggest a negative correlation between iNOS-induced NO and cytokine levels in the abolishment of ET.

Mapping the epigenomic landscape of human monocytes following innate immune activation reveals context-specific mechanisms driving endotoxin tolerance.

BACKGROUND: Monocytes are key mediators of innate immunity to infection, undergoing profound and dynamic changes in epigenetic state and immune function which are broadly protective but may be dysregulated in disease. Here, we aimed to advance understanding of epigenetic regulation following innate immune activation, acutely and in endotoxin tolerant states. METHODS: We exposed human primary monocytes from healthy donors (n = 6) to interferon-γ or differing combinations of endotoxin (lipopolysaccharide), including acute response (2 h) and two models of endotoxin tolerance: repeated stimulations (6 + 6 h) and prolonged exposure to endotoxin (24 h). Another subset of monocytes was left untreated (naïve). We identified context-specific regulatory elements based on epigenetic signatures for chromatin accessibility (ATAC-seq) and regulatory non-coding RNAs from total RNA sequencing. RESULTS: We present an atlas of differential gene expression for endotoxin and interferon response, identifying widespread context specific changes. Across assayed states, only 24-29% of genes showing differential exon usage are also differential at the gene level. Overall, 19.9% (6,884 of 34,616) of repeatedly observed ATAC peaks were differential in at least one condition, the majority upregulated on stimulation and located in distal regions (64.1% vs 45.9% of non-differential peaks) within which sequences were less conserved than non-differential peaks. We identified enhancer-derived RNA signatures specific to different monocyte states that correlated with chromatin accessibility changes. The endotoxin tolerance models showed distinct chromatin accessibility and transcriptomic signatures, with integrated analysis identifying genes and pathways involved in the inflammatory response, detoxification, metabolism and wound healing. We leveraged eQTL mapping for the same monocyte activation states to link potential enhancers with specific genes, identifying 1,946 unique differential ATAC peaks with 1,340 expression associated genes. We further use this to inform understanding of reported GWAS, for example involving FCHO1 and coronary artery disease. CONCLUSION: This study reports context-specific regulatory elements based on transcriptomic profiling and epigenetic signatures for enhancer-derived RNAs and chromatin accessibility in immune tolerant monocyte states, and demonstrates the informativeness of linking such elements and eQTL to inform future mechanistic studies aimed at defining therapeutic targets of immunosuppression and diseases.

TCR repertoire landscape reveals macrophage-mediated clone deletion in endotoxin tolerance.

BACKGROUND: Endotoxin tolerance (ET) is a protective mechanism in the process of sepsis, septic shock, and their sequelae including uncontrolled inflammation. Accumulating evidence has shown that peripheral T cells contribute to the induction of ET. However, what and how T-cell development contributes to ET inductions remain unclear. METHODS: Mice were intraperitoneally injected with LPS at a concentration of 5 mg/kg to establish an LPS tolerance model and were divided into two groups: a group examined 72 h after LPS injection (72-h group) and a group examined 8 days after LPS injection (8-day group). Injection of PBS was used as a control. We performed high-throughput sequencing to analyze the characteristics and changes of CD4+SP TCRß CDR3 repertoires with respect to V direct to J rearrangement during the ET induction. Moreover, the proportion and proliferation, as well as surface molecules such as CD80 and CD86, of F4/80+ macrophages were analyzed using FCM. Furthermore, ACT assay was designed and administered by the tail vein into murine LPS-induced mouse model to evaluate the role of F4/80+ macrophages on the development of CD4+SP thymocytes in ET condition. RESULTS: We found that the frequency and characteristics of the TCRß chain CDR3 changed obviously under condition of ET, indicating the occurrence of TCR rearrangement and thymocyte diversification. Moreover, the absolute numbers of F4/80+ macrophages, but not other APCs, were increased in thymic medulla at 72-h group, accompanied by the elevated function-related molecules of F4/80+ macrophages. Furthermore, adoptively transferred OVA332-339 peptide-loaded macrophages into Rag-1-/- mice induced the clone deletion of OVA-specific CD4+SP, thereby ameliorating the pathology in lung tissue in LPS challenge. CONCLUSIONS: These data reveal that the frequency and characteristics of the TCRß chain CDR3 undergo dynamic programming under conditions of LPS tolerance. Furthermore, the peripheral macrophages may be a key factor which carry peripheral antigen to thymic medulla and affect the negative selection of T-cell population, thereby contributing to the formation of ET. These results suggest that the clone selection in thymus in ET may confer protection against microbial sepsis.

Immunosuppressive effects of circulating bile acids in human endotoxemia and septic shock: patients with liver failure are at risk.

BACKGROUND: Sepsis-induced immunosuppression is a frequent cause of opportunistic infections and death in critically ill patients. A better understanding of the underlying mechanisms is needed to develop targeted therapies. Circulating bile acids with immunosuppressive effects were recently identified in critically ill patients. These bile acids activate the monocyte G-protein coupled receptor TGR5, thereby inducing profound innate immune dysfunction. Whether these mechanisms contribute to immunosuppression and disease severity in sepsis is unknown. The aim of this study was to determine if immunosuppressive bile acids are present in endotoxemia and septic shock and, if so, which patients are particularly at risk. METHODS: To induce experimental endotoxemia in humans, ten healthy volunteers received 2 ng/kg E. coli lipopolysaccharide (LPS). Circulating bile acids were profiled before and after LPS administration. Furthermore, 48 patients with early (shock onset within < 24 h) and severe septic shock (norepinephrine dose > 0.4 µg/kg/min) and 48 healthy age- and sex-matched controls were analyzed for circulating bile acids. To screen for immunosuppressive effects of circulating bile acids, the capability to induce TGR5 activation was computed for each individual bile acid profile by a recently published formula. RESULTS: Although experimental endotoxemia as well as septic shock led to significant increases in total bile acids compared to controls, this increase was mild in most cases. By contrast, there was a marked and significant increase in circulating bile acids in septic shock patients with severe liver failure compared to healthy controls (61.8 µmol/L vs. 2.8 µmol/L, p = 0.0016). Circulating bile acids in these patients were capable to induce immunosuppression, as indicated by a significant increase in TGR5 activation by circulating bile acids (20.4% in severe liver failure vs. 2.8% in healthy controls, p = 0.0139). CONCLUSIONS: Circulating bile acids capable of inducing immunosuppression are present in septic shock patients with severe liver failure. Future studies should examine whether modulation of bile acid metabolism can improve the clinical course and outcome of sepsis in these patients.

Thiosulfinate-Enriched Allium sativum Extract Exhibits Differential Effects between Healthy and Sepsis Patients: The Implication of HIF-1α.

Garlic (Allium sativum) has historically been associated with antioxidant, immunomodulatory, and microbiocidal properties, mainly due to its richness in thiosulfates and sulfur-containing phytoconstituents. Sepsis patients could benefit from these properties because it involves both inflammatory and refractory processes. We evaluated the effects of thiosulfinate-enriched Allium sativum extract (TASE) on the immune response to bacterial lipopolysaccharide (LPS) by monocytes from healthy volunteers (HVs) and patients with sepsis. We also explored the TASE effects in HIF-1α, described as the key transcription factor leading to endotoxin tolerance in sepsis monocytes through IRAK-M expression. Our results showed TASE reduced the LPS-triggered reactive oxygen species (ROS) production in monocytes from both patients with sepsis and HVs. Moreover, this extract significantly reduced tumor necrosis factor (TNF)-α, interleukin-1ß, and interleukin-6 production in LPS-stimulated monocytes from HVs. However, TASE enhanced the inflammatory response in monocytes from patients with sepsis along with increased expression of human leukocyte antigen-DR. Curiously, these dual effects of TASE on immune response were also found when the HV cohort was divided into low- and high-LPS responders. Although TASE enhanced TNFα production in the LPS-low responders, it decreased the inflammatory response in the LPS-high responders. Furthermore, TASE decreased the HIF-1α pathway-associated genes IRAK-M, VEGFA and PD-L1 in sepsis cells, suggesting HIF-1α inhibition by TASE leads to higher cytokine production in these cells as a consequence of IRAK-M downregulation. The suppression of this pathway by TASE was confirmed in vitro with the prolyl hydroxylase inhibitor dimethyloxalylglycine. Our data revealed TASE's dual effect on monocyte response according to status/phenotype and suggested the HIF-1α suppression as the possible underlying mechanism.

Endotoxin tolerance induced by Porphyromonas gingivalis lipopolysaccharide alters macrophage polarization.

Endotoxin tolerance refers to a state refractory to subsequent lipopolysaccharide (LPS) stimulations following a primary LPS exposure. To study the relationship between endotoxin tolerance and macrophage polarization, endotoxin tolerance was induced by 1 µg/mL LPS from the periodontal pathogen, Porphyromonas gingivalis (P. gingivalis), in peritoneal macrophages (PMs) and bone marrow-derived macrophages (BMDMs). Repeated P. gingivalis LPS challenges increased the quantities of CD206+ PMs, while the number of CD86+CD206+ PMs was reduced compared with the non-tolerant group (p < 0.05). However, there were no changes in BMDMs (p > 0.05). Down regulations of TNF-α, IL-12, nitric oxide and MMP-2 production, and upregulated IL-10, MMP-9 levels and arginase-1 activities occurred in tolerant PMs and BMDMs (p < 0.05). P. gingivalis LPS-tolerant PMs and BMDMs also enhanced scrape-wound healing abilities of 15p-1 cells (p < 0.05). Expressions of phospho-signal transducer and activator of transcription 6 (p-STAT6) and protein tyrosine phosphatase 1B (PTP1B) were increased, while p-MEK1/2 levels were downregulated in tolerant PMs and BMDMs (p < 0.05). IL-10 production in tolerant Stat6 knockdown RAW264.7 cells was lower than tolerant control cells (p < 0.05). P. gingivalis LPS-tolerant macrophages represented an intermediate state between M1/M2 polarization, which functioned as M2-like cells, and led to limited inflammatory responses and enhanced wound healing activities. The PTP1B-MEK1/2-STAT6 signaling pathway might be involved in the polarization of tolerant macrophages.

Placental extracellular vesicles-associated microRNA-519c mediates endotoxin adaptation in pregnancy.

BACKGROUND: Pregnancy represents a unique challenge for the maternal-fetal immune interface, requiring a balance between immunosuppression, which is essential for the maintenance of a semiallogeneic fetus, and proinflammatory host defense to protect the maternal-fetal interface from invading organisms. Adaptation to repeated inflammatory stimuli (endotoxin tolerance) may be critical in preventing inflammation-induced preterm birth caused by exaggerated maternal inflammatory responses to mild or moderate infections that are common during pregnancy. However, the exact mechanisms contributing to the maintenance of tolerance to repeated infections are not completely understood. MicroRNAs play important roles in pregnancy with several microRNAs implicated in gestational tissue function and in pathologic pregnancy conditions. MicroRNA-519c, a member of the chromosome 19 microRNA cluster, is a human-specific microRNA mainly expressed in the placenta. However, its role in pregnancy is largely unknown. OBJECTIVE: This study aimed to explore the role of "endotoxin tolerance" failure in the pathogenesis of an exaggerated inflammatory response often seen in inflammation-mediated preterm birth. In this study, we investigated the role of microRNA-519c, a placenta-specific microRNA, as a key regulator of endotoxin tolerance at the maternal-fetal interface. STUDY DESIGN: Using a placental explant culture system, samples from term and second-trimester placentas were treated with lipopolysaccharide. After 24 hours, the conditioned media were collected for analysis, and the placental explants were re-exposed to repeated doses of lipopolysaccharide for 3 days. The supernatant was analyzed for inflammatory markers, the presence of extracellular vesicles, and microRNAs. To study the possible mechanism of action of the microRNAs, we evaluated the phosphodiesterase 3B pathway involved in tumor necrosis factor alpha production using a microRNA mimic and phosphodiesterase 3B small interfering RNA transfection. Finally, we analyzed human placental samples from different gestational ages and from women affected by inflammation-associated pregnancies. RESULTS: Our data showed that repeated exposure of the human placenta to endotoxin challenges induced a tolerant phenotype characterized by decreased tumor necrosis factor alpha and up-regulated interleukin-10 levels. This reaction was mediated by the placenta-specific microRNA-519c packaged within placental extracellular vesicles. Lipopolysaccharide treatment increased the extracellular vesicles that were positive for the exosome tetraspanin markers, namely CD9, CD63, and CD81, and secreted primarily by trophoblasts. Primary human trophoblast cells transfected with a microRNA-519c mimic decreased phosphodiesterase 3B, whereas a lack of phosphodiesterase 3B, achieved by small interfering RNA transfection, led to decreased tumor necrosis factor alpha production. These data support the hypothesis that the anti-inflammatory action of microRNA-519c was mediated by a down-regulation of the phosphodiesterase 3B pathway, leading to inhibition of tumor necrosis factor alpha production. Furthermore, human placentas from normal and inflammation-associated pregnancies demonstrated that a decreased placental microRNA-519c level was linked to infection-induced inflammatory pathologies during pregnancy. CONCLUSION: We identified microRNA-519c, a human placenta-specific microRNA, as a novel regulator of immune adaptation associated with infection-induced preterm birth at the maternal-fetal interface. Our study serves as a basis for future experiments to explore the potential use of microRNA-519c as a biomarker for infection-induced preterm birth.

Synergistic Signaling of TLR and IFNα/ß Facilitates Escape of IL-18 Expression from Endotoxin Tolerance.

Rationale: IL-18 is a member of the IL-1 cytokine family, and elevated blood IL-18 concentrations associate with disease activity in macrophage activation syndrome (MAS) and poor clinical outcomes in severe inflammatory and septic conditions.Objectives: Although recent investigations provide mechanistic evidence for a contribution of IL-18 to inflammation and hyperinflammation in sepsis and MAS, we sought to study regulatory mechanisms underlying human IL-18 expression.Methods: Samples from in vivo and in vitro endotoxin rechallenge experiments, patients with inflammatory disease, and isolated human monocytes treated with various stimulants and drugs were tested for cytokine gene and protein expression. Serum IL-18 expression with or without JAK/STAT inhibition was analyzed in two MAS mouse models and in a patient with recurrent MAS.Measurements and Main Results: Peripheral blood and monocytic IL-18 expression escaped LPS-induced immunoparalysis. LPS-stimulated primary human monocytes revealed specific IL-18 expression kinetics controlled by IFNα/ß signaling. JAK/STAT inhibition or IFNß neutralization during LPS stimulation blunted cytokine expression. Similarly, microtubule-destabilizing drugs abrogated LPS-induced IL18 expression, but this effect could be fully reversed by addition of IFNα/ß. Ex vivo analysis of inflammatory disease patients' whole blood revealed strong correlation of type I IFN score and IL18 expression, whereas JAK/STAT inhibition strongly reduced IL-18 serum levels in two MAS mouse models and in a patient with recurrent MAS.Conclusions: Our data indicate that IL-18 (but not IL-1ß) production from human monocytes requires cooperative Toll-like receptor and IFNα/ß signaling. Interference with IFNα/ß expression or signaling following JAK/STAT inhibition may control catastrophic hyperinflammation in MAS.

Impaired antigen-specific lymphocyte priming in mice after Toll-like receptor 4 activation via induction of monocytic myeloid-derived suppressor cells.

In sepsis, the pathology involves a shift from a proinflammatory state toward an immunosuppressive phase. We previously showed that an agonistic anti-TLR4 antibody induced long-term endotoxin tolerance and suppressed antigen-specific secondary IgG production when primed prior to immunization with antigen. These findings led us to speculate that TLR4-induced innate tolerance due to primary infection causes an immunosuppressive pathology in sepsis. Therefore, the mechanism underlying impaired antigen-specific humoral immunity by the TLR4 antibody was investigated. We showed, in a mouse model, that primary antigen-specific IgG responses were impaired in TLR4 antibody-induced tolerized mice, which was the result of reduced numbers of antigen-specific GC B cells and plasma cells. Ovalbumin-specific CD4 and CD8 T-cell responses were impaired in TLR4 antibody-injected OT-I and -II transgenic mice ex vivo. Adoptive transfer studies demonstrated suppression of OVA-specific CD4 and CD8 T-cell responses by the TLR4 antibody in vivo. The TLR4 antibody induced Gr1+ CD11b+ myeloid-derived suppressor cell (MDSC) expansion with suppression of T-cell activation. Monocytic MDSCs were more suppressive and exhibited higher expression of PD-L1 and inducible nitric oxidase compared with granulocytic MDSCs. In conclusion, immune tolerance conferred by TLR4 activation induces the expansion of monocytic MDSCs, which impairs antigen-specific T-cell priming and IgG production.

Oxylipin Profiles as Functional Characteristics of Acute Inflammatory Responses in Astrocytes Pre-Treated with IL-4, IL-10, or LPS.

Functional phenotypes, which cells can acquire depending on the microenvironment, are currently the focus of investigations into new anti-inflammatory therapeutic approaches. Glial cells, microglia, and astrocytes are major participants in neuroinflammation, but their roles differ, as microglia are cells of mesodermal origin, while astrocytes are cells of ectodermal origin. The inflammatory phenotype of cells can be modulated by ω-6- and ω-3-polyunsaturated fatty acid-derived oxylipins, although data on changes in oxylipin profiles in different cell adaptations to pro- and anti-inflammatory stimuli are scarce. Our study aimed to compare UPLC-MS/MS-measured oxylipin profiles in various rat astrocyte adaptation states. We used cells treated for 24 h with lipopolysaccharide (LPS) for classical pro-inflammatory adaptation and with interleukin 4 (IL-4) or 10 (IL-10) for alternative anti-inflammatory adaptation, with the resulting phenotypes characterized by quantitative real-time PCR (RT-PCR). We also tested long-term, low-concentration LPS treatment (endotoxin treatment) as a model of astrocyte adaptations. The functional response of astrocytes was estimated by acute (4 h) LPS-induced cell reactivity, measured by gene expression markers and oxylipin synthesis. We discovered that, as well as gene markers, oxylipin profiles can serve as markers of pro- (A1-like) or anti-inflammatory (A2-like) adaptations. We observed predominant involvement of ω-6 polyunsaturated fatty acid (PUFA) and the cyclooxygenase branch for classical (LPS) pro-inflammatory adaptations and ω-3 PUFA and the lipoxygenase branch for alternative (IL-4) anti-inflammatory adaptations. Treatment with IL-4, but not IL-10, primes the ability of astrocytes to activate the innate immunity signaling pathways in response to LPS. Endotoxin-treated astrocytes provide an alternative anti-inflammatory adaptation, which makes cells less sensitive to acute LPS stimulation than the IL-4 induced adaptation. Taken together, the data reveal that oxylipin profiles associate with different states of polarization to generate a pro-inflammatory or anti-inflammatory phenotype. This association manifests itself both in native cells and in their responses to a pro-inflammatory stimulus.

The effects of CD14 and IL-27 on induction of endotoxin tolerance in human monocytes and macrophages.

Upon repeated exposure to endotoxin or lipopolysaccharide (LPS), myeloid cells enter a refractory state called endotoxin tolerance as a homeostatic mechanism. In innate immune cells, LPS is recognized by co-receptors Toll-like receptor 4 (TLR4) and CD-14 to initiate an inflammatory response for subsequent cytokine production. One such cytokine, interleukin (IL)-27, is produced by myeloid cells in response to bacterial infection. In monocytes, IL-27 has proinflammatory functions such as up-regulating TLR4 expression for enhanced LPS-mediated cytokine production; alternatively, IL-27 induces inhibitory functions in activated macrophages. This study investigated the effects of IL-27 on the induction of endotoxin tolerance in models of human monocytes compared with macrophages. Our data demonstrate that IL-27 inhibits endotoxin tolerance by up-regulating cell surface TLR4 expression and soluble CD14 production to mediate stability of the surface LPS-TLR4-CD14 complex in THP-1 cells. In contrast, elevated basal expression of membrane-bound CD14 in phorbol 12-myristate 13-acetate (PMA)-THP-1 cells, primary monocytes, and primary macrophages may promote CD14-mediated endocytosis and be responsible for the preservation of an endotoxin-tolerized state in the presence of IL-27. Overall, the efficacy of IL-27 in inhibiting endotoxin tolerance in human THP-1 monocytes and PMA-THP-1 macrophages is affected by membrane-bound and soluble CD14 expression.

Prolonged lipopolysaccharide exposure induces transient immunosuppression in BV2 microglia.

Continuous pre-exposure of immune cells to low level of inflammatory stimuli makes them hyporesponsive to subsequent exposure. This pathophysiological adaptation; known as endotoxin tolerance is a general paradigm behind several disease pathogenesis. Current study deals with this immunosuppression with respect to BV2 microglia. We attempted to investigate their immune response under prolonged endotoxin exposure and monitor the same upon withdrawal of the stimuli. BV2 microglia cells were maintained under continual exposure of lipopolysaccharide (LPS) for weeks with regular passage after 72 hr (prolonged LPS exposed cells [PLECs]). PLECs were found to be immunosuppressed with diminished expression of proinflammatory cytokines (IL6, IL1ß, TNF-α, and iNOS) and production of nitric oxide, as compared to once LPS exposed cells. Upon remaintenance of cells in normal media without LPS exposure (LPS withdrawal cells [LWCs]), the induced immunosuppression reversed and cells started responding to inflammatory stimuli; revealed by significant expression of proinflammatory cytokines. LWCs showed functional similarities to never LPS exposed cells (NLECs) in phagocytosis activity and their response to anti-inflammatory agents like dexamethasone. Despite their immunoresponsiveness, PLECs were inflamed and showed higher autophagy rate than NLECs. Additionally, we investigated the role of inhibitor of apoptotic proteins (IAPs) in PLECs to understand whether IAPs aids in the survival of microglial cells under stress conditions. Our results revealed that cIAP1 and cIAP2 are induced in PLECs which might play a role in retaining the viability. Furthermore, antagonism of IAPs has significantly induced cell death in PLECs suggesting the role of IAPs in microglial survival under stress condition. Conclusively, our data suggest that continuous exposure of BV2 microglia cells to LPS results in transient immunosuppression and indicates the involvement of IAPs in retaining their viability under inflammatory stress.

Inhibition of indoleamine 2,3-dioxygenase attenuates endotoxin-mediated tolerance in granulosa cells through kynurenine pathway.

Ovarian granulosa cells (GCs) have been shown to have innate immune capabilities, which modulate their native endocrine functions through toll-like receptors (TLRs). We have recently shown that GCs exposed to lipopolysaccharide (LPS; 1.0 µg/mL) transiently regulate proinflammatory cytokine expression (interleukin 1ß [IL-1ß], IL-6, and tumor necrosis factor α) through chromatin remodeling. In the present study, we have demonstrated that GCs become tolerant to LPS on repeated exposure of LPS. To understand the mechanism of this endotoxin tolerance (ET) phenomenon in buffalo GCs, we have further studied the genome-wide transcriptomic analyses in buffalo GCs (unpublished data) and identified indoleamine 2,3-dioxygenase 1 (IDO1) gene, known to be involved in tryptophan catabolism, was found to be highly upregulated in endotoxin-tolerant GCs. Real-time gene expression analyses also showed similar results. Further analyses of tryptophan and tryptophan metabolite, kynurenine, showed that tryptophan was found to be depleted with the accumulation of kynurenine in the endotoxin-tolerant GCs. The effect of IDO1 induced ET was reversed when cells were pretreated with IDO1 inhibitor (1-methyl tryptophan, 1 mM). To the best of our knowledge, this is the first report describing the role of IDO1 gene in ET in GCs mimicked by repeated endotoxin exposure in vitro. In summary, the present study convincingly demonstrated that the tryptophan catabolism, through the kynurenine pathway, plays a crucial role as an immunomodulatory mechanism of ET in GCs. The finding could be exploited in developing potential therapeutics to treat impaired GCs function due to the ET underlying prolonged uterine or systemic infection leads to accumulation of endotoxin in follicular fluid.

Recent advances in endotoxin tolerance.

Endotoxin tolerance is defined as a reduced capacity of a cell to respond endotoxin (lipopolysaccharide, LPS) challenge after an initial encounter with endotoxin in advance. The body becomes tolerant to subsequent challenge with a lethal dose of endotoxin and cytokines release and cell/tissue damage induced by inflammatory reaction are significantly reduced in the state of endotoxin tolerance. The main characteristics of endotoxin tolerance are downregulation of inflammatory mediators such as tumor necrosis factor α (TNF-α), interleukin-1ß (IL-1ß), and C-X-C motif chemokine 10 (CXCL10) and upregulation of anti-inflammatory cytokines such as IL-10 and transforming growth factor ß (TGF-ß). Therefore, endotoxin tolerance is often regarded as the regulatory mechanism of the host against excessive inflammation. Endotoxin tolerance is a complex pathophysiological process and involved in multiple cellular signal pathways, receptor alterations, and biological molecules. However, the exact mechanism remains elusive up to date. To better understand the underlying cellular and molecular mechanisms of endotoxin tolerance, it is crucial to investigate the comprehensive cellular signal pathways, signaling proteins, cell surface molecules, proinflammatory and anti-inflammatory cytokines, and other mediators. Endotoxin tolerance plays an important role in reducing the mortality of sepsis, endotoxin shock, and other endotoxin-related diseases. Recent reports indicated that endotoxin tolerance is also related to other diseases such as cystic fibrosis, acute coronary syndrome, liver ischemia-reperfusion injury, and cancer. The aim of this review is to discuss the recent advances in endotoxin tolerance mainly based on the cellular and molecular mechanisms by outline the current state of the knowledge of the involvement of the toll-like receptor 4 (TLR4) signaling pathways, negative regulate factor, microRNAs, apoptosis, chromatin modification, and gene reprogramming of immune cells in endotoxin tolerance. We hope to provide a new idea and scientific basis for the rational treatment of endotoxin-related diseases such as endotoxemia, sepsis, and endotoxin shock clinically.

Responses of perivascular macrophages to circulating lipopolysaccharides in the subfornical organ with special reference to endotoxin tolerance.

BACKGROUND: Circulating endotoxins including lipopolysaccharides (LPS) cause brain responses such as fever and decrease of food and water intake, while pre-injection of endotoxins attenuates these responses. This phenomenon is called endotoxin tolerance, but the mechanisms underlying it remain unclear. The subfornical organ (SFO) rapidly produces proinflammatory cytokines including interleukin-1ß (IL-1ß) in response to peripherally injected LPS, and repeated LPS injection attenuates IL-1ß production in the SFO, indicating that the SFO is involved in endotoxin tolerance. The purpose of this study is to investigate features of the IL-1ß source cells in the SFO of LPS-non-tolerant and LPS-tolerant mice. METHODS: We first established the endotoxin-tolerant mouse model by injecting LPS into adult male mice (C57BL/6J). Immunohistochemistry was performed to characterize IL-1ß-expressing cells, which were perivascular macrophages in the SFO. We depleted perivascular macrophages using clodronate liposomes to confirm the contribution of IL-1ß production. To assess the effect of LPS pre-injection on perivascular macrophages, we transferred bone marrow-derived cells obtained from male mice (C57BL/6-Tg (CAG-EGFP)) to male recipient mice (C57BL/6N). Finally, we examined the effect of a second LPS injection on IL-1ß expression in the SFO perivascular macrophages. RESULTS: We report that perivascular macrophages but not parenchymal microglia rapidly produced the proinflammatory cytokine IL-1ß in response to LPS. We found that peripherally injected LPS localized in the SFO perivascular space. Depletion of macrophages by injection of clodronate liposomes attenuated LPS-induced IL-1ß expression in the SFO. When tolerance developed to LPS-induced sickness behavior in mice, the SFO perivascular macrophages ceased producing IL-1ß, although bone marrow-derived perivascular macrophages increased in number in the SFO and peripherally injected LPS reached the SFO perivascular space. CONCLUSIONS: The current data indicate that perivascular macrophages enable the SFO to produce IL-1ß in response to circulating LPS and that its hyporesponsiveness may be the cause of endotoxin tolerance.

Immunobiology and application of toll-like receptor 4 agonists to augment host resistance to infection.

Infectious diseases remain a threat to critically ill patients, particularly with the rise of antibiotic-resistant bacteria. Septic shock carries a mortality of up to ∼40% with no compelling evidence of promising therapy to reduce morbidity or mortality. Septic shock survivors are also prone to nosocomial infections. Treatment with toll-like receptor 4 (TLR4) agonists have demonstrated significant protection against common nosocomial pathogens in various clinically relevant models of infection and septic shock. TLR4 agonists are derived from a bacteria cell wall or synthesized de novo, and more recently novel small molecule TLR4 agonists have also been developed. TLR4 agonists augment innate immune functions including expansion and recruitment of innate leukocytes to the site of infection. Recent studies demonstrate TLR4-induced leukocyte metabolic reprogramming of cellular metabolism to improve antimicrobial function. Metabolic changes include sustained augmentation of macrophage glycolysis, mitochondrial function, and tricarboxylic acid cycle flux. These findings set the stage for the use of TLR4 agonists as standalone therapeutic agents or antimicrobial adjuncts in patient populations vulnerable to nosocomial infections.

Innate Immune Function and Organ Failure Recovery in Adults With Sepsis.

PURPOSE: Sepsis stimulates pro- and anti-inflammatory immune responses. The innate immune response is critical to organ injury repair. We tested for an association between innate immune function and organ function recovery in a prospective cohort of immune-competent adults with sepsis. METHODS: We conducted a prospective observational cohort study enrolling immune-competent adults with sepsis. We tested innate immune function by quantification of lipopolysaccharide (LPS)-stimulated tumor necrosis factor (TNF) α production capacity in whole blood samples on hospital days 1, 4, and 6. The primary outcome was organ function recovery on day 4 defined as a 4-point decrease in the composite cardiovascular and respiratory Sequential Organ Failure Assessment (SOFA) score components or a SOFA score ≤2. RESULTS: Patients with sepsis who recovered organ function by day 4 (n = 11) had similar baseline characteristics when compared to those with ongoing organ failure (n = 13). Tumor necrosis factor α production capacity was similar between the 2 groups on hospital days 1 and 4 but significantly different on day 6. Patients who regained organ function recovery had significantly higher TNF-α production capacity on day 6 ( P = .01), which persisted after adjustment for age, Acute Physiology and Chronic Health Evaluation III score, and steroid administration ( P = .03). There was no difference in TNF-α production capacity over time in those who survived to hospital discharge versus nonsurvivors. CONCLUSION: Increasing TNF-α production capacity is associated with improved organ failure recovery. Further studies are needed to evaluate a causal association between innate immune suppression and organ failure recovery as well as predictive accuracy for hospital survival. Impaired TNF-α production as a marker of sepsis-associated innate immune dysfunction may be a feasible target for immune stimulation to decrease time to organ failure recovery.

Myalgic encephalomyelitis or chronic fatigue syndrome: how could the illness develop?

A model of the development and progression of chronic fatigue syndrome (myalgic encephalomyelitis), the aetiology of which is currently unknown, is put forward, starting with a consideration of the post-infection role of damage-associated molecular patterns and the development of chronic inflammatory, oxidative and nitrosative stress in genetically predisposed individuals. The consequences are detailed, including the role of increased intestinal permeability and the translocation of commensal antigens into the circulation, and the development of dysautonomia, neuroinflammation, and neurocognitive and neuroimaging abnormalities. Increasing levels of such stress and the switch to immune and metabolic downregulation are detailed next in relation to the advent of hypernitrosylation, impaired mitochondrial performance, immune suppression, cellular hibernation, endotoxin tolerance and sirtuin 1 activation. The role of chronic stress and the development of endotoxin tolerance via indoleamine 2,3-dioxygenase upregulation and the characteristics of neutrophils, monocytes, macrophages and T cells, including regulatory T cells, in endotoxin tolerance are detailed next. Finally, it is shown how the immune and metabolic abnormalities of chronic fatigue syndrome can be explained by endotoxin tolerance, thus completing the model.