Endotoxin tolerance represents a distinctive state of alternative polarization (M2) in human mononuclear cells

Classical (M1) and alternative (M2) polarization of mononuclear cells (MNCs) such as monocyte and macrophages is known to occur in response to challenges within a microenvironment, like the encounter of a pathogen. LPS, also known as endotoxin, is a potent inducer of inflammation and M1 polarization. LPS can also generate an effect in MNCs known as endotoxin tolerance, defined as the reduced capacity of a cell to respond to LPS activation after an initial exposure to this stimulus. Using systems biology approaches in PBMCs, monocytes, and monocyte-derived macrophages involving microarrays and advanced bioinformatic analysis, we determined that gene responses during endotoxin tolerance were similar to those found during M2 polarization, featuring gene and protein expression critical for the development of key M2 MNC functions, including reduced production of proinflammatory mediators, expression of genes involved in phagocytosis, as well as tissue remodeling. Moreover, expression of different metallothionein gene isoforms, known for their role in the control of oxidative stress and in immunomodulation, were also found to be consistently upregulated during endotoxin tolerance. These results demonstrate that after an initial inflammatory stimulus, human MNCs undergo an M2 polarization probably to control hyperinflammation and heal the affected tissue.     

Immune Responsive Gene 1 (IRG1) Promotes Endotoxin Tolerance by Increasing A20 Expression in Macrophages through Reactive Oxygen Species

Sepsis-associated immunosuppression (SAIS) is regarded as one of main causes for the death of septic patients at the late stage because of the decreased innate immunity with a more opportunistic infection. LPS-tolerized macrophages, which are re-challenged by LPS after prior exposure to LPS, are regarded as the common model of hypo-responsiveness for SAIS. However, the molecular mechanisms of endotoxin tolerance and SAIS remain to be fully elucidated. In addition, negative regulation of the Toll-like receptor (TLR)-triggered innate inflammatory response needs further investigation. Here we show that expression of immune responsive gene 1 (IRG1) was highly up-regulated in the peripheral blood mononuclear cells of septic patients and in LPS-tolerized mouse macrophages. IRG1 significantly suppressed TLR-triggered production of proinflammatory cytokines TNF-α, IL-6, and IFN-β in LPS-tolerized macrophages, with the elevated expression of reactive oxygen species (ROS) and A20. Moreover, ROS enhanced A20 expression by increasing the H3K4me3 modification of histone on the A20 promoter domain, and supplement of the ROS abrogated the IRG1 knockdown function in breaking endotoxin tolerance by increasing A20 expression. Our results demonstrate that inducible IRG1 promotes endotoxin tolerance by increasing A20 expression through ROS, indicating a new molecular mechanism regulating hypoinflammation of sepsis and endotoxin tolerance.     

Endotoxin tolerance in rats: expression of TNF-alpha, IL-6, IL-10, VCAM-1 AND HSP 70 in lung and liver during endotoxin shock.

Endotoxin can induce a state of tolerance against its own pathological effects, commonly referred to as endotoxin tolerance. This phenomenon has been found to be associated with reduced serum levels of cytokines such as TNF-alpha, IL-1, IL-6 and IL-10. In the present study the expression of TNF-alpha, IL-6, IL-10, the adhesion molecule VCAM-1 and the heat shock protein 70 was determined in vivo in lung and liver of LPS-tolerant and naive rats by means of semiquantitative RT-PCR after i.v. LPS injection. TNFalpha, IL-6, IL-10, HSP 70 and VCAM-1 were induced in lung and liver after LPS injection. In liver and lung of endotoxin-tolerant rats TNF-alpha and IL-6 were induced to a lower degree after LPS treatment when compared to non-tolerant controls. The LPS-induced IL-10 expression was also slightly attenuated in the lung of tolerant rats, but in the liver no differences between tolerant and non-tolerant animals were observed. HSP 70 and VCAM-1 were expressed after systemic LPS treatment in liver and lung. The degree of induction, however, was the same in tolerant and untreated controls. The presented data show that endotoxin tolerance is reflected by a reduced cytokine expression in lung and liver in vivo. On the other hand, levels of expression of the adhesion molecule VCAM-1 and the stress protein HSP 70 do not appear to be changed by endotoxin tolerance.     

SMAD4 is Involved in the Development of Endotoxin Tolerance in Microglia

Initial exposure of macrophages to LPS induces hyporesponsiveness to a second challenge with LPS, a phenomenon termed LPS tolerance. Smad4 plays important roles in the induction of LPS tolerance. However, the function of Smad4 in microglia remains unknown. Here we show that expression of Smad4 was highly up-regulated in LPS-tolerized mouse cerebral cortex. Smad4 was mostly colocalized with microglia, rarely with neurons. Using a microglia cell line, BV2, we find that LPS activates endogenous Smad4, inducing its migration into the nucleus and increasing its expression. Smad4 significantly suppressed TLR-triggered production of proinflammatory cytokines (IL-6), increased anti-inflammatory cytokine in LPS-tolerized microglia. Moreover, IL-6 concentrations in culture supernatants after second LPS challenge are higher in SMAD4 small interfering RNA (siRNA) BV2 cells than control siRNA BV2 cells, indicating failure to induce tolerance in absence of Smad4 signaling. In our study, we conclude that both in vivo and in vitro, Smad4 signaling is required for maximal induction of endotoxin tolerance.     

Syndecan-4 deficiency leads to high mortality of lipopolysaccharide-injected mice

Syndecan-4 is a transmembrane heparan sulfate proteoglycan belonging to the syndecan family. Following intraperitoneal injection of lipopolysaccharide (LPS), syndecan-4-deficient mice exhibited high mortality compared with wild-type controls. Severe endotoxin shock was observed in the deficient mice: systolic blood pressure and left ventricular fractional shortening were lower in the deficient mice than in the wild-type controls 9 h after LPS injection. Although histological examinations revealed no apparent differences between two groups, the plasma level of interleukin (IL)-1beta was higher in the deficient mice than in the wild-type controls 9 h after LPS injection. Consistent with the regulatory roles of syndecan-4, its expression in monocytes and endothelial cells of microvasculature increased in the wild-type mice after LPS administration. Although IL-1beta was produced to the same extent by macrophages from syndecan-4-deficient and wild-type mice after LPS stimulation, inhibition of its production by transforming growth factor-beta1 was impaired in the syndecan-4-deficient macrophages. These results indicate that syndecan-4 could be involved in prevention of endotoxin shock, at least partly through the inhibitory action of transforming growth factor-beta1 on IL-1beta production.     

Cutting edge: endotoxin tolerance in mouse peritoneal macrophages correlates with down-regulation of surface toll-like receptor 4 expression

Monocytes/macrophages exposed to LPS show reduced responses to second stimulation with LPS, which is termed LPS tolerance. In this study, we investigated molecular mechanism of LPS tolerance in macrophages. Mouse peritoneal macrophages pre-exposed to LPS exhibited reduced production of inflammatory cytokines in a time- and dose-dependent manner. Activation of neither IL-1 receptor-associated kinase nor NF-kappaB was observed in macrophages that became tolerant by LPS pretreatment, indicating that the proximal event in Toll-like receptor 4 (TLR4)-MyD88-dependent signaling is affected in tolerant macrophages. Although TLR4 mRNA expression significantly decreased within a few hours of LPS pretreatment and returned to the original level at 24 h, the surface TLR4 expression began to decrease within 1 h, with a gradual decrease after that, and remained suppressed over 24 h. A decrease in inflammatory cytokine production in tolerant macrophages well correlates with down-regulation of the surface TLR4 expression, which may explain one of the mechanisms for LPS tolerance.     

miR-146a Is Critical for Endotoxin-induced Tolerance: IMPLICATION IN INNATE IMMUNITY*

The human toll-like receptor 4 (TLR4) pathway is activated in response to lipopolysaccharide (LPS), and subsequent signal transductions lead to the production of cytokines such as tumor necrosis factor-α (TNF-α) by innate immune cells. Defects in innate immune response may contribute to the overproduction of TNF-α leading to systemic inflammation and diseases. Thus, the innate immune response needs to be tightly regulated by elaborate mechanisms to control its onset and termination. LPS tolerance is a state of hyporesponsiveness to subsequent LPS challenge and is achieved by monocytic cells after prolonged exposure to LPS. In this report, kinetics of endotoxin-responsive microRNAs expression analysis revealed a unique pattern of gradual increase for miR-146a starting 4 h after LPS stimulation in THP-1 cells and continued up to 35-fold over 24 h. Conversely, TNF-α increased up to 4 h and then decreased gradually implicating a negative correlation with miR-146a progression. The characteristic up-regulation of miR-146a toward subsequent LPS challenge in THP-1 cells was studied. Strikingly, microRNA expression analysis during the tolerized state of THP-1 cells showed only miR-146a overexpression suggesting its important role in LPS tolerance. In addition, LPS tolerance was dependent on a LPS-priming dose and associated miR-146a up-regulation. LPS-tolerized cells were observed to regain responsiveness in TNF-α production 22 h after LPS removal correlating with a decrease in miR-146a level. Transfection of miR-146a into THP-1 cells mimicked LPS priming, whereas transfection of miR-146a inhibitor largely abolished LPS tolerance. Thus our studies demonstrated that miR-146a is critical for the in vitro monocytic cell-based endotoxin tolerance.     

Immune status evaluation of patients with chronic heart failure

Chronic heart failure (CHF) may be considered a state of immune activation and persistent inflammation expressed by increased circulating levels of pro- and anti-inflammatory cytokines. The purpose of the study was to investigate the immune status in patients with CHF compared to normal individuals. We measured serum cytokine levels as well as cytokine production after ex vivo LPS stimulation of whole blood taken from 14 patients with CHF and 14 healthy volunteers. We used 500 pg/ml of LPS for an incubation period of 4h to stimulate 100 microL of whole blood. Patients with CHF had significantly higher levels of TNF-RI, and TNF-RII in serum compared to normal individuals. TNF-alpha, IL-6, and IL-10 did not differ significantly. After LPS stimulation, patients with CHF had significantly higher levels of TNF-alpha and IL-10, and significantly lower IL-6 levels compared to normal individuals. TNF-alpha receptors did not differ significantly. Patients with CHF may be found in a pro- as well as an anti-inflammatory state. They also do not develop endotoxin tolerance in an ex vivo laboratory model using whole blood stimulated with LPS. They may have increased TNF-alpha and IL-10 production after LPS stimulation of whole blood, which may contribute to a worsening of heart function, more severe disease presentation and a worse outcome during infections.     

Toxoplasma gondii interferes with lipopolysaccharide-induced mitogen-activated protein kinase activation by mechanisms distinct from endotoxin tolerance

We show in this study that Toxoplasma gondii infection induces rapid activation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase 1/2, and stress-activated protein kinase/c-Jun N-terminal kinase MAPK, followed promptly by their deactivation in mouse macrophages. Nevertheless, when infected cells were subsequently subjected to LPS triggering, MAPK activation was severely defective, in particular in the case of p38 MAPK, which is required for LPS-triggered TNF-alpha and IL-12 production. Similar effects occurred during endotoxin tolerance, but the phenomena were distinct. LPS pretriggering failed to activate the major p38 MAPK kinase, MAPK kinase 3/6. Toxoplasma infection, in contrast, resulted in sustained activation of this kinase. Furthermore, endotoxin pre-exposure blocked IkappaBalpha degradation upon subsequent LPS triggering, but this was not the case for Toxoplasma preinfection. Endotoxin-mediated down-regulation of the LPS receptor, Toll-like receptor 4, has been suggested as one possible mechanism contributing to tolerance, and we found in this study that LPS down-modulated Toll-like receptor 4 expression. In contrast, Toxoplasma infection induced up-regulation of this pattern recognition receptor. Our results show that T. gondii blocks LPS-triggered cytokine production in part through MAPK inactivation, and that this occurs through pathways distinct from endotoxin-induced tolerance.     

Cholera toxin B pretreatment of macrophages and monocytes diminishes their proinflammatory responsiveness to lipopolysaccharide

The cholera toxin B chain (CTB) has been reported to suppress T cell-dependent autoimmune diseases and to potentiate tolerance of the adaptive immune system. We have analyzed the effects of CTB on macrophages in vitro and have found that preincubation with CTB (10 microg/ml) suppresses the proinflammatory reaction to LPS challenge, as demonstrated by suppressed production of TNF-alpha, IL-6, IL-12(p70), and NO (p < 0.01) in cells of macrophage lines. Pre-exposure to CTB also suppresses LPS-induced TNF-alpha and IL-12(p70) formation in human PBMC. Both native and recombinant CTB exhibited suppressive activity, which was shared by intact cholera toxin. In cells of the human monocyte line Mono Mac 6, exposure to CTB failed to suppress the production of IL-10 in response to LPS. Control experiments excluded a role of possible contamination of CTB by endotoxin or intact cholera toxin. The suppression of TNF-alpha production occurred at the level of mRNA formation. Tolerance induction by CTB was dose and time dependent. The suppression of TNF-alpha and IL-6 production could be counteracted by the addition of Abs to IL-10 and TGF-beta. IFN-gamma also antagonized the actions of CTB on macrophages. In contrast to desensitization by low doses of LPS, tolerance induction by CTB occurred silently, i.e., in the absence of a measurable proinflammatory response. These findings identify immune-deviating properties of CTB at the level of innate immune cells and may be relevant to the use of CTB in modulating immune-mediated diseases.     

Mechanisms of endotoxin tolerance in human intestinal microvascular endothelial cells

Lipopolysaccharide (endotoxin) tolerance is well described in monocytes and macrophages, but is less well characterized in endothelial cells. Because intestinal microvascular endothelial cells exhibit a strong immune response to LPS challenge and play a critical regulatory role in gut inflammation, we sought to characterize the activation response of these cells to repeated LPS exposure. Primary cultures of human intestinal microvascular endothelial cells (HIMEC) were stimulated with LPS over 6-60 h and activation was assessed using U937 leukocyte adhesion, expression of E-selectin, ICAM-1, VCAM-1, IL-6, IL-8, manganese superoxide dismutase, HLA-DR, and CD86. Effect of repeat LPS stimulation on HIMEC NF-kappaB and mitogen-activated protein kinase (MAPK) activation, generation of superoxide anion, and Toll-like receptor 4 expression was characterized. LPS pretreatment of HIMEC for 24-48 h significantly decreased leukocyte adhesion after subsequent LPS stimulation. LPS pretreatment inhibited expression of E-selectin, VCAM-1, IL-6, and CD86, while ICAM-1, IL-8, and HLA-DR were not altered. Manganese superoxide dismutase expression increased with repeated LPS stimulation, with a reduction in intracellular superoxide. NF-kappaB activation was transiently inhibited by LPS pretreatment for 6 h, but not at later time points. In contrast, p44/42 MAPK, p38 MAPK, and c-Jun N-terminal kinase activation demonstrated inhibition by LPS pretreatment 24 or 48 h prior. Toll-like receptor 4 expression on HIMEC was not altered by LPS. HIMEC exhibit endotoxin tolerance after repeat LPS exposure in vitro, characterized by diminished activation and intracellular superoxide anion concentration, and reduced leukocyte adhesion. HIMEC possess specific mechanisms of immunoregulatory hyporesponsiveness to repeated LPS exposure.     

Participation of beta-adrenergic receptors on macrophages in modulation of LPS-induced cytokine release

For several years it is known that beta-adrenergic receptor agonists have anti-inflammatory effects. However, little is known about the role of beta-adrenergic receptors on macrophages in the modulation of cytokine production by beta-agonists during inflammation. In this study, the presence of beta-receptors on PMA-differentiated U937 human macrophages, and the participation of these receptors in the modulation of LPS-mediated cytokine production by beta-agonists was investigated. Total beta-receptor expression on undifferentiated (monocyte) and PMA-differentiated U937 cells was established using receptor binding studies on membrane fractions with a radio ligand. The expression of beta-receptors proved to be significantly lower on monocytes than on macrophages, additionally a predominant expression of beta 2-receptors was found. Production of the cytokines TNF-alpha, IL-6, and IL-10 by LPS-stimulated differentiated U937 cells was measured in time. Peak concentrations for TNF-alpha, IL-6 and IL-10 occurred at 3, 12 and 9 hrs, respectively. When differentiated U937 cells were incubated with both LPS and the beta-agonist clenbuterol the production of TNF-alpha and IL-6 was significantly reduced. However the production of IL-10 was increased. To study the mechanism of modulation of cytokine production in more detail, U937 macrophages were incubated with LPS/clenbuterol in combination with selective beta 1- and beta 2-antagonists. These results indicated that the beta 2- and not the beta 1-receptor is involved in the anti-inflammatory activity of clenbuterol.     

Over-expression of hsp-70 inhibits bacterial lipopolysaccharide-induced production of cytokines in human monocyte-derived macrophages

Cytokines released from monocytes and macrophages are major mediators of inflammation. Heat shock significantly inhibits cytokine production from these cells. To investigate whether this inhibitory effect was mediated by heat-shock proteins (HSP), we transfected human peripheral blood monocyte-derived macrophages (MDM) with HSP-70 cDNA and examined Brucella melitensis lipopolysaccharide (LPS)-induced cytokine production in transfected cells. Over-expression of HSP-70 protein in the gene-transfected MDM had no effect on cytokine synthesis unless LPS was added. LPS-induced increases in production of tumour necrosis factor alpha (TNF-alpha), interleukin 1beta (IL-1beta), IL-10 and IL-12 were significantly inhibited by the over-expression of HSP-70. However, over-expression of HSP-70 did not block LPS-induced increase in IL-6 synthesis. To further confirm these results, an antisense HSP-70 DNA oligomer was used to block HSP-70 synthesis. The inhibitory effect of HSP-70 on LPS-induced cytokine production in gene- transfected cells was completely reversed after treatment of cells with 5 microM antisense HSP-70. The same concentration of antisense HSP-70 also partially reversed heat-shock-induced inhibition of LPS-stimulated cytokine production. These results suggest that HSP-70 is involved in the regulation of LPS-induced cytokine production and that this family of proteins plays a role in mitigating adverse effects of endotoxin during infection or other pathological stresses.     

MicroRNA-27a Negatively Modulates the Inflammatory Response in Lipopolysaccharide-Stimulated Microglia by Targeting TLR4 and IRAK4

microRNA, a family of small non-coding RNA, plays significant roles in regulating gene expression, mainly via binding to the 3′-untranslated region of target genes. Although the role of miRNA in regulating neuroinflammation via the innate immune pathway has been studied, its role in the production of inflammatory mediators during microglial activation is poorly understood. In this study, we investigated the effect of miR-27a on lipopolysaccharide (LPS)-induced microglial inflammation. miR-27a expression was found to be rapidly decreased in microglia by real-time polymerase chain reaction (real-time PCR) after LPS stimulation. Over-expression of miR-27a significantly decreased the production of inflammatory cytokines, such as interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and nitric oxide (NO), whereas knockdown of miR-27a increased the expression of these inflammatory factors. We also demonstrated by loss- and gain-of-function studies that miR-27a directly suppressed the expression of toll-like receptor 4 (TLR4) and interleukin-1 receptor-associated kinase 4 (IRAK4)—a pivotal adaptor kinase in the TLR4/MyD88 signaling pathway—by directly binding their 3′-UTRs: knocking down TLR4 or IRAK4 in microglia significantly decreased TLR4 or IRAK4 expression and inhibited the downstream production of inflammatory mediators. Moreover, the inflammatory cytokines IL-6 and IL-1β were regulated by IRAK4, whereas TNF-α and NO were more dependent on TLR4 activation. Thus, miR-27a might regulate the LPS-induced production of inflammatory cytokines in microglia independently of TLR4 and IRAK4. Taken together, our results suggest that miR-27a is associated with microglial activation and the inflammatory response.     

Enhanced IL-10 production by TLR4- and TLR2-primed dendritic cells upon TLR restimulation

LPS tolerance has been investigated extensively in monocytes/macrophages. However, the LPS restimulation studies are not well documented in dendritic cells (DCs). In the present study, we investigated influences of TLR restimulation using murine bone marrow-derived DCs. Purified bone marrow-derived DCs (>98% CD11c+ B220-) were stimulated with TLR4 and TLR2 ligands for 24 h and then cultured with medium alone for 48 h as a resting interval (TLR4,2-primed DCs). The TLR4-MD2 expression was markedly reduced immediately after the TLR stimulation, but was restored following the resting interval. The TLR4,2-primed DCs exhibited significantly enhanced IL-10 production, but markedly diminished IL-12p40 production upon TLR4 restimulation compared with naive (unprimed) DCs. TLR4-mediated activation of p38 MAPK was markedly suppressed, whereas that of ERK1/2 was enhanced in the TLR4,2-primed DCs compared with naive DCs. Blocking the activation of ERK1/2 with U0126 reduced the enhanced IL-10 production by the TLR4,2-primed DCs upon the TLR4 restimulation. The U0126 showed no significant effects on the IL-12p40 production. Thus, the enhanced ERK1/2 activation appears to be, at least in part, responsible for the enhanced IL-10 production in the TLR4,2-primed DCs. In addition, TNFR-associated factor 3 expression was significantly up-regulated in the TLR4,2-primed DCs compared with that in naive DCs. We demonstrated in this study that DCs primed with TLR4 and TLR2 ligands and rested for 48 h showed enhanced IL-10 production upon TLR4 restimulation. The enhanced IL-10 production by the TLR4,2-primed DCs may be attributed to the altered balance of intracellular signaling pathways via p38 MAPK, ERK1/2, and TNFR-associated factor 3 upon TLR restimulation.