Potentilla discolor ameliorates LPS-induced inflammatory responses through suppressing NF-κB and AP-1 pathways.

Potentilla discolor Bunge (PD) is a traditional Chinese medicine which has been widely used for the treatment of various inflammatory diseases (e.g., diarrhea, fever and furuncle). However, few studies focused on its effect on classical inflammation. This study aimed to investigate the anti-inflammatory effect and potential mechanism of the ethanol extract of the whole herbs of PD (EPD) in lipopolysaccharide (LPS)-induced inflammatory models. The obtained results showed that EPD decreased supernatant NO, tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1) in LPS-activated RAW264.7 cells and mouse peritoneal macrophages. Moreover, its effect on NO was attributed to the suppression of iNOS expression rather than its activity. At the transcriptional level, EPD suppressed iNOS, TNF-α and MCP-1 mRNA expressions in LPS-stimulated RAW264.7 cells. Further study showed that EPD didn't affect the phosphorylation and degradation of IκBα, but yet impeded the nuclear translocation of p65 to inhibit NF-κB activation. Meanwhile, it also prevented JNK, ERK1/2 and p38 phosphorylation to dampen the activation of AP-1. In endotoxemia mouse model, EPD not only decreased interleukin-6, TNF-α and MCP-1 levels in serum, but also potently ameliorated diarrhea. These findings provide the theoretical basis for PD to treat inflammatory diseases, especially intestinal inflammation.

The Acute Hepatic NF-κB-Mediated Proinflammatory Response to Endotoxemia Is Attenuated in Intrauterine Growth-Restricted Newborn Mice.

Intrauterine growth restriction (IUGR) is a relevant predictor for higher rates of neonatal sepsis worldwide and is associated with an impaired neonatal immunity and lower immune cell counts. During the perinatal period, the liver is a key immunological organ responsible for the nuclear factor kappa B (NF-κB)-mediated innate immune response to inflammatory stimuli, but whether this role is affected by IUGR is unknown. Herein, we hypothesized that the newborn liver adapts to calorie-restriction IUGR by inducing changes in the NF-κB signaling transcriptome, leading to an attenuated acute proinflammatory response to intraperitoneal lipopolysaccharide (LPS). We first assessed the hepatic gene expression of key NF-κB factors in the IUGR and normally grown (NG) newborn mice. Real-time quantitative PCR (RT-qPCR) analysis revealed an upregulation of both IκB proteins genes (Nfkbia and Nfkbib) and the NF-κB subunit Nfkb1 in IUGR vs. NG. We next measured the LPS-induced hepatic expression of acute proinflammatory genes (Ccl3, Cxcl1, Il1b, Il6, and Tnf) and observed that the IUGR liver produced an attenuated acute proinflammatory cytokine gene response (Il1b and Tnf) to LPS in IUGR vs. unexposed (CTR). Consistent with these results, LPS-exposed hepatic tumor necrosis factor alpha (TNF-α) protein concentrations were lower in IUGR vs. LPS-exposed NG and did not differ from IUGR CTR. Sex differences at the transcriptome level were observed in the IUGR male vs. female. Our results demonstrate that IUGR induces key modifications in the NF-κB transcriptomic machinery in the newborn that compromised the acute proinflammatory cytokine gene and protein response to LPS. Our results bring novel insights in understanding how the IUGR newborn is immunocompromised due to fundamental changes in NF-κB key factors.

Endothelial SOCS3 maintains homeostasis and promotes survival in endotoxemic mice.

SOCS3 is the main inhibitor of the JAK/STAT3 pathway. This pathway is activated by interleukin 6 (IL-6), a major mediator of the cytokine storm during shock. To determine its role in the vascular response to shock, we challenged mice lacking SOCS3 in the adult endothelium (SOCS3iEKO) with a nonlethal dose of lipopolysaccharide (LPS). SOCS3iEKO mice died 16-24 hours postinjection after severe kidney failure. Loss of SOCS3 led to an LPS-induced type I IFN-like program and high expression of prothrombotic and proadhesive genes. Consistently, we observed intraluminal leukocyte adhesion and neutrophil extracellular trap-osis (NETosis), as well as retinal venular leukoembolization. Notably, heterozygous mice displayed an intermediate phenotype, suggesting a gene dose effect. In vitro studies were performed to study the role of SOCS3 protein levels in the regulation of the inflammatory response. In human umbilical vein endothelial cells, pulse-chase experiments showed that SOCS3 protein had a half-life less than 20 minutes. Inhibition of SOCS3 ubiquitination and proteasomal degradation led to protein accumulation and a stronger inhibition of IL-6 signaling and barrier function loss. Together, our data demonstrate that the regulation of SOCS3 protein levels is critical to inhibit IL-6-mediated endotheliopathy during shock and provide a promising therapeutic avenue to prevent multiorgan dysfunction through stabilization of endothelial SOCS3.

Activation of Neutrophil Granulocytes by Platelet-Activating Factor Is Impaired During Experimental Sepsis.

Platelet-activating factor (PAF) is an important mediator of the systemic inflammatory response. In the case of sepsis, proper activation and function of neutrophils as the first line of cellular defense are based on a well-balanced physiological response. However, little is known about the role of PAF in cellular changes of neutrophils during sepsis. Therefore, this study investigates the reaction patterns of neutrophils induced by PAF with a focus on membrane potential (MP), intracellular pH, and cellular swelling under physiological and pathophysiological conditions and hypothesizes that the PAF-mediated response of granulocytes is altered during sepsis. The cellular response of granulocytes including MP, intracellular pH, cellular swelling, and other activation markers were analyzed by multiparametric flow cytometry. In addition, the chemotactic activity and the formation of platelet-neutrophil complexes after exposure to PAF were investigated. The changes of the (electro-)physiological response features were translationally verified in a human ex vivo whole blood model of endotoxemia as well as during polymicrobial porcine sepsis. In neutrophils from healthy human donors, PAF elicited a rapid depolarization, an intracellular alkalization, and an increase in cell size in a time- and dose-dependent manner. Mechanistically, the alkalization was dependent on sodium-proton exchanger 1 (NHE1) activity, while the change in cellular shape was sodium flux- but only partially NHE1-dependent. In a pathophysiological altered environment, the PAF-induced response of neutrophils was modulated. Acidifying the extracellular pH in vitro enhanced PAF-mediated depolarization, whereas the increases in cell size and intracellular pH were largely unaffected. Ex vivo exposure of human whole blood to lipopolysaccharide diminished the PAF-induced intracellular alkalization and the change in neutrophil size. During experimental porcine sepsis, depolarization of the MP was significantly impaired. Additionally, there was a trend for increased cellular swelling, whereas intracellular alkalization remained stable. Overall, an impaired (electro-)physiological response of neutrophils to PAF stimulation represents a cellular hallmark of those cells challenged during systemic inflammation. Furthermore, this altered response may be indicative of and causative for the development of neutrophil dysfunction during sepsis.

Splenic Nerve Neuromodulation Reduces Inflammation and Promotes Resolution in Chronically Implanted Pigs.

Neuromodulation of the immune system has been proposed as a novel therapeutic strategy for the treatment of inflammatory conditions. We recently demonstrated that stimulation of near-organ autonomic nerves to the spleen can be harnessed to modulate the inflammatory response in an anesthetized pig model. The development of neuromodulation therapy for the clinic requires chronic efficacy and safety testing in a large animal model. This manuscript describes the effects of longitudinal conscious splenic nerve neuromodulation in chronically-implanted pigs. Firstly, clinically-relevant stimulation parameters were refined to efficiently activate the splenic nerve while reducing changes in cardiovascular parameters. Subsequently, pigs were implanted with a circumferential cuff electrode around the splenic neurovascular bundle connected to an implantable pulse generator, using a minimally-invasive laparoscopic procedure. Tolerability of stimulation was demonstrated in freely-behaving pigs using the refined stimulation parameters. Longitudinal stimulation significantly reduced circulating tumor necrosis factor alpha levels induced by systemic endotoxemia. This effect was accompanied by reduced peripheral monocytopenia as well as a lower systemic accumulation of CD16+CD14high pro-inflammatory monocytes. Further, lipid mediator profiling analysis demonstrated an increased concentration of specialized pro-resolving mediators in peripheral plasma of stimulated animals, with a concomitant reduction of pro-inflammatory eicosanoids including prostaglandins. Terminal electrophysiological and physiological measurements and histopathological assessment demonstrated integrity of the splenic nerves up to 70 days post implantation. These chronic translational experiments demonstrate that daily splenic nerve neuromodulation, via implanted electronics and clinically-relevant stimulation parameters, is well tolerated and is able to prime the immune system toward a less inflammatory, pro-resolving phenotype.

Connection between Periodontitis-Induced Low-Grade Endotoxemia and Systemic Diseases: Neutrophils as Protagonists and Targets.

Periodontitis is considered a promoter of many systemic diseases, but the signaling pathways of this interconnection remain elusive. Recently, it became evident that certain microbial challenges promote a heightened response of myeloid cell populations to subsequent infections either with the same or other pathogens. This phenomenon involves changes in the cell epigenetic and transcription, and is referred to as ''trained immunity''. It acts via modulation of hematopoietic stem and progenitor cells (HSPCs). A main modulation driver is the sustained, persistent low-level transmission of lipopolysaccharide from the periodontal pocket into the peripheral blood. Subsequently, the neutrophil phenotype changes and neutrophils become hyper-responsive and prone to boosted formation of neutrophil extracellular traps (NET). Cytotoxic neutrophil proteases and histones are responsible for ulcer formations on the pocket epithelium, which foster bacteremia and endoxemia. The latter promote systemic low-grade inflammation (SLGI), a precondition for many systemic diseases and some of them, e.g., atherosclerosis, diabetes etc., can be triggered by SLGI alone. Either reverting the polarized neutrophils back to the homeostatic state or attenuation of neutrophil hyper-responsiveness in periodontitis might be an approach to diminish or even to prevent systemic diseases.

Ablation of Aquaporin-9 Ameliorates the Systemic Inflammatory Response of LPS-Induced Endotoxic Shock in Mouse.

Septic shock is the most severe complication of sepsis, being characterized by a systemic inflammatory response following bacterial infection, leading to multiple organ failure and dramatically high mortality. Aquaporin-9 (AQP9), a membrane channel protein mainly expressed in hepatocytes and leukocytes, has been recently associated with inflammatory and infectious responses, thus triggering strong interest as a potential target for reducing septic shock-dependent mortality. Here, we evaluated whether AQP9 contributes to murine systemic inflammation during endotoxic shock. Wild type (Aqp9+/+; WT) and Aqp9 gene knockout (Aqp9-/-; KO) male mice were submitted to endotoxic shock by i.p. injection of lipopolysaccharide (LPS; 40 mg/kg) and the related survival times were followed during 72 h. The electronic paramagnetic resonance and confocal microscopy were employed to analyze the nitric oxide (NO) and superoxide anion (O2-) production, and the expression of inducible NO-synthase (iNOS) and cyclooxigenase-2 (COX-2), respectively, in the liver, kidney, aorta, heart and lung of the mouse specimens. LPS-treated KO mice survived significantly longer than corresponding WT mice, and 25% of the KO mice fully recovered from the endotoxin treatment. The LPS-injected KO mice showed lower inflammatory NO and O2- productions and reduced iNOS and COX-2 levels through impaired NF-κB p65 activation in the liver, kidney, aorta, and heart as compared to the LPS-treated WT mice. Consistent with these results, the treatment of FaO cells, a rodent hepatoma cell line, with the AQP9 blocker HTS13268 prevented the LPS-induced increase of inflammatory NO and O2-. A role for AQP9 is suggested in the early acute phase of LPS-induced endotoxic shock involving NF-κB signaling. The modulation of AQP9 expression/function may reveal to be useful in developing novel endotoxemia therapeutics.

Anti-Infective and Anti-Inflammatory Mode of Action of Peptide 19-2.5.

The polypeptide Pep19-2.5 (Aspidasept®) has been described to act efficiently against infection-inducing bacteria by binding and neutralizing their most potent toxins, i.e., lipopolysaccharides (LPS) and lipoproteins/peptides (LP), independent of the resistance status of the bacteria. The mode of action was described to consist of a primary Coulomb/polar interaction of the N-terminal region of Pep19-2.5 with the polar region of the toxins followed by a hydrophobic interaction of the C-terminal region of the peptide with the apolar moiety of the toxins. However, clinical development of Aspidasept as an anti-sepsis drug requires an in-depth characterization of the interaction of the peptide with the constituents of the human immune system and with other therapeutically relevant compounds such as antibiotics and non-steroidal anti-inflammatory drugs (NSAIDs). In this contribution, relevant details of primary and secondary pharmacodynamics, off-site targets, and immunogenicity are presented, proving that Pep19-2.5 may be readily applied therapeutically against the deleterious effects of a severe bacterial infection.

Prominent Indomethacin-Induced Enteropathy in Fcgriib Defi-cient lupus Mice: An Impact of Macrophage Responses and Immune Deposition in Gut.

A high dose of NSAIDs, a common analgesic, might induce lupus activity through several NSAIDs adverse effects including gastrointestinal permeability defect (gut leakage) and endotoxemia. Indomethacin (25 mg/day) was orally administered for 7 days in 24-wk-old Fc gamma receptor IIb deficient (FcgRIIb-/-) mice, an asymptomatic lupus model (increased anti-dsDNA without lupus nephritis), and age-matched wild-type (WT) mice. Severity of indomethacin-induced enteropathy in FcgRIIb-/- mice was higher than WT mice as demonstrated by survival analysis, intestinal injury (histology, immune-deposition, and intestinal cytokines), gut leakage (FITC-dextran assay and endotoxemia), serum cytokines, and lupus characteristics (anti-dsDNA, renal injury, and proteinuria). Prominent responses of FcgRIIb-/- macrophages toward lipopolysaccharide (LPS) compared to WT cells due to the expression of only activating-FcgRs without inhibitory-FcgRIIb were demonstrated. Extracellular flux analysis indicated the greater mitochondria activity (increased respiratory capacity and respiratory reserve) in FcgRIIb-/- macrophages with a concordant decrease in glycolysis activity when compared to WT cells. In conclusion, gut leakage-induced endotoxemia is more severe in indomethacin-administered FcgRIIb-/- mice than WT, possibly due to the enhanced indomethacin toxicity from lupus-induced intestinal immune-deposition. Due to a lack of inhibitory-FcgRIIb expression, mitochondrial function, and cytokine production of FcgRIIb-/- macrophages were more prominent than WT cells. Hence, lupus disease-activation from NSAIDs-enteropathy-induced gut leakage is possible.

Frontline Science: The expression of integrin αD ß2 (CD11d/CD18) on neutrophils orchestrates the defense mechanism against endotoxemia and sepsis.

Neutrophil-macrophage interplay is a fine-tuning mechanism that regulates the innate immune response during infection and inflammation. Cell surface receptors play an essential role in neutrophil and macrophage functions. The same receptor can provide different outcomes within diverse leukocyte subsets in different inflammatory conditions. Understanding the variety of responses mediated by one receptor is critical for the development of anti-inflammatory treatments. In this study, we evaluated the role of a leukocyte adhesive receptor, integrin αD ß2 , in the development of acute inflammation. αD ß2 is mostly expressed on macrophages and contributes to the development of chronic inflammation. In contrast, we found that αD -knockout dramatically increases mortality in the cecal ligation and puncture sepsis model and LPS-induced endotoxemia. This pathologic outcome of αD -deficient mice is associated with a reduced number of monocyte-derived macrophages and an increased number of neutrophils in their lungs. However, the tracking of adoptively transferred fluorescently labeled wild-type (WT) and αD-/- monocytes in WT mice during endotoxemia demonstrated only a moderate difference between the recruitment of these two subsets. Moreover, the rescue experiment, using i.v. injection of WT monocytes to αD -deficient mice followed by LPS challenge, showed only slightly reduced mortality. Surprisingly, the injection of WT neutrophils to the bloodstream of αD-/- mice markedly increased migration of monocyte-derived macrophage to lungs and dramatically improves survival. αD -deficient neutrophils demonstrate increased necrosis/pyroptosis. αD ß2 -mediated macrophage accumulation in the lungs promotes efferocytosis that reduced mortality. Hence, integrin αD ß2 implements a complex defense mechanism during endotoxemia, which is mediated by macrophages via a neutrophil-dependent pathway.

Intracellular immune sensing promotes inflammation via gasdermin D-driven release of a lectin alarmin.

Inflammatory caspase sensing of cytosolic lipopolysaccharide (LPS) triggers pyroptosis and the concurrent release of damage-associated molecular patterns (DAMPs). Collectively, DAMPs are key determinants that shape the aftermath of inflammatory cell death. However, the identity and function of the individual DAMPs released are poorly defined. Our proteomics study revealed that cytosolic LPS sensing triggered the release of galectin-1, a ß-galactoside-binding lectin. Galectin-1 release is a common feature of inflammatory cell death, including necroptosis. In vivo studies using galectin-1-deficient mice, recombinant galectin-1 and galectin-1-neutralizing antibody showed that galectin-1 promotes inflammation and plays a detrimental role in LPS-induced lethality. Mechanistically, galectin-1 inhibition of CD45 (Ptprc) underlies its unfavorable role in endotoxin shock. Finally, we found increased galectin-1 in sera from human patients with sepsis. Overall, we uncovered galectin-1 as a bona fide DAMP released as a consequence of cytosolic LPS sensing, identifying a new outcome of inflammatory cell death.

Macrophage ICAM-1 functions as a regulator of phagocytosis in LPS induced endotoxemia.

OBJECTIVE: Intracellular adhesion molecule-1 (ICAM-1), a transmembrane glycoprotein belonging to the immunoglobulin superfamily, plays a critical role in mediating cell-cell interaction and outside-in cell signaling during the immune response. ICAM-1 is expressed on the cell surface of several cell types including endothelial cells, epithelial cells, leucocytes, fibroblasts, and neutrophils. Despite ICAM-1 has been detected on macrophage, little is known about the function and mechanism of macrophage ICAM-1. METHODS: To investigate the role of lipopolysaccharide (LPS) in ICAM-1 regulation, both the protein and cell surface expression of ICAM-1 were measured. The phagocytosis of macrophage was evaluated by flow cytometry and Confocal microscopy. Small interfering RNA and neutralizing antibody of ICAM-1 were used to assess the effect of ICAM-1 on macrophage phagocytosis. TLR4 gene knockout mouse and cytoplasmic and mitochondrial ROS scavenger were used for the regulation of ICAM-1 expression. ROS was determined using flow cytometry. RESULTS: In this study, we reported that macrophage can be stimulated to increase both the protein and cell surface expression of ICAM-1 by LPS. Macrophage ICAM-1 expression was correlated with enhanced macrophage phagocytosis. We found that using ICAM-1 neutralizing antibody or ICAM-1 silencing to attenuate the function or expression of ICAM-1 could decrease LPS-induced macrophage phagocytosis. Furthermore, we found that knocking out of TLR4 led to inhibited cytoplasmic and mitochondrial ROS production, which in turn, attenuated ICAM-1 expression at both the protein and cell surface levels. CONCLUSION: This study demonstrates that the mechanism of ICAM-1-mediated macrophage phagocytosis is depending on TLR4-mediated ROS production and provides significant light on macrophage ICAM-1 in endotoxemia.

BAFF Blockade Attenuates Inflammatory Responses and Intestinal Barrier Dysfunction in a Murine Endotoxemia Model.

B cell-activating factor (BAFF) production is increased in septic patients. However, the specific role of BAFF in sepsis remains unknown. This study was designed to investigate the expression and function of BAFF in an experimental endotoxemia model and to identify the potential mechanisms. We established an endotoxemia mouse (6-8 weeks, 20-22 g) model by administering 30 mg/kg lipopolysaccharide (LPS). BAFF levels in the circulating system and organ tissues were measured 4 and 8 h after LPS injection. Survival rates in the endotoxemia mice were monitored for 72 h after BAFF blockade. The effects of BAFF blockade on systemic and local inflammation, organ injuries, and intestinal barrier function were also evaluated 4 h after LPS treatment. BAFF production was systemically and locally elevated after LPS challenge. BAFF blockade improved the survival rate, systemic inflammation, and multi-organ injuries. Moreover, BAFF blockade attenuated both intestinal inflammation and impaired intestinal permeability. BAFF blockade upregulated ZO-1 and occludin protein levels via the NF-κB/MLCK/MLC signaling pathway. These results suggested that BAFF blockade protects against lethal endotoxemia at least partially by alleviating inflammation, multi-organ injuries, and improving intestinal barrier function and provides a novel focus for further research on sepsis and experimental evidence for clinical therapy.

Identification of a brainstem locus that inhibits tumor necrosis factor.

In the brain, compact clusters of neuron cell bodies, termed nuclei, are essential for maintaining parameters of host physiology within a narrow range optimal for health. Neurons residing in the brainstem dorsal motor nucleus (DMN) project in the vagus nerve to communicate with the lungs, liver, gastrointestinal tract, and other organs. Vagus nerve-mediated reflexes also control immune system responses to infection and injury by inhibiting the production of tumor necrosis factor (TNF) and other cytokines in the spleen, although the function of DMN neurons in regulating TNF release is not known. Here, optogenetics and functional mapping reveal cholinergic neurons in the DMN, which project to the celiac-superior mesenteric ganglia, significantly increase splenic nerve activity and inhibit TNF production. Efferent vagus nerve fibers terminating in the celiac-superior mesenteric ganglia form varicose-like structures surrounding individual nerve cell bodies innervating the spleen. Selective optogenetic activation of DMN cholinergic neurons or electrical activation of the cervical vagus nerve evokes action potentials in the splenic nerve. Pharmacological blockade and surgical transection of the vagus nerve inhibit vagus nerve-evoked splenic nerve responses. These results indicate that cholinergic neurons residing in the brainstem DMN control TNF production, revealing a role for brainstem coordination of immunity.

Nicotine uncovers endotoxic-like cardiovascular manifestations in female rats: Estrogen and nitric oxide dependency.

Endotoxic manifestations are diminished in female populations due to immune boosting actions of sex steroids. Considering that tobacco constituents including nicotine inhibit estrogen synthesis, we tested the hypothesis that nicotine exposure unveils cardiovascular anomalies of endotoxemia in female rats. Studies were undertaken in conscious female rats treated with i.v. lipopolysaccharide (LPS, 10 mg/kg) in absence and presence of nicotine. In contrast to no effects for LPS when used alone, dose-related decreases in blood pressure (BP) and serum estrogen were noted in endotoxic rats treated consequently with nicotine (25, 50, or 100 µg/kg i.v.). Signs of cardiac autonomic dysfunction appeared in LPS/nicotine-treated rats such as (i) decreased time-domain indices of heart rate variability (HRV), e.g. standard deviation of R-R intervals (SDNN) and root mean square of successive differences in R-R interval durations (rMSSD), and (ii) reduced total power of the frequency spectrum and shifted cardiac sympathovagal balance toward sympathetic dominance. Nicotine reversed the LPS-evoked modest rises in serum TNFα and IL-1ß while had no effect on associated arterial baroreflex dysfunction, inferring no roles for inflammation or baroreflexes in LPS-nicotine interaction. Estrogen or aminoguanidine (iNOS inhibitor), but not pentoxifylline (TNFα inhibitor), abolished LPS/nicotine hypotension. Together, nicotine acts probably via reducing estrogen availability to uncover nitric oxide-dependent hypotension and autonomic dysregulation in endotoxic female rats.

Maturation of the Acute Hepatic TLR4/NF-κB Mediated Innate Immune Response Is p65 Dependent in Mice.

Compared to adults, neonates are at increased risk of infection. There is a growing recognition that dynamic qualitative and quantitative differences in immunity over development contribute to these observations. The liver plays a key role as an immunologic organ, but whether its contribution to the acute innate immune response changes over lifetime is unknown. We hypothesized that the liver would activate a developmentally-regulated acute innate immune response to intraperitoneal lipopolysaccharide (LPS). We first assessed the hepatic expression and activity of the NF-κB, a key regulator of the innate immune response, at different developmental ages (p0, p3, p7, p35, and adult). Ontogeny of the NF-κB subunits (p65/p50) revealed a reduction in Rela (p65) and Nfkb1 (p105, precursor to p50) gene expression (p0) and p65 subunit protein levels (p0 and p3) vs. older ages. The acute hepatic innate immune response to LPS was associated by the degradation of the NF-κB inhibitory proteins (IκBα and IκBß), and nuclear translocation of the NF-κB subunit p50 in all ages, whereas nuclear translocation of the NF-κB subunit p65 was only observed in the p35 and adult mouse. Consistent with these findings, we detected NF-κB subunit p65 nuclear staining exclusively in the LPS-exposed adult liver compared with p7 mouse. We next interrogated the LPS-induced hepatic expression of pro-inflammatory genes (Tnf, Icam1, Ccl3, and Traf1), and observed a gradually increase in gene expression starting from p0. Confirming our results, hepatic NF-κB subunit p65 nuclear translocation was associated with up-regulation of the Icam1 gene in the adult, and was not detected in the p7 mouse. Thus, an inflammatory challenge induces an NF-κB-mediated hepatic innate immune response activation across all developmental ages, but nuclear translocation of the NF-κB subunit p65 and associated induction of pro-inflammatory genes occurred only after the first month of life. Our results demonstrate that the LPS-induced hepatic innate immune response is developmentally regulated by the NF-κB subunit p65 in the mouse.

DUSP11 Attenuates Lipopolysaccharide-Induced Macrophage Activation by Targeting TAK1.

Dual-specificity phosphatase 11 (DUSP11, also named as PIR1) is a member of the atypical DUSP protein tyrosine phosphatase family. DUSP11 is only known to be an RNA phosphatase that regulates noncoding RNA stability. To date, the role of DUSP11 in immune cell signaling and immune responses remains unknown. In this study, we generated and characterized the immune cell functions of DUSP11-deficient mice. We identified TGF-ß-activated kinase 1 (TAK1) as a DUSP11-targeted protein. DUSP11 interacted directly with TAK1, and the DUSP11-TAK1 interaction was enhanced by LPS stimulation in bone marrow-derived macrophages. DUSP11 deficiency enhanced the LPS-induced TAK1 phosphorylation and cytokine production in bone marrow-derived macrophages. Furthermore, DUSP11-deficient mice were more susceptible to LPS-induced endotoxic shock. The LPS-induced serum levels of IL-1ß, TNF-α, and IL-6 were significantly elevated in DUSP11-deficient mice compared with those of wild-type mice. The data indicate that DUSP11 inhibits LPS-induced macrophage activation by targeting TAK1.

Early-Life Maternal Deprivation Predicts Stronger Sickness Behaviour and Reduced Immune Responses to Acute Endotoxaemia in a Pig Model.

Early-life adversity may have programming effects on neuroendocrine and immune adaptation mechanisms in humans and socially living animals. Using a pig model, we investigated the effect of daily 2-h maternal and littermate deprivation from postnatal days 2-15, either alone (DA) or in a group of littermates (DG) on the neuroendocrine, immunological and behavioural responses of piglets challenged with the bacterial endotoxin lipopolysaccharide (LPS) on day 42. LPS increased plasma concentrations of cortisol, tumour necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and interleukin-10 (IL-10) and induced typical signs of sickness in all piglets. DA+DG piglets showed stronger signs of sickness compared to control (C) piglets. Plasma TNF-α concentrations were significantly lower in DA+DG males. In addition, the TNF-α/IL-10 ratio was significantly lower in DA than in DG and C males. Gene expression analyses showed lower hypothalamic TNF-α mRNA expression and diminished mRNA expression of the mineralocorticoid receptor (MR) and IL-10 in the amygdala of DA+DG piglets in response to LPS. Interestingly, males showed a higher MR- and a lower IL-10 mRNA expression in the amygdala than females. The present data suggest that repeated maternal deprivation during early life may alter neuroendocrine and immune responses to acute endotoxaemia in a sex-specific manner.

IRF-7 Mediates Type I IFN Responses in Endotoxin-Challenged Mice.

IRF-7 mediates robust production of type I IFN via MyD88 of the TLR9 pathway in plasmacytoid dendritic cells (pDCs). Previous in vitro studies using bone marrow-derived dendritic cells lacking either Irf7 or Irf3 have demonstrated that only IRF-3 is required for IFN-ß production in the TLR4 pathway. Here, we show that IRF-7 is essential for both type I IFN induction and IL-1ß responses via TLR4 in mice. Mice lacking Irf7 were defective in production of both IFN-ß and IL-1ß, an IFN-ß-induced pro-inflammatory cytokine, after LPS challenge. IFN-ß production in response to LPS was impaired in IRF-7-deficient macrophages, but not dendritic cells. Unlike pDCs, IRF-7 is activated by the TRIF-, but not MyD88-, dependent pathway via TBK-1 in macrophages after LPS stimulation. Like pDCs, resting macrophages constitutively expressed IRF-7 protein. This basal IRF-7 protein was completely abolished in either Ifnar1-/- or Stat1-/- macrophages, which corresponded with the loss of LPS-stimulated IFN-ß induction in these macrophages. These findings demonstrate that macrophage IRF-7 is critical for LPS-induced type I IFN responses, which in turn facilitate IL-1ß production in mice.

Short communication: Characterizing ovine serum stress biomarkers during endotoxemia.

Breeding stress-resilient livestock is a potential strategy to help mitigate the negative effect of environmental and pathogenic stressors. The hypothalamic-pituitary-adrenal axis and immune system are activated during stress events and release mediators into the circulation that help restore physiological homeostasis. The purpose of this study was to assess a comprehensive set of circulatory mediators released in response to an acute immune stress challenge to identify candidate biomarkers that can be used for the selection of stress-resilient animals. Fifteen female lambs were stress challenged with an intravenous bolus of lipopolysaccharide (LPS; 400 ng/kg), and blood was collected from the jugular vein at 0, 2, 4, and 6 h after LPS challenge to identify and monitor candidate stress biomarkers; temperature was also recorded over time. Biomarker responses were evaluated with a repeated-measures model to compare time points with baseline values. As expected, all sheep had a monophasic febrile response to LPS challenge, and cortisol increased and returned to baseline by 6 h. The cytokines tumor necrosis factor-α, IL-6, IFN-γ (proinflammatory), and IL-10 (anti-inflammatory) increased, but only tumor necrosis factor-α returned to baseline during the monitoring period. The cytokines IL-1α, IL-1ß, IL-17α (proinflammatory), and IL-4 (anti-inflammatory) did not respond to LPS challenge. All chemokines (CCL2, CCL3, CCL4, CXCL10, and IL-8) responded to LPS challenge; however, only CCL2, CCL3, CCL4, and CXCL10 increased over time, and only CCL3, CCL4, and CXCL10 returned to baseline during the monitoring period. MicroRNA (miR-145, miR-233, and miR-1246) also increased and remained elevated during the study. In summary, the LPS challenge induced a strong stress response in Rideau-Dorset sheep that could be monitored with a distinct profile of circulatory biomarkers.