Early Pro-inflammatory Response Research Articles

Hif-1α-Induced Expression of Il-1β Protects against Mycobacterial Infection in Zebrafish.

Drug-resistant mycobacteria are a rising problem worldwide. There is an urgent need to understand the immune response to tuberculosis to identify host targets that, if targeted therapeutically, could be used to tackle these currently untreatable infections. In this study we use an Il-1β fluorescent transgenic line to show that there is an early innate immune proinflammatory response to well-established zebrafish models of inflammation and Mycobacterium marinum infection. We demonstrate that host-derived hypoxia signaling, mediated by the Hif-1α transcription factor, can prime macrophages with increased levels of Il-1β in the absence of infection, upregulating neutrophil antimicrobial NO production, leading to greater protection against infection. Our data link Hif-1α to proinflammatory macrophage Il-1β transcription in vivo during early mycobacterial infection and importantly highlight a host protective mechanism, via antimicrobial NO, that decreases disease outcomes and that could be targeted therapeutically to stimulate the innate immune response to better deal with infections.

The Stress Hormone Cortisol Enhances Interferon-υ-Mediated Proinflammatory Responses of Human Immune Cells.

Cortisol is a prototypical human stress hormone essential for life, yet the precise role of cortisol in the human stress response to injury or infection is still uncertain. Glucocorticoids (GCs) such as cortisol are widely understood to suppress inflammation and immunity. However, recent research shows that GCs also induce delayed immune effects manifesting as immune stimulation. In this study, we show that cortisol enhances the immune-stimulating effects of a prototypical proinflammatory cytokine, interferon-υ (IFN-υ). We tested the hypothesis that cortisol enhances IFN-υ-mediated proinflammatory responses of human mononuclear phagocytes (monocyte/macrophages [MOs]) stimulated by bacterial endotoxin (lipopolysaccharide [LPS]). Human MOs were cultured for 18 hours with or without IFN-υ and/or cortisol before LPS stimulation. MO differentiation factors granulocyte-macrophage colony stimulating factor (GM-CSF) or M-CSF were added to separate cultures. We also compared the inflammatory response with an acute, 4-hour MO incubation with IFN-υ plus cortisol and LPS to a delayed 18-hour incubation with cortisol before LPS exposure. MO activation was assessed by interleukin-6 (IL-6) release and by multiplex analysis of pro- and anti-inflammatory soluble mediators. After the 18-hour incubation, we observed that cortisol significantly increased LPS-stimulated IL-6 release from IFN-υ-treated undifferentiated MOs. In GM-CSF-pretreated MOs, cortisol increased IFN-υ-mediated IL-6 release by >4-fold and release of the immune stimulant IFN-α2 (IFN-α2) by >3-fold, while suppressing release of the anti-inflammatory mediator, IL-1 receptor antagonist to 15% of control. These results were reversed by either the GC receptor antagonist RU486 or by an IFN-υ receptor type 1 antibody antagonist. Cortisol alone increased expression of the IFN-υ receptor type 1 on undifferentiated and GM-CSF-treated MOs. In contrast, an acute 4-hour incubation of MOs with IFN-υ and cortisol showed classic suppression of the IL-6 response to LPS. These results reveal a surprisingly robust proinflammatory interaction between the human stress response hormone cortisol and the immune activating cytokine IFN-υ. The results support an emerging physiological model with an adaptive role for cortisol, wherein acute release of cortisol suppresses early proinflammatory responses but also primes immune cells for an augmented response to a subsequent immune challenge. These findings have broad clinical implications and provide an experimental framework to examine individual differences, mechanisms, and translational implications of cortisol-enhanced immune responses in humans.

Molecular characterization and expression analysis of the interleukin 1b gene in Pacific cod (Gadus macrocephalus)

Interleukin 1b (IL-1b) is a member of the cytokine family that serves as major mediators for early pro-inflammatory responses. During artificial breeding of Pacific cod (Gadus macrocephalus), outbreaks of NNV in the early development stages could lead to high mortality. Recent research reported that IL-1b participated in the host-virus interaction against NNV infection, however, no IL-1b gene has been identified from Pacific cod so far. In this study, we described the identification and characterization of a IL-1b from Pacific cod transcriptome at both transcript and protein level. Sequence alignment and phylogenetic analysis were conducted to determine and confirm their evolutionary relationship. Expression analysis revealed that IL-1b was expressed in all tested tissues and the western blot analysis confirmed the same expression profiles using purified recombinant IL-1b protein. In addition, significant up-regulation was detected in IL-1b transcripts during early development stages at 30dph and 50dph. After challenged with poly I:C, IL-1b was significantly induced after 24 h. Taken together, these results strongly suggested that IL-1b might play an essential role in innate defense mechanism of Pacific cod.

Rocaglates as dual-targeting agents for experimental cerebral malaria

Cerebral malaria (CM) is a severe and rapidly progressing complication of infection by Plasmodium parasites that is associated with high rates of mortality and morbidity. Treatment options are currently few, and intervention with artemisinin (Art) has limited efficacy, a problem that is compounded by the emergence of resistance to Art in Plasmodium parasites. Rocaglates are a class of natural products derived from plants of the Aglaia genus that have been shown to interfere with eukaryotic initiation factor 4A (eIF4A), ultimately blocking initiation of protein synthesis. Here, we show that the rocaglate CR-1-31B perturbs association of Plasmodium falciparum eIF4A (PfeIF4A) with RNA. CR-1-31B shows potent prophylactic and therapeutic antiplasmodial activity in vivo in mouse models of infection with Plasmodium berghei (CM) and Plasmodium chabaudi (blood-stage malaria), and can also block replication of different clinical isolates of P. falciparum in human erythrocytes infected ex vivo, including drug-resistant P. falciparum isolates. In vivo, a single dosing of CR-1-31B in P. berghei-infected animals is sufficient to provide protection against lethality. CR-1-31B is shown to dampen expression of the early proinflammatory response in myeloid cells in vitro and dampens the inflammatory response in vivo in P. berghei-infected mice. The dual activity of CR-1-31B as an antiplasmodial and as an inhibitor of the inflammatory response in myeloid cells should prove extremely valuable for therapeutic intervention in human cases of CM.

Peripheral Blood Biomarkers of Disease Outcome in a Monkey Model of Rift Valley Fever Encephalitis.

Rift Valley Fever (RVF) is an emerging arboviral disease of livestock and humans. Although the disease is caused by a mosquito-borne virus, humans are infected through contact with, or inhalation of, virus-laden particles from contaminated animal carcasses. Some individuals infected with RVF virus (RVFV) develop meningoencephalitis, resulting in morbidity and mortality. Little is known about the pathogenic mechanisms that lead to neurologic sequelae, and thus, animal models that represent human disease are needed. African green monkeys (AGM) exposed to aerosols containing RVFV develop a reproducibly lethal neurological disease that resembles human illness. To understand the disease process and identify biomarkers of lethality, two groups of 5 AGM were infected by inhalation with either a lethal or a sublethal dose of RVFV. Divergence between lethal and sublethal infections occurred as early as 2 days postinfection (dpi), at which point CD8+ T cells from lethally infected AGM expressed activated caspase-3 and simultaneously failed to increase levels of major histocompatibility complex (MHC) class II molecules, in contrast to surviving animals. At 4 dpi, lethally infected animals failed to demonstrate proliferation of total CD4+ and CD8+ T cells, in contrast to survivors. These marked changes in peripheral blood cells occur much earlier than more-established indicators of severe RVF disease, such as granulocytosis and fever. In addition, an early proinflammatory (gamma interferon [IFN-γ], interleukin 6 [IL-6], IL-8, monocyte chemoattractant protein 1 [MCP-1]) and antiviral (IFN-α) response was seen in survivors, while very late cytokine expression was found in animals with lethal infections. By characterizing immunological markers of lethal disease, this study furthers our understanding of RVF pathogenesis and will allow the testing of therapeutics and vaccines in the AGM model.IMPORTANCE Rift Valley Fever (RVF) is an important emerging viral disease for which we lack both an effective human vaccine and treatment. Encephalitis and neurological disease resulting from RVF lead to death or significant long-term disability for infected people. African green monkeys (AGM) develop lethal neurological disease when infected with RVF virus by inhalation. Here we report the similarities in disease course between infected AGM and humans. For the first time, we examine the peripheral immune response during the course of infection in AGM and show that there are very early differences in the immune response between animals that survive infection and those that succumb. We conclude that AGM are a novel and suitable monkey model for studying the neuropathogenesis of RVF and for testing vaccines and therapeutics against this emerging viral pathogen.

Bilateral tactile hypersensitivity and neuroimmune responses after spared nerve injury in mice lacking vasoactive intestinal peptide

Vasoactive intestinal peptide (VIP) is one of the neuropeptides showing the strongest up-regulation in the nociceptive pathway after peripheral nerve injury and has been proposed to support neuropathic pain. Nevertheless, the story may be more complicated considering the known suppressive effects of the peptide on the immune reactivity of microglial cells, which have been heavily implicated in the onset and maintenance of pain after nerve injury. We here used mice deficient in VIP and the model of spared nerve injury, characterized by persistent tactile hypersensitivity. While tactile hypersensitivity developed similarly to wild type mice for the ipsilateral hindpaw, only transgenic mice showed a mirror-image tactile hypersensitivity in the contralateral hindpaw. This exacerbated neuropathic pain phenotype appeared to be mediated through a local mechanism acting at the level of the lumbar spinal cord as a distant nerve lesion in the front limb did not lead to hindpaw hypersensitivity in VIP-deficient mice. Innocuous tactile hindpaw stimulation was found to increase a neuronal activation marker in the bilateral superficial laminae of the lumbar dorsal horn of VIP-deficient, but not wild type mice, after SNI. A deeper study into the immune responsiveness to the nerve lesion also proved that VIP-deficient mice had a stronger early pro-inflammatory cytokine response and a more pronounced microglial reactivity compared to wild type controls. The latter was also observed at four weeks after spared nerve injury, a time at which bilateral tactile hypersensitivity persisted in VIP-deficient mice. These data suggest an action of VIP in neuropathic states that is more complicated than previously assumed. Future research is now needed for a deeper understanding of the relative contribution of receptors and fiber populations involved in the VIP-neuropathic pain link.

Immunological Defects in Neonatal Sepsis and Potential Therapeutic Approaches.

Despite advances in critical care medicine, neonatal sepsis remains a major cause of morbidity and mortality worldwide, with the greatest risk affecting very low birth weight, preterm neonates. The presentation of neonatal sepsis varies markedly from its presentation in adults, and there is no clear consensus definition of neonatal sepsis. Previous work has demonstrated that when neonates become septic, death can occur rapidly over a matter of hours or days and is generally associated with inflammation, organ injury, and respiratory failure. Studies of the transcriptomic response by neonates to infection and sepsis have led to unique insights into the early proinflammatory and host protective responses to sepsis. Paradoxically, this early inflammatory response in neonates, although lethal, is clearly less robust relative to children and adults. Similarly, the expression of genes involved in host protective immunity, particularly neutrophil function, is also markedly deficient. As a result, neonates have both a diminished inflammatory and protective immune response to infection which may explain their increased risk to infection, and their reduced ability to clear infections. Such studies imply that novel approaches unique to the neonate will be required for the development of both diagnostics and therapeutics in this high at-risk population.

Cystathionine metabolic enzymes play a role in the inflammation resolution of human keratinocytes in response to sub-cytotoxic formaldehyde exposure

Low-level formaldehyde exposure is inevitable in industrialized countries. Although daily-life formaldehyde exposure level is practically impossible to induce cell death, most of mechanistic studies related to formaldehyde toxicity have been performed in cytotoxic concentrations enough to trigger cell death mechanism. Currently, toxicological mechanisms underlying the sub-cytotoxic exposure to formaldehyde are not clearly elucidated in skin cells. In this study, the genome-scale transcriptional analysis in normal human keratinocytes (NHKs) was performed to investigate cutaneous biological pathways associated with daily life formaldehyde exposure. We selected the 175 upregulated differentially expressed genes (DEGs) and 116 downregulated DEGs in NHKs treated with 200μM formaldehyde. In the Gene Ontology (GO) enrichment analysis of the 175 upregulated DEGs, the endoplasmic reticulum (ER) unfolded protein response (UPR) was identified as the most significant GO biological process in the formaldeyde-treated NHKs. Interestingly, the sub-cytotoxic formaldehyde affected NHKs to upregulate two enzymes important in the cellular transsulfuration pathway, cystathionine γ-lyase (CTH) and cystathionine-β-synthase (CBS). In the temporal expression analysis, the upregulation of the pro-inflammatory DEGs such as MMP1 and PTGS2 was detected earlier than that of CTH, CBS and other ER UPR genes. The metabolites of CTH and CBS, l-cystathionine and l-cysteine, attenuated the formaldehyde-induced upregulation of pro-inflammatory DEGs, MMP1, PTGS2, and CXCL8, suggesting that CTH and CBS play a role in the negative feedback regulation of formaldehyde-induced pro-inflammatory responses in NHKs. In this regard, the sub-cytotoxic formaldehyde-induced CBS and CTH may regulate inflammation fate decision to resolution by suppressing the early pro-inflammatory response.

Mycoplasma hyopneumoniae and Mycoplasma flocculare differential domains from orthologous surface proteins induce distinct cellular immune responses in mice

Mycoplasma hyopneumoniae and Mycoplasma flocculare are two genetically close species found in the swine respiratory tract. Despite their similarities, while M. hyopneumoniae is the causative agent of porcine enzootic pneumonia, M. flocculare is a commensal bacterium. Genomic and transcriptional comparative analyses so far failed to explain the difference in pathogenicity between these two species. We then hypothesized that such difference might be, at least in part, explained by amino acid sequence and immunological or functional differences between ortholog surface proteins. In line with that, it was verified that approximately 85% of the ortholog surface proteins from M. hyopneumoniae 7448 and M. flocculare present one or more differential domains. To experimentally assess possible immunological implications of this kind of difference, the extracellular differential domains from one pair of orthologous surface proteins (MHP7448_0612, from M. hyopneumoniae, and MF_00357, from M. flocculare) were expressed in E. coli and used to immunize mice. The recombinant polypeptides (rMHP61267–169 and rMF35767–196, respectively) induced distinct cellular immune responses. While, rMHP61267–169 induced both Th1 and Th2 responses, rMF35767-196 induced just an early pro-inflammatory response. These results indicate that immunological properties determined by differential domains in orthologous surface protein might play a role in pathogenicity, contributing to elicit specific and differential immune responses against each species.

Fas Regulates Macrophage Polarization and Fibrogenic Phenotype in a Model of Chronic Ethanol-Induced Hepatocellular Injury

The role of Fas-mediated apoptosis and its effect on proinflammatory cytokine production in early alcoholic liver disease has not been addressed. Wild-type mice (C57Bl/6) or mice with a functional mutation in the Fas ligand (B6.gld) were given either high-fat control diet or ethanol diet by intragastric cannulation for 2 or 4 weeks. Liver injury, hepatic lipid accumulation, and proinflammatory cytokine production associated with chronic ethanol consumption were largely prevented in B6.gld mice compared with wild-type mice. Conversely, B6.gld mice given ethanol exhibited increases in collagen deposition, hepatic collagen gene expression, and profibrogenic cytokines (eg, transforming growth factor-β and IL-13) and alterations in matrix remodeling proteins (eg, matrix metalloproteinases and tissue inhibitor of metalloproteinases) compared with wild-type mice. Hepatic F4/80(+) macrophage populations were increased significantly in B6.gld mice compared with wild-type mice; hepatic CD3(+) cell populations were not significantly different. Importantly, a shift toward the expression of M2/Th2 cytokines (eg, IL-4 and IL-13) after ethanol exposure was observed in B6.gld mice compared with classical M1 cytokine expression in wild-type mice under similar conditions. In isolated macrophages, stimulation of Fas receptor minimally enhances lipopolysaccharide-induced M1 cytokine production and significantly limits M2 cytokine production. These data support the hypothesis that Fas-mediated signaling is important for an early ethanol-induced proinflammatory response but limits the profibrogenic response, regulating collagen production in response to chronic ethanol.

Perioperative Simvastatin Therapy in Major Colorectal Surgery: A Prospective, Double-Blind Randomized Controlled Trial

Statins have numerous potential benefits relevant to abdominal surgery, and their use has been associated with a reduction in the systemic inflammatory response syndrome, wound infection, and anastomotic leak after colorectal surgery. However, this clinical evidence is limited to retrospective studies. The aim of this study was to prospectively investigate whether perioperative statin therapy can decrease the incidence of complications after major colorectal surgery. A prospective, double-blind, parallel-group, randomized controlled trial was conducted at 3 tertiary hospitals in New Zealand, between October 2011 and August 2013. Adult patients undergoing elective colorectal resection for any indication or reversal of Hartmann's procedure were randomized with a 1:1 patient allocation ratio to receive either 40 mg oral simvastatin or placebo once daily for 3 to 7 days preoperatively until 14 days postoperatively. The primary outcome was the overall incidence of complications for 30 days postoperatively. Secondary outcomes included the systemic and peritoneal cytokine response (interleukin [IL]-1α, IL-1β, IL-6, IL-8, IL-10, tumor necrosis factor [TNF]α) on postoperative day1. There were 132 patients included in the study (65 simvastatin, 67 placebo). There were no significant differences between the 2 groups at baseline with regard to patient, operation, and disease characteristics. There were no significant differences between the 2 groups in the incidence, grade, and type of postoperative complications (simvastatin: 44 [68%] vs placebo: 50 [75%], odds ratio 0.71 [95% CI 0.33 to 1.52], p= 0.444). Plasma concentrations of IL-6, IL-8, and TNFα, and peritoneal concentrations of IL-6 and IL-8, were significantly lower in the simvastatin group postoperatively. Perioperative simvastatin therapy in major colorectal surgery attenuates the early proinflammatory response to surgery, but there were no differences in postoperative complications.

Temporal Progression of Pneumonic Plague in Blood of Nonhuman Primate: A Transcriptomic Analysis.

Early identification of impending illness during widespread exposure to a pathogenic agent offers a potential means to initiate treatment during a timeframe when it would be most likely to be effective and has the potential to identify novel therapeutic strategies. The latter could be critical, especially as antibiotic resistance is becoming widespread. In order to examine pre-symptomatic illness, African green monkeys were challenged intranasally with aerosolized Yersinia pestis strain CO92 and blood samples were collected in short intervals from 45 m till 42 h post-exposure. Presenting one of the first genomic investigations of a NHP model challenged by pneumonic plague, whole genome analysis was annotated in silico and validated by qPCR assay. Transcriptomic profiles of blood showed early perturbation with the number of differentially expressed genes increasing until 24 h. By then, Y. pestis had paralyzed the host defense, as suggested by the functional analyses. Early activation of the apoptotic networks possibly facilitated the pathogen to overwhelm the defense mechanisms, despite the activation of the pro-inflammatory mechanism, toll-like receptors and microtubules at the port-of-entry. The overexpressed transcripts encoding an early pro-inflammatory response particularly manifested in active lymphocytes and ubiquitin networks were a potential deviation from the rodent models, which needs further verification. In summary, the present study recognized a pattern of Y. pestis pathogenesis potentially more applicable to the human system. Independent validation using the complementary omics approach with comprehensive evaluation of the organs, such as lungs which showed early bacterial infection, is essential.

AMPK and mTOR: sensors and regulators of immunometabolic changes during Salmonella infection in the chicken

Non-typhoidal Salmonella enterica induce an early pro-inflammatory response in chickens, but the response is short-lived, asymptomatic of clinical disease, results in a persistent colonization of the gastrointestinal (GI) tract, and can transmit infections to naïve hosts via fecal shedding of bacteria. The underlying mechanisms that facilitate this persistent colonization of the ceca of chickens by Salmonella are unknown. We have begun to concentrate on the convergence of metabolism and immune function as playing a major role in regulating the host responsiveness to infection. It is now recognized that the immune system monitors the metabolic state of tissues and responds by modulating metabolic function. The aim in this review is to summarize the literature that has defined a series of genotypic and phenotypic alterations in the regulatory host immune-metabolic signaling pathways in the local cecal microenvironment during the first 4 d following infection with Salmonella enterica serovar Enteritidis. Using chicken-specific kinomic immune-metabolism peptide arrays and quantitative real-time-PCR of cecal tissue during the early (4 to 48 h) and late stages (4 to 17 d) of a Salmonella infection in young broiler chickens, the local immunometabolic microenvironment has been ascertained. Distinct immune and metabolic pathways are altered between 2 to 4 d post-infection that dramatically changed the local immunometabolic environment. Thus, the tissue immunometabolic phenotype of the cecum plays a major role in the ability of the bacterium to establish a persistent cecal colonization. In general, our findings show that AMPK and mTOR are key players linking specific extracellular milieu and intracellular metabolism. Phenotypically, the early response (4 to 48 h) to Salmonella infection is pro-inflammatory, fueled by glycolysis and mTOR-mediated protein synthesis, whereas by the later phase (4 to 5 d), the local environment has undergone an immune-metabolic reprogramming to an anti-inflammatory state driven by AMPK-directed oxidative phosphorylation. Therefore, metabolism appears to provide a potential critical control point that can impact infection. Further understanding of metabolic control of immunity during infection should provide crucial information of the development of novel therapeutics based on metabolic modulators that enhance protection or inhibit infection.

MICU1 Regulation of Mitochondrial Calcium Uptake is Crucial for Liver Regeneration

Liver regeneration is a complex and well-orchestrated process where after partial hepatectomy (PHx) the initial mass of the organ is restored through both proliferative and hepatoprotective signaling cascades. Among the myriad of agonists that drives this process many involve Ca2+ signaling. Mechanisms through which cytosolic and mitochondrial Ca2+ signaling impact the onset of regeneration and progression of hepatocytes through the cell cycle are not well understood. Mitochondrial Ca2+ handling is critical in maintaining cellular Ca2+ homeostasis. MICU1 (mitochondrial calcium uptake-1) has been identified as a critical regulatory protein of MCU (mitochondrial calcium uniporter) mediated Ca2+ uptake. Here we test the hypothesis that deregulation of mitochondrial Ca2+ transport by knockdown (KD) of MICU1 compromises liver regeneration. We performed 70% PHX in mice with acute liver-specific MICU1 deletion. By 30hrs post-PHX, KD mice showed evidence of major liver damage and impaired function as shown by liver histology and dramatic increases in serum ALT and bilirubin as compared to control. Hepatocyte proliferation as measured by BrdU incorporation was undetectable in remnant livers in KD mice. This correlated with a total lack of cyclin D1 expression 30hrs post-PHX. Events associated with the early priming phase (1hr) of regeneration were mostly unaffected by the loss of MICU1. However, a sustained tissue IL6 and TNFα elevation in KD mice by 6hrs suggested a failure to resolve early pro-inflammatory responses. Experiments on isolated hepatocytes indicate that MICU1 ablation decreases the mitochondrial Ca2+ uptake threshold and enhances their susceptibility to permeability transition due to calcium overload. Treatment with the PTP inhibitor NIM811 protected KD mice from liver damage and restored hepatocyte proliferation after PHX. In summary, regulation of mitochondrial calcium uptake through MICU1 is pivotal in protecting against mitochondrial calcium overload to permit progression of liver regeneration.

A Role for the Non-Canonical Wnt-β-Catenin and TGF-β Signaling Pathways in the Induction of Tolerance during the Establishment of a Salmonella enterica Serovar Enteritidis Persistent Cecal Infection in Chickens.

Non-typhoidal Salmonella enterica induce an early pro-inflammatory response in chickens. However, the response is short-lived, asymptomatic of disease, resulting in a persistent colonization of the ceca, and fecal shedding of bacteria. The underlying mechanisms that control this persistent infection of chickens by Salmonella are unknown. Recently, we found an expansion of the Treg population and subsequent increased in vitro immunosuppressive functions of the CD4+CD25+ cells isolated from the ceca of the Salmonella-infected chickens by day 4 post-infection that increased steadily throughout the course of the 14 days of infection, whereas the number of CD4+CD25+ cells in the non-infected controls remained steady throughout the study. CD4+CD25+ cells from cecal tonsils of S. enteritidis-infected birds had greater expression of IL-10 mRNA content than the CD4+CD25+ cells from the non-infected controls at all the time points studied. These results suggest the development of a tolerogenic immune response in the cecum of Salmonella-infected chickens may contribute to the persistance of Salmonella cecal colonization. Using a chicken-specific kinome peptide immune array, we have analyzed the signaling pathways altered during the establishment of this tolerogenic state. This analysis has revealed a role for the non-canonical Wnt signaling pathway in the cecum at 4 days post-infection. Infection induced the significant (p < 0.01) phosphorylation of the G-protein-coupled transmembrane protein, Frizzled 1 (FZD1), resulting in an influx of intracellular Ca2+ and the phosphorylation of the Ca2+-dependent effector molecules calcium/calmodulin-dependent kinase II (CamKII), β-catenin, protein kinase C, and the activation of the transcription factor, NFAT. Nuclear translocation of NFAT resulted in a significant increase in the expression of the anti-inflammatory cytokines IL-10 and TGF-β. Increased expression of TGF-β4 mRNA activates the TGF-β signaling pathway that phosphorylates the receptor-activated Smads, Smad2 and Smad3. Combined with the results from our Treg studies, these studies describe kinome-based phenotypic changes in the cecum of chickens during Salmonella Enteritidis infection starting 4 days post-infection that leads to an anti-inflammatory, tolerogenic local environment, and results in the establishment of persistent intestinal colonization.

The mitochondria-targeted anti-oxidant MitoQ decreases ischemia-reperfusion injury in a murine syngeneic heart transplant model

BackgroundFree radical production and mitochondrial dysfunction during cardiac graft reperfusion is a major factor in post-transplant ischemia-reperfusion (IR) injury, an important underlying cause of primary graft dysfunction. We therefore assessed the efficacy of the mitochondria-targeted anti-oxidant MitoQ in reducing IR injury in a murine heterotopic cardiac transplant model.MethodsHearts from C57BL/6 donor mice were flushed with storage solution alone, solution containing the anti-oxidant MitoQ, or solution containing the non–anti-oxidant decyltriphenylphosphonium control and exposed to short (30 minutes) or prolonged (4 hour) cold preservation before transplantation. Grafts were transplanted into C57BL/6 recipients and analyzed for mitochondrial reactive oxygen species production, oxidative damage, serum troponin, beating score, and inflammatory markers 120 minutes or 24 hours post-transplant.ResultsMitoQ was taken up by the heart during cold storage. Prolonged cold preservation of donor hearts before IR increased IR injury (troponin I, beating score) and mitochondrial reactive oxygen species, mitochondrial DNA damage, protein carbonyls, and pro-inflammatory cytokine release 24 hours after transplant. Administration of MitoQ to the donor heart in the storage solution protected against this IR injury by blocking graft oxidative damage and dampening the early pro-inflammatory response in the recipient.ConclusionsIR after heart transplantation results in mitochondrial oxidative damage that is potentiated by cold ischemia. Supplementing donor graft perfusion with the anti-oxidant MitoQ before transplantation should be studied further to reduce IR-related free radical production, the innate immune response to IR injury, and subsequent donor cardiac injury.

The type III secretion effector NleF of enteropathogenic Escherichia coli activates NF-κB early during infection.

The enteric pathogens enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli employ a type 3 secretion system (T3SS) to manipulate the host inflammatory response during infection. Previously, it has been reported that EPEC, in a T3SS-dependent manner, induces an early proinflammatory response through activation of NF-κB via extracellular signal-regulated kinases 1 and 2 (ERK1/2) and protein kinase Cζ (PKCζ). However, the activation of NF-κB during infection has not yet been attributed to an effector. At later time points postinfection, NF-κB signaling is inhibited through the translocation of multiple effectors, including NleE and NleC. Here we report that the highly conserved non-LEE (locus of enterocyte effacement)-encoded effector F (NleF) shows both diffuse and mitochondrial localization during ectopic expression. Moreover, NleF induces the nuclear translocation of NF-κB p65 and the expression of interleukin 8 (IL-8) following ectopic expression and during EPEC infection. Furthermore, the proinflammatory activity and localization of NleF were dependent on the C-terminal amino acids LQCG. While the C-terminal domain of NleF has previously been shown to be essential for interaction with caspase-4, caspase-8, and caspase-9, the proinflammatory activity of NleF was independent of interaction with caspase-4, -8, or -9. In conclusion, EPEC, through the T3SS-dependent translocation of NleF, induces a proinflammatory response in an NF-κB-dependent manner in the early stages of infection.

Interferon regulatory factor 5-dependent immune responses in the draining lymph node protect against West Nile virus infection.

Upon activation of Toll-like and RIG-I-like receptor signaling pathways, the transcription factor IRF5 translocates to the nucleus and induces antiviral immune programs. The recent discovery of a homozygous mutation in the immunoregulatory gene guanine exchange factor dedicator of cytokinesis 2 (Dock2mu/mu) in several Irf5-/- mouse colonies has complicated interpretation of immune functions previously ascribed to IRF5. To define the antiviral functions of IRF5 in vivo, we infected backcrossed Irf5-/-×Dock2wt/wt mice (here called Irf5-/- mice) and independently generated CMV-Cre Irf5fl/fl mice with West Nile virus (WNV), a pathogenic neurotropic flavivirus. Compared to congenic wild-type animals, Irf5-/- and CMV-Cre Irf5fl/fl mice were more vulnerable to WNV infection, and this phenotype was associated with increased infection in peripheral organs, which resulted in higher virus titers in the central nervous system. The loss of IRF5, however, was associated with only small differences in the type I interferon response systemically and in the draining lymph node during WNV infection. Instead, lower levels of several other proinflammatory cytokines and chemokines, as well as fewer and less activated immune cells, were detected in the draining lymph node 2 days after WNV infection. WNV-specific antibody responses in Irf5-/- mice also were blunted in the context of live or inactivated virus infection and this was associated with fewer antigen-specific memory B cells and long-lived plasma cells. Our results with Irf5-/- mice establish a key role for IRF5 in shaping the early innate immune response in the draining lymph node, which impacts the spread of virus infection, optimal B cell immunity, and disease pathogenesis. Although the roles of IRF3 and IRF7 in orchestrating innate and adaptive immunity after viral infection are established, the function of the related transcription factor IRF5 remains less certain. Prior studies in Irf5-/- mice reported conflicting results as to the contribution of IRF5 in regulating type I interferon and adaptive immune responses. The lack of clarity may stem from a recently discovered homozygous loss-of-function mutation of the immunoregulatory gene Dock2 in several colonies of Irf5-/- mice. Here, using a mouse model with a deficiency in IRF5 and wild-type Dock2 alleles, we investigated how IRF5 modulates West Nile virus (WNV) pathogenesis and host immune responses. Our in vivo studies indicate that IRF5 has a key role in shaping the early proinflammatory cytokine response in the draining lymph node, which impacts immunity and control of WNV infection.

Alpha/Beta Interferon Receptor Signaling Amplifies Early Proinflammatory Cytokine Production in the Lung during Respiratory Syncytial Virus Infection

ABSTRACTType I interferons (IFNs) are produced early upon virus infection and signal through the alpha/beta interferon (IFN-α/β) receptor (IFNAR) to induce genes that encode proteins important for limiting viral replication and directing immune responses. To investigate the extent to which type I IFNs play a role in the local regulation of inflammation in the airways, we examined their importance in early lung responses to infection with respiratory syncytial virus (RSV). IFNAR1-deficient (IFNAR1−/−) mice displayed increased lung viral load and weight loss during RSV infection. As expected, expression of IFN-inducible genes was markedly reduced in the lungs of IFNAR1−/− mice. Surprisingly, we found that the levels of proinflammatory cytokines and chemokines in the lungs of RSV-infected mice were also greatly reduced in the absence of IFNAR signaling. Furthermore, low levels of proinflammatory cytokines were also detected in the lungs of IFNAR1−/− mice challenged with noninfectious innate immune stimuli such as selected Toll-like receptor (TLR) agonists. Finally, recombinant IFN-α was sufficient to potentiate the production of inflammatory mediators in the lungs of wild-type mice challenged with innate immune stimuli. Thus, in addition to its well-known role in antiviral resistance, type I IFN receptor signaling acts as a central driver of early proinflammatory responses in the lung. Inhibiting the effects of type I IFNs may therefore be useful in dampening inflammation in lung diseases characterized by enhanced inflammatory cytokine production.IMPORTANCE The initial response to viral infection is characterized by the production of interferons (IFNs). One group of IFNs, the type I IFNs, are produced early upon virus infection and signal through the IFN-α/β receptor (IFNAR) to induce proteins important for limiting viral replication and directing immune responses. Here we examined the importance of type I IFNs in early responses to respiratory syncytial virus (RSV). Our data suggest that type I IFN production and IFNAR receptor signaling not only induce an antiviral state but also serve to amplify proinflammatory responses in the respiratory tract. We also confirm this conclusion in another model of acute inflammation induced by noninfectious stimuli. Our findings are of relevance to human disease, as RSV is a major cause of infant bronchiolitis and polymorphisms in the IFN system are known to impact disease severity.

Suppressed cytokine expression immediatey following traumatic brain injury in neonatal rats indicates an expeditious endogenous anti-inflammatory response

The timing of therapeutic intervention in traumatic brain injury (TBI) is critical. Although immediate cell death cascades have become established in adult TBI, the pathophysiology underlying neonatal TBI is poorly understood. The objective of the present study was to determine the role of cytokine regulation following TBI in neonatal rats. Seven-day-old Sprague-Dawley rats were subjected to TBI using the controlled cortical impact (CCI) injury model. Age-matched littermates that did not receive TBI served as the controls. Immediately following TBI, rats were euthanized, and the brains were divided into the ipsilateral and contralateral hemispheres then flash frozen. A BioRad 23-Plex panel was used to measure cytokine levels. Surprisingly, the data revealed that 18 of the 23 cytokines analyzed were significantly downregulated in the hemisphere contralateral to the TBI impacted hemisphere. IL-5, IL-6 and MIP-3a were significantly suppressed in both ipsilateral and contralateral hemispheres of neonatal TBI rats compared to the control rats. A parallel study processing the plasma of the same cohort of neonatal rats revealed no difference in the same cytokines analyzed in the brain tissue, suggesting highly localized cytokine suppression in the brain during early injury. In stark contrast to the reported early pro-inflammatory response exhibited in adult TBI, the present neonatal TBI study demonstrated a reversed cytokine profile of downregulation. These results suggest a robust, immediate anti-inflammatory response mounted by the contralateral hemisphere of the young brain.