Blunted Cytokine Response Research Articles

MDA5 ISGylation is crucial for immune signaling to control viral replication and pathogenesis.

The posttranslational modification (PTM) of innate immune sensor proteins by ubiquitin or ubiquitin-like proteins is crucial for regulating antiviral host responses. The cytoplasmic dsRNA receptor melanoma differentiation-associated protein 5 (MDA5) undergoes several PTMs including ISGylation within its first caspase activation and recruitment domain (CARD), which promotes MDA5 signaling. However, the relevance of MDA5 ISGylation for antiviral immunity in an infected organism has been elusive. Here, we generated knock-in mice (MDA5K23R/K43R) in which the two major ISGylation sites, K23 and K43, in MDA5 were mutated. Primary cells derived from MDA5K23R/K43R mice exhibited abrogated endogenous MDA5 ISGylation and an impaired ability of MDA5 to form oligomeric assemblies leading to blunted cytokine responses to MDA5 RNA-agonist stimulation or infection with encephalomyocarditis virus (EMCV) or West Nile virus. Phenocopying MDA5-/- mice, the MDA5K23R/K43R mice infected with EMCV displayed increased mortality, elevated viral titers, and an ablated induction of cytokines and chemokines compared to WT mice. Molecular studies identified human HERC5 (and its functional murine homolog HERC6) as the primary E3 ligases responsible for MDA5 ISGylation and activation. Taken together, these findings establish the importance of CARD ISGylation for MDA5-mediated RNA virus restriction, promoting potential avenues for immunomodulatory drug design for antiviral or anti-inflammatory applications.

The shifting lipidomic landscape of blood monocytes and neutrophils during pneumonia.

The lipidome of immune cells during infection has remained unexplored, although evidence of the importance of lipids in the context of immunity is mounting. In this study, we performed untargeted lipidomic analysis of blood monocytes and neutrophils from patients hospitalized for pneumonia and age- and sex-matched noninfectious control volunteers. We annotated 521 and 706 lipids in monocytes and neutrophils, respectively, which were normalized to an extensive set of internal standards per lipid class. The cellular lipidomes were profoundly altered in patients, with both common and distinct changes between the cell types. Changes involved every level of the cellular lipidome: differential lipid species, class-wide shifts, and altered saturation patterns. Overall, differential lipids were mainly less abundant in monocytes and more abundant in neutrophils from patients. One month after hospital admission, lipidomic changes were fully resolved in monocytes and partially in neutrophils. Integration of lipidomic and concurrently collected transcriptomic data highlighted altered sphingolipid metabolism in both cell types. Inhibition of ceramide and sphingosine-1-phosphate synthesis in healthy monocytes and neutrophils resulted in blunted cytokine responses upon stimulation with lipopolysaccharide. These data reveal major lipidomic remodeling in immune cells during infection, and link the cellular lipidome to immune functionality.

TLR7 activation at epithelial barriers promotes emergency myelopoiesis and lung antiviral immunity.

Monocytes are heterogeneous innate effector leukocytes generated in the bone marrow and released into circulation in a CCR2-dependent manner. During infection or inflammation, myelopoiesis is modulated to rapidly meet the demand for more effector cells. Danger signals from peripheral tissues can influence this process. Herein we demonstrate that repetitive TLR7 stimulation via the epithelial barriers drove a potent emergency bone marrow monocyte response in mice. This process was unique to TLR7 activation and occurred independently of the canonical CCR2 and CX3CR1 axes or prototypical cytokines. The monocytes egressing the bone marrow had an immature Ly6C-high profile and differentiated into vascular Ly6C-low monocytes and tissue macrophages in multiple organs. They displayed a blunted cytokine response to further TLR7 stimulation and reduced lung viral load after RSV and influenza virus infection. These data provide insights into the emergency myelopoiesis likely to occur in response to the encounter of single-stranded RNA viruses at barrier sites.

Dolphin leukocytes exhibit an attenuated cytokine response and increase heme oxygenase activity upon exposure to lipopolysaccharides

Cetaceans exhibit physiological adaptations that allowed the transition to aquatic life, including a robust antioxidant defense system that prevents injury from repeated exposure to ischemia/reperfusion events associated with breath-hold diving. The signaling cascades that characterize ischemic inflammation in humans are well characterized. In contrast, cetaceans' molecular and biochemical mechanisms that confer tolerance to inflammatory events are poorly understood. Heme oxygenase (HO) is a cytoprotective protein with anti-inflammatory properties. HO catalyzes the first step in the oxidative degradation of heme. The inducible HO-1 isoform is regulated by various stimuli, including hypoxia, oxidant stress, and inflammatory cytokines. The objective of this study was to compare the response of HO-1 and cytokines to a proinflammatory challenge in leukocytes isolated from humans and bottlenose dolphins (Tursiops truncatus). We measured changes in HO activity, and abundance and expression of interleukin 1 beta (IL-1β), interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), and heme oxygenase 1 (HMOX1) in leukocytes treated with lipopolysaccharide (LPS) for 24 and 48 h. HO activity increased (p < 0.05) in dolphin (48 h) but not human cells. TNF-α expression increased in human (24 h, 48 h), but not dolphin cells following LPS stimulation. LPS-induced cytokine expression was lower in dolphin than in human leukocytes, suggesting a blunted cytokine response in bottlenose dolphin leukocytes treated with LPS. Results suggest species-specific regulation of inflammatory cytokines in leukocytes treated with LPS, which may lead to differential responses to a pro-inflammatory challenge between marine and terrestrial mammals.

EXTH-40. MULTIDIMENSIONAL INTERROGATION OF GLIOBLASTOMA MICROENVIRONMENT TREATMENT RESPONSE IN EXPLANT CULTURES

Abstract The immune tumor microenvironment (iTME) of glioblastoma (GBM) contains microglial, macrophage, other myeloid cell populations and as adaptive immune cells. Recent therapeutic strategies for GBM aim at targeting iTME components to induce antitumoral immunity. A patient-tailored, ex vivo drug testing and response analysis platform would facilitate personalized therapy planning, provide insights into treatment-induced immune mechanisms in the iTME, and enable the discovery of biomarkers of response and resistance. Here, we generated patient-derived, live 3D GBM bioreactors from different tumor regions to assess iTME treatment responses to microglia modulators and immune checkpoint inhibitors. Intact GBM tissue specimens from the tumor center and periphery were cultured for 7 days in the presence or absence of anti-PD1, anti-CD47 antibodies or their combination. Tissues were analyzed by CODEX highly multiplexed microscopy using an immune-centered 54-marker panel, and changes in cytokine and chemokine levels in culture supernatants were investigated. A computational pipeline for integrative therapy response assessment was implemented. Explant cultures from n=8 IDH wt GBM were subjected to this integrative personalized analysis. Tissue integrity after 3D bioreactor cultures was comparable to tissue taken directly after surgery. FFPE CODEX workflow was feasible with adequate staining quality in bioreactor cultures. 850'000 single cells were segmented and clustered. Cellular composition between tumor center and the peripheral invasion zone differed significantly in immune phenotypes, cytokine profile and response to innate, adaptive or combinatorial local immunotherapies. Multiplexed cytokine analysis revealed IFNγ response signatures in a subset of center samples, whereas the peripheral invasion zone displayed a blunted cytokine response. This cytokine signature corresponded to cellular composition shifts within specific cellular neighborhoods. CD4 and CD8 T cells were invigorated and left their vascular niche. Our study demonstrates that local immunotherapies enable an active antitumoral immune response within the tumor center, and provides a multidimensional personalized framework for immunotherapy response assessment.

Racial Differences in the Biochemical Effects of Stress in Pregnancy.

Prenatal stress has been linked to preterm birth via inflammatory dysregulation. We conducted a cross-sectional study on female participants who delivered live, singleton infants at University of Pittsburgh Medical Center Magee Women’s Hospital. Participants (n = 200) were stratified by cumulative risk scores using a combination of individual factors (maternal education, diabetes, hypertension, smoking, relationship status, obesity, depression) and neighborhood deprivation scores. We hypothesized that inflammatory cytokines levels differ by risk group and race. Multiplex analyses of IL-6, IL-8, IL-10, IL-13 and TNF-alpha were run. We found that Black birthing people had more risk factors for chronic stress and had lower levels of IL-6 compared to White birthing people. When stratified by risk group and race, low-risk Black birthing people had lower levels of IL-6 compared to low-risk White birthing people, and high-risk Black birthing people had lower levels of IL-8 compared to high-risk White birthing people. Higher area deprivation scores were associated with lower IL-6 levels. Our results suggest that the relationship between chronic stress and inflammatory cytokines is modified by race. We theorize that Black birthing people encounter repetitive stress due to racism and social disadvantage which may result in stress pathway desensitization and a blunted cytokine response to future stressors.

Improving our mechanistic understanding of the indirect effects of CMV infection in transplant recipients.

Cytomegalovirus (CMV) is an immunomodulatory virus that indirectly increases the risk for bacterial, fungal, and viral infections. However, the pathogenesis of this phenomenon is poorly understood. We determined whether inflammatory responses to different Toll-like receptor (TLR) ligands are blunted during CMV infection in solid-organ transplant (SOT) patients. Peripheral blood mononuclear cells from 38 SOT patients with and without CMV were incubated in the presence of various viral, fungal, and bacterial TLR ligands. Cytokines were measured in the supernatant by multiplex enzyme-linked immunosorbent assay. Patients had blunted cytokine responses to bacterial, fungal, and viral ligands during CMV infection when compared to the absence of CMV infection. This was independent of viral load, clinical presentation of CMV infection or immunosuppression, supporting the clinical observation in SOT recipients that CMV infection increases susceptibility to bacterial, fungal, and other viral infections. Moreover, in the absence of CMV infection, patients with subsequent CMV infection had lower cytokines in response to TLR ligands compared to those without subsequent CMV infection, suggesting that inherent differences in patients not directly related to CMV also contribute to this increased susceptibility. In summary, these data provide novel ex vivo evidence to support indirect effects of CMV.

IAPs Regulate Distinct Innate Immune Pathways to Co-ordinate the Response to Bacterial Peptidoglycans

Inhibitors of apoptosis (IAPs) proteins are critical regulators of innate immune signaling pathways and therefore have potential as drug targets. X-linked IAP (XIAP) and cellular IAP1 and IAP2 (cIAP1 and cIAP2) are E3 ligases that have been shown to be required for signaling downstream of NOD2, an intracellular receptor for bacterial peptidoglycan. We used genetic and biochemical approaches to compare the responses of IAP-deficient mice and cells to NOD2 stimulation. In all cell types tested, XIAP is the only IAP required for signaling immediately downstream of NOD2, while cIAP1 and cIAP2 are dispensable for NOD2-induced nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) activation. However, mice lacking cIAP1 or TNFR1 have a blunted cytokine response to NOD2 stimulation. We conclude that cIAPs regulate NOD2-dependent autocrine TNF signaling invivo and highlight the importance of physiological context in the interplay of innate immune signaling pathways.

Retinal Caveolin-1 Modulates Neuroprotective Signaling.

Caveolin-1 (Cav-1), the scaffolding protein of caveolae, is expressed in several retinal cell types and is associated with ocular pathologies. Cav-1 modulates neuroinflammatory/neuroprotective responses to central nervous system injury. We have shown that loss of Cav-1 results in a blunted cytokine response in retinas challenged with inflammatory stimuli. As neuroinflammatory and neuroprotective signaling overlap in their cytokine production and downstream signaling pathways, we hypothesized that loss of Cav-1 may also suppress neuroprotective signaling in the retina. To test this, we subjected mice in which Cav-1 was deleted specifically in the retina to a neurodegenerative insult induced by sodium iodate (NaIO3) and measured STAT3 activation, a measure of neuroprotective signaling. Our results show that Cav-1 ablation blunts STAT3 activation induced by NaIO3. STAT3 activation in response to intravitreal administration of the IL-6 family cytokine, leukemia inhibitory factor (LIF), was not affected by Cav-1 deletion indicating a competent gp130 receptor response. Thus, Cav-1 modulates neuroprotective signaling by regulating the endogenous production of neuroprotective factors.

Male adolescent rats display blunted cytokine responses in the CNS after acute ethanol or lipopolysaccharide exposure

Alcohol induces widespread changes in cytokine expression, with recent data from our laboratory having demonstrated that, during acute ethanol intoxication, adult rats exhibit consistent increases in interleukin (IL)-6 mRNA expression in several brain regions, while showing reductions in IL-1 and TNFα expression. Given evidence indicating that adolescence may be an ontogenetic period in which some neuroimmune processes and cells may not yet have fully matured, the purpose of the current experiments was to examine potential age differences in the central cytokine response of adolescent (P31–33days of age) and adult (69–71days of age) rats to either an acute immune (lipopolysaccharide; LPS) or non-immune challenge (ethanol). In Experiment 1, male Sprague–Dawley rats were given an intraperitoneal (i.p.) injection of either sterile saline, LPS (250μg/kg), or ethanol (4-g/kg), and then trunk blood and brain tissue were collected 3h later for measurement of blood ethanol concentrations (BECs), plasma endotoxin, and central mRNA expression of several immune-related gene targets. In Experiment 2, the response to intragastrically (i.g.) administered ethanol was examined and compared to animals given tap water (i.g.). Results showed that LPS stimulated robust increases in expression of IL-1, IL-6, TNFα, and IκBα in the hippocampus, PVN, and amygdala, and that these increases were generally less pronounced in adolescents relative to adults. Following an i.p. ethanol challenge, IL-6 and IκBα expression was significantly increased in both ages in the PVN and amygdala, and adults exhibited even greater increases in IκBα than adolescents. I.g. administration of ethanol also increased IL-6 and IκBα expression in all three brain regions, with hippocampal IL-6 elevated even more so in adults compared to adolescents. Furthermore, assessment of plasma endotoxin concentrations revealed (i) whereas robust increases in plasma endotoxin were observed in adults injected with LPS, no corresponding elevations were seen in adolescents after LPS; and (ii) neither adolescents nor adults demonstrated increases in plasma endotoxin concentrations following i.p. or i.g. ethanol administration. Analysis of BECs indicated that, for both routes of exposure, adolescents exhibited lower BECs than adults. Taken together, these data suggest that categorically different mechanisms are involved in the central cytokine response to antigen exposure versus ethanol administration. Furthermore, these findings confirm once again that acute ethanol intoxication is a potent activator of brain cytokines, and calls for future studies to identify the mechanisms underlying age-related differences in the cytokine response observed during ethanol intoxication.

Resistance to invasive pneumococcal infection in mice with acute allergic lung inflammation (MPF1P.766)

Abstract A recent publication by Talbot et al (N Engl J Med 2005) first identified asthma as a risk factor for invasive pneumococcal disease (IPD). However, more recent reports suggest that mortality is unaffected following IPD in patients with asthma and that COPD, an obstructive condition similar to asthma, protects against the development of complicated pneumonia. To clarify the effects of asthma on the immune response to a pneumococcal infection in the lung, we used an acute murine model of ovalbumin (OVA)-induced allergic lung inflammation (ALI) and hypothesized that mice with acute ALI would show increased susceptibility to IPD. Surprisingly, mice with acute ALI demonstrated enhanced resistance to infection. Resistance in mice with ALI correlated with a greater capacity for bacterial clearance from the lungs within eight hours post infection. This early clearance resulted in decreased bacterial invasiveness into the lung tissue as well as a blunted cytokine response to infection. Despite an increase in alveolar macrophage levels following OVA challenge, the increased bacterial clearance appeared to be mediated by MHCII- SiglecF+ eosinophils. These findings suggest that while eosinophils are pathogenic in asthma they could be protective against IPD, highlighting the need for a clearer understanding of the risk of IPD in patients with different asthma phenotypes.

Screen of whole blood responses to flagellin identifies TLR5 variation associated with outcome in melioidosis

Melioidosis is a severe infection caused by the flagellated bacterium Burkholderia pseudomallei. The nonsense polymorphism TLR51174C>T is associated with improved outcome in Thais with melioidosis. We hypothesized that other TLR5 variants may modulate the host response and determine outcome in melioidosis. We genotyped 12 TLR5 variants selected de novo from the HapMap database and examined the association of each with cytokines induced by flagellin stimulation of whole blood from healthy Thai subjects. We found a blunted cytokine response for three related markers that were in linkage disequilibrium with a non-synonymous variant, TLR51846T>C. Carriers of TLR51846T>C had broadly impaired cytokine responses induced by flagellin. TLR51846T>C was associated with protection against death in melioidosis patients (OR 0.62, 95% CI: 0.42-0.93, p=0.021). We observed no impairment in TLR51846C-dependent NF-κB activation, however, suggesting an alternative mechanism for the effect. We found that TLR51846T>C was in strong linkage disequilibrium with TLR51174C>T. Many of the blunted cytokine responses observed and the association of TLR51846T>C with survival in melioidosis patients may be attributable to TLR51174C>T, but we could not exclude an independent effect of TLR51846T>C. These data identify novel associations for TLR51846T>C, enhance our understanding of TLR5 genetic architecture in Thais, and provide future directions of study.

Potential role for alternatively activated macrophages in the secondary bacterial infection during recovery from influenza

Purpose Secondary bacterial infections are a common complication of influenza. Innate immune host defenses appear to be impaired following influenza, leading to susceptibility to subsequent bacterial infections. Alternatively activated macrophages (AAM) in the lungs may play a critical role in eliciting the hypersusceptibility to secondary bacterial pneumonia. Methods C57BL6 mice were challenged with sublethal doses of the mouse-adapted A/PR/8/34 (PR8) influenza virus or saline and allowed to recover. At complete recovery (day 14), mice were re-challenged with sublethal doses of Streptococcus pneumoniae serotype 3 (Sp3). Results PR8-recovered mice developed a rapidly fatal pulmonary infection to a 100-fold sublethal pneumococcal challenge, whereas PR8-naive mice demonstrated no mortality or illness. The cytokines which induce AAM (IL-4 and IL-13) and the expression of genes associated with AAM (Arginase-1, FIZZ1, and YM1) were elevated after PR8 infection. Flow cytometry suggests that alveolar macrophages demonstrate the AAM-phenotype, as indicated by MGL-1 and MHCII expression, in response to PR8 infection. Recovery from PR8 was associated with blunted cytokine responses to TLR ligands. Conclusions The mechanisms of immune regulation during recovery from influenza are being elucidated. We provide evidence that pulmonary AAM are induced during influenza infection and may contribute to the elicitation of hypersusceptibility to a secondary bacterial infection.

Lysostaphin Reduces the Production of Inflammatory Cytokines in Staphylococcus aureus Challenged Mice, and Prevents Systemic Shock

Lysostaphin is being developed as a treatment for serious staphylococcal infections. Mice challenged with S. aureus produce inflammatory cytokines including, TNF-� and IL-6, and over-production of these cytokines can lead to shock and contribute to the lethality of staphylococcal infections. Two major components of the staphylococcal cell wall, peptidoglycan and lipoteichoic acid, are known to synergize to induce shock and organ failure in animal models, and we wished to determine whether the rapid lysostaphin-mediated degradation of peptidoglycan during treatment of systemic S. aureus infection could affect shock-associated parameters. We found that lysostaphin treatment of S. aureus-infected mice, which reduces bacteremia and organ infection, also reduced the serum levels of inflammatory cytokines and reversed the symptoms of S. aureus-induced shock. We compared the cytokine response of mice challenged with S. aureus to that of mice challenged with S. aureus and then treated with lysostaphin or nafcillin. Lysostaphin-treated mice, as compared with untreated mice or nafcillin-treated mice, had a blunted cytokine responses to S. aureus challenge. Core body temperature was used as a real time indicator for systemic shock in mice. Mice infected with S. aureus demonstrated a rapid drop in core body temperature, which was reversed by lysostaphin treatment. These studies demonstrated that lysostaphin treatment did not contribute to the induction of shock by rapidly releasing staphylococcal cell wall components, but rather it blunted the inflammatory cytokine response and ameliorated shock related symptoms. Keyword: S. aureus, lysostaphin, shock, cytokine release.

Alloimmunization to transfused HOD red blood cells is not increased in mice with sickle cell disease

Increased rates of red blood cell (RBC) alloimmunization in patients with sickle cell disease may be due to transfusion frequency, genetic predisposition, or immune dysregulation. To test the hypothesis that sickle cell pathophysiology influences RBC alloimmunization, we utilized two transgenic mouse models of sickle cell disease. Transgenic sickle mice, which express human α and β(S) globin, were transfused with fresh or 14-day-stored RBCs containing the HOD (hen egg lysozyme, ovalbumin, and human Duffy(b) ) antigen; some recipients were inflamed with poly(I : C) before transfusion. Anti-HOD alloantibody responses were subsequently measured by enzyme-linked immunosorbent assay and flow crossmatch; a cohort of recipients had posttransfusion serum cytokines measured by bead array. Both Berkeley and Townes homozygous (SS) and heterozygous (AS) mice had similar rates and magnitude of anti-HOD RBC alloimmunization after fresh HOD RBC transfusion compared with control animals; under no tested condition did homozygous SS recipients make higher levels of alloantibodies than control animals. Unexpectedly, homozygous SS recipients had blunted cytokine responses and lower levels of anti-HOD alloantibodies after transfusion of 14-day stored RBCs, compared with control animals. In sum, homozygous β(S) expression and the ensuing disease state are not alone sufficient to enhance RBC alloimmunization to transfused HOD RBCs in two distinct humanized murine models of sickle cell disease under the conditions examined. These data suggest that other factors may contribute to the high rates of RBC alloimmunization observed in humans with sickle cell disease.

Human Intestinal Lamina Propria CD1c+ Dendritic Cells Display an Activated Phenotype at Steady State and Produce IL-23 in Response to TLR7/8 Stimulation

Intestinal dendritic cells (DCs) play key roles in mediating tolerance to commensal flora and inflammatory responses against mucosal pathogens. The mechanisms by which intestinal "conditioning" influences human DC responses to microbial stimuli remain poorly understood. Infections with viruses, such as HIV-1, that target mucosal tissue result in intestinal epithelial barrier breakdown and increased translocation of commensal bacteria into the lamina propria (LP). It is unclear whether innate LP DC responses to concurrent viral and bacterial stimuli influence mucosal HIV-1 pathogenesis. In this study, direct ex vivo phenotype and in vitro constitutive cytokine production of CD1c+ DCs in human intestinal LP were compared with those in peripheral blood (PB). To evaluate innate responses to viral and bacterial stimuli, intracellular cytokine production by LP and PB DCs following stimulation with ligands for TLRs 2, 4, 5, and 7/8 was evaluated. At steady state, LP CD1c+ DCs expressed higher levels of activation markers (CD40, CD83, CD86, HLA-DR, and CCR7) than did PB CD1c+ DCs, and higher frequencies of LP CD1c+ DCs constitutively produced IL-6 and -10 and TNF-alpha. LP DCs had blunted cytokine responses to TLR4 ligand and TLR5 ligand stimulation relative to PB DCs, yet similarly produced IL-10 in response to TLR2 ligand. Only synthetic TLR7/8 ligand, a mimic of viral ssRNA, induced IL-23 production by LP CD1c+ DCs, and this proinflammatory cytokine response was synergistically enhanced following combined TLR7/8 and TLR4 stimulation. These findings highlight a potential mechanism by which viruses like HIV-1 may subvert homeostatic mechanisms and induce inflammation in the intestinal mucosa.

Toll-Like Receptor 2 and 4 Expression After Severe Injury is Not Involved in the Dysregulation of the Innate Immune System

Severe injury after trauma is associated with a diminished production of different proinflammatory cytokines after stimulation with bacterial cell wall components. The cellular mechanisms, leading to a decreased responsiveness especially of monocytes after multiple injuries have not yet been elucidated in detail. The expression of Toll-like receptors (TLR) on leukocytes is essential for recognition of bacterial components. We investigated the expression of TLR2 and 4 in correlation with gram-negative and gram-positive stimuli-dependent cytokine liberation after severe injury in comparison with that in healthy volunteers. In a prospective clinical experimental study, 12 trauma patients with an Injury Severity Score above 21 points and 14 healthy volunteers were analyzed. Heparinized whole blood samples of patients were collected within 48 hours after trauma and incubated in vitro with or without lipopolysaccharide (LPS) and peptidoglycan (PGN). TLR2 and TLR4 expression on monocytes was analyzed by flow cytometry. LPS- and PGN-induced tumor necrosis factor alpha (TNFalpha) and interleukin-8 production was measured by means of enzyme-linked immunosorbent assay. Both LPS- and PGN-induced TNFalpha liberation were significantly reduced in severely injured patients. The surface expression of TLR2 was also significantly decreased on monocytes collected from trauma patients, whereas the expression of TLR4 remained unchanged. There was only a negative correlation between TLR2 expression and the liberation of TNFalpha after stimulation with LPS or PGN. We conclude that diminished cytokine production after trauma cannot be explained simply by changes in TLR2 or TLR4 expression and that subsequent signaling cascades or additional receptors are involved in the blunted cytokine response after trauma.

Herpes simplex virus 1 interaction with Toll-like receptor 2 contributes to lethal encephalitis.

Human neonates infected with herpes simplex virus 1 (HSV-1) develop one of three distinct patterns of infection: (i) infection limited to the skin, eye or mouth; (ii) infection of the CNS; or (iii) disseminated infection. The disseminated form usually involves the liver, adrenal gland, and lung, and resembles the clinical picture of bacterial sepsis. This spectrum of symptoms in HSV-1-infected neonates suggests that inflammatory cytokines play a significant role in the pathogenesis of the disease. Recent studies suggest that the Toll-like receptors (TLRs) may play an important role in the induction of inflammatory cytokines in response to viruses. TLRs are mammalian homologues of Toll, a Drosophila protein that is essential for host defense against infection. Engagement of TLRs by bacterial, viral, or fungal components leads to the production and release of cytokines and other antimicrobial products. Here, we demonstrate that TLR2 mediates the inflammatory cytokine response to HSV-1 by using both transfected cell lines and knockout mice. Studies of infected mice revealed that HSV-1 induced a blunted cytokine response in TLR2(-/-) mice. Brain levels of monocyte chemoattractant protein 1 chemokine were significantly lower in TLR2(-/-) mice than in either wild-type or TLR4(-/-) mice. TLR2(-/-) mice had reduced mortality compared with wild-type mice. The differences between TLR2(-/-) mice and both wild-type and TLR4(-/-) mice in the induction of monocyte chemoattractant protein 1, brain inflammation, or mortality could not be accounted for on the basis of virus levels. Thus, these studies suggest the TLR2-mediated cytokine response to HSV-1 is detrimental to the host.

Glutamine: Role in the Fetus and Low-birthweight Infant

After completing this article, readers should be able to: 1. Describe the glutamine-glutamate shuttle. 2. Delineate the effects of glutamine supplementation during total parenteral nutrition, chemotherapy, and radiotherapy in animals. 3. Describe the beneficial effects of glutamine supplementation in low-birthweight infants and in critically ill adults. 4. List the primary actions of glutamine in the intestine. The importance of glutamine for optimal growth of cells in culture has been known since the 1950s, when Eagle published his landmark paper in Science .1 His work demonstrated that of all the amino acids, cell growth in culture relied most on glutamine. Numerous subsequent studies have established the importance of glutamine for a variety of metabolic processes in the entire organism and are beginning to demonstrate that nutritional supplementation of this amino acid may be highly beneficial under certain circumstances. Glutamine and glutamate are structurally similar five-carbon amino acids. Glutamine is a neutral amino acid at pH 7.0 that contains an alpha-amino group and a deltaamide group. In glutamate, the amide nitrogen of glutamine is replaced by a carboxy-group, conferring to it a net negative charge at pH 7.0. This at least partially accounts for the transport of these two amino acids across cell membranes via different transport systems. The presence or absence of the amide group is responsible for different metabolic interactions of these amino acids. The array of metabolic processes in which glutamine is involved is highlighted by its relationship to its metabolites and the citric acid cycle (Fig. 1⇓ ).Glutamine, via glutamate, is readily converted to alpha-ketoglutarate, an integral component of the citric acid cycle. Deamidation of glutamine via glutaminase produces glutamate, a precursor of gamma-amino butyric acid (GABA), a neurotransmission inhibitor. Proline also is produced by the cyclization of glutamate. Proline is an important amino acid component of collagen and …

Laparoscopic insufflation with room air causes exaggerated interleukin-6 response.

The gut is a central organ in the postoperative stress reaction. We previously reported that measuring gut-mucosal cytokines may more accurately reflect the response to operative stress. Additionally, we have shown that the gut demonstrates a blunted cytokine response after laparoscopy as compared with laparotomy. To further investigate whether this differential response is caused by exposure of the peritoneal cavity to general atmospheric air during laparotomy, 80 A/J mice were randomized equally into four groups: CD (carbon dioxide [CO2] pneumoperitoneum), RA (room air pneumoperitoneum), AP (anesthesia and port insertion only), and PC (pure control, no intervention). Pneumoperitoneum was established and maintained at 3 mmHg for 60 min. All the mice were killed 4 h after the intervention. Jejunal mucosa and serum samples were collected and analyzed for interleukin-6 (IL-6) levels. Results were analyzed by analysis of variance (ANOVA). Gut-mucosal IL-6 in the RA group was significantly higher than in all other groups: RA, 1,354.5 +/- 117.9* vs. CD, 964.3 +/- 114.0 vs. AP, 960.2 +/- 86.2 vs. PC, 908.0 +/- 83.6; *p < 0.05. The CD group did not show a significant increase in gut-mucosal IL-6 as compared with the AP and PC groups. Similarly, RA resulted in significant increases in serum IL-6 as compared with AP and PC, whereas CD showed no significant difference: RA, 161.3 +/- 66.2* vs. 95.1 +/- 1 vs. AP, 10.6 +/- 5.3 vs. PC, undetectable; *p < 0.05. There was no difference in serum IL-6 level between CD or any of the other groups. Exposure of the peritoneal cavity to atmospheric air, independently of the trauma of abdominal access, causes an exaggerated serum and gut mucosal IL-6 response 4 h after intervention. The beneficial effect of CO2 laparoscopy may be caused by the exclusion of general atmospheric air from the peritoneal cavity.