Hyper innate responses in neonates lead to increased morbidity and mortality after infection

Neonates suffer high morbidity and mortality in infection, presumably because of the lack of a fully developed adaptive and innate immune system. Evidence of poor innate responses in neonates has been shown by using a model that sensitizes the host to Toll-like receptor (TLR)-mediated inflammation with d-galactosamine (d-GalN). However, we show that neonatal mice demonstrate much stronger inflammatory responses than adult mice in response to LPS stimulation, and such hypersensitivity extends to other TLR agonists including actual viral infection. Our study reveals that the ensuing inflammatory reaction after d-GalN sensitization reflects preferential toxicity of d-GalN to adult liver cells, rather than accurately reflecting the TLR response to LPS. We show further that an uncontrolled proinflammatory innate response due to inadequate T cells makes neonates more vulnerable to TLR agonists or viral infection. Remarkably, through transfer of T cells into neonates or depletion of T cells in adult mice, we show that T cells are sufficient and necessary to control the early inflammatory response to LPS. Therefore, neonates might suffer from the unleashed innate responses caused by an insufficient number of T cells, which leads to increased morbidity and mortality.     

Glutathione protects mice from lethal sepsis by limiting inflammation and potentiating host defense

Neutrophils have a dual role in sepsis-defending against infection and mediating organ failure. Because glutathione (GSH) is lower in sepsis, the hypothesis that GSH depletion might impair the migratory response of neutrophils to infection was tested. In a mouse model of polymicrobial sepsis induced by cecal ligation and puncture, GSH depletion inhibited peritoneal neutrophil infiltration, increased bacterial colonies, augmented pulmonary neutrophil infiltrate, and worsened survival. The reduced peritoneal influx of neutrophils was explained by a reduced in vivo neutrophil migration in response to locally administered chemokines and by reduced chemotactic activity and chemokine levels in peritoneal lavage fluid. Conversely, the GSH precursor N-acetyl-l-cysteine augmented neutrophil infiltration in the peritoneum but not in the lung, decreased bacterial colonies, and improved survival. Thus, migration of neutrophils to a site of infection and to a distant site is differently regulated, and optimal GSH levels are important for an efficient response to sepsis.     

Reparixin,a CXCR1/2 inhibitor in islet allotransplantation

Quality of life in Type 1 diabetic patients may be improved with islet transplantation, but lifelong immunosuppression is required to prevent rejection. Allo-immune response is a key player in graft dysfunction and although the adaptive immune response is well characterized, the effect of the innate immune reaction after transplantation is only recently becoming appreciated. In this study, we address how the innate response affects long-term outcomes in a murine islet allotransplant model. CTLA-4 Ig treatment is known to significantly prolong kidney subcapsular islet allograft survival and enhance glucose tolerance. The combination of CTLA-4 Ig with reparixin, which blocks against inflammatory neutrophil infiltration, yielded no long-term graft survival in an intrahepatic allotransplant model but had similar long-term graft survival in the kidney subcapsular model. Seven days after transplant, serum blood IFN-γ levels were significantly lower in the CTLA-4 Ig with reparixin treatment group compared to controls. IL-12p70 cytokine levels were increased with combination treatment, a positive modulation of the inflammatory response to the allograft. Furthermore, KC GRO, also known as CXCL1, was decreased in serum 7 d after transplant. Histologically, we found that immune cell infiltrate, CD4+ and CD8+ T cell populations along with both CXCR1+ and CXCR2+ cell populations were decreased within the CTLA-4 Ig and reparixin islet transplant graft. Overall these data provide insight into the down regulation of T-cell recruitment by CTLA-4 Ig and decreased neutrophil activation and recruitment with reparixin after long-term islet graft survival.     

Effect of obstructive jaundice on neutrophil chemotactic activity: An in vivo assessment in zymosan-induced peritonitis model in rats

The effect of obstructive jaundice on local neutrophil accumulation in response to inflammatory stimulus was investigated in rats. Obstructive jaundice was produced by bile duct ligation for 7 days. Zymosan (200 mg) was injected intraperitoneally and 4h later myeloperoxidase activity in the peritoneal fluid was measured to quantify neutrophil recruitment. Zymosan-induced neutrophil recruitment was significantly greater (more than two-fold) in bile duct-ligated rats than in sham-ligated or normal animals. Depletion of peritoneal cells significantly suppressed neutrophil recruitment after zymosan injection in all three groups, with no significant differences between the groups. In normal rats, replacement of their peritoneal cells by those from bile duct-ligated rats did not enhance zymosan-induced neutrophil recruitment. In contrast, bile duct-ligated rats treated with peritoneal cell replacement from normals showed significantly increased neutrophil recruitment after zymosan injection. In vitro neutrophil chemotaxis in response to formyl-Met-Leu-Phe was significantly enhanced in bile duct-ligated rats, compared with that in sham-ligated animals. The results suggest that local neutrophil recruitment in response to inflammation may be enhanced in obstructive jaundice and that increased neutrophil chemotactic activity, not macrophage activity, may play a prime role in the mechanism.     

Different Doses of Lipopolysaccharides Regulate the Lung Inflammation of Asthmatic Mice via TLR4 Pathway in Alveolar Macrophages

Allergic asthma is a complicated genetic disorder caused by interaction of the acquired and innate immune responses. Acquired immune responses to protein antigens could induce type 2 T lymphocyte-driven responses and result in atopic asthma. Recent studies demonstrated that endotoxin, LPS and air pollution-induced innate immunity induce asthma through Toll-like receptors (TLR). However, the definite mechanism of LPS-induced asthma is still not known. Here, we investigated the effects of different doses of LPS in a mouse model of allergic asthma to define the molecular mechanism of LPS-induced asthma. We found that low doses of LPS in OVA induced significant inflammatory infiltration in lung tissue of asthmatic mice. Histologic studies demonstrated that lungs of these asthmatic mice were characterized by the recruitment of both eosinophils and neutrophils, increased airway mucus secretion and the elevated levels of Th2 cytokines. A high dose of LPS in OVA can induce a Th1 associated response, histologically characterized by neutrophil recruitment, the absence of airway mucus secretion and an increase of IFN-γ production. Regardless of high or low dose of LPS, TLR4 in alveolar macrophages (AM) was up-regulated in lungs of asthmatic mice. Our data demonstrated that the dose of LPS exposure determines the type of inflammatory response and a low dose of LPS together with OVA augments the antigen-induced lung inflammation in asthma. This study demonstrates that the TLR4 signaling pathway plays a vital role in the development of asthma and indicates the tight connection between endotoxin exposure and asthma prevalence in the clinic.     

Toll-like receptors and their role in gastrointestinal disease

The innate immune response to invading pathogens is centred upon a family of non-clonal, germline-encoded pattern recognition receptors (PRRs), the Toll-like receptors (TLRs). These provide specificity for a vast range of microbial pathogens, and offer an immediate anti-microbial response system. Thirteen mammalian TLRs have been described; 10 are expressed in humans, each responsible for the recognition of distinct, invariant microbial structures originating from bacteria, viruses, fungi and protozoa. The two most thoroughly studied are TLR4 and TLR2, the PRRs for Gram-negative and Gram-positive bacterial products, respectively. TLR4 is also the major receptor recognising endogenous ligands released from damaged or dying cells. Activation of a TLR by its relevant ligand rapidly ignites a complex intracellular signaling cascade that ultimately results in upregulation of inflammatory genes and production of proinflammatory cytokines, interferons and recruitment of myeloid cells. It also stimulates expression, upon antigen presenting cells, of co-stimulatory molecules required to induce an adaptive immune response. Whilst a robust TLR response is critical for survival and defence against invading pathogens, inappropriate signaling in response to alterations in the local microflora environment can be detrimental. Such 'unhelpful TLR responses' could form the basis for a large number of gastrointestinal and liver disorders, including inflammatory bowel disease, viral hepatitis, autoimmune liver diseases and hepatic fibrosis. As our understanding of TLRs expands, the pathogenesis of a number of gastrointestinal disorders will be further elucidated, and this offers potential for specific therapies aimed directly at TLR signaling.     

Oral Selective TLR8 Agonist Selgantolimod Induces Multiple Immune Cell Responses in Humans

TLR8 agonists have the potential for use as immunomodulatory components in therapeutic modalities for viral infections such as chronic HBV (CHB) and HIV. In this study, using peripheral blood samples from a phase 1a clinical trial, we examined the acute effects of a single oral administration of a selective TLR8 agonist on immune cell phenotypes. Administration of the TLR8 agonist selgantolimod (SLGN) in healthy individuals resulted in alteration in frequencies of peripheral blood monocytes, pDCs, mDCs and MAIT cells. Frequencies of mDCs and lymphoid cells significantly reduced after 8 h of SLGN administration, whereas pDC frequencies significantly increased, with changes possibly reflecting migration of different cell types between peripheral and tissue compartments in response to the agonist. Myeloid cell activation was evident by an upregulated expression of co-stimulatory molecules CD40 and CD86 accompanied by the production of IL-6 and IL-18 from these cells. Concomitantly, there was induction of the early activation marker CD69 on innate and adaptive lymphoid cells, including MAIT and NK cell subsets. Further, these activated lymphoid cells had enhanced expression of the effector molecules granzyme B and perforin. Microarray analysis of isolated lymphocytes and monocytes from baseline and post-SLGN treatment revealed changes in expression of genes involved in cellular response to cytokine stimulus, innate immune response, myeloid cell differentiation and antigen receptor-mediated signaling pathway. In a preliminary analysis of samples from CHB patients treated with selgantolimod, activation of innate and adaptive lymphocytes was evident. In conclusion, this first in-human study shows that selgantolimod administration in humans results in activation of multiple immune cell responses with antiviral potential.     

Regulation of innate immune responses by Toll-like receptors

Innate immune response in Drosophila is mediated by signaling through Toll receptors. In mammals, Toll-like receptors (TLRs), comprising a large family, recognize a specific pattern of microbial components. So far, the roles of TLR2, TLR4, TLR5, TLR6, and TLR9 have been revealed. The recognition of microbial components by TLRs leads to activation of innate immunity, which provokes inflammatory responses and finally the development of adaptive immunity. The inflammatory response depends on a TLR-mediated MyD88-dependent cascade. However, there seems to exist additional cascades in TLR signaling. In the case of TLR4 signaling, an MyD88-independent pathway is now being characterized. In addition to the activation of innate immune responses, TLR-mediated signaling leads to suppression of the activity of innate immune cells, represented by "lipopolysaccharide (LPS) tolerance". Progress in elucidating the molecular mechanisms for LPS tolerance has been made through the analysis of TLR-mediated signaling pathways. Thus, the activity for innate immune responses is known to be finely regulated by TLRs.     

Platelet Toll-like receptor expression and activation induced by lipopolysaccharide and sepsis

Platelets and Toll-like receptor (TLR) signalling play a role in the immune response during sepsis. Although preclinical knowledge about the role of platelet TLR signalling is increasing, data during human sepsis are less abundant. Moreover, controversy remains about the effect of the TLR4 agonist lipopolysaccharide (LPS) on platelet activation. We therefore assessed platelet TLR expression during human and murine sepsis. Moreover, we investigated the effect of TLR4 signalling on platelet activation and TLR expression. Platelets from healthy controls stimulated with LPS did not show classical platelet activation (P-selectin, CD63 and phosphatidylserine expression), potentiation of subthreshold agonist stimulation nor platelet-leukocyte complex formation. LPS stimulation however did increase maximal mitochondrial respiration in a TLR4-dependent manner. Platelet stimulation with LPS did not alter TLR expression. Platelet stimulation with thrombin receptor activating peptide increased TLR5 and TLR9, but not TLR2 or TLR4 expression. Platelets from patients with sepsis and mice with experimental sepsis showed platelet activation, but unaltered TLR expression. These results indicate that sepsis-induced platelet activation is not associated with altered platelet TLR expression and, although platelets are responsive to LPS, stimulation of platelet TLR4 does not result in classical platelet activation.     

Expression of immune genes in skin of channel catfish immunized with live theronts of Ichthyophthirius multifiliis

The objective of this study was to evaluate differential expression of innate and adaptive immune genes, including immunoglobulin, immune cell receptor, cytokine, inflammatory protein , toll‐like receptors (TLR) and recombination‐activating gene (RAG) in skin from channel catfish, Ictalurus punctatus after immunization with live theronts of Ichthyophthirius multifiliis (Ich) by intraperitoneal injection. The immunized catfish showed significantly higher survival rate (95%) than those of mock‐immunized control fish (0% survival) after the theront challenge. The gene expression of innate immune system, such as cytokines (IL‐1β type a, IL‐1β type b, IFN‐γ, TGF1‐β and TNF‐α) and inflammatory proteins (NF‐kB and iNOS 2), showed significant upregulation at day 1 (D1) post‐immunization. Expression of TLR genes exhibited a rapid increase from hour 4 (h4) to D10 post‐immunization. Genes of the adaptive response, such as the cell receptor MHC I, CD8⁺, CD4⁺ and TCR‐α, showed upregulation at D1, D6 and D10. The TCR‐β expression increased rapidly at h4 and remained upregulated until D10. Immunoglobulin IgM upregulation was detected from h4 until D2 while IgD expression was increased from D1 until D10. Rapid upregulation of innate and adaptive immune genes in skin of catfish following live theront vaccination was demonstrated in this study ultimately resulting in significant protection against Ich infection.     

Sex differences in resident immune cell phenotype underlie more efficient acute inflammatory responses in female mice

Females are protected against mortality arising from severe sepsis; however, the precise mechanisms that confer this survival advantage in females over males are unclear. Resident leukocytes in resting tissues have a significant influence on circulating cytokine levels and recruitment of blood leukocytes during acute inflammatory responses. Whether the phenotype of resident leukocytes is distinct in females is unknown. In the present study, we show that the numbers of leukocytes occupying the naive peritoneal and pleural cavities is higher in female than in male mice and rats, comprising more T and B lymphocytes and macrophages. The altered immune cell composition of the female peritoneum is controlled by elevated tissue chemokine expression. Female resident macrophages also exhibit greater TLR expression and enhanced phagocytosis and NADPH oxidase-mediated bacterial killing. However, macrophage-derived cytokine production is diminished by proportionally more resident immunomodulatory CD4+ T lymphocytes. Ovarian hormones regulate macrophage phenotype, function, and numbers, but have no significant impact on T-lymphocyte populations in females. We have identified a fundamental sex difference in phenotype of resident leukocytes. We propose that the distinct resident leukocyte population in females allows aggressive recognition and elimination of diverse infectious stimuli without recruitment of circulating neutrophils or excessive cytokine production.     

Toll-like receptors stimulate human neutrophil function

The first immune cell to arrive at the site of infection is the neutrophil. Upon arrival, neutrophils quickly initiate microbicidal functions, including the production of antimicrobial products and proinflammatory cytokines that serve to contain infection. This allows the acquired immune system enough time to generate sterilizing immunity and memory. Neutrophils detect the presence of a pathogen through germ line-encoded receptors that recognize microbe-associated molecular patterns. In vertebrates, the best characterized of these receptors are Toll-like receptors (TLRs). We have determined the expression and function of TLRs in freshly isolated human neutrophils. Neutrophils expressed TLR1, 2, 4, 5, 6, 7, 8, 9, and 10-all the TLRs except TLR3. Granulocyte-macrophage colony-stimulating factor (GM-CSF) treatment increased TLR2 and TLR9 expression levels. The agonists of all TLRs expressed in neutrophils triggered or primed cytokine release, superoxide generation, and L-selectin shedding, while inhibiting chemotaxis to interleukin-8 (IL-8) and increasing phagocytosis of opsonized latex beads. The response to the TLR9 agonist nonmethylated CpG-motif-containing DNA (CpG DNA) required GM-CSF pretreatment, which also enhanced the response to the other TLR agonists. Finally, using quantitative polymerase chain reaction (QPCR), we demonstrate a chemokine expression profile that suggests that TLR-stimulated neutrophils recruit innate, but not acquired, immune cells to sites of infection.     

Toll样受体及其在抗结核感染免疫中的作用

Toll样受体(Toll-like receptors,TLR)是表达在哺乳动物细胞表面的一类重要的模式识别受体,是进化中比较保守的一个受体家族,TLR能特异地识别病原体相关分子模式,不仅在激活天然免疫中发挥着莺要的作用,而且还调节获得性免疫,是连接天然免疫与特异性免疫的主要桥梁.研究表明,TLR是介导宿主对结核杆菌的识别及抗结核免疫反应的关键分子,与抗结核感染免疫有关的主要是TLR2和TLR4.对TLR的研究有助于阐明结核病的发病机制并为其治疗提供崭新的策略.     

Inactivation of heparan sulfate 2-O-sulfotransferase accentuates neutrophil infiltration during acute inflammation in mice

Neutrophil recruitment and extravasation at sites of inflammation provide a mechanism for host defense. We showed previously that heparan sulfate, a type of sulfated glycosaminoglycan, facilitates neutrophil recruitment based on the reduction of neutrophil infiltration in mice in which the overall sulfation of the chains was reduced by selective inactivation of N-acetylglucosamine N-deacetylase-N-sulfotransferase (Ndst1) in endothelial cells. Here we show that inactivation of uronyl 2-O-sulfotransferase in endothelial cells (Hs2st), an enzyme that acts downstream from Ndst1, results in enhanced neutrophil recruitment in several models of acute inflammation. Enhanced neutrophil infiltration resulted in part from reduced rolling velocity under flow both in vivo and in vitro, which correlated with stronger binding of neutrophil L-selectin to mutant endothelial cells. Hs2st-deficient endothelial cells also displayed a striking increase in binding of IL-8 and macrophage inflammatory protein-2. The enhanced binding of these mediators of neutrophil recruitment resulted from a change in heparan sulfate structure caused by increased N-sulfation and 6-O-sulfation of glucosamine units in response to the decrease in 2-O-sulfation of uronic acid residues. This gain-of-function phenotype provides formidable evidence demonstrating the importance of endothelial heparan sulfate in inflammation and suggests a novel enzyme target for enhancing the innate immune response.     

Toll-Like Receptor 2 Deficiency Increases Resistance to Pseudomonas aeruginosa Pneumonia in the Setting of Sepsis-Induced Immune Dysfunction

Background.?Sepsis is characterized by a dysregulated inflammatory response followed by immunosuppression that favors the development of secondary infections. Toll-like receptors (TLRs) are major regulators of the host's response to infections. How variability in TLR signaling may impact the development of sepsis-induced immune dysfunction has not been established. We sought to establish the role of TLR2, TLR4, and TLR5 in postseptic mice with Pseudomonas aeruginosa pneumonia. Methods.?We used an experimental model of sublethal polymicrobial sepsis induced by cecal ligation and puncture (CLP). Wild-type, tlr2(-/-), tlr4(-/-), tlr5(-/-), tlr2?4(-/-) mice that underwent CLP were secondarily subjected to P. aeruginosa pulmonary infection. Results.?Postseptic wild-type and tlr4(-/-) and tlr5(-/-) mice displayed high susceptibility to P. aeruginosa pneumonia. In contrast, TLR2-deficient mice, either tlr2(-/-)or tlr2?4(-/-), that underwent CLP were resistant to the secondary pulmonary infection. As compared to wild-type mice, tlr2(-/-) mice displayed improvement in bacterial clearance, decreased bacteremic dissemination, and attenuated lung damage. Furthermore, tlr2(-/-) mice exhibited a pulmonary proinflammatory cytokine balance, with increased production of tumor necrosis factor α and decreased release of interleukin 10. Conclusions.?In a model of secondary P. aeruginosa pneumonia in postseptic mice, TLR2 deficiency improves survival by promoting efficient bacterial clearance and restoring a proinflammatory cytokine balance in the lung.     

A new paradigm for the treatment of sepsis: is it time to consider combination therapy?

Despite the advances in supportive care and the availability of potent antimicrobial agents, mortality from sepsis, a leading cause of death in intensive care units, has not improved. Over the last decade, clinical trials with numerous adjunctive therapies, including antiendotoxin antibodies and inhibitors of the inflammatory response, have yielded disappointing results. Recently, treatment with recombinant human activated protein C reduced mortality 6% compared with controls. Given the likelihood that many processes in the complex pathophysiology of sepsis are simultaneously activated, it is unlikely that therapy directed at any one of them, as has been done in the past, will dramatically improve survival. Rather, a combination of therapies directed at many arms of the septic process, much like the strategy used for cancer and HIV infection, is required. Given the likelihood that sepsis represents an excessive innate immune response to microbial products, vigorous attempts must be made to develop rapid assays that reflect the level of innate immune activation. Such assays could be used to identify patients who would benefit from therapy and to monitor their response so that overtreatment does not completely abrogate host defense mechanisms and render these patients susceptible to fatal infection. It is now time to test a new therapeutic paradigm based on an improved understanding of the pathophysiology of the septic process and the recognition that we may have reached the limits of adjunctive monotherapy.     

Toll-like receptor 4 plays a crucial role in the immune-adrenal response to systemic inflammatory response syndrome

Sepsis and septic shock are leading killers in the noncoronary intensive care unit, and they remain worldwide health concerns. The initial host defense against bacterial infections involves Toll-like receptors (TLRs), which detect and respond to microbial ligands. In addition, a coordinated response of the adrenal and immune systems is crucial for survival during severe inflammation. Previously, we demonstrated a link between the innate immune system and the endocrine stress response involving TLR-2. Like TLR-2, TLR-4 is also expressed in human and mouse adrenals. In the present work, by using a low dose of LPS to mimic systemic inflammatory response syndrome, we have revealed marked cellular alterations in adrenocortical tissue and an impaired adrenal corticosterone response in TLR-4-/- mice. Our findings demonstrate that TLR-4 is a key mediator in the crosstalks between the innate immune system and the endocrine stress response. Furthermore, TLR polymorphisms could contribute to the underlying mechanisms of impaired adrenal stress response in patients with bacterial sepsis.     

Toll样受体与支气管哮喘

Toll样受体(Toll-like receptors,TLR)是近年发现的一组天然受体,其不仅依赖胚系基因编码的保守序列识别病原微生物和激活天然免疫,而且调节获得性免疫和诱导免疫耐受,与支气管哮喘密切相关.其宿主通过TLR识别大量病原体相关结构,利用细胞信号转导途径激活机体免疫细胞,一方面参与早期的宿主防御,在天然免疫应答中发挥重要作用;另一方面通过分泌细胞因子、趋化因子、黏附分子等参与炎症反应,最终激活获得性免疫系统.本文就近年来有关TLR的结构功能、信号通路及TLR与支气管哮喘关系作一综述.     

Pulmonary defense mechanisms against pneumonia and sepsis

PURPOSE OF REVIEW: Pneumonia and sepsis are major causes of morbidity and mortality. Lung defense against pathogens involves innate and acquired immune responses. In this review, we focus on lung pathogens associated with sepsis and the innate immune response to them. In addition to discussing typical lung pathogens, the structural defenses, antimicrobial particles, complement, and cellular components of the immune response against these pathogens are also explored. RECENT FINDINGS: The rising importance of pathogen-associated molecular pattern recognition molecules (such as Toll-like receptors) is discussed, as is the pivotal role of the dendritic cell in linking the innate and adaptive immune response. Although the adaptive response is delayed, it is more specific than the innate response and confers long-lasting memory allowing rapid and efficient clearance of pathogens on subsequent re-exposure. Sepsis interferes with both the innate and adaptive immune responses by inducing marked apoptosis of dendritic cells and lymphocytes, suppressing both arms of the immune response. This immunosuppression is a major hallmark of this disorder. SUMMARY: Clearly, understanding the immune response in the lung is critical to the development of future therapeutics for pneumonia and sepsis. This review concludes with novel discoveries in the lung immune response, which may lead to future avenues of treatment.     

Plant homologue constitutive photomorphogenesis 9 (COP9) signalosome subunit CSN5 regulates innate immune responses in macrophages

COP9 plays a role in plant innate immunity. The role of COP9 in mammalian innate immune responses is unknown. Here, we show that the COP9 signalosome subunit 5 (CSN5) is required for activation of proinflammatory kinases p38 and Erk and for down-regulation of the expression of genes regulated by nuclear factor E2-related factor 2. Mice with myeloid-specific CSN5 deficiency have lower mortality in polymicrobial sepsis. CSN5 is required for both Toll-like receptor (TLR) and reactive oxygen species-mediated deneddylation of Cul3, which is essential for Cul3/Keap1-mediated degradation of nuclear factor E2-related factor 2. On the basis of our results COP9 subunit CSN5 is considered to be an essential component of mammalian innate immunity.