Cutting edge: myeloid differentiation factor 88 deficiency improves resistance against sepsis caused by polymicrobial infection

Toll-like receptors (TLRs) are important for the activation of innate immune cells upon encounter of microbial pathogens. The present study investigated the potential roles of TLR2, TLR4, and the signaling protein myeloid differentiation factor 88 (MyD88) in polymicrobial septic peritonitis. Whereas both TLR2 and TLR4 were dispensable for host defense against septic peritonitis, MyD88-deficient mice were protected in this infection model. Recruitment of neutrophils to the septic focus and bacterial clearance were normal in MyD88-deficient mice. In contrast, the systemic inflammatory response was strongly attenuated in the absence of MyD88. Surprisingly, MyD88 deficiency did not alter cytokine and chemokine production in spleen, but markedly reduced the inflammatory response in liver and lung. Production of monocyte chemoattractant protein-1 and macrophage-inflammatory protein-1alpha was entirely independent of MyD88. These results imply a central role of MyD88 for the systemic immune pathology of polymicrobial sepsis and show that cytokine production in spleen and induction of certain chemokines are MyD88 independent.     

Local delivery of CpG oligodeoxynucleotides induces rapid changes in the genital mucosa and inhibits replication,but not entry,of herpes simplex virus type 2

Mucosal surfaces are the entry sites for the vast majority of infectious pathogens and provide the first line of defense against infection. In addition to the epithelial barrier, the innate immune system plays a key role in recognizing and rapidly responding to invading pathogens via innate receptors, such as Toll-like receptors (TLR). Bacterial CpG DNA, a potent activator of innate immunity, is recognized by TLR9. Here, we confirm that local mucosal, but not systemic, delivery of CpG oligodeoxynucleotides (ODN) to the genital tract protects mice from a subsequent lethal vaginal herpes simplex virus type 2 (HSV-2) challenge. Since these effects were so local in action, we examined the genital mucosa. Local delivery of CpG ODN induced rapid proliferation and thickening of the genital epithelium and caused significant recruitment of inflammatory cells to the submucosa. Local CpG ODN treatment also resulted in inhibition of HSV-2 replication but had no effect on HSV-2 entry into the genital mucosa. CpG ODN-induced protection against HSV-2 was not associated with early increases in gamma interferon (IFN-gamma) secretion in the genital tract, and CpG ODN-treated IFN-gamma(-/-) mice were protected from subsequent challenge with a lethal dose of HSV-2. Treatment of human HEK-293 cells transfected with murine TLR9 showed that the antiviral activity of CpG ODN was mediated through TLR9. These studies suggest that local induction of mucosal innate immunity can provide protection against sexually transmitted infections, such as HSV-2 or possibly human immunodeficiency virus, at the mucosal surfaces.     

Innate Immune Detection of Flagellin Positively and Negatively Regulates Salmonella Infection

Salmonella enterica serovar Typhimurium is a flagellated bacterium and one of the leading causes of gastroenteritis in humans. Bacterial flagellin is required for motility and also a prime target of the innate immune system. Innate immune recognition of flagellin is mediated by at least two independent pathways, TLR5 and Naip5-Naip6/NlrC4/Caspase-1. The functional significance of each of the two independent flagellin recognition systems for host defense against wild type Salmonella infection is complex, and innate immune detection of flagellin contributes to both protection and susceptibility. We hypothesized that efficient modulation of flagellin expression in vivo permits Salmonella to evade innate immune detection and limit the functional role of flagellin-specific host innate defenses. To test this hypothesis, we used Salmonella deficient in the anti-sigma factor flgM, which overproduce flagella and are attenuated in vivo. In this study we demonstrate that flagellin recognition by the innate immune system is responsible for the attenuation of flgM⁻ S. Typhimurium, and dissect the contribution of each flagellin recognition pathway to bacterial clearance and inflammation. We demonstrate that caspase-1 controls mucosal and systemic infection of flgM⁻ S. Typhimurium, and also limits intestinal inflammation and injury. In contrast, TLR5 paradoxically promotes bacterial colonization in the cecum and systemic infection, but attenuates intestinal inflammation. Our results indicate that Salmonella evasion of caspase-1 dependent flagellin recognition is critical for establishing infection and that evasion of TLR5 and caspase-1 dependent flagellin recognition helps Salmonella induce intestinal inflammation and establish a niche in the inflamed gut.     

Flagellin-deficient Legionella mutants evade caspase-1- and Naip5-mediated macrophage immunity

Macrophages from C57BL/6J (B6) mice restrict growth of the intracellular bacterial pathogen Legionella pneumophila. Restriction of bacterial growth requires caspase-1 and the leucine-rich repeat-containing protein Naip5 (Birc1e). We identified mutants of L. pneumophila that evade macrophage innate immunity. All mutants were deficient in expression of flagellin, the primary flagellar subunit, and failed to induce caspase-1-mediated macrophage death. Interestingly, a previously isolated flagellar mutant (fliI) that expresses, but does not assemble, flagellin did not replicate in macrophages, and induced macrophage death. Thus, flagellin itself, not flagella or motility, is required to initiate macrophage innate immunity. Immunity to Legionella did not require MyD88, an essential adaptor for toll-like receptor 5 (TLR5) signaling. Moreover, flagellin of Legionella and Salmonella induced cytotoxicity when delivered to the macrophage cytosol using Escherichia coli as a heterologous host. It thus appears that macrophages sense cytosolic flagellin via a TLR5-independent pathway that leads to rapid caspase-1-dependent cell death and provides defense against intracellular bacterial pathogens.     

Blocking of the TLR5 activation domain hampers protective potential of flagellin DNA vaccine

Flagellin is a key component of the flagella of many pathogens, including Pseudomonas aeruginosa. Flagellin is an attractive vaccine candidate because it is readily produced and manipulated as a recombinant protein and has intrinsic adjuvant activity mediated through TLR5. Although DNA vaccines encoding native Pseudomonas B-type (FliC) or A-type (FlaA) flagellin are strongly immunogenic, the resultant Ab response interferes with the interaction of homologous flagellin with TLR5. This reduces the ability of the host to clear homologous, but not heterologous, flagellin-expressing P. aeruginosa. To circumvent this problem, a DNA vaccine encoding a mutant FliC R90A flagellin was developed. The mutant Ag encoded by this vaccine was highly immunogenic, but its ability to interact with TLR5 was reduced by >100-fold. Vaccination with this flagellin mutant DNA vaccine induced cross-reactive Abs against both FliC and FlaA, but few Abs capable of interfering with TLR5 activation. The flagellin mutant DNA vaccine provided excellent protection against both FliC- and FlaA-expressing P. aeruginosa. These findings suggest that vaccines against flagellated pathogens should avoid inducing Abs against TLR5 and raise the possibility that flagellated bacteria evade host elimination by facilitating the production of Abs that reduce the host's ability to mount an innate immune response.     

Topical flagellin protects the injured corneas from Pseudomonas aeruginosa infection

Among bacterial pathogens, Pseudomonas (P.) aeruginosa infection is the most sight threatening. The corneal innate immune responses are key mediators of the host’s defense to P. aeruginosa. Using a mouse model of Pseudomonas keratitis, we evaluated the protective effects of topical application of flagellin, a ligand for Toll-Like receptor 5 (TLR5), on the development of Pseudomonas keratitis and elucidated the underlying mechanisms. Topical application of purified flagellin 6 and 24 h prior to P. aeruginosa inoculation on injured mouse corneas significantly attenuated clinical symptoms of P. aeruginosa keratitis, decreased bacterial burden, and suppressed infection induced inflammation in the B6 mouse cornea. Topical application of flagellin on wounded cornea induced PMN infiltration and markedly upregulated cathelicidin-related antimicrobial peptide (CRAMP) expression. In PMN depleted mice, flagellin promoted bacterial clearance in the cornea compared to that of the PBS treated mice, but was unable to prevent corneal perforation and systemic bacterial dissemination and sepses. Deletion of CRAMP increased corneal susceptibility to P. aeruginosa and abolished flagellin-induced protection in B6 mice. Our findings illustrate the profound protective effect of flagellin on the cornea innate defense, a response that can be exploited for prophylactic purposes to prevent contact lens associated Pseudomonas keratitis.     

High variability of TLR4 gene in different ethnic groups in Iran

Infectious diseases exert a constant evolutionary pressure on the innate immunity genes. TLR4, an important member of the TLR family, specifically recognizes conserved structures of various infectious pathogens. Two functional TLR4 polymorphisms, Asp299Gly and Thr399Ile, modulate innate host defense against infections, and their prevalence between various populations has been proposed to be influenced by local infectious pressures. If this assumption is true, strong local infectious pressures would lead to a homogeneous pattern of these ancient TLR4 polymorphisms in geographically-close populations, while a weak selection or genetic drift may result in a diverse pattern. We evaluated TLR4 polymorphisms in 15 ethnic groups in Iran, to assess whether infections exerted selective pressures on different haplotypes containing these variants. The Iranian subpopulations displayed a heterogeneous pattern of TLR4 polymorphisms, comprising various percentages of Asp299Gly and Thr399Ile, alone or in combination. The Iranian sample, as a whole, showed an intermediate mixed pattern when compared with commonly-found patterns in Africa, Europe, Eastern Asia and the Americas. These findings suggest a weak, or absent, selection pressure on TLR4 polymorphisms in the Middle-East that does not support the assumption of an important role of these polymorphisms in the host defense against local pathogens.     

Toll-like receptors are key participants in innate immune responses

During an infection, one of the principal challenges for the host is to detect the pathogen and activate a rapid defensive response. The Toll-like family of receptors (TLRs), among other pattern recognition receptors (PRR), performs this detection process in vertebrate and invertebrate organisms. These type I transmembrane receptors identify microbial conserved structures or pathogen-associated molecular patterns (PAMPs). Recognition of microbial components by TLRs initiates signaling transduction pathways that induce gene expression. These gene products regulate innate immune responses and further develop an antigen-specific acquired immunity. TLR signaling pathways are regulated by intracellular adaptor molecules, such as MyD88, TIRAP/Mal, between others that provide specificity of individual TLR- mediated signaling pathways. TLR-mediated activation of innate immunity is involved not only in host defense against pathogens but also in immune disorders. The involvement of TLR-mediated pathways in auto-immune and inflammatory diseases is described in this review article.     

Prevention and mitigation of acute radiation syndrome in mice by synthetic lipopeptide agonists of Toll-like receptor 2 (TLR2)

Bacterial lipoproteins (BLP) induce innate immune responses in mammals by activating heterodimeric receptor complexes containing Toll-like receptor 2 (TLR2). TLR2 signaling results in nuclear factor-kappaB (NF-κB)-dependent upregulation of anti-apoptotic factors, anti-oxidants and cytokines, all of which have been implicated in radiation protection. Here we demonstrate that synthetic lipopeptides (sLP) that mimic the structure of naturally occurring mycoplasmal BLP significantly increase mouse survival following lethal total body irradiation (TBI) when administered between 48 hours before and 24 hours after irradiation. The TBI dose ranges against which sLP are effective indicate that sLP primarily impact the hematopoietic (HP) component of acute radiation syndrome. Indeed, sLP treatment accelerated recovery of bone marrow (BM) and spleen cellularity and ameliorated thrombocytopenia of irradiated mice. sLP did not improve survival of irradiated TLR2-knockout mice, confirming that sLP-mediated radioprotection requires TLR2. However, sLP was radioprotective in chimeric mice containing TLR2-null BM on a wild type background, indicating that radioprotection of the HP system by sLP is, at least in part, indirect and initiated in non-BM cells. sLP injection resulted in strong transient induction of multiple cytokines with known roles in hematopoiesis, including granulocyte colony-stimulating factor (G-CSF), keratinocyte chemoattractant (KC) and interleukin-6 (IL-6). sLP-induced cytokines, particularly G-CSF, are likely mediators of the radioprotective/mitigative activity of sLP. This study illustrates the strong potential of LP-based TLR2 agonists for anti-radiation prophylaxis and therapy in defense and medical scenarios.     

Toll-like receptor 2 plays a role in the early inflammatory response to murine pneumococcal pneumonia but does not contribute to antibacterial defense

Toll-like receptors (TLR) are crucial pattern recognition receptors in innate immunity. The importance of TLR2 in host defense against Gram-positive bacteria has been suggested by the fact that this receptor recognizes major Gram-positive cell wall components, such as peptidoglycan and lipoteichoic acid. To determine the role of TLR2 in pulmonary Gram-positive infection, we first established that TLR2 is indispensable for alveolar macrophage responsiveness toward Streptococcus pneumoniae. Nonetheless, TLR2 gene-deficient mice intranasally inoculated with S. pneumoniae at doses varying from nonlethal (with complete clearance of the infection) to lethal displayed only a modestly reduced inflammatory response in their lungs and an unaltered antibacterial defense when compared with normal wild-type mice. These data suggest that TLR2 plays a limited role in the innate immune response to pneumococcal pneumonia, and that additional pattern recognition receptors likely are involved in host defense against this common respiratory pathogen.     

Innate immunity: cutaneous expression of Toll-like receptors

Toll receptors were first identified as an essential molecule for embryonic patterning in Drosophila and were subsequently shown to be a key in antibacterial and antifungal immunity in adult flies. Toll receptors have been conserved throughout evolution. In mammals, TLRs have been implicated in both inflammatory responses and innate host defense to pathogens. The 11 different TLRs recognize conserved molecular patterns of microbial pathogens termed pathogen-specific molecular patterns (PAMPs), that permit to confer responsiveness to a wide variety of pathogens. Endogenous ligands are also able to activate TLRs. All adult tissue is capable to express at least one of member of TLR family, but a largest repertoire of TLRs is found in tissues exposed to the external environment. The TLR activation induce the NF-kappaB translocation to the nucleus and cytokine secretion. Since the primary function of skin is to provide an effective barrier against outside agression, it is likely that keratinocytes may play a role in a rapid and efficient host defence system, and the fact that keratinocytes are capable of expressing a wide variety of TLRs is subsequently not surprising.     

Flagellin-Induced Corneal Antimicrobial Peptide Production and Wound Repair Involve a Novel NF-κB–Independent and EGFR-Dependent Pathway

Background

The bacterial protein flagellin plays a major role in stimulating mucosal surface innate immune response to bacterial infection and uniquely induces profound cytoprotection against pathogens, chemicals, and radiation. This study sought to determine signaling pathways responsible for the flagellin-induced inflammatory and cytoprotective effects on human corneal epithelial cells (HCECs).

Methodology/Principal Findings

Flagellin purified from Pseudomonas aeruginosa (strain PAK) or live bacteria were used to challenge cultured HCECs. The activation of signaling pathways was assessed with Western blot, and the secretion of cytokine/chemokine and production of antimicrobial peptides (AMPs) were measured with ELISA and dot blot, respectively. Effects of flagellin on wound healing were assessed in cultured porcine corneas. L94A (a site mutation in TLR5 binding region) flagellin and PAK expressing L94A flagellin were unable to stimulate NF-κB activation, but were potent in eliciting EGFR signaling in a TGF-α–related pathway in HCECs. Concomitant with the lack of NF-κB activation, L94A flagellin was ineffective in inducing IL-6 and IL-8 production in HCECs. Surprisingly, the secretion of two inducible AMPs, LL-37 and hBD2, was not affected by L94A mutation. Similar to wild-type flagellin, L94A induced epithelial wound closure in cultured porcine cornea through maintaining EGFR-mediated signaling.

Conclusions/Significance

Our data suggest that inflammatory response mediated by NF-κB can be uncoupled from epithelial innate defense machinery (i.e., AMP expression) and major epithelial proliferation/repair pathways mediated by EGFR, and that flagellin and its derivatives may have broad therapeutic applications in cytoprotection and in controlling infection in the cornea and other mucosal tissues.     

Flagellin perception: a paradigm for innate immunity

There are surprising similarities between how animals and plants perceive pathogens. In animals, innate immunity is based on the recognition of pathogen-associated molecular patterns. This is mediated by the Toll-like receptor (TLR) family, which rapidly induce the innate immunity response, a first line of defence against infectious disease. Plants have highly sensitive perception systems for general elicitors and they respond to these stimuli with a defence response. One of these general elicitors is flagellin, the main component of the bacterial flagellum. Genetic analysis in Arabidopsis has shown that FLS2, which encodes a receptor-like kinase, is essential for flagellin perception. FLS2 shares homology with the TLR family, and TLR5 is responsible for flagellin perception in mammals.     

The innate immune system in the intestine

The innate immune system provides the first line of host defense against invading pathogens. Innate immune responses are initiated by germline-encoded PRR, which recognize specific structures expressed by microorganisms. TLR are a family of PRR which sense a wide range of microorganisms, including bacteria, fungi, protozoa and viruses. TLR are also expressed in the intestine and are critical for intestinal homeostasis. Recently, cytoplasmic PRR, such as NLR and RLR, have been shown to detect pathogens that have invaded the cytosol. One of the NLR, NOD2, is thought to be involved in the pathogenesis of Crohn's disease. This review focuses on the innate immune responses triggered by PRR in the intestine.     

Control of Pseudomonas aeruginosa in the lung requires the recognition of either lipopolysaccharide or flagellin

Acute lung infection due to Pseudomonas aeruginosa is an increasingly serious problem that results in high mortality especially in the compromised host. In this study, we set out to ascertain what components of the TLR system are most important for innate immunity to this microorganism. We previously demonstrated that TLR2,4-/- mice were not hypersusceptible to infection by a wild-type P. aeruginosa strain. However, we now find that mice lacking both TLR2 and TLR4 (TLR2,4-/- mice) are hypersusceptible to infection following challenge with a P. aeruginosa mutant devoid of flagellin production. We demonstrate that this hypersusceptibility is largely due to a lack of innate defense by the host that fails to control bacterial replication in the lung. Further evidence that a response to flagellin is a key factor in the failure of TLR2,4-/- mice to control the infection with the mutant strain was obtained by demonstrating that the intrapulmonary administration of flagellin over a 18 h period following infection, saved 100% of TLR2,4-/- mice from death. We conclude that the interactions of either TLR4 with LPS or TLR5 with flagellin can effectively defend the lung from P. aeruginosa infection and the absence of a response by both results in hypersusceptibility to this infection.     

Monocyte and macrophage heterogeneity and Toll-like receptors in the lung

Mononuclear phagocytes are crucial components of the innate host defense system. Cells such as macrophages and monocytes phagocytose and process pathogens, produce inflammatory mediators, and link the innate and the adaptive immune systems. The role of innate immune receptors such as Toll-like receptors (TLRs) in the recognition of pathogens is critical for mounting a precise and targeted immune response. This review focuses attention on the development of monocytes and macrophages, various populations of macrophages, and the expression and function of TLRs on macrophages.     

Selective regulation of IL-10 signaling and function by zymosan

Balanced activity of pro- and anti-inflammatory cytokines during innate immune responses is required to allow effective host defense while avoiding tissue damage and autoimmunity. Induction of cytokine production after recognition of pathogen-associated molecular patterns (PAMPs) by innate immune cells has been well demonstrated, but modulation of cytokine function by PAMPs is not well understood. In this study we show that stimulation of macrophages with zymosan, which contains PAMPs derived from yeast, rapidly extinguished macrophage responses to IL-10, a suppressive cytokine that limits inflammatory tissue damage but also compromises host defense. The mechanism of inhibition involved protein kinase Cbeta and internalization of IL-10R, and was independent of TLR2 and phagocytosis. Inhibition of IL-10 signaling and function required direct contact with zymosan, and cells in an inflammatory environment that had not contacted zymosan remained responsive to the paracrine activity of zymosan-induced IL-10. These results reveal a mechanism that regulates IL-10 function such that antimicrobial functions of infected macrophages are not suppressed, but the activation of surrounding noninfected cells and subsequent tissue damage are limited. The fate of individual cells in an inflammatory microenvironment is thus specified by dynamic interactions among host cells, microbes, and cytokines that determine the balance between protection and pathology.     

Cutting edge: the immunostimulatory activity of the lung surfactant protein-A involves Toll-like receptor 4

The collectin surfactant protein-A (SP-A) is involved in the innate host defense and the regulation of inflammatory processes in the lung. In this work we investigated the molecular mechanisms related to the immunostimulatory activity of SP-A using macrophages from C3H/HeJ mice, which carry an inactivating mutation in the Toll-like receptor (TLR)4 gene, and TLR4-transfected Chinese hamster ovary cells. We demonstrate that SP-A-induced activation of the NF-kappaB signaling pathway and up-regulation of cytokine synthesis such as TNF-alpha and IL-10 are critically dependent on the TLR4 functional complex. These findings support the concept that TLR4 is a pattern recognition receptor that signals in response to both foreign pathogens and endogenous host mediators.     

Innate responses of corneal epithelial cells against Aspergillus fumigatus challenge

Toll-like receptors (TLRs) are key components of the innate immune system that detects microbial infection and triggers host defensive responses. To determine the roles of TLR2 and TLR4 in corneal epithelial cells in mediating innate responses against Aspergillus fumigatus , telomerase-immortalized human corneal epithelial cells (THCE) were challenged by TLR2 ligand zymosan, TLR4 ligand lipopolysaccharide and A. fumigatus hyphae, respectively. Culture media were collected at different time points and enzyme-linked immunosorbent assay was performed to detect the levels of inflammatory cytokines interleukin-1β (IL-1β) and IL-6. We found that THCE responded to the challenge of TLR2 or TLR4 ligand by expressing and secreting inflammatory cytokines into the culture media. And exposure of THCE to A. fumigatus hyphae resulted in the upregulation of IL-1β and IL-6. Treatment with TLR2- or TLR4-siRNA plasmid reduced TLR2 or TLR4 expression level in THCE when compared with controls, and caused a significant decrease in A. fumigatus -induced IL-1β and IL-6 production. Our results suggested that THCE can respond to TLR2 and TLR4 ligand challenge by secreting IL-1β and IL-6. They recognize A. fumigatus hyphae via TLR2 and TLR4 and initiate innate immune responses. Corneal epithelial cells play a role in innate defense against fungal infection through the mediation of inflammatory cytokines production.     

Toll-like receptors and corneal innate immunity

The ocular surface is constantly exposed to a wide array of microorganisms. The ability of the cornea to recognize pathogens as foreign and eliminate them is critical to retain its transparency, hence preservation of sight. In the eye, as in other parts of the body, the early response against invading pathogens is provided by innate immunity. Corneal innate immune system uses a series of pattern recognition receptors to detect the presence of pathogens thus allowing for rapid host defense responses to invading microbes. A key component of such receptors is the "Toll-like receptors" (TLRs), which have come to occupy the center stage in innate immunity against invading pathogens. An increasing number of studies have shown that TLRs are expressed by a variety of tissues and cells of the eye and play an important role in ocular defense against microbial infection. Here in this review we summarize the current knowledge about TLR expression in human eye with main emphasis on the cornea, and discuss the future directions of the field.