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1.  Flagellin induces antibody responses through a TLR5- and inflammasome-independent pathway1 
Flagellin is a potent immunogen that activates the innate immune system via TLR5 and Naip5/6, and generates strong T and B cell responses. The adaptor protein MyD88 is critical for signaling by TLR5, as well as IL-1 and IL-18 receptors, major downstream mediators of the Naip5/6 Nlrc4-inflammasome. Herein we define roles of known flagellin receptors and MyD88 in antibody responses generated towards flagellin. We used mice genetically deficient in flagellin recognition pathways to characterize innate immune components that regulate isotype specific antibody responses. Using purified flagellin from Salmonella, we dissected the contribution of innate flagellin recognition pathways to promote antibody responses towards flagellin and co-administered ovalbumin in C57BL/6 mice. We demonstrate IgG2c responses towards flagellin were TLR5- and inflammasome-dependent; IgG1 was the dominant isotype and partially TLR5- and inflammasome-dependent. Our data indicates a substantial flagellin-specific IgG1 response was induced through a TLR5-, inflammasome-, and MyD88-independent pathway. IgA anti-FliC responses were TLR5- & MyD88-dependent and caspase-1-independent. Unlike C57BL/6 mice, flagellin immunized A/J mice induced co-dominant IgG1 and IgG2a responses. Furthermore, MyD88-independent flagellin-induced antibody responses were even more pronounced in A/J MyD88−/− mice, and IgA anti-FliC responses were suppressed by MyD88. Flagellin also worked as an adjuvant toward co-administered ovalbumin, but it only promoted IgG1 anti-OVA responses. Our results demonstrate that a novel pathway for flagellin recognition contributes to antibody production. Characterization of this pathway will be useful for understanding immunity to flagellin and the rationale design of flagellin-based vaccines.
PMCID: PMC3925749  PMID: 24442437
2.  Toll-like receptors in kidney disease 
Purpose of review
The innate immune system is our first line of disease against infection and injury, and responsible for initiating inflammatory and immune responses to resolve infections and repair injured tissues. This review focuses on the Toll-like receptors (TLRs) of the innate immune system and their role in recognizing infection and injury, and regulating inflammatory responses in the kidney.
Recent findings
There is increasing data to support a role for TLRs in immune complex-mediated glomerulonephritis. TLR7 has emerged as a key regulator of autoantibody production in murine lupus nephritis. In addition, studies have implicated TLR recognition of endogenous molecules released during cellular necrosis as critical regulators of sterile inflammation and injury. Tonic interactions between TLRs and environmental agonists derived from commensal microbes and endogenous sources may also influence autoimmune disease and inflammatory disorders affecting the kidney.
Future studies to decipher the contribution of TLRs and other innate immune receptors in the regulation of inflammation, immune responses, and injury in the kidney will pave the way for novel therapeutic interventions.
PMCID: PMC2896868  PMID: 19352178
autoimmunity; endogenous agonists; inflammation; innate immunity; sterile injury; Toll-like receptors
3.  Innate Immune Detection of Flagellin Positively and Negatively Regulates Salmonella Infection 
PLoS ONE  2013;8(8):e72047.
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.
PMCID: PMC3747147  PMID: 23977202
4.  Nucleotide-binding oligomerization domain containing-like receptor family, caspase recruitment domain (CARD) containing 4 (NLRC4) regulates intrapulmonary replication of aerosolized Legionella pneumophila 
BMC Infectious Diseases  2013;13:371.
Legionella pneumophila (Lp) flagellin activates signaling pathways in murine macrophages that control Lp replication. Nucleotide-binding oligomerization domain (NOD) containing-like receptor (NLR) family, caspase recruitment domain (CARD) containing 4 (NLRC4) and Toll-like Receptor (TLR5) both recognize Lp flagellin in vitro, but whether these two receptors play redundant or separate functional roles in vivo is unknown.
The immune response of Nlrc4−/−, Nlrc4−/−/Tlr5−/−, and wild type C57Bl/6 mice was analyzed after in vivo infection with aerosolized Lp.
Lp clearance from the lungs was delayed in Nlrc4−/− mice over seven days in comparison to wild type controls. Nlrc4−/−/Tlr5−/− mice had no additional defect. In contrast to TLR5, NLRC4 did not regulate recruitment of neutrophils to the lung. Although there were no differences among the mouse strains in the lung transcriptome at 4 hours, Nlrc4−/− and Nlrc4−/−Tlr5−/− mice had increased lung inflammation at 72 hours in comparison to WT. Nlrc4−/−/Tlr5−/− mice also had altered cytokine production at both 4 and 24 hours post infection when compared to wild-type (WT) and Nlrc4−/− mice. Lp replication in murine alveolar macrophages was NLRC4-dependent and TLR5-independent.
These studies reveal that NLRC4 and TLR5 mediate different roles in the inflammatory response to Lp flagellin in an aerosolized infection model and NLRC4 regulates replication in both lungs and alveolar macrophages.
PMCID: PMC3751498  PMID: 23937571
Legionella pneumophila; Pneumonia; Alveolar macrophage; NLRC4; TLR5
5.  Homeostatic Regulation of Salmonella-Induced Mucosal Inflammation and Injury by IL-23 
PLoS ONE  2012;7(5):e37311.
IL-12 and IL-23 regulate innate and adaptive immunity to microbial pathogens through influencing the expression of IFN-γ, IL-17, and IL-22. Herein we define the roles of IL-12 and IL-23 in regulating host resistance and intestinal inflammation during acute Salmonella infection. We find that IL-23 alone is dispensable for protection against systemic spread of bacteria, but synergizes with IL-12 for optimal protection. IL-12 promotes the production of IFN-γ by NK cells, which is required for resistance against Salmonella and also for induction of intestinal inflammation and epithelial injury. In contrast, IL-23 controls the severity of inflammation by inhibiting IL-12A expression, reducing IFN-γ and preventing excessive mucosal injury. Our studies demonstrate that IL-23 is a homeostatic regulator of IL-12-dependent, IFN-γ-mediated intestinal inflammation.
PMCID: PMC3356277  PMID: 22624013
6.  NOD1 and NOD2 regulation of pulmonary innate immunity to Legionella pneumophila 
European journal of immunology  2010;40(12):3519-3527.
The role of nucleotide-binding oligomerization domain-1 (NOD1) and nucleotide-binding oligomerization domain-2 (NOD2), cytoplasmic receptors which detect bacterial cell wall molecules, in pulmonary innate immune responses is poorly understood. We determined that both NOD1 and NOD2 detect heat-killed Legionella and stimulate NF-κb and IFN-β promoter activity using an in vitro luciferase reporter system. We next infected NOD1- and NOD2-deficient animals with aerosolized Legionella pneumophila. At 3 days post infection, Nod1–/– mice had impaired bacterial clearance compared to WT controls. In addition, at 4 h and 24 h, Nod1–/– mice had impaired neutrophil recruitment to the alveolar space. In contrast, increased lung neutrophils were seen in the Nod2–/– animals at 24 h. Analysis of cytokine production at 4 h post infection revealed a significant decrease in proinflammatory cytokines in the Nod1–/– animals when compared to WT animals. In contrast, increased 4-h proinflammatory cytokines were seen in the Nod2–/– animals. Furthermore, the lungs of both Nod1–/– and Nod2–/– mice had significantly increased pro-inflammatory cytokine levels at 24 h, suggesting possible suppressive roles for later stages of infection. Together, our data suggest that although both NOD1 and NOD2 can detect Legionella, these receptors modulate the in vivo pulmonary immune response differently.
PMCID: PMC3063513  PMID: 21108472
Legionella pneumophila; NOD1; NOD2; Pneumonia
7.  The angiogenic response of the aorta to injury and inflammatory cytokines requires macrophages 
The purpose of this study was to define early events during the angiogenic response of the aortic wall to injury. Rat aortic rings produced neovessels in collagen culture but lost this capacity over time. These quiescent rings responded to vascular endothelial growth factor (VEGF) but not to a cocktail of macrophage-stimulatory cytokines and chemokines that was angiogenically active on fresh rings. Analysis of cytokine receptor expression revealed selective loss in quiescent rings of the proangiogenic chemokine receptor CXCR2, which was expressed predominantly in aortic macrophages. Pharmacologic inhibition of CXCR2 impaired angiogenesis from fresh rings but had no effect on VEGF-induced angiogenesis from quiescent explants. Angiogenesis was also impaired in cultures of aortic rings from CXCR2-deficient mice. Reduced CXCR2 expression in quiescent rat aortic rings correlated with marked macrophage depletion. Pharmacologic ablation of macrophages from aortic explants blocked formation of neovessels in vitro and reduced aortic ring-induced angiogenesis in vivo. The angiogenic response of macrophage-depleted rings was completely restored by adding exogenous macrophages. Moreover, angiogenesis from fresh rings was promoted by macrophage colony stimulating factor (CSF-1) and inhibited with anti-CSF-1 antibody. Thus aortic angiogenic sprouting following injury is strongly influenced by conditions that modulate resident macrophage numbers and function.
PMCID: PMC2713030  PMID: 18832730
chemokines; inflammation; leukocytes; monocytes; neovascularization
8.  Iron metabolism at the host pathogen interface: lipocalin 2 and the pathogen-associated iroA gene cluster 
The host innate immune defense protein lipocalin 2 binds bacterial enterobactin siderophores to limit bacterial iron acquisition. To counteract this host defense mechanism bacteria have acquired the iroA gene cluster, which encodes enzymatic machinery and transporters that revitalize enterobactin in the form of salmochelin. The iroB enzyme introduces glucosyl residues at the C5 site on 2,3-dihydroxybenzoylserine moieties of enterobactin and thereby prevents lipocalin 2 binding. Additional strategies to evade lipocalin 2 have evolved in other bacteria, such as Mycobacteria tuberculosis and Bacillus anthracis. Targeting these specialized bacterial evasion strategy may provide a mechanism to reinvigorate lipocalin 2 in defense against specific pathogens.
PMCID: PMC2695446  PMID: 17714976
innate immunity; host defense; lipocalin 2; iron; siderophores
9.  Activating transcription factor 3 is a negative regulator of allergic pulmonary inflammation 
The Journal of Experimental Medicine  2008;205(10):2349-2357.
We recently demonstrated the pivotal role of the transcription factor (TF) activating TF 3 (ATF3) in dampening inflammation. We demonstrate that ATF3 also ameliorates allergen-induced airway inflammation and hyperresponsiveness in a mouse model of human asthma. ATF3 expression was increased in the lungs of mice challenged with ovalbumin allergen, and this was associated with its recruitment to the promoters of genes encoding Th2-associated cytokines. ATF3-deficient mice developed significantly increased airway hyperresponsiveness, pulmonary eosinophilia, and enhanced chemokine and Th2 cytokine responses in lung tissue and in lung-derived CD4+ lymphocytes. Although several TFs have been associated with enhanced inflammatory responses in the lung, ATF3 attenuates the inflammatory responses associated with allergic airway disease.
PMCID: PMC2556774  PMID: 18794337
10.  The Innate Immune Response to Salmonella enterica Serovar Typhimurium by Macrophages Is Dependent on TREM2-DAP12▿  
Infection and Immunity  2008;76(6):2439-2447.
Macrophage recognition of Salmonella enterica serovar Typhimurium leads to a cascade of signaling events, including the activation of Src family and Syk kinases and the production of reactive oxygen species (ROS), which are critical for host innate defense during early stages of bacterial infection. ROS production depends on the NADPH oxidase, but little is known about the innate immune receptors and proximal adapters that regulate Salmonella-induced ROS. Herein, we demonstrate that serovar Typhimurium induces ROS through a pathway that requires both triggering receptor expressed on myeloid cells 2 (TREM2) and DAP12. This pathway is highly analogous to the pathways utilized by Fc receptors and integrins to regulate ROS production. Oral infection of mice with serovar Typhimurium demonstrates that the DAP12-dependent pathway regulates cecal colonization during early stages of Salmonella infection. Thus, DAP12 is an important regulator of Salmonella-induced ROS production in macrophages, and TREM2 is essential for linking DAP12 to the innate response to serovar Typhimurium.
PMCID: PMC2423085  PMID: 18391000
11.  Conservation of Toll-Like Receptor Signaling Pathways in Teleost Fish 
In mammals, Toll-like receptors (TLR) recognize ligands, including pathogen-associated molecular patterns (PAMPs), and respond with ligand-specific induction of genes. In this study, we establish evolutionary conservation in teleost fish of key components of the TLR-signaling pathway that act as switches for differential gene induction, including MYD88, TIRAP, TRIF, TRAF6, IRF3, and IRF7. We further explore this conservation with a molecular phylogenetic analysis of MYD88. To the extent that current genomic analysis can establish, each vertebrate has one ortholog to each of these genes. For molecular tree construction and phylogeny inference, we demonstrate a methodology for including genes with only partial primary sequences without disrupting the topology provided by the high-confidence full-length sequences. Conservation of the TLR-signaling molecules suggests that the basic program of gene regulation by the TLR-signaling pathway is conserved across vertebrates. To test this hypothesis, leukocytes from a model fish, rainbow trout (Oncorhynchus mykiss), were stimulated with known mammalian TLR agonists including: diacylated and triacylated forms of lipoprotein, flagellin, two forms of LPS, synthetic double-stranded RNA, and two imidazoquinoline compounds (loxoribine and R848). Trout leukocytes responded in vitro to a number of these agonists with distinct patterns of cytokine expression that correspond to mammalian responses. Our results support the key prediction from our phylogenetic analyses that strong selective pressure of pathogenic microbes has preserved both TLR recognition and signaling functions during vertebrate evolution.
PMCID: PMC1524722  PMID: 17330145
pro-inflammatory cytokine; interferon; MYD88; TIRAP; TRIF; TRAF6; IRF3; phylogeny; molecular tree; PHYLIP
12.  A conserved surface on Toll-like receptor 5 recognizes bacterial flagellin 
The molecular basis for Toll-like receptor (TLR) recognition of microbial ligands is unknown. We demonstrate that mouse and human TLR5 discriminate between different flagellins, and we use this difference to map the flagellin recognition site on TLR5 to 228 amino acids of the extracellular domain. Through molecular modeling of the TLR5 ectodomain, we identify two conserved surface-exposed regions. Mutagenesis studies demonstrate that naturally occurring amino acid variation in TLR5 residue 268 is responsible for human and mouse discrimination between flagellin molecules. Mutations within one conserved surface identify residues D295 and D367 as important for flagellin recognition. These studies localize flagellin recognition to a conserved surface on the modeled TLR5 structure, providing detailed analysis of the interaction of a TLR with its ligand. These findings suggest that ligand binding at the β sheets results in TLR activation and provide a new framework for understanding TLR–agonist interactions.
PMCID: PMC2118731  PMID: 17283206
13.  CD4+ T Cells and Toll-Like Receptors Recognize Salmonella Antigens Expressed in Bacterial Surface Organelles  
Infection and Immunity  2005;73(3):1350-1356.
A better understanding of immunity to infection is revealed from the characteristics of microbial ligands recognized by host immune responses. Murine infection with the intracellular bacterium Salmonella generates CD4+ T cells that specifically recognize Salmonella proteins expressed in bacterial surface organelles such as flagella and membrane vesicles. These natural Salmonella antigens are also ligands for Toll-like receptors (TLRs) or avidly associated with TLR ligands such as lipopolysaccharide (LPS). PhoP/PhoQ, a regulon controlling Salmonella virulence and remodeling of LPS to resist innate immunity, coordinately represses production of surface-exposed antigens recognized by CD4+ T cells and TLRs. These data suggest that genetically coordinated surface modifications may provide a growth advantage for Salmonella in host tissues by limiting both innate and adaptive immune recognition.
PMCID: PMC1064935  PMID: 15731032
14.  A Common Dominant TLR5 Stop Codon Polymorphism Abolishes Flagellin Signaling and Is Associated with Susceptibility to Legionnaires' Disease 
The Journal of Experimental Medicine  2003;198(10):1563-1572.
Although Toll-like receptors (TLRs) are critical mediators of the immune response to pathogens, the influence of polymorphisms in this gene family on human susceptibility to infection is poorly understood. We demonstrated recently that TLR5 recognizes flagellin, a potent inflammatory stimulus present in the flagellar structure of many bacteria. Here, we show that a common stop codon polymorphism in the ligand-binding domain of TLR5 (TLR5392STOP) is unable to mediate flagellin signaling, acts in a dominant fashion, and is associated with susceptibility to pneumonia caused by Legionella pneumophila, a flagellated bacterium. We also show that flagellin is a principal stimulant of proinflammatory cytokine production in lung epithelial cells. Together, these observations suggest that TLR5392STOP increases human susceptibility to infection through an unusual dominant mechanism that compromises TLR5's essential role as a regulator of the lung epithelial innate immune response.
PMCID: PMC2194120  PMID: 14623910
inflammation; immunity; genetic predisposition to disease; genetic markers; bacterial infections

Results 1-14 (14)