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1.  Participation of MyD88 and Interleukin-33 as Innate Drivers of Th2 Immunity to Trichinella spiralis 
Infection and Immunity  2013;81(4):1354-1363.
Trichinella spiralis is a highly destructive parasitic nematode that invades and destroys intestinal epithelial cells, injures many different tissues during its migratory phase, and occupies and transforms myotubes during the final phase of its life cycle. We set out to investigate the role in immunity of innate receptors for potential pathogen- or danger-associated molecular patterns (PAMPs or DAMPs). Focusing on the MyD88-dependent receptors, which include Toll-like receptors (TLRs) and interleukin-1 (IL-1) family members, we found that MyD88-deficient mice expelled worms normally, while TLR2/4-deficient mice showed accelerated worm expulsion, suggesting that MyD88 was active in signaling pathways for more than one receptor during intestinal immunity. A direct role for PAMPs in TLR activation was not supported in a transactivation assay involving a panel of murine and human TLRs. Mice deficient in the IL-1 family receptor for the DAMP, IL-33 (called ST2), displayed reduced intestinal Th2 responses and impaired mast cell activation. IL-33 was constitutively expressed in intestinal epithelial cells, where it became concentrated in nuclei within 2 days of infection. Nuclear localization was an innate response to infection that occurred in intestinal regions where worms were actively migrating. Th2 responses were also compromised in the lymph nodes draining the skeletal muscles of ST2-deficient mice, and this correlated with increased larval burdens in muscle. Our results support a mechanism in which the immune system recognizes and responds to tissue injury in a way that promotes Th2 responses.
PMCID: PMC3639596  PMID: 23403558
2.  Heat Shock Protein gp96 regulates Toll-Like Receptor 9 proteolytic processing and conformational stability 
Nucleic acid-sensing Toll-like receptors (TLRs) initiate innate immune responses to foreign RNA and DNA, yet can detect and respond to host DNA. To avoid autoimmune pathologies, nucleic acid sensing TLRs are tightly regulated. TLR9 primarily resides in the endoplasmic reticulum, traffics to endosomes, is proteolytically processed and responds to DNA. The heat shock protein gp96 is one of several accessory proteins that regulate intracellular trafficking of TLR9. In the absence of gp96, TLR9 fails to exit the endoplasmic reticulum, and therefore gp96-deficient macrophages fail to respond to CpG DNA. However, absence of gp96 precludes studies on potential chaperoning functions of gp96 for TLR9. Here we demonstrate that pharmacologic interference with gp96 function inhibits TLR9 signaling. TLR9 remains associated with gp96 during intracellular trafficking, and gp96-specific inhibitors increase TLR9 sensitivity to proteolytic degradation. We propose that gp96 is critical for both TLR9 egress from the ER, and for protein conformational stability in the endosomal compartment. These studies highlight the importance of examining gp96-specific inhibitors for modulating TLR9 activation, and the treatment autoimmune diseases.
PMCID: PMC3358552  PMID: 22554506
innate immunity; heat shock protein; Toll-like receptor 9
3.  Discordant Results Obtained with Francisella tularensis during In Vitro and In Vivo Immunological Studies Are Attributable to Compromised Bacterial Structural Integrity 
PLoS ONE  2013;8(3):e58513.
Francisella tularensis (Ft) is a highly infectious intracellular pathogen and the causative agent of tularemia. Because Ft can be dispersed via small droplet-aerosols and has a very low infectious dose it is characterized as a category A Select Agent of biological warfare. Respiratory infection with the attenuated Live Vaccine Strain (LVS) and the highly virulent SchuS4 strain of Ft engenders intense peribronchiolar and perivascular inflammation, but fails to elicit select pro-inflammatory mediators (e.g., TNF, IL-1β, IL-6, IL-12, and IFN-γ) within the first ∼72 h. This in vivo finding is discordant with the principally TH1-oriented response to Ft frequently observed in cell-based studies wherein the aforementioned cytokines are produced. An often overlooked confounding factor in the interpretation of experimental results is the influence of environmental cues on the bacterium's capacity to elicit certain host responses. Herein, we reveal that adaptation of Ft to its mammalian host imparts an inability to elicit select pro-inflammatory mediators throughout the course of infection. Furthermore, in vitro findings that non-host adapted Ft elicits such a response from host cells reflect aberrant recognition of the DNA of structurally-compromised bacteria by AIM2-dependent and -independent host cell cytosolic DNA sensors. Growth of Ft in Muller-Hinton Broth or on Muller-Hinton-based chocolate agar plates or genetic mutation of Ft was found to compromise the structural integrity of the bacterium thus rendering it capable of aberrantly eliciting pro-inflammatory mediators (e.g., TNF, IL-1β, IL-6, IL-12, and IFN-γ). Our studies highlight the profound impact of different growth conditions on host cell response to infection and demonstrate that not all in vitro-derived findings may be relevant to tularemia pathogenesis in the mammalian host. Rational development of a vaccine and immunotherapeutics can only proceed from a foundation of knowledge based upon in vitro findings that recapitulate those observed during natural infection.
PMCID: PMC3595284  PMID: 23554897
4.  A TLR 9 cytoplasmic tyrosine motif is selectively required for proinflammatory cytokine production1 
Compartmentalization of nucleic acid sensing TLR9 has been implicated as a mechanism to prevent recognition of self nucleic acid structures. Furthermore, recognition of CpG DNA in different endosomal compartments leads to the production of the proinflammatory cytokine TNF-α, or type I IFN. We previously characterized a tyrosine-based motif at amino acid 888–891 in the cytoplasmic tail of TLR9 important for appropriate intracellular localization. Here we show that this motif is selectively required for the production of TNF, but not IFN. In response to CpG DNA stimulation, the proteolytically processed 80 kDa fragment is tyrosine phosphorylated. Although tyrosine 888 is not itself phosphorylated, the structure of this motif is necessary for both TLR9 phosphorylation and TNF-α production in response to CpG DNA. We conclude that bifurcation in TLR9 signaling is regulated by a critical tyrosine motif in the cytoplasmic tail.
PMCID: PMC3253180  PMID: 22174451
5.  Antimicrobial peptide inhibits poly(I:C) induced immune responses§ 
Viral proteins and nucleic acids stimulate Toll-like receptors (TLRs) to elicit production of cytokines, chemokines, and interferons. Due to their immunostimulatory activity, several TLR agonists are being developed as vaccine adjuvants and cancer immunotherapeutics. However, TLR signaling is modified by disease state, which could enhance or impair therapeutic efficacy. For example, in the skin of psoriasis patients, the human cationic antimicrobial peptide LL37 is highly expressed and binds to host DNA. Association with LL37 enhances DNA uptake into intracellular compartments where it stimulates TLR9-dependent overproduction of interferons. Poly(I:C), an analogue of viral double-stranded (ds) RNA is recognized by TLR3, and is currently in preclinical trials as an inducer of type I interferon. If LL37 similarly enhanced interferon production, use of poly(I:C) might be contraindicated in certain conditions where LL37 is elevated. Here we show that TLR3 signaling was not enhanced, but was dramatically inhibited, by LL37 or mouse cathelicidin-related antimicrobial peptide (mCRAMP) in macrophages, microglial cells, and dendritic cells. Inhibition correlated with formation of a strong complex between antimicrobial peptides and poly(I:C), which partially inhibited poly(I:C) binding to TLR3. Therefore, after injury or during existing acute or chronic inflammation, when LL37 levels are elevated, the therapeutic activity of poly(I:C) will be compromised. Our findings highlight the importance of using caution when therapeutically delivering nucleic acids as immunomodulators.
PMCID: PMC3233200  PMID: 22048772
poly(I:C); antimicrobial peptide; Toll-like receptor 3; innate immunity; proinflammatory cytokines
6.  TLR9 is important for protection against intestinal damage and for intestinal repair 
Scientific Reports  2012;2:574.
Toll-like receptors (TLRs) are innate receptors critical for host defense, and play a role in normal biological processes. For example, host DNA, a TLR9 ligand, stimulates epithelial repair following skin wounding. TLR signaling also plays a crucial role in regulating intestinal homeostasis. We therefore asked whether TLR9 is important for intestinal wound repair using a dextran sulfate sodium (DSS)-induced intestinal damage and repair model. We showed that TLR9-deficient mice are more susceptible to DSS, and exhibited delayed wound repair at both the clinical and histologic levels. TLR9-deficient mice showed reduced gene expression of hairy enhancer of split 1, an intestinal progenitor cell differentiation factor, and vascular endothelial growth factor, a growth factor important for epithelial cell restitution. Therefore, we conclude that TLR stimulation may play a normal role in regulating intestinal homeostasis and could potentially be a novel therapeutic target to enhance intestinal wound repair in inflammatory bowel diseases.
PMCID: PMC3418518  PMID: 22893852
7.  Negative Regulation by a Soluble Form of Toll-Like Receptor 9 
European Journal of Immunology  2011;41(8):2176-2184.
Nucleic acids structures are highly conserved through evolution and when self nucleic acids are aberrantly detected by Toll-Like Receptors (TLRs) they contribute to autoimmune disease. For this reason, multiple regulatory mechanisms exist to prevent response to self nucleic acids. TLR9 is a nucleic acid sensing TLR that is regulated at multiple levels including association with accessory proteins, intracellular localization and proteolytic processing. In the endolysosomal compartment TLR9 is proteolytically processed to an 80 kilodalton form (p80) and this processing is a prerequisite for activation. Here we identified a soluble form of TLR9 generated by a novel proteolytic event that cleaved TLR9 between amino acids 724–735. Similar to p80, sTLR9 was generated in endosomes. However, generation of sTLR9 was independent of the cysteine protease cathepsin B active at acidic pH, but partially dependent on cathepsin S, a protease active at neutral pH. Most importantly, sTLR9 inhibited TLR9-dependent signaling. Together, these data support a model where an intrinsic proteolytic processing mechanism negatively regulates TLR9 signaling. Proper balance between the independent proteolytic events likely contributes to regulation of TLR9 mediated innate immunity and prevention of autoimmune disease.
PMCID: PMC3379887  PMID: 21604257
Toll-like receptor; proteolysis; soluble-TLR9
8.  Multifunctional role of dextran sulfate sodium for in vivo modeling of intestinal diseases 
BMC Immunology  2012;13:41.
Inflammatory bowel diseases (IBDs) are chronic, relapsing disorders that affect the gastrointestinal tract of millions of people and continue to increase in incidence each year. While several factors have been associated with development of IBDs, the exact etiology is unknown. Research using animal models of IBDs is beginning to provide insights into how the different factors contribute to disease development. Oral administration of dextran sulfate sodium (DSS) to mice induces a reproducible experimental colitis that models several intestinal lesions associated with IBDs. The murine DSS colitis model can also be adapted to quantify intestinal repair following injury. Understanding the mechanistic basis behind intestinal repair is critical to development of new therapeutics for IBDs because of their chronic relapsing nature.
The murine DSS colitis model was adapted to provide a system enabling the quantification of severe intestinal injury with impaired wound healing or mild intestinal injury with rapid restoration of mucosal integrity, by altering DSS concentrations and including a recovery phase. We showed that through a novel format for presentation of the clinical disease data, the temporal progression of intestinal lesions can be quantified on an individual mouse basis. Additionally, parameters for quantification of DSS-induced alterations in epithelial cell populations are included to provide insights into mechanisms underlying the development of these lesions. For example, the use of the two different model systems showed that toll-like receptor 9, a nucleic acid-sensing pattern recognition receptor, is important for protection only following mild intestinal damage and suggests that this model is superior for identifying proteins necessary for intestinal repair.
We showed that using a murine DSS-induced experimental colitis model system, and presenting data in a longitudinal manner on a per mouse basis, enhanced the usefulness of this model, and provided novel insights into the role of an innate immune receptor in intestinal repair. By elucidating the mechanistic basis of intestinal injury and repair, we can begin to understand the etiology of IBDs, enabling development of novel therapeutics or prophylactics.
PMCID: PMC3488029  PMID: 22853702
Dextran sulfate sodium; Inflammatory bowel disease; Intestinal repair; Toll-like receptor 9
9.  Early Response of Mucosal Epithelial Cells During Toxoplasma gondii Infection 
The innate immune response of mucosal epithelial cells during pathogen invasion plays a central role in immune regulation in the gut. Toxoplasma gondii (T. gondii) is a protozoan intracellular parasite that is usually transmitted through oral infection. Although much of the information on immunity to T. gondii has come from intraperitoneal infection models, more recent studies have revealed the importance of studying immunity following infection through the natural per-oral route. Oral infection studies have identified many of the key players in the intestinal response; however, they have relied on responses detected days to weeks following infection. Much less is known about how the gut epithelial layer senses and reacts during initial contact with the pathogen. Given the importance of epithelial cells during pathogen invasion, this study uses an in vitro approach to isolate the key players and examine the early response of intestinal epithelial cells during infection by T. gondii. We show that human intestinal epithelial cells infected with T. gondii elicit rapid MAPK phosphorylation, NF-κB nuclear translocation, and secretion of interleukin (IL)-8. Both ERK1/2 activation and IL-8 secretion responses were shown to be MyD88 dependent and TLR2 was identified to be involved in the recognition of the parasite regardless of the parasite genotype. Furthermore, we were able to identify additional T. gondii-regulated genes in the infected cells using a pathway-focused array. Together, our findings suggest that intestinal epithelial cells were able to recognize T. gondii during infection, and the outcome is important for modulating intestinal immune responses.
PMCID: PMC3374343  PMID: 19917706
The Journal of biological chemistry  2006;281(46):35585-35592.
Toll like receptors (TLRs) are essential for host defense. While several TLRs reside on the cell surface, nucleic acid recognizing TLRs are intracellular. For example, the receptor for CpG containing bacterial and viral DNA, TLR9, is retained in the endoplasmic reticulum (ER). Recent evidence suggests that the localization of TLR9 is critical for appropriate ligand recognition. Here we define which structural features of the TLR9 molecule control its intracellular localization. Both the cytoplasmic and ectodomains of TLR9 contain sufficient information while the transmembrane domain plays no role in intracellular localization. We identify a 14 amino acid stretch that directs TLR9 intracellularly and confers intracellular localization to the normally cell surface expressed TLR4. Truncation or mutation of the cytoplasmic tail of TLR9 reveals a vesicle localization motif that targets early endosomes. We propose a model whereby modification of the cytoplasmic tail of TLR9 results in trafficking to early endosomes where it encounters CpG DNA.
PMCID: PMC2758030  PMID: 16990271
11.  TLR9 is Localized in the Endoplasmic Reticulum Prior to Stimulation 
In mammals, ten Toll like receptors (TLR) recognize conserved pathogen associated molecular patterns (PAMP), resulting in the induction of inflammatory innate immune responses. One of these, TLR9, is activated intracellularly by bacterial DNA and synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotides. Following treatment with CpG ODN, TLR9 is found in LAMP-1 positive lysosomes and we asked which intracellular compartment contains TLR9 prior to CpG exposure. Surprisingly, we found by microscopy and supporting biochemical evidence that both transfected and endogenously expressed human TLR9 is retained in the endoplasmic reticulum (ER). By contrast, human TLR4 trafficked to the cell surface, indicating that ER retention is not a property common to all TLRs. Since TLR9 is observed in endocytic vesicles following exposure to CpG ODN, our data indicate that a special mechanism must exist for translocating TLR9 to the signaling compartments that contain the CpG DNA.
PMCID: PMC2757936  PMID: 15240708
Human; B lymphocytes; Toll-Like Receptors
12.  TLR9 traffics through the Golgi complex to localize to endolysosomes and respond to CpG DNA 
Immunology and cell biology  2008;87(3):209-217.
Toll-like receptor 9 (TLR9) promiscuously binds self and microbial DNA, but only microbial DNA elicits an inflammatory response. How TLR9 discriminates between self and foreign DNA is unclear, but inappropriate localization of TLR9 permits response to self DNA, suggesting that TLR9 localization and trafficking are critical components. The molecular mechanisms controlling the movement of TLR9 may provide new insight into the recognition of DNA in normal and in pathological conditions such as autoimmune systemic lupus erythematosus. We previously showed that TLR9 is retained in the endoplasmic reticulum (ER) and it moves to endolysosomes to recognize CpG DNA. Other studies have suggested that TLR9 bypasses the Golgi complex to access endolysosomes. Here, we demonstrate that TLR9 translocates from ER to endolysosomes via the Golgi complex and that Golgi export is required for optimal TLR9 signaling. Six to thirteen percent of TLR9 constitutively exits the ER, moves through the Golgi complex and resides in LAMP-1 positive vesicles. TLR9 bound to CpG DNA had glycan modifications indicative of Golgi processing confirming that TLR9 travels through the Golgi complex to access CpG DNA in endolysosomes. Together, these data support a model where TLR9 uses traditional secretory pathways and does not bypass the Golgi complex.
PMCID: PMC2753824  PMID: 19079358
Brefeldin A; CpG DNA; Endolysosome; Golgi; TLR9; trafficking
13.  MARCO, TLR2, and CD14 Are Required for Macrophage Cytokine Responses to Mycobacterial Trehalose Dimycolate and Mycobacterium tuberculosis 
PLoS Pathogens  2009;5(6):e1000474.
Virtually all of the elements of Mycobacterium tuberculosis (Mtb) pathogenesis, including pro-inflammatory cytokine production, granuloma formation, cachexia, and mortality, can be induced by its predominant cell wall glycolipid, trehalose 6,6′-dimycolate (TDM/cord factor). TDM mediates these potent inflammatory responses via interactions with macrophages both in vitro and in vivo in a myeloid differentiation factor 88 (MyD88)-dependent manner via phosphorylation of the mitogen activated protein kinases (MAPKs), implying involvement of toll-like receptors (TLRs). However, specific TLRs or binding receptors for TDM have yet to be identified. Herein, we demonstrate that the macrophage receptor with collagenous structure (MARCO), a class A scavenger receptor, is utilized preferentially to “tether” TDM to the macrophage and to activate the TLR2 signaling pathway. TDM-induced signaling, as measured by a nuclear factor-kappa B (NF-κB)-luciferase reporter assay, required MARCO in addition to TLR2 and CD14. MARCO was used preferentially over the highly homologous scavenger receptor class A (SRA), which required TLR2 and TLR4, as well as their respective accessory molecules, in order for a slight increase in NF-κB signaling to occur. Consistent with these observations, macrophages from MARCO−/− or MARCO−/−SRA−/− mice are defective in activation of extracellular signal-related kinase 1/2 (ERK1/2) and subsequent pro-inflammatory cytokine production in response to TDM. These results show that MARCO-expressing macrophages secrete pro-inflammatory cytokines in response to TDM by cooperation between MARCO and TLR2/CD14, whereas other macrophage subtypes (e.g. bone marrow–derived) may rely somewhat less effectively on SRA, TLR2/CD14, and TLR4/MD2. Macrophages from MARCO−/− mice also produce markedly lower levels of pro-inflammatory cytokines in response to infection with virulent Mtb. These observations identify the scavenger receptors as essential binding receptors for TDM, explain the differential response to TDM of various macrophage populations, which differ in their expression of the scavenger receptors, and identify MARCO as a novel component required for TLR signaling.
Author Summary
The causative agent of tuberculosis, Mycobacterium tuberculosis, has a lipid-rich cell wall that contains a high percentage of mycolic acids. These mycolic acids contribute to both the impermeable nature of the cell wall and to the immunostimulatory properties of the bacterium. Indeed, it has been known for over 50 years that trehalose 6,6′-dimycolate (TDM/cord factor) is the major immunogenic lipid of M. tuberculosis, which induces potent pro-inflammatory responses from macrophages, although the receptor has not been identified. We have demonstrated that the toll-like receptor (TLR) pathway is required for pro-inflammatory cytokine production in response to TDM; however, the TLRs alone, or in conjunction with known co-receptors, are not sufficient to induce a response. We demonstrate that the macrophage receptor MARCO, a scavenger receptor, is utilized preferentially to “tether” TDM to the macrophage and activate the TLR2 signaling pathway, and is used preferentially over the related SRA. Macrophages from MARCO−/− mice are defective in activation of TDM-induced signaling and subsequent pro-inflammatory cytokine production in response to both TDM-coated beads and virulent M. tuberculosis. By identifying the macrophage receptors involved in initial recognition we can now explain variable responses to TDM between different macrophage populations (which differ in scavenger receptor expression), and have identified a novel co-receptor that may be involved in lipid presentation to TLRs.
PMCID: PMC2688075  PMID: 19521507
14.  TLR9 regulates Th1 responses and cooperates with TLR2 in mediating optimal resistance to Mycobacterium tuberculosis 
The Journal of Experimental Medicine  2005;202(12):1715-1724.
To investigate the role of Toll-like receptor (TLR)9 in the immune response to mycobacteria as well as its cooperation with TLR2, a receptor known to be triggered by several major mycobacterial ligands, we analyzed the resistance of TLR9−/− as well as TLR2/9 double knockout mice to aerosol infection with Mycobacterium tuberculosis. Infected TLR9−/− but not TLR2−/− mice displayed defective mycobacteria-induced interleukin (IL)-12p40 and interferon (IFN)-γ responses in vivo, but in common with TLR2−/− animals, the TLR9−/− mice exhibited only minor reductions in acute resistance to low dose pathogen challenge. When compared with either of the single TLR-deficient animals, TLR2/9−/− mice displayed markedly enhanced susceptibility to infection in association with combined defects in proinflammatory cytokine production in vitro, IFN-γ recall responses ex vivo, and altered pulmonary pathology. Cooperation between TLR9 and TLR2 was also evident at the level of the in vitro response to live M. tuberculosis, where dendritic cells and macrophages from TLR2/9−/− mice exhibited a greater defect in IL-12 response than the equivalent cell populations from single TLR9-deficient animals. These findings reveal a previously unappreciated role for TLR9 in the host response to M. tuberculosis and illustrate TLR collaboration in host resistance to a major human pathogen.
PMCID: PMC2212963  PMID: 16365150

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