Michael G. Drage

Human β-defensin-3 activates professional antigen-presenting cells via Toll-like receptors 1 and 2

There is increasing evidence that innate and adaptive immune responses are intimately linked. This linkage is in part mediated through the recognition of conserved microbial products by Toll-like receptors (TLRs). Detection of microbial products by TLRs can result in induction of inflammatory cytokines and activation of professional antigen-presenting cells, thereby enhancing adaptive immune responses. Here, we show that human β-defensin-3 (hBD-3), an innate antimicrobial peptide, can induce expression of the costimulatory molecules CD80, CD86, and CD40, on monocytes and myeloid dendritic cells in a TLR-dependent manner. Activation of monocytes by hBD-3 is mediated by interaction with TLRs 1 and 2, resulting in signaling that requires myeloid differentiating factor 88 and results in IL-1 receptor-associated kinase-1 phosphorylation. In studies with HEK cells engineered to express various TLRs, we show that activation of NF-κB by hBD-3 depends on the expression of both TLR1 and TLR2. Thus, human TLR signaling is not restricted to recognition of microbial patterns but also can be initiated by host-derived peptides such as hBD-3.

TLR2 and its co-receptors determine responses of macrophages and dendritic cells to lipoproteins of Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) signals through Toll-like receptor 2 (TLR2) to regulate antigen presenting cells (APCs). Mtb lipoproteins, including LpqH, LprA, LprG and PhoS1, are TLR2 agonists, but their co-receptor requirements are unknown. We studied Mtb lipoprotein-induced responses in TLR2(-/-), TLR1(-/-), TLR6(-/-), CD14(-/-) and CD36(-/-) macrophages. Responses to LprA, LprG, LpqH and PhoS1 were completely dependent on TLR2. LprG, LpqH, and PhoS1 were dependent on TLR1, but LprA did not require TLR1. None of the lipoproteins required TLR6, although a redundant contribution by TLR6 cannot be excluded. CD14 contributed to detection of LprA, LprG and LpqH, whereas CD36 contributed only to detection of LprA. Studies of lung APC subsets revealed lower TLR2 expression by CD11b(high)/CD11c(low) lung macrophages than CD11b(low)/CD11c(high) alveolar macrophages, which correlated with hyporesponsiveness of lung macrophages to LpqH. Thus, lung APC subsets differ in TLR expression, which may determine differences in responses to Mtb.

Mycobacterium tuberculosis lipoprotein LprG (Rv1411c) binds triacylated glycolipid agonists of Toll-like receptor 2

Knockout of lprG results in decreased virulence of Mycobacterium tuberculosis (MTB) in mice. MTB lipoprotein LprG has TLR2 agonist activity, which is thought to be dependent on its N-terminal triacylation. Unexpectedly, here we find that nonacylated LprG retains TLR2 activity. Moreover, we show LprG association with triacylated glycolipid TLR2 agonists lipoarabinomannan, lipomannan and phosphatidylinositol mannosides (which share core structures). Binding of triacylated species was specific to LprG (not LprA) and increased LprG TLR2 agonist activity; conversely, association of glycolipids with LprG enhanced their recognition by TLR2. The crystal structure of LprG in complex with phosphatidylinositol mannoside revealed a hydrophobic pocket that accommodates the three alkyl chains of the ligand. In conclusion, we demonstrate a glycolipid binding function of LprG that enhances recognition of triacylated MTB glycolipids by TLR2 and may affect glycolipid assembly or transport for bacterial cell wall biogenesis.

Mycobacterium bovis BCG decreases MHC-II expression in vivo on murine lung macrophages and dendritic cells during aerosol infection

Mycobacterium tuberculosis and M. bovis BCG infect APCs. In vitro, mycobacteria inhibit IFN-gamma-induced MHC-II expression by macrophages, but the effects of mycobacteria on lung APCs in vivo remain unclear. To assess MHC-II expression on APCs infected in vivo, mice were aerosol-infected with GFP-expressing BCG. At 28 d, approximately 1% of lung APCs were GFP+ by flow cytometry and CFU data. Most GFP+ cells were CD11b(high)/CD11c(neg-mid) lung macrophages (58-68%) or CD11b(high)/CD11c(high) DCs (28-31%). Lung APC MHC-II expression was higher in infected mice than naïve mice. Within infected lungs, however, MHC-II expression was lower in GFP+ cells than GFP- cells for both macrophages and DCs. MHC-II expression was also inhibited on purified lung macrophages and DCs that were infected with BCG in vitro. Thus, lung APCs that harbor mycobacteria in vivo have decreased MHC-II expression relative to uninfected APCs from the same lung, possibly contributing to evasion of T cell responses.

Hippocampal neurons and glia in epileptic EL mice

Reactive changes in hippocampal astrocytes are frequently encountered in association with temporal lobe epilepsy in humans and with drug or kindling-induced seizures in animal models. These reactive changes generally involve increases in astrocyte size and number and often occur together with neuronal loss and synaptic rearrangements. In addition to producing astrocytic changes, seizure activity can also produce reactive changes in microglia, the resident macrophages of brain. In this study, we examined the effects of recurrent seizure activity on hippocampal neurons and glia in the epileptic EL mouse, a natural model of human multifactorial idiopathic epilepsy and complex partial seizures. Timm staining was used to evaluate infrapyramidal mossy fiber organization and the optical dissector method was used to count Nissl-stained neurons in hippocampus of adult (about one year of age) EL mice and nonepileptic C57BL/6J (B6) and DDY mice. Immunostaining forglial fibrillary acidic protein (GFAP) and Iba1, an actin cross-linking molecule restricted to macrophages and microglia, was used to evaluate astrocytes and microglia, respectively. The EL mice experienced about 25–30 complex partial seizures with secondary generalization during routine weekly cage changing. No significant differences were found among the mouse strains for Timm staining scores or for neuronal counts in the CA1 and CA3 pyramidal fields or in the hilus. However, the number of GFAP-positive astrocytes was significantly elevated in the stratum radiatum and hilus of EL mice, while microglia appeared hyper-ramified and were more intensely stained in EL mice than in the B6 or DDY mice in the hilus, parietal cortex, and pyriform cortex. The results indicate that recurrent seizure activity in EL mice is associated with abnormalities in hippocampal astrocytes and brain microglia, but is not associated with obvious neuronal loss or mossy fiber synaptic rearrangements. The EL mouse can be a useful model for evaluating neuron-glia interactions related to idiopathic epilepsy.

Mesenchymal gene program-expressing ovarian cancer spheroids exhibit enhanced mesothelial clearance.

Metastatic dissemination of ovarian tumors involves the invasion of tumor cell clusters into the mesothelial cell lining of peritoneal cavity organs; however, the tumor-specific factors that allow ovarian cancer cells to spread are unclear. We used an in vitro assay that models the initial step of ovarian cancer metastasis, clearance of the mesothelial cell layer, to examine the clearance ability of a large panel of both established and primary ovarian tumor cells. Comparison of the gene and protein expression profiles of clearance-competent and clearance-incompetent cells revealed that mesenchymal genes are enriched in tumor populations that display strong clearance activity, while epithelial genes are enriched in those with weak or undetectable activity. Overexpression of transcription factors SNAI1, TWIST1, and ZEB1, which regulate the epithelial-to-mesenchymal transition (EMT), promoted mesothelial clearance in cell lines with weak activity, while knockdown of the EMT-regulatory transcription factors TWIST1 and ZEB1 attenuated mesothelial clearance in ovarian cancer cell lines with strong activity. These findings provide important insights into the mechanisms associated with metastatic progression of ovarian cancer and suggest that inhibiting pathways that drive mesenchymal programs may suppress tumor cell invasion of peritoneal tissues.

Mycobacterium tuberculosis Lipoproteins Directly Regulate Human Memory CD4 T Cell Activation via Toll-Like Receptors 1 and 2

ABSTRACT The success of Mycobacterium tuberculosis as a pathogen relies on its ability to regulate the host immune response. M. tuberculosis can manipulate adaptive T cell responses indirectly by modulating antigen-presenting cell (APC) function or by directly interacting with T cells. Little is known about the role of M. tuberculosis molecules in direct regulation of T cell function. Using a biochemical approach, we identified lipoproteins LprG and LpqH as major molecules in M. tuberculosis lysate responsible for costimulation of primary human CD4+ T cells. In the absence of APCs, activation of memory CD4+ T cells with LprG or LpqH in combination with anti-CD3 antibody induces Th1 cytokine secretion and cellular proliferation. Lipoprotein-induced T cell costimulation was inhibited by blocking antibodies to Toll-like receptor 2 (TLR2) and TLR1, indicating that human CD4+ T cells can use TLR2/TLR1 heterodimers to directly respond to M. tuberculosis products. M. tuberculosis lipoproteins induced NF-κB activation in CD4+ T cells in the absence of TCR co-engagement. Thus, TLR2/TLR1 engagement alone by M. tuberculosis lipoprotein triggered intracellular signaling, but upregulation of cytokine production and proliferation required co-engagement of the TCR. In conclusion, our results demonstrate that M. tuberculosis lipoproteins LprG and LpqH participate in the regulation of adaptive immunity not only by inducing cytokine secretion and costimulatory molecules in innate immune cells but also through directly regulating the activation of memory T lymphocytes.

Seizure-prone EL mice exhibit deficits in pup nursing and retrieval assessed using a novel method of maternal behavior phenotyping.

The selectively bred EL mouse strain exhibits hyperreactivity to environmental disturbance reflected by handling-induced seizures and motor hyperactivity in an exploratory task relative to a non-seizure-prone control strain. One possible mechanism for the nongenomic transmission of an adult seizure-prone/hyperactive phenotype is the quality of parenting provided to immature offspring. In particular, the quality of maternal behavior has been implicated as an environmental determinant in rodent biochemical and behavioral development. A complication in testing this hypothesis is that human handling for husbandry and testing itself triggers seizures in seizure-prone EL mice. Thus, the present study evaluated potential EL versus control strain differences in maternal behavior using a novel apparatus for passive observation of undisturbed mice. Nonmaternal behaviors were also measured to control for any nonspecific differences in activity or exploration. EL dams were slower than DDY controls to initiate pup retrieval and spent less time nursing/crouching over pups than DDY mice. EL dams also exhibited a profile of sustained exploration and grooming over time relative to the profile of DDY controls. These results suggest that EL mothers exhibit an overabundance of motor activities that compete with crouching/nursing and retrieval behaviors required for viability of the litter.

Sexual dysfunction and sudden death in epileptic male EL mice: inheritance and prevention with the ketogenic diet.

Summary:  Purpose: The EL mouse is an animal model for multifactorial idiopathic epilepsy. Although EL mice have been studied extensively for >45 years, the etiology of male sudden death and its relation to seizures have not been defined. Here we investigated the cause of EL male sudden death and its relation to epilepsy.

Phenotyping the untouchables: environmental enhancement of behavioral and physiological activation in seizure-prone El mice

The onset and frequency of spontaneous and tail suspension-induced seizures in El mice appear to be influenced strongly by developmental and experiential factors over the first 3 months of life. To assess the impact of social factors on behavioral characteristics of El mice prior to the age of seizure susceptibility, locomotor activity and exploratory measures of arousal were recorded in 40-day-old El and control DDY mice exposed to group and isolation housing conditions. Once mice reached maturity, physiological reactivity to a tail suspension stressor was evaluated. The locomotor activity measure revealed circadian entrainment in both strains, nocturnal hyperactivity in El mice, and a locomotor activity-attenuating effect of group housing in El mice. In the two-compartment model of exploration, latency to enter, transitions to and from, and rearing in a brightly lit compartment were 50% higher in El relative to DDY mice, again suggesting a hyperactive phenotype. Finally, an acute 2-minute tail suspension stressor applied to 75-day-old mice implanted with radiotelemetry transmitters revealed a reactive tachycardia in El, but not DDY, mice. No seizures were observed during any of the experimental manipulations. Taken together, these results suggest that spontaneously occurring deviations in behavioral and cardiovascular measures of arousal characterize preseizure El mice and that motor features of hyperarousal can be exaggerated by the environmental manipulation of isolation housing.