Ian Marriott

Cultured astrocytes express toll-like receptors for bacterial products

It has become apparent that astrocytes may be important contributors to inflammatory immune responses within the brain in response to microbial challenges. To date, the mechanisms that underlie activation of this major glial cell type by such challenges have not been investigated. In the present study, we present evidence for members of a recently discovered family of receptors for highly conserved microbial components, the Toll‐like receptors (TLRs), in isolated cultures of primary murine astrocytes. We describe the low‐level constitutive expression of messenger RNA‐encoding TLR2, TLR4, TLR5, and TLR9 in resting cultures of these cells. Importantly, the level of expression of messenger RNA for each of these receptors is markedly elevated following exposure to specific bacteria‐derived ligands for these receptors. The functional expression of these receptor proteins is further supported by the ability of known ligands for each TLR to induce both message expression and protein secretion of the proinflammatory cytokine, interleukin‐6. In addition, the recent availability of antibodies to TLR2 and TLR4 has enabled us to demonstrate directly the presence of these receptors on astrocytes by Western blot and immunofluorescence analysis, respectively. Furthermore, we have confirmed the sensitivity of such receptor expression to ligand stimulation. The present demonstration of Toll‐like microbial pattern‐recognition receptors on primary astrocytes provides a mechanistic link between bacterial challenge and inflammatory immune responses that may be an important component of the pathologies of bacterially induced inflammatory CNS disorders.

Sexual dimorphism in innate immune responses to infectious organisms.

Gender has long been known to be a contributory factor in the incidence and progression of disorders associated with immune system dysregulation. More recently, evidence has accumulated that gender may also play an important role in infectious disease susceptibility. In general, females generate more robust and potentially protective humoral and cell-medated immune responses following antigenic challenge than their male counterparts. In contrast, males have frequently been observed to mount more aggressive and damaging inflammatory immune responses to microbial stimuli. In this article we review the evidence for sexual dimorphism in innate immune responses to infectious organisms and describe our recent studies that may provide a mechanism underlying gender-based differences in conditions such as bacterial sepsis.

Testosterone Reduces Macrophage Expression in the Mouse of Toll-Like Receptor 4, a Trigger for Inflammation and Innate Immunity

Abstract Though gender-based differences in the development of protective or pathological adaptive host responses have been widely noted, it is becoming apparent that sex may also influence the early perception of microbial challenges and the generation of inflammatory immune responses. These differences may be due to the actions of reproductive hormones, and such a hypothesis is supported by the presence of receptors for these hormones in a variety of immune cell types. Androgens such as testosterone have been shown to decrease immune functions, including cytokine production. However, the mechanisms by which testosterone limits such responses remain undefined. In this study, we have investigated the acute effects of testosterone on the level of expression of a key trigger for inflammation and innate immunity, Toll-like receptor 4 (TLR4), on isolated mouse macrophages. We show that in vitro testosterone treatment of macrophages, generated in the absence of androgen, elicits a modest but significant decrease in TLR4 expression and sensitivity to a TLR4-specific ligand. In addition, we have studied the effect of in vivo removal of endogenous testosterone on TLR4 expression and endotoxin susceptibility. We report that orchidectomized mice were significantly more susceptible to endotoxic shock and show that macrophages isolated from these animals have significantly higher TLR4 cell surface expression than those derived from sham gonadectomized mice. Importantly, these effects were not apparent in orchidectomized animals that received exogenous testosterone treatment. As such, these data may represent an important mechanism underlying the immunosuppressive effects of testosterone.

Salmonella efficiently enter and survive within cultured CD11c+ dendritic cells initiating cytokine expression.

While Salmonella infects macrophages, this cell population may not be the only one important for disseminating intracellular bacteria from mucosal sites. Dendritic cells (DC) are present in the Peyers patches and are mobilized following stimulation. Such characteristics would seem to be ideal for the dissemination of an intracellular, mucosal pathogen. However, it has been difficult to obtain sufficient numbers of DC to assess their ability to harbor Salmonella or to monitor DC in vivo. In the present study, this problem has been addressed by expanding DC in vivo using flt3 ligand, followed by the purification of CD11c+ cells using antibody‐coated magnetic beads or by fluorescence‐activated cell sorting. Salmonella dublin were found to be efficiently internalized, and to survive and replicate within purified CD11c+ DC, and also in CD11c+, CD8α+ or CD11c+, CD11b+ DC subpopulations. The ability of Salmonella to enter DC is of similar magnitude to that reported for macrophages, suggesting that this cell population could be an important host cell for dissemination of this pathogen from mucosal sites. Furthermore, infected DC responded to Salmonella by secretion of IL‐1, IL‐6 and IL‐12. As such, these cells may be important sources of these cytokines during the host response against Salmonella infection.

Staphylococcus aureus infection of mouse or human osteoblasts induces high levels of interleukin-6 and interleukin-12 production

Staphylococcus aureus is the principal causative agent of the inflammatory bone disease osteomyelitis. Unfortunately, the pathogenesis of this often chronic infection is poorly understood and is complicated by the recent observation that bone-forming osteoblasts can harbor S. aureus. Such an infection presents a significant challenge for the host immune response, because osteoblasts are not known to initiate protective cell-mediated immune responses. Cultured mouse and human osteoblasts infected with S. aureus were found to express high levels of interleukin (IL)-6 and IL-12p75, on the basis of complementary investigations demonstrating both S. aureus-induced up-regulation of expression of IL-6 and IL-12p40 mRNA and secretion of IL-6 and IL-12p75 by these cells. Additionally, a quantitative bioassay demonstrated that IL-12p75 secreted after infection was biologically active. These studies are the first to demonstrate induced IL-12p75 expression by osteoblasts and suggest a previously unrecognized role for osteoblasts in initiating immune responses after S. aureus infection.

Substance P activates NF-κB independent of elevations in intracellular calcium in murine macrophages and dendritic cells

Professional antigen presenting cells, such as macrophages, can be activated by intracellular calcium-dependent as well as calcium-independent mechanisms, depending upon the stimulus used. In this report, we addressed the mechanism of substance P-induced intracellular signalling in murine macrophages and dendritic cells. While no increases in intracellular calcium concentration were detected in macrophages or dendritic cells using sensitive fluorimetric techniques, substance P did induce rapid enhanced activation of NF-kappaB, a transcriptional activator known to regulate pro-inflammatory cytokines. These data provide an important mechanism by which substance P may augment the production of pro-inflammatory molecules.

The role of Toll-like receptors in CNS response to microbial challenge

The recent discovery of the family of Toll‐like receptors has vastly expanded our understanding of the mechanisms by which the innate immune system recognizes and responds to a wide variety of microbial and endogenous pathogens. Toll‐like receptors are transmembrane proteins that upon ligation with their cognate ligands trigger the production of cytokines, enzymes and other inflammatory agents. In the CNS Toll‐like receptors are expressed predominantly by glial cells. In particular, the vastly abundant astrocytes are likely to be the major contributors to inflammatory responses within the CNS. Studies of the murine brain abscess model revealed that Toll‐like receptor 2 plays a pivotal role in the generation of immune responses to Staphylococcus aureus. Although Toll‐like receptor signaling is essential in antimicrobial defense, it may also lead to bystander injury of CNS tissue.

IL-4 and IFN-γ Up-Regulate Substance P Receptor Expression in Murine Peritoneal Macrophages

While the ability of macrophages to express authentic substance P receptors (i.e., NK-1 receptors) has been inferred from radioreceptor binding assays and functional assays and, most recently, by identification of NK-1 receptor mRNA expression, we know little about NK-1 expression at the protein level or what host factors might up-regulate expression of this receptor. In the present study we demonstrate that the cytokines IL-4 and IFN-γ can increase the expression of NK-1 receptors on murine peritoneal macrophages. Specifically, we show that IL-4 and IFN-γ can elicit increases in the level of mRNA encoding the NK-1 receptor by up to 12- and 13-fold, respectively. Furthermore, these cytokines can significantly increase the expression of the NK-1 receptor protein as measured by Western blot and FACS analysis using specific Abs developed in our laboratory. In addition, we have demonstrated the ability of both IL-4 and IFN-γ to enhance the ability of macrophages to bind substance P as measured by radiolabeled binding assay. The observation that the level of expression of this receptor protein can be enhanced by cytokines that promote either cell-mediated (Th1) or humoral (Th2) immune responses supports the idea that this receptor can be induced during either type of immune response. As such, these results may point to a more ubiquitous role for substance P in the generation of optimal immune responses than previously appreciated.

Sex-based differences in immune function and responses to vaccination

Females typically develop higher antibody responses and experience more adverse reactions following vaccination than males. These differences are observed in response to diverse vaccines, including the bacillus Calmette-Guerin vaccine, the measles, mumps and rubella vaccine, the yellow fever virus vaccine and influenza vaccines. Sex differences in the responses to vaccines are observed across diverse age groups, ranging from infants to aged individuals. Biological as well as behavioral differences between the sexes are likely to contribute to differences in the outcome of vaccination between the sexes. Immunological, hormonal, genetic and microbiota differences between males and females may also affect the outcome of vaccination. Identifying ways to reduce adverse reactions in females and increase immune responses in males will be necessary to adequately protect both sexes against infectious diseases.

Expression of functional NK‐1 receptors in murine microglia

Cells of myeloid origin such as microglia have the potential to contribute significantly to the development of inflammatory responses in the CNS. The ability of the neuropeptide substance P to augment proinflammatory responses by other myeloid cell types such as macrophages and dendritic cells is well recognized. In the present study, we demonstrate the presence of mRNA encoding NK‐1 (substance P) receptors in murine microglia cell lines. Importantly, we have utilized specific antibodies developed by our laboratory to detect the expression of the NK‐1 receptor protein in murine microglia cell lines by Western blot analysis and flow cytometry. Furthermore, we have investigated the presence of this receptor on primary murine microglia and report the presence of authentic NK‐1 receptors as determined by Western blot analysis and flow cytometry. In addition, we demonstrate that NK‐1 receptors expressed on microglia are functional as demonstrated by the ability of nanomolar concentrations of substance P to initiate activation of the transcriptional activator, NF‐κB. Given the weight of evidence supporting the role of substance P–substance P receptor interactions in the initiation of optimal proinflammatory responses by myeloid cells, the demonstration of authentic and functional NK‐1 receptors in microglia identifies this neuropeptide as a potentially important contributor to CNS inflammatory responses during disease states. GLIA 37:258–267, 2002.