Gareth D. Healey

Humoral and Cell-Mediated Adaptive Immune Responses Are Required for Protection against Burkholderia pseudomallei Challenge and Bacterial Clearance Postinfection

ABSTRACT Burkholderia pseudomallei, the causative agent of melioidosis, is a gram-negative bacillus endemic to areas of southeast Asia and northern Australia. Presently, there is no licensed vaccine for B. pseudomallei and the organism is refractive to antibiotic therapy. The bacterium is known to survive and multiply inside both phagocytic and nonphagocytic host cells and may be able to spread directly from cell to cell. Current vaccine delivery systems are unlikely to induce the correct immune effectors to stimulate a protective response to the organism. In this study, we have developed a procedure to utilize dendritic cells as a vaccine delivery vector to induce cell-mediated immune responses to B. pseudomallei. Dendritic cells were produced by culturing murine bone marrow progenitor cells in medium containing granulocyte-macrophage colony-stimulating factor and tumor necrosis factor alpha. Purified dendritic cells were pulsed with heat-killed whole-cell B. pseudomallei and used to immunize syngeneic mice. Strong cellular immune responses were elicited by this immunization method, although antibody responses were low. Booster immunizations of either a second dose of dendritic cells or heat-killed B. pseudomallei were administered to increase the immune response. Immunized animals were challenged with fully virulent B. pseudomallei, and protection was demonstrated in those with strong humoral and cell-mediated immunity. These results indicate the importance of both cell-mediated and humoral immune mechanisms in protection against intracellular pathogens.

Epithelial and Stromal Cells of Bovine Endometrium Have Roles in Innate Immunity and Initiate Inflammatory Responses to Bacterial Lipopeptides In Vitro via Toll-Like Receptors TLR2, TLR1, and TLR6

Bacteria often infect the endometrium of cattle to cause endometritis, uterine disease, and infertility. Lipopeptides are commonly found among bacteria and are detected by the Toll-like receptor (TLR) cell surface receptor TLR2 on immune cells. Heterodimers of TLR2 with TLR1 or TLR6 activate MAPK and nuclear factor-κB intracellular signaling pathways to stimulate inflammatory responses. In the endometrium, epithelial and stromal cells are the first to encounter invading bacteria, so the present study explored whether endometrial cells can also mount inflammatory responses to bacterial lipopeptides via TLRs. The supernatants of pure populations of primary bovine endometrial epithelial and stromal cells accumulated the cytokine IL-6 and the chemokine IL-8 in response to triacylated or diacylated bacterial lipopeptides. The accumulation of IL-6 and IL-8 in response to triacylated lipopeptides was reduced by small interfering RNA targeting TLR2 or TLR1 but not TLR6, whereas cellular responses to diacylated lipopeptide were reduced by small interfering RNA targeting TLR2, TLR1, or TLR6. Both lipopeptides induced rapid phosphorylation of ERK1/2, p38, and nuclear factor-κB in endometrial cells, and inhibitors of ERK1/2 or p38 limited the accumulation of IL-6. The ovarian steroids estradiol and progesterone had little impact on inflammatory responses to lipopeptides. The endometrial epithelial and stromal cell responses to lipopeptides via TLR2, TLR1, and TLR6 provide a mechanism linking a wide range of bacterial infections to inflammation of the endometrium.

Differential Endometrial Cell Sensitivity to a Cholesterol-Dependent Cytolysin Links Trueperella pyogenes to Uterine Disease in Cattle

ABSTRACT Purulent disease of the uterus develops in 40% of dairy cows after parturition, when the epithelium of the endometrium is disrupted to expose the underlying stroma to bacteria. The severity of endometrial pathology is associated with isolation of Trueperella pyogenes. In the present study, T. pyogenes alone caused uterine disease when infused into the uterus of cattle where the endometrial epithelium was disrupted. The bacterium secretes a cholesterol-dependent cytolysin, pyolysin (PLO), and the plo gene was identical and the plo gene promoter was highly similar amongst 12 clinical isolates of T. pyogenes. Bacteria-free filtrates of the T. pyogenes cultures caused hemolysis and endometrial cytolysis, and PLO was the main cytolytic agent, because addition of anti-PLO antibody prevented cytolysis. Similarly, a plo-deletion T. pyogenes mutant did not cause hemolysis or endometrial cytolysis. Endometrial stromal cells were notably more sensitive to PLO-mediated cytolysis than epithelial or immune cells. Stromal cells also contained more cholesterol than epithelial cells, and reducing stromal cell cholesterol content using cyclodextrins protected against PLO. Although T. pyogenes or plo-deletion T. pyogenes stimulated accumulation of inflammatory mediators, such as IL-1beta, IL-6, and IL-8, from endometrium, PLO did not stimulate inflammatory responses by endometrial or hematopoietic cells, or in vitro organ cultures of endometrium. The marked sensitivity of stromal cells to PLO-mediated cytolysis provides an explanation for how T. pyogenes acts as an opportunistic pathogen to cause pathology of the endometrium once the protective epithelium is lost after parturition.

Administration of Antibody to the Lung Protects Mice against Pneumonic Plague

ABSTRACT Intratracheal delivery of aerosolized monoclonal antibodies with specificity for Yersinia pestis LcrV and F1 antigens protected mice in a model of pneumonic plague. These data support the utility of inhaled antibodies as a fast-acting postexposure treatment for plague.

Immunity and Inflammation in the Uterus

Microbes often infect the uterus and particularly the endometrium of animals. Infections are most commonly associated with natural service, pregnancy and the post-partum period, leading to inflammation with the elaboration of cytokines, chemokines and prostaglandins. Clinical diseases such as metritis, endometritis and abortion are important causes of infertility. The adaptive immune response to infection has been characterized previously, so the present review aims to highlight the emerging role for innate immunity in the endometrium. The detection of microbes and the innate immune response depends on the detection of pathogen-associated molecular patterns by pattern recognition receptors. The main families of pattern recognition receptors are Toll-like receptors (TLRs), nucleotide oligomerization domain-like receptors, retinoic acid-inducible gene I-like receptors and C-type lectin receptors. These receptors are most often expressed by hematopoietic cells, but the epithelial and stromal cells of the endometrium also possess functional receptors. For example, endometrial cells express TLR4 for recognition of the lipopolysaccharide endotoxin of Gram-negative bacteria, leading to secretion of IL-6, IL-8 and prostaglandin E(2) . It is likely that the epithelial and stromal cells provide a first line of defence in the endometrium to alert hematopoietic cells to the presence of microbes within the uterus.

Innate immunity and inflammation of the bovine female reproductive tract in health and disease

Mammalian reproductive physiology and the development of viviparity co-evolved with inflammation and immunity over millennia. Many inflammatory mediators contribute to paracrine and endocrine signalling, and the maintenance of tissue homeostasis in the female reproductive tract. However, inflammation is also a feature of microbial infections of the reproductive tract. Bacteria and viruses commonly cause endometritis, perturb ovarian follicle development and suppress the endocrine activity of the hypothalamus and pituitary in cattle. Innate immunity is an evolutionary ancient system that orchestrates host cell inflammatory responses aimed at eliminating pathogens and repairing damaged tissue. Pattern recognition receptors on host cells bind pathogen-associated molecular patterns and damage-associated molecular patterns, leading to the activation of intracellular MAPK and NFκB signalling pathways and the release of inflammatory mediators. Inflammatory mediators typically include the interleukin cytokines IL1β and IL6, chemokines such as IL8, interferons and prostaglandins. This review outlines the mechanisms of inflammation and innate immunity in the bovine female reproductive tract during health and disease condition.

Explants of Intact Endometrium to Model Bovine Innate Immunity and Inflammation Ex Vivo

Bacterial infections commonly cause bovine endometritis and infertility via innate immune pathways. However, mechanistic studies using isolated cells or chopped tissue may be compromised by the disruption of endometrial architecture and release of damage‐associated molecular patterns. So, this study aimed to establish an ex vivo model of intact bovine endometrium to study innate immunity and inflammation.

Protection against Heterologous Burkholderia pseudomallei Strains by Dendritic Cell Immunization

ABSTRACT Burkholderia pseudomallei, the causative agent of melioidosis, is a gram-negative bacterium which can cause either chronic infections or acute lethal sepsis in infected individuals. The disease is endemic in Southeast Asia and northern Australia, but little is known about the mechanisms of protective immunity to the bacterium. In this study, we have developed a procedure to utilize dendritic cells in combination with CpG oligodeoxynucleotides as a vaccine delivery vector to induce protective immune responses to various strains of B. pseudomallei. Our results show that strong cell-mediated immune responses were generated, while antibody responses, although low, were detectable. Upon virulent challenge with B. pseudomallei strain K96243, NCTC 4845, or 576, animals immunized with dendritic cells that were pulsed with heat-killed K96243 and matured in the presence of CpG 1826 showed significant levels of protection. These results show that a vaccine strategy that actively targets dendritic cells can evoke protective immune responses.

RNA interference of STAT6 rapidly attenuates ongoing interleukin‐13‐mediated events in lung epithelial cells

Signal transducer and activator of transcription 6 (STAT6) expression in lung epithelial cells plays a central role in asthma pathogenesis, with its activation driving the development of airway hyper‐reactivity and local inflammation. Therefore, inhibition of local STAT6 expression provides a rationale for therapeutic intervention in bronchial asthma. Given the absence of specific inhibitory drugs, we tested the ability of small interfering RNAs (siRNAs) to target STAT6 gene expression through the molecular process of RNA interference (RNAi). At pico‐molar concentrations, STAT6‐specific siRNAs potently inhibited STAT6 mRNA expression in lung epithelial cells (50% inhibitory concentration range = 134–861 pm) without inducing cellular interferon responses. Detectable STAT6 protein expression was rapidly abolished within 48 hr of treatment (t1/2 range = < 12–37 hr) and this was unaffected by pretreatment with STAT6‐activating cytokines. Furthermore, STAT6 suppression by RNAi produced downstream functional inhibitory effects in that interleukin (IL)‐13‐ or IL‐4‐driven eotaxin chemokine family [chemokine (C‐C motif) ligand 11 (CCL11), CCL24 and CCL26] mRNA expression was markedly inhibited. Induction of detectable CCL26 protein synthesis was completely ablated by pretreating cells with STAT6‐specific siRNA. The therapeutic potential of this approach is further demonstrated by novel findings that cells pre‐exposed to IL‐13 or IL‐4 and subsequently treated with STAT6‐targeting siRNA exhibited a rapid and significant attenuation of ongoing CCL26 protein expression, suggesting that chronic asthma‐associated lung inflammation will be responsive to this approach.

Ovarian steroids do not affect bovine endometrial cytokine or chemokine responses to Escherichia coli or LPS in vitro

The risk of bacterial infection of the endometrium causing uterine disease in cattle is increased in the progesterone-dominated luteal phase of the ovarian cycle, while oestrogens or oestrus are therapeutic or protective against disease. The first line of defence against bacteria, such as Escherichia coli that cause inflammation of the endometrium, is the innate immune system, which recognises bacterial lipopolysaccharide (LPS). This study tested the hypothesis that cyclic variation in ovarian hormone concentrations alters innate immune responses within the bovine endometrium. Ex vivo organ cultures of endometrium, and in vitro cultures of endometrial epithelial and stromal cells, and peripheral blood mononuclear cells (PBMCs), all mounted inflammatory responses to E. coli or LPS, with secretion of inflammatory mediators interleukin 1β (IL1β), IL6 and IL8, and increased expression of mRNA encoding IL1B, IL6, CXCL8 (IL8) and CCL5. However, these inflammatory responses, typical of innate immunity, were not affected by the stage of ovarian cycle in which the endometrium was collected for organ culture, or by exogenous oestradiol or progesterone. Although a dexamethasone-positive control reduced inflammation stimulated by E. coli or LPS, treatment with oestradiol or progesterone, or inhibitors of oestradiol or progesterone nuclear receptors, did not affect endometrial cell or PBMC secretion of IL1β, IL6 or IL8, or IL1B, IL6, CXCL8 and CCL5 gene expression. In conclusion, the stage of the oestrus cycle or ovarian steroids did not modulate the innate immune response in the bovine endometrium in vitro.