Lisa Rothwell

Differential cytokine expression in avian cells in response to invasion by Salmonella typhimurium, Salmonella enteritidis and Salmonella gallinarum

Salmonella enterica is a facultative intracellular pathogen that is capable of causing disease in a range of hosts. Although human salmonellosis is frequently associated with consumption of contaminated poultry and eggs, and the serotypes Salmonella gallinarum and Salmonella pullorum are important world-wide pathogens of poultry, little is understood of the mechanisms of pathogenesis of Salmonella in the chicken. Type III secretion systems play a key role in host cell invasiveness and trigger the production of pro-inflammatory cytokines during invasion of mammalian hosts. This results in a polymorphonuclear cell influx that contributes to the resulting enteritis. In this study, a chicken primary cell culture model was used to investigate the cytokine responses to entry by the broad host range serotypes S. enteritidis and S. typhimurium, and the host specific serotype S. gallinarum, which rarely causes disease outside its main host, the chicken. The cytokines interleukin (IL)-1ss, IL-2, IL-6 and interferon (IFN)-gamma were measured by quantitative RT-PCR, and production of IL-6 and IFN-gamma was also determined through bioassays. All serotypes were invasive and had little effect on the production of IFN-gamma compared with non-infected cells; S. enteritidis invasion caused a slight down-regulation of IL-2 production. For IL-1ss production, infection with S. typhimurium had little effect, whilst infection with S. gallinarum or S. enteritidis caused a reduction in IL-1ss mRNA levels. Invasion of S. typhimurium and S. enteritidis caused an eight- to tenfold increase in production of the pro-inflammatory cytokine IL-6, whilst invasion by S. gallinarum caused no increase. These findings correlate with the pathogenesis of Salmonella in poultry. S. typhimurium and S. enteritidis invasion produces a strong inflammatory response, that may limit the spread of Salmonella largely to the gut, whilst S. gallinarum does not induce an inflammatory response and may not be limited by the immune system, leading to the severe systemic disease fowl typhoid.

Cloning and Characterization of Chicken IL-10 and Its Role in the Immune Response to Eimeria maxima

We isolated the full-length chicken IL-10 (chIL-10) cDNA from an expressed sequence tag library derived from RNA from cecal tonsils of Eimeria tenella-infected chickens. It encodes a 178-aa polypeptide, with a predicted 162-aa mature peptide. Chicken IL-10 has 45 and 42% aa identity with human and murine IL-10, respectively. The structures of the chIL-10 gene and its promoter were determined by direct sequencing of a bacterial artificial chromosome containing chIL-10. The chIL-10 gene structure is similar to (five exons, four introns), but more compact than, that of its mammalian orthologues. The promoter is more similar to that of Fugu IL-10 than human IL-10. Chicken IL-10 mRNA expression was identified mainly in the bursa of Fabricius and cecal tonsils, with low levels of expression also seen in thymus, liver, and lung. Expression was also detected in PHA-activated thymocytes and LPS-stimulated monocyte-derived macrophages, with high expression in an LPS-stimulated macrophage cell line. Recombinant chIL-10 was produced and bioactivity demonstrated through IL-10-induced inhibition of IFN-γ synthesis by mitogen-activated lymphocytes. We measured the expression of mRNA for chIL-10 and other signature cytokines in gut and spleen of resistant (line C.B12) and susceptible (line 15I) chickens during the course of an E. maxima infection. Susceptible chickens showed higher levels of chIL-10 mRNA expression in the spleen, both constitutively and after infection, and in the small intestine after infection than did resistant chickens. These data indicate a potential role for chIL-10 in changing the Th bias during infection with an intracellular protozoan, thereby contributing to susceptibility of line 15I chickens.

Differential cytokine mRNA expression in heterophils isolated from Salmonella-resistant and -susceptible chickens

We recently showed that increased in vitro heterophil functional efficiency translates to increased in vivo resistance to a systemic Salmonella enteritidis (SE) infection utilizing a parental pair of broiler chickens (lines A and B) and the F1 reciprocal crosses (C and D). Heterophils produce cytokines and modulate acute protection against Salmonella in young poultry. Therefore, we hypothesize that heterophils from SE‐resistant chickens (A and D) have the ability to produce an up‐regulated pro‐inflammatory cytokine response compared to that of heterophils from SE‐susceptible chickens (B and C). In this study, heterophils were isolated from day‐old chickens and treated with either RPMI‐1640 (as the control), or phagocytic agonists (SE, or SE opsonized with either normal chicken serum or immune serum against SE) and cytokine mRNA expression assessed using real‐time quantitative reverse transcription–polymerase chain reaction. Heterophils from SE‐resistant chickens (A and D) had significantly higher levels of pro‐inflammatory cytokine (interleukin (IL)‐6, IL‐8, and IL‐18) mRNA expression upon treatment with all agonists compared to heterophils from SE‐susceptible lines (B and C). Further, heterophils from SE‐resistant chickens had significantly decreased mRNA expression levels of transforming growth factor‐β4, an anti‐inflammatory cytokine, when compared to heterophils from SE‐susceptible chickens. These data indicate cytokine gene expression in heterophils may be a useful parameter in determining resistance to Salmonella, as indicated by our previous in vivo SE studies. Therefore, heterophil functional efficiency and cytokine production may be useful biomarkers for poultry breeders to consider when developing new immunocompetent lines of birds.

Differential Regulation of Cytokine Gene Expression by Avian Heterophils During Receptor-Mediated Phagocytosis of Opsonized and Nonopsonized Salmonella enteritidis

Internalization of pathogens by phagocytic cells triggers the innate immune response, which in turn regulates the acquired response. Phagocytes express a variety of receptors that are involved in recognition of pathogens, including (1) pattern recognition receptors (PRR), which recognize conserved motifs, (2) complement receptors (CR), which recognize complement-opsonized pathogens, and (3) Fc receptors (FcR), which recognize antibody-opsonized pathogens. Recognition of microbes is accompanied by the induction of multiple cell processes, including the production of proinflammatory and anti-inflammatory cytokines and chemokines. The objective of the present experiments was to use probes to known avian proinflammatory and anti-inflammatory cytokines and TaqMan technology to ascertain levels of cytokine gene expression in avian heterophils following receptor-mediated phagocytosis of either nonopsonized Salmonella enteritidis (SE), serum-opsonized SE, or IgG-opsonized SE. Expression of interleukin-6 (IL-6) and IL-8, considered in mammals as a proinflammatory chemokine, were upregulated following exposure to the nonopsonized or the opsonized SE. However, mRNA expression for IL-18 and interferon-gamma (IFN-gamma) was downregulated, and the expression of mRNA for the anti-inflammatory cytokine transforming growth factor-beta4 (TGF-beta 4) was upregulated. Interestingly, IL-1beta mRNA expression was significantly upregulated in heterophils that phagocytized either the nonopsonized SE via PRRs or IgG-opsonized SE via FcRs, whereas serum-opsonized SE phagocytized by CRs induced a downregulation of IL-1beta mRNA. These results suggest that signaling interactions initiated by receptor recognition of the microbe surface differentially regulate the induction of inflammatory cytokines in avian heterophils.

Campylobacter jejuni-Induced Cytokine Responses in Avian Cells

ABSTRACT Campylobacter jejuni is a major cause of human inflammatory enteritis. During the course of human disease numerous proinflammatory cytokines are produced. Little is known, however, about the cytokine responses produced during the interaction of this bacterium with the avian host. Campylobacter has been considered a commensal of the avian host. Any differences in innate responses to this pathogen between the human and avian hosts should lead to a greater understanding of the disease process in humans. We have demonstrated expression of proinflammatory cytokines and chemokines in response to Campylobacter infection in avian primary chick kidney cells and the avian macrophage cell line HD11. The data indicate that Campylobacter can stimulate the avian host in a proinflammatory manner. The data strongly suggest that the lack of pathology in vivo is not due to an inability of Campylobacter to stimulate a proinflammatory response from avian cells.

Generation and characterization of chicken bone marrow‐derived dendritic cells

Dendritic cells (DCs) are bone marrow‐derived professional antigen‐presenting cells. The in vitro generation of DCs from either bone marrow or blood is routine in mammals. Their distinct morphology and phenotype and their unique ability to stimulate naïve T cells are used to define DCs. In this study, chicken bone marrow cells were cultured in the presence of recombinant chicken granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and recombinant chicken interleukin‐4 (IL‐4) for 7 days. The cultured population showed the typical morphology of DCs, with the surface phenotype of major histocompatibility complex (MHC) class II+ (high), CD11c+ (high), CD40+ (moderate), CD1·1+ (moderate), CD86+ (low), CD83− and DEC‐205−. Upon maturation with lipopolysaccharide (LPS) or CD40L, surface expression of CD40, CD1·1, CD86, CD83 and DEC‐205 was greatly increased. Endocytosis and phagocytosis were assessed by fluorescein isothiocyanate (FITC)‐dextran uptake and fluorescent bead uptake, respectively, and both decreased after stimulation. Non‐stimulated chicken bone marrow‐derived DCs (chBM‐DCs) stimulated both allogeneic and syngeneic peripheral blood lymphocytes (PBLs) to proliferate in a mixed lymphocyte reaction (MLR). LPS‐ or CD40L‐stimulated chBM‐DCs were more effective T‐cell stimulators in MLR than non‐stimulated chBM‐DCs. Cultured chBM‐DCs could be matured to a T helper type 1 (Th1)‐promoting phenotype by LPS or CD40L stimulation, as determined by mRNA expression levels of Th1 and Th2 cytokines. We have therefore cultured functional chBM‐DCs in a non‐mammalian species for the first time.

The turkey, compared to the chicken, fails to mount an effective early immune response to Histomonas meleagridis in the gut.

Histomonosis is a disease of poultry caused by Histomonas meleagridis. Chickens usually recover while the mortality rate in turkeys is high. The immunological response of both species towards H. meleagridis was investigated. Parasites migrated in greater numbers to the turkey liver compared with that of chicken. Chicken mounted an effective caecal innate response, with increased expression of IL‐1β, CXCLi2 and IL‐6 mRNA, resulting in control of parasite numbers. The turkey failed to mount such an effective innate response in the caecal tonsil, allowing greater numbers to migrate to the liver, where a sustained, uncontrolled immune response was mounted, evidenced by the upregulation of mRNA for IL‐1β, CXCLi2, IFN‐γ, IL‐13, IL‐4 and IL‐10. Expression levels of mRNA of the chicken and turkey β‐defensin AvBD2 suggest that this response was not limited to the cytokines. There was an influx of CD4+, CD8α+, CD28+ and CD44+ cells into the livers of both species, coinciding with parasite movement. These influxes were more pronounced in the turkey, correlating with a decrease in numbers of the same cells in the spleen, which was not observed in the chicken. Antibody levels in the chicken increased more than those in the turkey, supporting evidence of an adaptive response.

Avian coccidiosis: changes in intestinal lymphocyte populations associated with the development of immunity to Eimeria maxima

The effect of infection and subsequent challenge with Eimeria maxima on the populations of lymphocytes in the small intestine of Light Sussex chickens was assessed by immunohistochemistry. T cells were characterized for CD3, CD4, CD8, TCR1 (γδ heterodimer) or TCR2 (αβ1 heterodimer) markers, and B cells for the expression of IgM, IgA andlgG. After a primary inoculum there were, in both the epithelium and the lamina propria, two distinct increases in the numbers of T lymphocytes. The first peaked on days 3–5 and the second, greater influx, on day 11 after infection. CD4+ and CD8+ cells were represented in both peaks but, whereas CD4+ cells were found almost exclusively in the lamina propria, CD8+ cells were present in both sites. The area staining positive for CD8+ cells was somewhat greater than the value obtained for CD4+ cells. In the epithelium there was an early, small increase in TCR1+‐staining, followed by a larger rise to the second peak, at which time there was also an increase in the lamina propria. Staining for TCR2+ cells followed the same pattern with a reversed distribution between epithelium and lamina propria. Changes after challenge were minimal and confined to the epithelium. The most notable changes in the expression of immunoglobulins were, in the lamina propria, a biphasic increase in the amount ofIgM+‐staining in the course of primary infection (corresponding approximately to that of the T cells), and in IgA + cells shortly after challenge.

Campylobacter colonization of the chicken induces a proinflammatory response in mucosal tissues

Campylobacter jejuni is a major cause of human inflammatory enteritis, but colonizes the gastrointestinal tract of poultry to a high level in a commensal manner. In vitro, C. jejuni induces the production of cytokines from both human and avian-model epithelial cell and macrophage infections. This suggests that, in vivo, Campylobacter could induce proinflammatory signals in both hosts. We investigated whether a proinflammatory cytokine response can be measured in both day-of-hatch and 2-week-old Light Sussex chickens during infection with C. jejuni. A significant induction of proinflammatory chemokine transcript was observed in birds of both ages, compared with levels in mock-infected controls. This correlated with an influx of heterophils but was not associated with any pathology. These results suggest that in poultry there may be a controlled inflammatory process during colonization.

Macrophages Isolated from Chickens Genetically Resistant or Susceptible to Systemic Salmonellosis Show Magnitudinal and Temporal Differential Expression of Cytokines and Chemokines following Salmonella enterica Challenge

ABSTRACT Macrophages from inbred chickens that are resistant to salmonellosis show greater and more rapid expression of proinflammatory chemokines and cytokines, including the key Th1-inducing cytokine interleukin-18, upon Salmonella challenge than those from susceptible birds. This suggests the possibility that salmonellosis resistant-line macrophages signal more effectively and rapidly and are more able to induce protective Th1 adaptive responses.