Comparison of cytokine gene expression in bovine tissues with acute or chronic inflammation

Cytokines are small molecular weight glycoproteins secreted by a variety of cells. These proteins have diverse activities and serve as signal mediators in immune reactions. Among the stimulators of cytokine induction, bacteria and their products are well-known. However, the regulatory mechanisms of cytokine expression associated with bacterial diseases are complicated and not completely elucidated;In the first study, mammary parenchymal tissues taken from Holstein cows at 6, 12, and 24 hours post-challenge with Escherichia coli were used as an acute bacterial infection model. In the study, we determined that IL-1alpha and TNF-alpha were the predominant cytokines expressed in mammary parenchymal tissues during the early phase of E. coli-induced mastitis. In contrast to these cytokines, expression of mRNA for IL-1beta, IL-6, and IFN-gamma in E. coli-infected mammary gland were low at the time points examined. The second study was designed to investigate the effects of CD18, an adhesion molecule, on cytokine gene expression in bovine pulmonary tissues after respiratory infection with Pasteurella haemolytica. This study suggested that CD18 may contribute to mRNA expression of IFN-gamma and TNF-alpha in the lung of P. haemolytica -infected cattle at 4 hours post-challenge. Based on histological evidence, this observation correlated with the infiltration of neutrophils into the lung tissue. For the third study, ileal tissues from cattle chronically infected with Mycobacterium avium subsp. paratuberculosis were examined for the chronic expression of cytokine-specific mRNA. Ileal tissues collected from cattle with paratuberculosis expressed significantly (P \u3c 0.05) more IL-1alpha, IL-1beta, IL-6 and IFN-gamma mRNA than paratuberculosis negative cattle. The expression level of TNF-alpha, however, was not different between M. avium subsp. paratuberculosis -infected and non-infected animals. Histologically, the ileal tissues from paratuberculosis positive cows harbored 3 times more macrophages than control cattle;In combination, these studies suggest that the patterns of cytokine expression and the predominant subset of leukocytes in cattle may vary depending on a variety of factors that include pathogens, tissues and time after infection. We believe that developing an understanding of the role of cytokines in disease can be a step towards controlling infectious disease

Synergistic effect of interleukin 1 alpha on nontypeable Haemophilus influenzae-induced up-regulation of human beta-defensin 2 in middle ear epithelial cells

BACKGROUND: We recently showed that beta-defensins have antimicrobial activity against nontypeable Haemophilus influenzae (NTHi) and that interleukin 1 alpha (IL-1 alpha) up-regulates the transcription of beta-defensin 2 (DEFB4 according to new nomenclature of the Human Genome Organization) in human middle ear epithelial cells via a Src-dependent Raf-MEK1/2-ERK signaling pathway. Based on these observations, we investigated if human middle ear epithelial cells could release IL-1 alpha upon exposure to a lysate of NTHi and if this cytokine could have a synergistic effect on beta-defensin 2 up-regulation by the bacterial components. METHODS: The studies described herein were carried out using epithelial cell lines as well as a murine model of acute otitis media (OM). Human cytokine macroarray analysis was performed to detect the released cytokines in response to NTHi exposure. Real time quantitative PCR was done to compare the induction of IL-1 alpha or beta-defensin 2 mRNAs and to identify the signaling pathways involved. Direct activation of the beta-defensin 2 promoter was monitored using a beta-defensin 2 promoter-Luciferase construct. An IL-1 alpha blocking antibody was used to demonstrate the direct involvement of this cytokine on DEFB4 induction. RESULTS: Middle ear epithelial cells released IL-1 alpha when stimulated by NTHi components and this cytokine acted in an autocrine/paracrine synergistic manner with NTHi to up-regulate beta-defensin 2. This synergistic effect of IL-1 alpha on NTHi-induced beta-defensin 2 up-regulation appeared to be mediated by the p38 MAP kinase pathway. CONCLUSION: We demonstrate that IL-1 alpha is secreted by middle ear epithelial cells upon exposure to NTHi components and that it can synergistically act with certain of these molecules to up-regulate beta-defensin 2 via the p38 MAP kinase pathway

Lysozyme M deficiency leads to an increased susceptibility to Streptococcus pneumoniae-induced otitis media

<p>Abstract</p> <p>Background</p> <p>Lysozyme is an antimicrobial innate immune molecule degrading peptidoglycan of the bacterial cell wall. Lysozyme shows the ubiquitous expression in wide varieties of species and tissues including the tubotympanum of mammals. We aim to investigate the effects of lysozyme depletion on pneumococcal clearance from the middle ear cavity.</p> <p>Methods</p> <p>Immunohistochemistry was performed to localize lysozyme in the Eustachian tube. Lysozyme expression was compared between the wild type and the lysozyme M^-/-mice using real time quantitative RT-PCR and western blotting. Muramidase activity and bactericidal activity of lysozyme was measured using a lysoplate radial diffusion assay and a liquid broth assay, respectively. To determine if depletion of lysozyme M increases a susceptibility to pneumococal otitis media, 50 CFU of <it>S. pneumoniae </it>6B were transtympanically inoculated to the middle ear and viable bacteria were counted at day 3 and 7 with clinical grading of middle ear inflammation.</p> <p>Results</p> <p>Immunolabeling revealed that localization of lysozyme M and lysozyme P is specific to some/particular cell types of the Eustachian tube. Lysozyme P of lysozyme M^-/-mice was mainly expressed in the submucosal gland but not in the tubal epithelium. Although lysozyme M^-/-mice showed compensatory up-regulation of lysozyme P, lysozyme M depletion resulted in a decrease in both muramidase and antimicrobial activities. Deficiency in lysozyme M led to an increased susceptibility to middle ear infection with <it>S. pneumoniae </it>6B and resulted in severe middle ear inflammation, compared to wild type mice.</p> <p>Conclusion</p> <p>The results suggest that lysozyme M plays an important role in protecting the middle ear from invading pathogens, particularly in the early phase. We suggest a possibility of the exogenous lysozyme as an adjuvant therapeutic agent for otitis media, but further studies are necessary.</p

Induction of beta defensin 2 by NTHi requires TLR2 mediated MyD88 and IRAK-TRAF6-p38MAPK signaling pathway in human middle ear epithelial cells

<p>Abstract</p> <p>Background</p> <p>All mucosal epithelia, including those of the tubotympanium, are secreting a variety of antimicrobial innate immune molecules (AIIMs). In our previous study, we showed the bactericidal/bacteriostatic functions of AIIMs against various otitis media pathogens. Among the AIIMs, human β-defensin 2 is the most potent molecule and is inducible by exposure to inflammatory stimuli such as bacterial components or proinflammatory cytokines. Even though the β-defensin 2 is an important AIIM, the induction mechanism of this molecule has not been clearly established. We believe that this report is the first attempt to elucidate NTHi induced β-defensin expression in airway mucosa, which includes the middle ear.</p> <p>Methods</p> <p>Monoclonal antibody blocking method was employed in monitoring the TLR-dependent NTHi response. Two gene knock down methods – dominant negative (DN) plasmid and small interfering RNA (siRNA) – were employed to detect and confirm the involvement of several key genes in the signaling cascade resulting from the NTHi stimulated β-defensin 2 expression in human middle ear epithelial cell (HMEEC-1). The student's <it>t</it>-test was used for the statistical analysis of the data.</p> <p>Results</p> <p>The experimental results showed that the major NTHi-specific receptor in HMEEC-1 is the Toll-like receptor 2 (TLR2). Furthermore, recognition of NTHi component(s)/ligand(s) by TLR2, activated the Toll/IL-1 receptor (TIR)-MyD88-IRAK1-TRAF6-MKK3/6-p38 MAPK signal transduction pathway, ultimately leading to the induction of β-defensin 2.</p> <p>Conclusion</p> <p>This study found that the induction of β-defensin 2 is highest in whole cell lysate (WCL) preparations of NTHi, suggesting that the ligand(s) responsible for this up-regulation may be soluble macromolecule(s). We also found that this induction takes place through the TLR2 dependent MyD88-IRAK1-TRAF6-p38 MAPK pathway, with the primary response occurring within the first hour of stimulation. In combination with our previous studies showing that IL-1α-induced β-defensin 2 expression takes place through a MyD88-independent Raf-MEK1/2-ERK MAPK pathway, we found that both signaling cascades act synergistically to up-regulate β-defensin 2 levels. We propose that this confers an essential evolutionary advantage to the cells in coping with infections and may serve to amplify the innate immune response through paracrine signaling.</p

Comparison of cytokine gene expression in bovine tissues with acute or chronic inflammation

Cytokines are small molecular weight glycoproteins secreted by a variety of cells. These proteins have diverse activities and serve as signal mediators in immune reactions. Among the stimulators of cytokine induction, bacteria and their products are well-known. However, the regulatory mechanisms of cytokine expression associated with bacterial diseases are complicated and not completely elucidated;In the first study, mammary parenchymal tissues taken from Holstein cows at 6, 12, and 24 hours post-challenge with Escherichia coli were used as an acute bacterial infection model. In the study, we determined that IL-1alpha and TNF-alpha were the predominant cytokines expressed in mammary parenchymal tissues during the early phase of E. coli-induced mastitis. In contrast to these cytokines, expression of mRNA for IL-1beta, IL-6, and IFN-gamma in E. coli-infected mammary gland were low at the time points examined. The second study was designed to investigate the effects of CD18, an adhesion molecule, on cytokine gene expression in bovine pulmonary tissues after respiratory infection with Pasteurella haemolytica. This study suggested that CD18 may contribute to mRNA expression of IFN-gamma and TNF-alpha in the lung of P. haemolytica -infected cattle at 4 hours post-challenge. Based on histological evidence, this observation correlated with the infiltration of neutrophils into the lung tissue. For the third study, ileal tissues from cattle chronically infected with Mycobacterium avium subsp. paratuberculosis were examined for the chronic expression of cytokine-specific mRNA. Ileal tissues collected from cattle with paratuberculosis expressed significantly (P < 0.05) more IL-1alpha, IL-1beta, IL-6 and IFN-gamma mRNA than paratuberculosis negative cattle. The expression level of TNF-alpha, however, was not different between M. avium subsp. paratuberculosis -infected and non-infected animals. Histologically, the ileal tissues from paratuberculosis positive cows harbored 3 times more macrophages than control cattle;In combination, these studies suggest that the patterns of cytokine expression and the predominant subset of leukocytes in cattle may vary depending on a variety of factors that include pathogens, tissues and time after infection. We believe that developing an understanding of the role of cytokines in disease can be a step towards controlling infectious disease.</p