The interplay between immune maturation, age, chronic viral infection and environment

Background The worldwide increase in life expectancy has been associated with an increase in age-related morbidities. The underlying mechanisms resulting in immunosenescence are only incompletely understood. Chronic viral infections, in particular infection with human cytomegalovirus (HCMV), have been suggested as a main driver in immunosenescence. Here, we propose that rhesus macaques could serve as a relevant model to define the impact of chronic viral infections on host immunity in the aging host. We evaluated whether chronic rhesus CMV (RhCMV) infection, similar to HCMV infection in humans, would modulate normal immunological changes in the aging individual by taking advantage of the unique resource of rhesus macaques that were bred and raised to be Specific Pathogen Free (SPF-2) for distinct viruses. Results Our results demonstrate that normal age-related immunological changes in frequencies, activation, maturation, and function of peripheral blood cell lymphocytes in humans occur in a similar manner over the lifespan of rhesus macaques. The comparative analysis of age-matched SPF-2 and non-SPF macaques that were housed under identical conditions revealed distinct differences in certain immune parameters suggesting that chronic pathogen exposure modulated host immune responses. All non-SPF macaques were infected with RhCMV, suggesting that chronic RhCMV infection was a major contributor to altered immune function in non-SPF macaques, although a causative relationship was not established and outside the scope of these studies. Further, we showed that immunological differences between SPF-2 and non-SPF macaques were already apparent in adolescent macaques, potentially predisposing RhCMV-infected animals to age-related pathologies. Conclusions Our data validate rhesus macaques as a relevant animal model to study how chronic viral infections modulate host immunity and impact immunosenescence. Comparative studies in SPF-2 and non-SPF macaques could identify important mechanisms associated with inflammaging and thereby lead to new therapies promoting healthy aging in humans

The Helicobacter pylori type IV secretion system promotes IL-8 synthesis in a model of pediatric airway epithelium via p38 MAP kinase.

Epidemiologic studies have reported an inverse relationship between childhood Helicobacter pylori infection and development of allergic asthma. Because lung epithelium plays an important role in allergic asthma pathogenesis, we hypothesized that H. pylori may directly influence airway epithelial cell innate immune function, particularly in early childhood. To test our hypothesis, we established an in vitro H. pylori infection model using primary tracheobronchial epithelial cell cultures derived from infant, juvenile and adult rhesus monkeys. Airway epithelial cell cultures were infected with wild-type or cag pathogenicity island mutant H. pylori strains, followed by evaluation of IL-8 and IL-6 protein synthesis. We found that H. pylori primarily increased IL-8 synthesis in a MOI and age-dependent fashion, with a greater than 4-fold induction in infant versus adult cultures. H. pylori-induced IL-8 synthesis in infant and juvenile cultures was significantly reduced by cag pathogenicity island mutants, indicating a requirement for the type IV secretion system. Although peptidoglycan recognition of nucleotide binding oligomerization domain-containing protein 1 (NOD1) and NF-kappaB have been implicated as key cytokine signaling molecules for H. pylori infection in gastric epithelium, NOD1 (ML130) or NF-kappaB (JSH-23) inhibitors minimally affected IL-8 synthesis in airway epithelial cell cultures following H. pylori infection. In contrast, inhibition of the p38 MAP kinase pathway (SB203580) resulted in almost complete suppression of H. pylori-induced IL-8 synthesis. Collectively, these results indicate that H. pylori can preferentially elicit IL-8 synthesis in a model of pediatric airway epithelium using the type IV secretion system via p38 MAP kinase

<i>Helicobacter pylori</i> strains used to infect airway epithelial cell cultures.

<p><i>Helicobacter pylori</i> strains used to infect airway epithelial cell cultures.</p

Activation of NOD1 promotes IL-8 synthesis in both infant and adult airway epithelial cell cultures.

<p><b>(A)</b> NOD1 real time PCR analysis for both infant (n = 4) and adult (n = 9) primary cultures. *p<0.05 as determined by unpaired t-test. <b>(B)</b> Fold change of IL-8 protein in infant (n = 5) or adult (n = 3) primary cultures following treatment with iE-DAP. *p<0.05 by two-way ANOVA followed by Bonferroni’s multiple comparison test. <b>(C</b>) IL-8 protein synthesis in infant (n = 8) or adult (n = 9) primary cultures following treatment with iE-DAP only or iE-DAP in the presence of ML130. *p<0.05 by two-way ANOVA followed by Bonferroni’s multiple comparison test, infant iE-DAP vs infant ML130, infant iE-DAP vs adult ML130. Error bars represent SEM.</p

<i>H</i>. <i>pylori</i> viability is essential for optimal IL-8 synthesis in primary airway epithelial cell cultures.

<p>Supernatants from juvenile primary airway epithelial cell cultures were collected following infection with <b>(A)</b> <i>H</i>. <i>pylori</i> PMSS1 (MOI = 2.5–15) for 24 hr or treatment with <b>(B)</b> 10⁸ cells/ml heat-killed <i>H</i>. <i>pylori</i> for 24 hr. Statistical significance was determined using unpaired t-test *p<0.05. Error bars represent SEM.</p

Age-dependent IL-8 synthesis in primary airway epithelial cell cultures following <i>H</i>. <i>pylori</i> infection.

<p>Supernatants from infant, juvenile or adult monkey primary airway epithelial cell cultures were collected following infection with <i>H</i>. <i>pylori</i> strain PMSS1. <b>(A and D)</b> Effect of 24 hr incubation with <i>H</i>. <i>pylori</i> PMSS1 on IL-8 and IL6 protein concentration in infant cultures (n = 10). <b>(B and E)</b> Effect of 24 hr incubation with <i>H</i>. <i>pylori</i> PMSS1 on IL-8 and IL-6 protein concentration in juvenile cultures (n = 3). <b>(C and F)</b> Effect of 24 hr incubation with <i>H</i>. <i>pylori</i> PMSS1 on IL-8 and IL-6 protein concentration in adult cultures (n = 4). Statistical significance was determined using one-way ANOVA followed by Bonferroni’s multiple comparison test. Error bars represent SEM. ** p <0.01, **** p< 0.0001</p

Effect of <i>cag</i>PAI mutation on H. pylori-induced proinflammatory cytokines in primary airway epithelial cell cultures.

<p>Primary airway epithelial cell cultures were infected with PMSS1 WT, <i>cagY</i> knockout or <i>cagA</i> knockout strains for 24 hr, followed by collection of supernatant for ELISA. <b>(A and D)</b> Effect of WT or mutant stains on IL-8 and IL-6 protein concentration in cultures of infant epithelial cells (n = 3). <b>(B and E)</b> Effect of WT or mutant stains on IL-8 and IL-6 protein concentration in cultures of juvenile airway epithelial cells (n = 3). <b>(C and F)</b> Effect of WT or mutant strains on IL-8 and IL-6 protein concentration in cultures of adult airway epithelial cells (n = 3). Statistical significance was determined by two-way ANOVA followed by Bonferroni’s multiple comparison test. Error bars represent SEM. **p < 0.01 (G) Western blot analysis for PMSS1 CagA phosphorylation following infection of the 16-HBE cell line. 16-HBE cell cultures were assessed at 0, 1, 6, 12 and 18 hrs post-infection with PMSS1 (MOI = 10). Arrows point to detected anti-phosphotyrosine labelled bands at 150 kDa for 6 and 12 hrs.</p

<i>H</i>. <i>pylor</i>i induced IL-8 synthesis is inhibited by the p38 MAP kinase inhibitor SB203580.

<p>Juvenile primary cultures were infected with <i>H</i>. <i>pylori</i> (PMSS1) in the presence of inhibitors for NOD1, NF-kappaB and p38 kinase. Supernatant were harvested after 24hr. <b>(A)</b> <i>H</i>. <i>pylori</i>-induced IL-8 synthesis following treatment with ML130 (n = 6). *p<0.05 (<i>H</i>. <i>pylori</i> vs media). <b>(B)</b> <i>H</i>. <i>pylori</i>-induced IL-8 synthesis following treatment with JSH-23 (n = 4). **p<0.01 (<i>H</i>. <i>pylori</i> vs media). <b>(C)</b> <i>H</i>. <i>pylori</i>-induced IL-8 synthesis following treatment with SB203580 (n = 6). *p<0.05 (<i>H</i>. <i>pylori</i> vs media), ***p<0.001 (<i>H</i>. <i>pylori</i> vs. <i>H</i>. <i>pylori</i> + SB203584). Statistical significance was determined by repeated measures one-way ANOVA.</p

The <i>Helicobacter pylori</i> type IV secretion system promotes IL-8 synthesis in a model of pediatric airway epithelium via p38 MAP kinase

<div><p>Epidemiologic studies have reported an inverse relationship between childhood <i>Helicobacter pylori</i> infection and development of allergic asthma. Because lung epithelium plays an important role in allergic asthma pathogenesis, we hypothesized that <i>H</i>. <i>pylori</i> may directly influence airway epithelial cell innate immune function, particularly in early childhood. To test our hypothesis, we established an <i>in vitro H</i>. <i>pylori</i> infection model using primary tracheobronchial epithelial cell cultures derived from infant, juvenile and adult rhesus monkeys. Airway epithelial cell cultures were infected with wild-type or cag pathogenicity island mutant <i>H</i>. <i>pylori</i> strains, followed by evaluation of IL-8 and IL-6 protein synthesis. We found that <i>H</i>. <i>pylori</i> primarily increased IL-8 synthesis in a MOI and age-dependent fashion, with a greater than 4-fold induction in infant versus adult cultures. <i>H</i>. <i>pylori</i>-induced IL-8 synthesis in infant and juvenile cultures was significantly reduced by cag pathogenicity island mutants, indicating a requirement for the type IV secretion system. Although peptidoglycan recognition of nucleotide binding oligomerization domain-containing protein 1 (NOD1) and NF-kappaB have been implicated as key cytokine signaling molecules for <i>H</i>. <i>pylori</i> infection in gastric epithelium, NOD1 (ML130) or NF-kappaB (JSH-23) inhibitors minimally affected IL-8 synthesis in airway epithelial cell cultures following <i>H</i>. <i>pylori</i> infection. In contrast, inhibition of the p38 MAP kinase pathway (SB203580) resulted in almost complete suppression of <i>H</i>. <i>pylori</i>-induced IL-8 synthesis. Collectively, these results indicate that <i>H</i>. <i>pylori</i> can preferentially elicit IL-8 synthesis in a model of pediatric airway epithelium using the type IV secretion system via p38 MAP kinase.</p></div