Alling pathways that are up-regulated by innate interferons; and different cytokines
Alling pathways that happen to be up-regulated by innate interferons; and different cytokines able to promote an inflammatory response or amplify a Th2 response. In preliminary function employing mouse MLE-12 cells, an immortalised line derived from distal AEC, we showed that expression of quite a few chemokines and proinflammatory cytokines was considerably up-regulated in cells that had been pre-treated with Th2 cytokines and then stimulated with poly I:C, whilst expression of big anti-viral response genes was either unchanged or was also substantially increased. This was unexpected and we consequently undertook additional function making use of low-passage human bronchial epithelial cells. The major response of AEC to viral infection will be the production of interferons, largely interferon-1 and the various type III IL-8 Biological Activity interferons (IFN-1/2/3) [30]. Becausethe magnitude of induction of interferons in AEC is relatively low in comparison with blood leucocytes [30], detection of secreted interferon proteins is tricky, so we assessed expression of those genes by quantitative real-time PCR. We discovered that in human AEC which had been pretreated with Th2 cytokines, expression of interferons was unchanged, when interferons exhibited modest but statistically considerable up-regulation. The innate interferons in turn stimulate expression of numerous other genes [29,31], including not only antiviral response genes but in addition chemokines as well as other proinflammatory cytokines, that are secreted at levels that readily permit detection by enzyme immunoassay. Hence we were capable to assess the latter in terms of both mRNA expression and protein concentrations in supernatants of AEC in culture. We noted enhanced expression and secretion of various chemokines, such as the neutrophil chemoattractant CXCL8, the T cell chemoattractants CXCL9, CXCL10 and CXCL11, also because the T cell/eosinophil chemoattractant CCL5. These results have been largely equivalent to the data for MLE-12 cells. Despite the fact that we observed no change in expression on the IL6 gene, which is constant with previously reported information [7], there wasHerbert et al. Translational Respiratory Medicine 2014, 2:11 transrespmed.com/content/2/1/Page 7 ofFigure four (See legend on next page.)Herbert et al. Translational Respiratory Medicine 2014, 2:11 transrespmed.com/content/2/1/Page 8 of(See figure on prior page.) Figure 4 Before-and-after plots displaying effects of prior exposure to Th2 cytokines on the expression of mRNA for anti-viral response genes by human AEC at Bax supplier baseline (left) or following stimulation with poly I:C (appropriate). Information are mean values for individual individuals, displaying expression relative to the housekeeping gene HPRT. p values for variations among cells cultured in media with or with no IL-4 and IL-13 were assessed by ratio paired t-test.some improve in levels of IL-6 protein, possibly indicating secretion of pre-formed cytokine. Interestingly, we observed decreased expression of mRNA for the Th2-promoting cytokine IL-33, once again analogous for the obtaining in MLE12 cells, whilst expression of TSLP was increased. Some of the increases in cytokine protein concentrations were not statistically significant, which might have been because culture supernatants were collected at four hours after stimulation, a relatively early time point for assessment of secretion of cytokine proteins. Ideally, we would have wished to carry out parallel experiments in which cells were collected at 4 hours after stimulation for assessment of mRNA and at 164 hours for assessment.