In addition, Selleck PND-1186 consistent with the results shown in Figure 3, it showed that procedure-dependent
effects occurred before 48 h and were more pronounced in the DBA/2J strain. Figure 4 Overall mean fold changes in mRNA expression throughout the time course. A. Mean expression changes in mock-treated and infected DBA/2J and C57BL/6J mice across all 10 target Sotrastaurin chemical structure host mRNAs in the 5-day time course of IAV infection. The analysis is based on the same data set as used for Figures 2 and 3. Mean fold change values and 95% confidence intervals (vertical lines) were calculated with the Dunnett’s Modified Tukey-Kramer test, using the dCt values (qRT-PCR) of all 10 host-encoded mRNAs as input. B. Schematic representation of the results shown in panel A. As reflected by the thickness of the lines, overall changes are more pronounced in the DBA/2J strain. Procedure-dependent effects are evident between 6 and 24 h in both strains, but infection-related changes begin to evolve and Napabucasin peak earlier in the DBA/2J strain. Discussion This analysis of sequential changes in pulmonary expression
of several mRNAs after real or simulated IAV infection revealed effects that can be ascribed to anesthesia and/or the intranasal inoculation procedure. The results clearly demonstrate that the appropriate control group treated with a simulated anesthesia/infection should always be included in studies of IAV infection in mice that cover approximately the first 24 h
post infection. What might be the underlying pathophysiological mechanisms? Anesthesia is known to influence cytokine expression in humans, but actually appears to have an anti-inflammatory effect as, for instance, suggested by reduction of circulating Il6 levels [7–9]. The intranasal infection why procedure appears to be a more likely candidate. Despite the relatively small volume of 20 μl that is used and the near physiological properties of PBS, we consider it likely that entry of PBS into the airway creates a stress response similar to that observed after fluid aspiration, including at least focal pulmonary hypoxia due to bronchospasm. Responsible mechanisms may both relate to stimulation of nerve endings in the airway epithelium and direct noxious stimulation of airway epithelial cells. Indeed, except for Irg1, three of the four mRNAs whose expression was regulated in response to mock treatment are known to be induced during a stress response or hypoxia at the cellular or tissue level (Retnla: [10]; Il6: e.g., [11]; Cxcl10: [12]). The fourth one, Irg1, is preferentially expressed in macrophages, is strongly induced during macrophage activation, and localizes to mitochondria [13, 14]. Its expression in stress or hypoxia has not been examined, and it would therefore be interesting to test whether it plays a role in these processes. The four interferon related genes (Stat1, Ifng, Ifnl2 and Mx1) were clearly induced in infected mice only.