Red-2 KO mice (Fig. 2E). Moreover, immunohistochemical evaluation of samples from Spred-2 KO mice showed drastically improved number of phosphorylated ERK, particularly in bronchial epithelial and inflammatory cells, compared with WT mice (Fig.2F and 2G).Crit Care Med. Author manuscript; accessible in PMC 2017 July 01.Ito et al.PageSpred-2 KO mice display an enhanced immune response throughout influenza virus infectionAuthor Manuscript Author Manuscript Author Manuscript Author ManuscriptTo enable elucidate the mechanism underlying the elevated mortality and serious inflammation noticed in Spred-2 KO mice, we examined the cytokine and chemokine profile in entire lungs for the duration of H1N1 challenge. Type-I IFN protein expression was substantially higher in H1N1-infected entire lung homogenates from Spred-2 KO mice compared with lungs from WT mice (Fig. 3A). In addition, H1N1-infected whole lungs from Spred-2 KO mice had substantially larger protein levels of CCL2 and CXCL1, molecules that play a essential part inside the recruitment of monocytes/macrophages and neutrophils into inflammatory lesions (Fig. 3A). In agreement together with the chemokine profile observed in entire lungs, flow cytometric evaluation demonstrated elevated macrophage (WT vs. Spred-2 KO; Day 3: 20.two 1.two vs. 27.1 0.eight, p0.01, Day 5: 31.0 three.three vs. 48.eight four.0, p0.05) and neutrophil (WT vs. Spred-2 KO; Day 3: 5.0 0.eight vs. 7.9 0.six, p0.05, Day 5: six.0 0.3 vs. 7.1 0.two, p0.05) recruitment through H1N1 infection in Spred-2 KO mice (Fig. 3B). Furthermore, the numbers of both CD4+(WT vs. Spred-2 KO; Day three: 3.0 0.5 vs. five.six 0.6, p0.05, Day 5: three.1 0.3 vs. four.7 0.three, p0.01) and CD8+ (WT vs. Spred-2 KO; Day 3: 3.0 0.1 vs. four.eight 0.six, p0.05, Day five: four.0 0.four vs. 4.9 0.2) T cells were significantly larger in Spred-2 KO mice, whereas no important differences were found inside the quantity of NK cells (WT vs. Spred-2 KO; Day three: three.0 0.1 vs. three.3 0.3, Day five: 4.9 0.7 vs. four.2 0.three) and NKT cells (WT vs. Spred-2 KO; Day three: 0.23 0.01 vs. 0.33 0.06, Day 5: 0.HEPACAM Protein MedChemExpress 32 0.04 vs. 0.28 0.06) in between each mouse strains (Fig.INPP5A Protein Synonyms 3B). Inhibition in the Raf/MEK/ERK signaling cascade leads to impaired pathogenesis of influenza virus infection To directly test no matter whether the Raf/MEK/ERK signaling pathway correlates with abrogated pathogenesis of influenza virus infection in Spred-2 KO mice, we utilised the specific MEK inhibitor U0126.PMID:23537004 Intranasal administration of U0126 in Spred-2 KO mice through influenza infection led to reduced mortality with decreased expression of phosphorylated-ERK (0.six.7 fold) in the lungs compared with the handle DMSO-treated group (Fig.4A and 4B). Furthermore, measurement of TCID50 indicated considerably reduce viral load within the lungs of mice receiving U0126 compared with DMSO controls at day five post-infection (Fig. 4C). Also, histological appearance demonstrated that the U0126-treated group displayed decreased inflammation within the lungs (Fig. 4D). We also found considerably impaired production of IFN- (WT vs. Spred-2 KO; Day 3: 72.0 13.1 vs. 23.1 6.4 p0.05, Day five: eight.09 1.3 vs. ten.5 2.5), IFN- (WT vs. Spred-2 KO; Day 3: 17.six two.8 vs. 11.three 1.two, Day 5: 11.3 1.two vs. four.35 0.eight, p0.01), CCL2 (WT vs. Spred-2 KO; Day three: 1.59 0.30 vs. 0.51 0.04, p0.05, Day five: 0.94 0.20 vs. 0.62 0.11), and CXCL1 (WT vs. Spred-2 KO; Day three: 2.02 0.48 vs. 0.81 0.17, p0.05, Day 5: 0.76 0.08 vs. 0.26 0.03, p0.01), compared with all the manage treated group (Fig. 4E). Conversely, U0126 treatment of WT mice led to no considerable reduction in mortality (information not shown). Differential r.