Neutralizing TNFα restores glucocorticoid sensitivity in a mouse model of neutrophilic airway inflammation.

Asthma is a heterogeneous disorder, evidenced by distinct types of inflammation resulting in different responsiveness to therapy with glucocorticoids (GCs). Tumor necrosis factor α (TNFα) is involved in asthma pathogenesis, but anti-TNFα therapies have not proven broadly effective. The effects of anti-TNFα treatment on steroid resistance have never been assessed. We investigated the role of TNFα blockade using etanercept in the responsiveness to GCs in two ovalbumin-based mouse models of airway hyperinflammation. The first model is GC sensitive and T helper type 2 (Th2)/eosinophil driven, whereas the second reflects GC-insensitive, Th1/neutrophil-predominant asthma subphenotypes. We found that TNFα blockade restores the therapeutic effects of GCs in the GC-insensitive model. An adoptive transfer indicated that the TNFα-induced GC insensitivity occurs in the non-myeloid compartment. Early during airway hyperinflammation, mice are GC insensitive specifically at the level of thymic stromal lymphopoietin (Tslp) transcriptional repression, and this insensitivity is reverted when TNFα is neutralized. Interestingly, TSLP knockout mice displayed increased inflammation in the GC-insensitive model, suggesting a limited therapeutic application of TSLP-neutralizing antibodies in subsets of patients suffering from Th2-mediated asthma. In conclusion, we demonstrate that TNFα reduces the responsiveness to GCs in a mouse model of neutrophilic airway inflammation. Thus antagonizing TNFα may offer a new strategy for therapeutic intervention in GC-resistant asthma.

Determining differentially expressed miRNAs and validating miRNA--target relationships using the SPRET/Ei mouse strain.

Micro RNAs (miRs) are involved in many biological processes. The challenge of identifying genes influenced by miRs is evidenced by the relatively few validated miR-target interactions. In this work, we used the Mus spretus SPRET/Ei strain as an in vivo system to identify new miR-target relations. Mus spretus diverged from Mus musculus over one million years ago, making it genetically and phenotypically divergent. SPRET/Ei mice are resistant to inflammation and several cancers, making them attractive for different research fields. Their phenotype is unique and is considerably different from that of almost all other laboratory mouse strains. We exploited the characteristics of SPRET/Ei mice as a tool to identify miR-target relationships. Hepatic genes and miRs differentially expressed between C57BL/6 and SPRET/Ei mice at basal levels were identified with an Affymetrix microarray and a multiplex qPCR, respectively. A total of 955 genes and 38 miRs were identified as differentially expressed. Increased miR expression might result in downregulation of its target mRNA and vice versa. Subsequently, we used our miR and mRNA data to identify possible in vivo miR-target interactions. Ingenuity pathway analysis (IPA) analysis revealed 380 possible miR-target interactions. Five miRs were selected for experimental validation by in vivo overexpression of the miRs. This resulted in the confirmation of six previously unknown miR-target interactions: miR-146a, Zdhhc2; miR-150, Elovl3, Kcnk5, and Nrd1d2; miR-155, Camta1; and miR-592, Steap2. In conclusion, we show that SPRET/Ei mice can be used as a platform for miR-target identification in vivo, and we used this platform to identify and experimentally confirm miR-target interactions.