Allergic asthma is a complex disease characterized in part by granulocytic inflammation of the airways. In addition to eosinophils, neutrophils (PMN) are also present, particularly in cases of severe asthma. We sought to identify the genetic determinants of neutrophilic inflammation in a mouse model of house dust mite (HDM)-induced asthma. We applied an HDM model of allergic asthma to the eight founder strains of the Collaborative Cross (CC) and 151 incipient lines of the CC (preCC). Lung lavage fluid was analyzed for PMN count and the concentration of CXCL1, a hallmark PMN chemokine. PMN and CXCL1 were strongly correlated in preCC mice. We used quantitative trait locus (QTL) mapping to identify three variants affecting PMN, one of which colocalized with a QTL for CXCL1 on chromosome (Chr) 7. We used lung eQTL data to implicate a variant in the gene Zfp30 in the CXCL1/PMN response. This genetic variant regulates both CXCL1 and PMN by altering Zfp30 expression, and we model the relationships between the QTL and these three endophenotypes. We show that Zfp30 is expressed in airway epithelia in the normal mouse lung and that altering Zfp30 expression in vitro affects CXCL1 responses to an immune stimulus. Our results provide strong evidence that Zfp30 is a novel regulator of neutrophilic airway inflammation.

Airway allergen exposure induces inflammation among individuals with atopy that is characterized by altered airway gene expression, elevated levels of T helper type 2 cytokines, mucus hypersecretion, and airflow obstruction. To identify the genetic determinants of the airway allergen response, we employed a systems genetics approach. We applied a house dust mite mouse model of allergic airway disease to 151 incipient lines of the Collaborative Cross, a new mouse genetic reference population, and measured serum IgE, airway eosinophilia, and gene expression in the lung. Allergen-induced serum IgE and airway eosinophilia were not correlated. We detected quantitative trait loci (QTL) for airway eosinophilia on chromosome (Chr) 11 (71.802-87.098 megabases [Mb]) and allergen-induced IgE on Chr 4 (13.950-31.660 Mb). More than 4,500 genes expressed in the lung had gene expression QTL (eQTL), the majority of which were located near the gene itself. However, we also detected approximately 1,700 trans-eQTL, and many of these trans-eQTL clustered into two regions on Chr 2. We show that one of these loci (at 147.6 Mb) is associated with the expression of more than 100 genes, and, using bioinformatics resources, fine-map this locus to a 53 kb-long interval. We also use the gene expression and eQTL data to identify a candidate gene, Tlcd2, for the eosinophil QTL. Our results demonstrate that hallmark allergic airway disease phenotypes are associated with distinct genetic loci on Chrs 4 and 11, and that gene expression in the allergically inflamed lung is controlled by both cis and trans regulatory factors.

Innate immunity is the first line of defense against microbial infections. Although polymorphisms in toll-like receptors (TLRs) and downstream signaling molecules (CD14, TLR2, TLR4, TLR5, and IRAK4) affect the innate immune response, these variants account for only a portion of the ability of the host to respond to bacteria, fungi, and viruses. To identify other genes involved in the innate immune response, we challenged 16 inbred murine strains with lipopolysaccharide (LPS) systemically and measured serum concentrations of pro-inflammatory cytokines IL-1b, IL-6, and TNFa, and the chemokine KC 6 hr posttreatment. Loci that segregate with strain phenotypes were identified by whole genome association (WGA) mapping of cytokine concentrations. Published gene expression profiles and quantitative trait loci (QTL) were then utilized to prioritize loci and genes that potentially regulate the host response to LPS. Sixteen loci were selected for further investigation by combining WGA analysis with previously published QTL for murine response to LPS or gram negative bacteria. Thirty-eight genes within these loci were then selected for further investigation on the basis of the significance of the identified locus, transcriptional response to LPS, and biological plausibility. RNA interference-mediated inhibition of 4 of 38 candidate genes was shown to block the production of IL-6 in J774A.1 macrophages. In summary, our analysis identified 4 genes that have not previously been implicated in innate immunity, namely, 1110058L19Rik, 4933415F23Rik, Fbxo9, and Ipo7. These genes could represent potential sepsis biomarkers or therapeutic targets that should be further investigated in human populations.

Our understanding of the role that host genetic factors play in the initiation and severity of infections caused by gram-negative bacteria is incomplete. To identify novel regulators of the host response to lipopolysaccharide (LPS), 11 inbred murine strains were challenged with LPS systemically. In addition to two strains lacking functional TLR4 (C3H/HeJ and C57BL/6J TLR42/2 ), three murine strains with functional TLR4 (C57BL/6J, 129/SvImJ, and NZW/LacJ) were found to be relatively resistant to systemic LPS challenge; the other six strains were classified as sensitive. RNA from lung, liver, and spleen tissue was profiled on oligonucleotide microarrays to determine if unique transcripts differentiate susceptible and resistant strains. Gene expression analysis identified the Hedgehog signaling pathway and a number of transcription factors (TFs) involved in the response to LPS. RNA interference-mediated inhibition of six TFs (C/EBP, Cdx-2, E2F1, Hoxa4, Nhlh1, and Tead2) was found to diminish IL-6 and TNF-a production by murine macrophages. Mouse lines with targeted mutations were used to verify the involvement of two novel genes in innate immunity. Compared with wild-type control mice, mice deficient in the E2F1 transcription factor were found to have a reduced inflammatory response to systemic LPS, and mice heterozygote for Ptch, a gene involved in Hedgehog signaling, were found to be more responsive to systemic LPS. Our analysis of gene expression data identified novel pathways and transcription factors that regulate the host response to systemic LPS. Our results provide potential sepsis biomarkers and therapeutic targets that should be further investigated in human populations.

Lipopolysaccharide is a major component of the cell wall of Gram-negative bacteria and a potent stimulator of innate immune response via TLR4. Studies on the LPS action both in vivo and in vitro have used different preparations of LPS, including ultra-pure LPS (LIST) and a less pure but less expensive form (Sigma) isolated from Escherichia coli serotype O111:B4. The difference between the effects of these compounds has not been well studied although this information is important in understanding TLR stimulation. In this study, we compared response of RAW264.7 macrophage cells treated LIST or Sigma LPS for 6 h and 24 h. Gene expression data were analyzed to identify specific genes and pathways that are in common and unique to the two LPS preparations. Seven hundred fifty-five genes were differentially expressed at 6 h in response to Sigma LPS and 973 were differentially expressed following LIST LPS treatment, with 503 in common. At 24 h, Sigma LPS induced or repressed 901 genes while 1646 genes were differentially regulated by LIST LPS treatment; 701 genes were shared by two forms of LPS. Although considerably more genes were differentially expressed in response to LIST LPS, similar molecular pathways and transcriptional networks were activated by the two LPS preparations. We also treated bone marrow-derived macrophages (BMMs) from three strains of mice with different concentrations of LIST and Sigma LPS and showed that BMMs produced more IL-6 and TNF-α in response to LIST LPS at low LPS concentrations but, at higher LPS concentrations, more cytokines were produced in response to stimulation by Sigma LPS. Together, these findings suggest that, despite activation of similar molecular pathways by LIST and Sigma LPS preparations, residual protein impurities in the Sigma LPS preparation may nevertheless influence the transcriptional profile attributed to TLR4 stimulation.

BackgroundAllergic airway diseases (AADs) such as asthma are characterized in part by granulocytic airway inflammation. The gene regulatory networks that govern granulocyte recruitment are poorly understood, but evidence is accruing that microRNAs (miRNAs) play an important role. To identify miRNAs that may underlie AADs, we used two complementary approaches that leveraged the genotypic and phenotypic diversity of the Collaborative Cross (CC) mouse population. In the first approach, we sought to identify miRNA expression quantitative trait loci (eQTL) that overlap QTL for AAD-related phenotypes. Specifically, CC founder strains and incipient lines of the CC were sensitized and challenged with house dust mite allergen followed by measurement of granulocyte recruitment to the lung. Total lung RNA was isolated and miRNA was measured using arrays for CC founders and qRT-PCR for incipient CC lines.ResultsAmong CC founders, 92 miRNAs were differentially expressed. We measured the expression of 40 of the most highly expressed of these 92 miRNAs in the incipient lines of the CC and identified 18 eQTL corresponding to 14 different miRNAs. Surprisingly, half of these eQTL were distal to the corresponding miRNAs, and even on different chromosomes. One of the largest-effect local miRNA eQTL was for miR-342-3p, for which we identified putative causal variants by bioinformatic analysis of the effects of single nucleotide polymorphisms on RNA structure. None of the miRNA eQTL co-localized with QTL for eosinophil or neutrophil recruitment. In the second approach, we constructed putative miRNA/mRNA regulatory networks and identified three miRNAs (miR-497, miR-351 and miR-31) as candidate master regulators of genes associated with neutrophil recruitment. Analysis of a dataset from human keratinocytes transfected with a miR-31 inhibitor revealed two target genes in common with miR-31 targets correlated with neutrophils, namely Oxsr1 and Nsf.ConclusionsmiRNA expression in the allergically inflamed murine lung is regulated by genetic loci that are smaller in effect size compared to mRNA eQTL and often act in trans. Thus our results indicate that the genetic architecture of miRNA expression is different from mRNA expression. We identified three miRNAs, miR-497, miR-351 and miR-31, that are candidate master regulators of genes associated with neutrophil recruitment. Because miR-31 is expressed in airway epithelia and is predicted to target genes with known links to neutrophilic inflammation, we suggest that miR-31 is a potentially novel regulator of airway inflammation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1732-9) contains supplementary material, which is available to authorized users.

Previous publications from our and other groups identified E2F1 as a transcription factor involved in the regulation of inflammatory response to Toll-like receptor ligands including LPS. In this study, we challenged E2F1-deficient mice with LPS systemically and demonstrated decreased survival despite attenuated inflammatory response compared with controls. Gene expression profiling of liver tissue identified a dampened transcriptional response in the coagulation cascade in B6;129(E2F1-/-) compared with B6x129 F2 mice. These data were further corroborated by increased prothrombin time, activated partial thromboplastin time, and fibrin split products in the blood of E2F1-deficient mice, suggesting disseminated intravascular coagulation as a consequence of uncontrolled sepsis providing at least a partial explanation for their decreased survival despite attenuated inflammatory response. To identify novel miRNAs involved in the innate immune response to LPS, we also performed miRNA profiling of liver tissue from B6;129(E2F1-/-) and B6x129 F2 mice treated with LPS systemically. Our analysis identified a set of miRNAs and their mRNA targets that are significantly differentially regulated in E2F1-deficient but not control mice including let-7g, miR-101b, miR-181b, and miR-455. These miRNAs represent novel regulators of the innate immune response. In summary, we used transcriptional and miRNA profiling to characterize the response of E2F1-deficient mice to systemic LPS.

Chronic LPS inhalation causes submucosal thickening and airway narrowing. To address the hypothesis that environmental airway disease is, in part, a fibroproliferative lung disease, we exposed C57BL/6 mice daily to LPS by inhalation for up to 2 months followed by 1 month of recovery. C57BL/6 mice exposed to daily inhaled LPS had significantly enhanced mRNA expression of TGF-beta1, TIMP-1, fibronectin-1, and pro-collagen types I, III, and IV and show prominent submucosal expression of the myofibroblast markers desmin and alpha-smooth muscle actin. To further characterize global gene expression in airway fibroproliferation, we performed microarray analysis on total lung RNA from mice exposed to LPS both acutely and chronically. This analysis revealed a subset of genes typically associated with lung injury and repair, and ECM homeostasis. To further identify candidate genes specifically involved in generic fibroproliferation, we interrogated this analysis with genes induced in C57BL/6 mouse lung by bleomycin. This analysis yielded a list of 212 genes in common suggesting that there is a common subset of genes that regulate fibroproliferation in the lung independent of etiologic agent and site of injury.