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The immune responses of type 2 T helper cells (Th2) play an important role in asthma and promote the differentiation of alternatively activated (M2) macrophages. M2 macrophages have been increasingly understood to contribute to Th2 immunity. We hypothesized that M2 macrophages are altered in asthma and modulate Th2 responses. The aim of this study was to characterize the phenotype and function of human monocyte-derived M2 and bronchoalveolar lavage fluid (BALF) macrophages from healthy control subjects and subjects with asthma. Phenotypic characteristics and effector function of M2 macrophages were examined using monocyte-derived and BALF macrophages obtained from subjects with asthma (n = 28) and healthy volunteers (n = 9) by flow cytometry and quantitative PCR. Resting monocyte-derived (M0) and M2 macrophages were generated by the addition of macrophage colony–stimulating factor or macrophage colony–stimulating factor plus IL-4, respectively. M2 macrophage cytokine expression and their impact on dendritic and CD4+ T cell activation were examined in vitro. High levels of CD206 and major histocompatibility complex class II expression identify macrophages with an M2 phenotype that are increased 2.9-fold in the BALF of subjects with asthma compared with control subjects. M2 macrophages have elevated IL-6, IL-10, and IL-12p40 production compared with conventional macrophages and modulate dendritic and CD4+ T cell interactions. Histamine receptor 1 and E-cadherin expression identify M2 macrophage subsets associated with increased airflow obstruction. M2 macrophages have a distinct cell surface and effector phenotype and are found in increased numbers in subjects with asthma. These findings suggest that M2 macrophages may play an important role in allergic asthma through their bidirectional interactions with immune and structural cells, and inflammatory mediators.

Genetic variants annotated to the hedgehog interacting protein (HHIP) are robustly associated with chronic obstructive pulmonary disease (COPD). Hhip haploinsufficiency in mice leads to increased susceptibility towards the development of emphysema following exposure to chronic cigarette smoke (CS). To explore the molecular pathways which contribute to increased susceptibility, we performed metabolomic profiling using high performance liquid chromatography tandem mass spectroscopy (LC/MS-MS) on plasma, urine, and lung tissue of Hhip+/− heterozygotes and wild type (Hhip+/+) C57/BL6 mice exposed to either room-air or CS for six months. Univariate comparisons between groups were made with a combined fold change ≥2 and Student’s t-test p-value despite comparable plasma levels was observed in Hhip+/− heterozygotes; a strong gene-by-smoking association was also observed. Correlations between MACL and markers of oxidative stress such as urinary methionine sulfoxide were observed in Hhip+/− but not in Hhip+/+ mice. Metabolite set enrichment analyses suggest reduced antioxidant capacity and alterations in macronutrient metabolism contribute to increased susceptibility to chronic CS-induced oxidative stress in Hhip haploinsufficiency states.

Genetic variants in Hedgehog interacting protein (HHIP) have consistently been associated with the susceptibility to develop chronic obstructive pulmonary disease and pulmonary function levels, including the forced expiratory volume in 1 s (FEV1), in general population samples by genome-wide association studies. However, in vivo evidence connecting Hhip to age-related FEV1 decline and emphysema development is lacking. Herein, using Hhip heterozygous mice (Hhip(+/-)), we observed increased lung compliance and spontaneous emphysema in Hhip(+/-) mice starting at 10 mo of age. This increase was preceded by increases in oxidative stress levels in the lungs of Hhip(+/-) vs. Hhip(+/+) mice. To our knowledge, these results provide the first line of evidence that HHIP is involved in maintaining normal lung function and alveolar structures. Interestingly, antioxidant N-acetyl cysteine treatment in mice starting at age of 5 mo improved lung function and prevented emphysema development in Hhip(+/-) mice, suggesting that N-acetyl cysteine treatment limits the progression of age-related emphysema in Hhip(+/-) mice. Therefore, reduced lung function and age-related spontaneous emphysema development in Hhip(+/-) mice may be caused by increased oxidative stress levels in murine lungs as a result of haploinsufficiency of Hhip.

A genetic locus within the FAM13A gene has been consistently associated with chronic obstructive pulmonary disease (COPD) in genome-wide association studies. However, the mechanisms by which FAM13A contributes to COPD susceptibility are unknown.To determine the biologic function of FAM13A in human COPD and murine COPD models and discover the molecular mechanism by which FAM13A influences COPD susceptibility.Fam13a null mice (Fam13a(-/-)) were generated and exposed to cigarette smoke. The lung inflammatory response and airspace size were assessed in Fam13a(-/-) and Fam13a(+/+) littermate control mice. Cellular localization of FAM13A protein and mRNA levels of FAM13A in COPD lungs were assessed using immunofluorescence, Western blotting, and reverse transcriptase-polymerase chain reaction, respectively. Immunoprecipitation followed by mass spectrometry identified cellular proteins that interact with FAM13A to reveal insights on FAM13A's function.In murine and human lungs, FAM13A is expressed in airway and alveolar type II epithelial cells and macrophages. Fam13a null mice (Fam13a(-/-)) were resistant to chronic cigarette smoke-induced emphysema compared with Fam13a(+/+) mice. In vitro, FAM13A interacts with protein phosphatase 2A and recruits protein phosphatase 2A with glycogen synthase kinase 3β and β-catenin, inducing β-catenin degradation. Fam13a(-/-) mice were also resistant to elastase-induced emphysema, and this resistance was reversed by coadministration of a β-catenin inhibitor, suggesting that FAM13A could increase the susceptibility of mice to emphysema development by inhibiting β-catenin signaling. Moreover, human COPD lungs had decreased protein levels of β-catenin and increased protein levels of FAM13A.We show that FAM13A may influence COPD susceptibility by promoting β-catenin degradation.

Lymphangioleiomyomatosis (LAM) is a destructive lung disease primarily affecting women. Genetic studies indicate that LAM cells carry inactivating tuberous sclerosis complex (TSC)-2 mutations, and metastasize to the lung. We previously discovered that estradiol increases the metastasis of TSC2-deficient cells in mice carrying xenograft tumors. Here, we investigate the molecular basis underlying the estradiol-induced lung metastasis of TSC2-deficient cells, and test the efficacy of Faslodex (an estrogen receptor antagonist) in a preclinical model of LAM. We used a xenograft tumor model in which estradiol induces the lung metastasis of TSC2-deficient cells. We analyzed the impact of Faslodex on tumor size, the extracellular matrix organization, the expression of matrix metalloproteinase (MMP)-2, and lung metastasis. We also examined the effects of estradiol and Faslodex on MMP2 expression and activity in tuberin-deficient cells in vitro. Estradiol resulted in a marked reduction of Type IV collagen deposition in xenograft tumors, associated with 2-fold greater MMP2 concentrations compared with placebo-treated mice. Faslodex normalized the Type IV collagen changes in xenograft tumors, enhanced the survival of the mice, and completely blocked lung metastases. In vitro, estradiol enhanced MMP2 transcripts, protein accumulation, and activity. These estradiol-induced changes in MMP2 were blocked by Faslodex. In TSC2-deficient cells, estradiol increased MMP2 concentrations in vitro and in vivo, and induced extracellular matrix remodeling. Faslodex inhibits the estradiol-induced lung metastasis of TSC2-deficient cells. Targeting estrogen receptors with Faslodex may be of efficacy in the treatment of LAM.

Multiple intergenic single-nucleotide polymorphisms (SNPs) near hedgehog interacting protein (HHIP) on chromosome 4q31 have been strongly associated with pulmonary function levels and moderate-to-severe chronic obstructive pulmonary disease (COPD). However, whether the effects of variants in this region are related to HHIP or another gene has not been proven. We confirmed genetic association of SNPs in the 4q31 COPD genome-wide association study (GWAS) region in a Polish cohort containing severe COPD cases and healthy smoking controls (P = 0.001 to 0.002). We found that HHIP expression at both mRNA and protein levels is reduced in COPD lung tissues. We identified a genomic region located ∼85 kb upstream of HHIP which contains a subset of associated SNPs, interacts with the HHIP promoter through a chromatin loop and functions as an HHIP enhancer. The COPD risk haplotype of two SNPs within this enhancer region (rs6537296A and rs1542725C) was associated with statistically significant reductions in HHIP promoter activity. Moreover, rs1542725 demonstrates differential binding to the transcription factor Sp3; the COPD-associated allele exhibits increased Sp3 binding, which is consistent with Sp3's usual function as a transcriptional repressor. Thus, increased Sp3 binding at a functional SNP within the chromosome 4q31 COPD GWAS locus leads to reduced HHIP expression and increased susceptibility to COPD through distal transcriptional regulation. Together, our findings reveal one mechanism through which SNPs upstream of the HHIP gene modulate the expression of HHIP and functionally implicate reduced HHIP gene expression in the pathogenesis of COPD.

Hedgehog interacting protein (HHIP) was implicated in chronic obstructive pulmonary disease (COPD) by genome-wide association studies (GWAS). However, it remains unclear how HHIP contributes to COPD pathogenesis. To identify genes regulated by HHIP, we performed gene expression microarray analysis in a human bronchial epithelial cell line (Beas-2B) stably infected with HHIP shRNAs. HHIP silencing led to differential expression of 296 genes; enrichment for variants nominally associated with COPD was found. Eighteen of the differentially expressed genes were validated by real-time PCR in Beas-2B cells. Seven of 11 validated genes tested in human COPD and control lung tissues demonstrated significant gene expression differences. Functional annotation indicated enrichment for extracellular matrix and cell growth genes. Network modeling demonstrated that the extracellular matrix and cell proliferation genes influenced by HHIP tended to be interconnected. Thus, we identified potential HHIP targets in human bronchial epithelial cells that may contribute to COPD pathogenesis.

Background The HHIP gene, encoding Hedgehog interacting protein, has been implicated in chronic obstructive pulmonary disease (COPD) by genome-wide association studies (GWAS), and our subsequent studies identified a functional upstream genetic variant that decreased HHIP transcription. However, little is known about how HHIP contributes to COPD pathogenesis. Methods We exposed Hhip haploinsufficient mice (Hhip+/-) to cigarette smoke (CS) for 6 months to model the biological consequences caused by CS in human COPD risk-allele carriers at the HHIP locus. Gene expression profiling in murine lungs was performed followed by an integrative network inference analysis, PANDA (Passing Attributes between Networks for Data Assimilation) analysis. Results We detected more severe airspace enlargement in Hhip+/- mice vs. wild-type littermates (Hhip+/+) exposed to CS. Gene expression profiling in murine lungs suggested enhanced lymphocyte activation pathways in CS-exposed Hhip+/- vs. Hhip+/+ mice, which was supported by increased numbers of lymphoid aggregates and enhanced activation of CD8+ T cells after CS-exposure in the lungs of Hhip+/-mice compared to Hhip+/+ mice. Mechanistically, results from PANDA network analysis suggested a rewired and dampened Klf4 signaling network in Hhip+/- mice after CS exposure. Conclusions In summary, HHIP haploinsufficiency exaggerated CS-induced airspace enlargement, which models CS-induced emphysema in human smokers carrying COPD risk alleles at the HHIP locus. Network modeling suggested rewired lymphocyte activation signaling circuits in the HHIP haploinsufficiency state. Electronic supplementary material The online version of this article (doi:10.1186/s13073-015-0137-3) contains supplementary material, which is available to authorized users.