Agonistic induction of PPARγ reverses cigarette smoke-induced emphysema.

The development of emphysema in humans and mice exposed to cigarette smoke is promoted by activation of an adaptive immune response. Lung myeloid dendritic cells (mDCs) derived from cigarette smokers activate autoreactive Th1 and Th17 cells. mDC-dependent activation of T cell subsets requires expression of the SPP1 gene, which encodes osteopontin (OPN), a pleiotropic cytokine implicated in autoimmune responses. The upstream molecular events that promote SPP1 expression and activate mDCs in response to smoke remain unknown. Here, we show that peroxisome proliferator-activated receptor γ (PPARG/Pparg) expression was downregulated in mDCs of smokers with emphysema and mice exposed to chronic smoke. Conditional knockout of PPARγ in APCs using Cd11c-Cre Pparg(flox/flox) mice led to spontaneous lung inflammation and emphysema that resembled the phenotype of smoke-exposed mice. The inflammatory phenotype of Cd11c-Cre Pparg(flox/flox) mice required OPN, suggesting an antiinflammatory mechanism in which PPARγ negatively regulates Spp1 expression in the lung. A 2-month treatment with a PPARγ agonist reversed emphysema in WT mice despite continual smoke exposure. Furthermore, endogenous PPARγ agonists were reduced in the plasma of smokers with emphysema. These findings reveal a proinflammatory pathway, in which reduced PPARγ activity promotes emphysema, and suggest that targeting this pathway in smokers could prevent and reverse emphysema.

Autoreactive T Cells in Human Smokers is Predictive of Clinical Outcome.

Cross-sectional studies have suggested a role for activation of adaptive immunity in smokers with emphysema, but the clinical application of these findings has not been explored. Here we examined the utility of detecting autoreactive T cells as a screening tool for emphysema in an at-risk population of smokers. We followed 156 former and current (ever)-smokers for 2 years to assess whether peripheral blood CD4 T cell cytokine responses to lung elastin fragments (EFs) could discriminate between those with and without emphysema, and to evaluate the relevance of autoreactive T cells to predict changes during follow-up in lung physiological parameters. Volunteers underwent baseline complete phenotypic assessment with pulmonary function tests, quantitative chest CT, yearly 6-min walk distance (6MWD) testing, and annual measurement of CD4 T cell cytokine responses to EFs. The areas under the receiver operating characteristic curve to predict emphysema for interferon gamma (IFN-γ), and interleukin 6 (IL-6) responses to EFs were 0.81 (95% CI of 0.74-0.88) and 0.79 (95% CI of 0.72-0.86) respectively. We developed a dual cytokine enzyme-linked immunocell spot assay, the γ-6 Spot, using CD4 T cell IFN-γ and IL-6 responses and found that it discriminated emphysema with 90% sensitivity. After adjusting for potential confounders, the presence of autoreactive T cells was predictive of a decrease in 6MWD over 2 years (decline in 6MWD, -19 m per fold change in IFN-γ; P = 0.026, and -26 m per fold change in IL-6; P = 0.003). In support of the human association studies, we cloned CD4 T cells with characteristic T helper (Th)1 and Th17 responses to EFs in the peripheral blood of ever-smokers with emphysema, confirming antigenicity of lung elastin in this population. These findings collectively suggest that the EF-specific autoreactive CD4 T cell assay, γ-6 Spot, could provide a non-invasive diagnostic tool with potential application to large-scale screening to discriminate emphysema in ever-smokers, and predict early relevant physiological outcomes in those at risk.

Cigarette smoke induction of osteopontin (SPP1) mediates T(H)17 inflammation in human and experimental emphysema.

Smoking-related lung diseases are among the leading causes of death worldwide, underscoring the need to understand their pathogenesis and develop new effective therapies. We have shown that CD1a+ antigen-presenting cells (APCs) from lungs of patients with emphysema can induce autoreactive T helper 1 (T(H)1) and T(H)17 cells. Similarly, the canonical cytokines interferon-γ (IFN-γ) and interleukin-17A (IL-17A) are specifically linked to lung destruction in smokers, but how smoke activates APCs to mediate emphysema remains unknown. Here, we show that, in addition to increasing IFN-γ expression, cigarette smoke increased the expression of IL-17A in both CD4+ and γδ T cells from mouse lung. IL-17A deficiency resulted in attenuation of, whereas lack of γδ T cells exacerbated, smoke-induced emphysema in mice. Adoptive transfer of lung APCs isolated from mice with emphysema revealed that this cell population was capable of transferring disease even in the absence of active smoke exposure, a process that was dependent on IL-17A expression. Spp1 (the gene for osteopontin) was highly expressed in the pathogenic lung APCs of smoke-exposed mice and was required for the T(H)17 responses and emphysema in vivo, in part through its inhibition of the expression of the transcription factor Irf7. Thus, the Spp1-Irf7 axis is critical for induction of pathological T(H)17 responses, revealing a major mechanism by which smoke activates lung APCs to induce emphysema and identifying a pathway that could be targeted for therapeutic purposes.

The Pax gene eyegone facilitates repression of eye development in Tribolium.

BACKGROUND: The Pax transcription factor gene eyegone (eyg) participates in many developmental processes in Drosophila, including the Notch signaling activated postembryonic growth of the eye primordium, global development of the adult head and the development of the antenna. In contrast to other Pax genes, the functional conservation of eyg in species other than Drosophila has not yet been explored. RESULTS: We investigated the role of eyg during the postembryonic development of the red flour beetle Tribolium castaneum. Our results indicate conserved roles in antennal but not in eye development. Besides segmentation defects in the antenna, Tribolium eyg knockdown animals were characterized by eye enlargement due to the formation of surplus ommatidia at the central anterior edge of the compound eye. This effect resulted from the failure of the developing gena to locally repress retinal differentiation, which underlies the formation of the characteristic anterior notch in the Tribolium eye. Neither varying the induction time point of eyg knockdown nor knocking down components of the Janus kinase/Signal Transducer and Activators of Transcription signaling pathway in combination with eyg reduced eye size like in Drosophila. CONCLUSIONS: Taken together, expression and knockdown data suggest that Tribolium eyg serves as a competence factor that facilitates the repression of retinal differentiation in response to an unknown signal produced in the developing gena. At the comparative level, our findings reveal diverged roles of eyg associated with the evolution of different modes of postembryonic head development in endopterygote insects as well as diversified head morphologies in darkling beetles.

Probing the Drosophila retinal determination gene network in Tribolium (II): The Pax6 genes eyeless and twin of eyeless.

The Pax6 genes eyeless (ey) and twin of eyeless (toy) are upstream regulators in the retinal determination gene network (RDGN), which instructs the formation of the adult eye primordium in Drosophila. Most animals possess a singleton Pax6 ortholog, but the dependence of eye development on Pax6 is widely conserved. A rare exception is given by the larval eyes of Drosophila, which develop independently of ey and toy. To obtain insight into the origin of differential larval and adult eye regulation, we studied the function of toy and ey in the red flour beetle Tribolium castaneum. We find that single and combinatorial knockdown of toy and ey affect larval eye development strongly but adult eye development only mildly in this primitive hemimetabolous species. Compound eye-loss, however, was provoked when ey and toy were RNAi-silenced in combination with the early retinal gene dachshund (dac). We propose that these data reflect a role of Pax6 during regional specification in the developing head and that the subsequent maintenance and growth of the adult eye primordium is regulated partly by redundant and partly by specific functions of toy, ey and dac in Tribolium. The results from embryonic knockdown and comparative protein sequence analysis lead us further to conclude that Tribolium represents an ancestral state of redundant control by ey and toy.

Probing the Drosophila retinal determination gene network in Tribolium (I): The early retinal genes dachshund, eyes absent and sine oculis.

The early retinal genes dachshund (dac), eyes absent (eya) and sine oculis (so) are key regulators of adult eye development in Drosophila. Expression data implicate homologs of all three transcription factor genes in vertebrate eye development. However, functional confirmation has thus far only been reported for so homologs. We therefore investigated expression and function of these genes in the red flour beetle Tribolium castaneum. Our results show that Tribolium so and eya are essential for both larval and adult eye development. Moreover, widespread co-expression and indistinguishable knockdown phenotypes suggests eya interaction with so in many tissues. dac expression, by contrast, overlaps with eya and so only in select tissues, which, however, includes the primordia of larval and adult eyes. Notwithstanding, dac knockdown has no detectable effect on larval eye development and causes strong but incomplete adult eye reduction. In a parallel study, we show that dac is essential for adult eye development in combination with the Pax6 transcription factors eyeless (ey) and twin of eyeless (toy). Taken together, our data reveal conservation but also evolutionary plasticity of the Drosophila retinal determination gene network in insects.