In ADPKD renal function is corrupted by the accumulation and growth of fluid-filled cysts. Disruption of the PKD1 gene product, polycystin-1, is the most frequent cause of ADPKD, but the mechanisms that predispose PKD1 to recurrent somatic mutation remain poorly understood. Because current evidence for sequence- and structure-dependent mutational susceptibility is strongest at the human PKD1 locus, we will focus here on mechanisms that may promote somatic PKD1 inactivation. Experimental evidence for polycistin-1 inactivation supports a two-hit pathway, with the first hit being an inherited germline pathogenic mutation in PKD1 and the second hit mutation arising later in a somatic cell to inactivate the gene or lower the gene's dosage to lift a barrier to cyst initiation. An affected kidney can have thousands of cysts, each of which is a clonal lineage arising from an independent mutational second-hit event. Why human PKD1 is prone to inactivation and why the rodent orthologs escape similar mutagenesis is a mystery that, once solved, promises to provide important insights into the molecular mechanisms governing cyst initiation. A step toward that goal came from the characterization of the guanine-rich sequence architecture of human PKD1 that distinguishes it from rodent Pkd1. These guanine-rich tracts are intrinsically susceptible to oxidative damage and can adopt non-duplex secondary structures such as guanine-quadruplex DNA. Although guanine quadruplexes serve important regulatory functions, both oxidized guanine lesions and guanine quadruplex structures can interfere with faithful DNA replication and repair, thereby increasing mutation risk. Here, we discuss PKD1 mutagenesis in the context of the renal inflammatory microenvironment, integrating established principles of sequence-dependent mutagenesis and guanine-rich DNA structure biology with mechanisms of cyst initiation in ADPKD. This synthesis supports a conceptual model in which intrinsic sequence-dependent mutational susceptibility at the human PKD1 locus may interact with localized inflammatory microenvironments characterized by oxidative stress and epithelial proliferation to contribute to recurrent somatic second-hit formation.

The "secondhit" pathway is responsible for biallelic inactivation of many tumor suppressors, where a pathogenic germline allele is joined by somatic mutation of the remaining functional allele. The mechanisms are unresolved, but the human PKD1 tumor suppressor is a good experimental model for identifying the molecular determinants. Inactivation of PKD1 results in autosomal dominant polycystic kidney disease, a very common disorder characterized by the accumulation of fluid-filled cysts and end-stage renal disease. Since human PKD1 follows second hit and mouse Pkd1 heterozygotes do not, we reasoned that there is likely a molecular difference that explains the elevated mutagenesis of the human gene. Here we demonstrate that guanine quadruplex DNA structures are abundant throughout human, but not mouse, PKD1 where they activate the DNA damage response. Our results suggest that guanine quadruplex DNAs provoke DNA breaks in PKD1, providing a potential mechanism for cystogenesis in autosomal dominant polycystic kidney disease specifically and for the inactivation of guanine quadruplex-rich tumor suppressors generally.

Autosomal dominant polycystic kidney disease results from the loss of the PKD1 gene product, polycystin 1. Regulatory mechanisms are unresolved, but an apparent G/C sequence bias in the gene is consistent with co-transcriptional R-loop formation. R-loops regulate gene expression and stability, and they form when newly synthesized RNA extensively pairs with the template DNA to displace the non-template strand. In this study, we tested two human PKD1 sequences for co-transcriptional R-loop formation in vitro. We observed RNase H-sensitive R-loop formation in intron 1 and 22 sequences, but only in one transcriptional orientation. Therefore, R-loops may participate in PKD1 expression or stability.

Mammalian antibody switch regions (∼1500 bp) are composed of a series of closely neighboring G4-capable sequences. Whereas numerous structural and genome-wide analyses of roles for minimal G4s in transcriptional regulation have been reported, Long G4-capable regions (LG4s)-like those at antibody switch regions-remain virtually unexplored. Using a novel computational approach we have identified 301 LG4s in the human genome and find LG4s prone to mutation and significantly associated with chromosomal rearrangements in malignancy. Strikingly, 217 LG4s overlap annotated enhancers, and we find the promoters regulated by these enhancers markedly enriched in G4-capable sequences suggesting G4s facilitate promoter-enhancer interactions. Finally, and much to our surprise, we also find single-stranded loops of minimal G4s within individual LG4 loci are frequently highly complementary to one another with 178 LG4 loci averaging >35 internal loop:loop complements of >8 bp. As such, we hypothesized (then experimentally confirmed) that G4 loops within individual LG4 loci directly basepair with one another (similar to characterized stem-loop kissing interactions) forming a hitherto undescribed, higher-order, G4-based secondary structure we term a 'G4 Kiss or G4K'. In conclusion, LG4s adopt novel, higher-order, composite G4 structures directly contributing to the inherent instability, regulatory capacity, and maintenance of these conspicuous genomic regions.

Our lab has previously showed that the immunoprotective lung collectin, SP-D suppresses proinflammatory dendritic cell activation. SP-D however was also shown to be an opsonin with importance in foreign antigen uptake by phagocytes. The role of SP-D in dendritic cell-driven T cell activation remains unclear. Uptake of intranasally instilled fluorescent apoptotic bodies by dendritic cells was studied by immunochemistry and FACS analysis using lung cells of WT C57BL/6 and SP-D-/- mice. Bone-marrow cells were isolated and cultured with mucosal differentiating cytokines including 200 ng/mL Flt3L for 10 days. WT C57BL/6 mice were sensitized to ovalbumin (OVA) and alum and splenocytes were harvested after an intranasal challenge 13 days later. Splenocytes were put into culture in vitro with the mitogen PHA or OVA at varying concentrations in the presence or absence SP-D (0.1-10 μg/mL). T cell proliferation was assessed by a BrdU ELISA. Lack of SP-D in gene deficient mice was associated with increased number of TNF-ɑ+ CD11b+ DCs in the lung. These activated DCs had a diminished capability of engulfing apoptotic bodies suggesting that SP-D is necessary for increasing phagocytic capabilities of lung DCs. CD11c+ BMDCs grown with Flt3L preferentially expressed CD103 (as opposed to CD11b) and MHC II. T cell proliferation was diminished by recombinant SP-D (starting at 0.1 μg/mL). These data indicate that SP-D plays a specific immunemodulatory role by inhibiting antigen and mitogen-induced T cell proliferation and decreasing inflammatory activation while increasing antigen sequestering function of DCs.

Acute exposure to O3 causes pulmonary inflammation and airway hyperreactivity but the underlying mechanisms are unclear. LGM2605 is the synthetic version of the flaxseed lignan secoisolaricirecinol diglucoside, an agent with anti-oxidant and anti-inflammatory properties. We hypothesized that LGM2605 would protect from O3-induced airway inflammation. We employed a longitudinal study design to comprehensively define the inflammatory response to inhaled O3 and oral LGM2605. Rhesus macaques (n=18) were segregated into LGM2605+air, placebo+O3, or LGM2605+O3 groups and underwent measurements of pulmonary function, blood and airway immune cell populations, and bronchial brush biopsy at baseline (day 1) followed by 7 days oral treatment with LGM2605 or placebo (in a peanut butter sandwich) at 25 mg/kg twice per day. Macaques were exposed to 0.3 ppm O3 or filtered air for 6 hours. Measurements were repeated approximately 12 hours post O3 exposure. O3 inhalation induced airway neutrophilia, eosinophilia, and airway hyperreactivity to methacholine 12 h later. O3 increased gene expression of pro-inflammatory cytokines IL-6, IL-8, and IL-25 in cells derived from bronchial brush biopsies. LGM2605 pre-treatment prevented airway neutrophilia (not eosinophilia) and hyperreactivity and reduced inflammatory gene expression. O3 induced structural changes in the immunoregulatory protein surfactant protein-D (SP-D) and increased airway group 2 innate lymphoid cell and CD1c+ myeloid dendritic cell counts. LGM2605 treatment significantly reduced these changes. We demonstrated that LGM2605, a novel anti-inflammatory synthetic compound protected from O3-induced airway hyperreactivity, neutrophilic inflammation, and innate immune changes. We propose that LGM2605 has clinical potential for the treatment of neutrophilic asthma.

BACKGROUND:Ozone (O3) inhalation elicits airway inflammation and impairs treatment responsiveness in asthmatic patients. The underlying immune mechanisms have been difficult to study because of the lack of relevant experimental models. Rhesus macaques spontaneously have asthma and have a similar immune system to human subjects. OBJECTIVES:We sought to investigate mucosal immune changes after O3 inhalation in a clinically relevant nonhuman primate asthma model and to study the effects of an antioxidant synthetic lignan (synthetic secoisolariciresinol diglucoside [LGM2605]). METHODS:A cohort of macaques (n = 17) previously characterized with airway hyperreactivity (AHR) to methacholine was assessed (day 1). Macaques were treated (orally) with LGM2605 (25 mg/kg) or placebo twice per day for 7 days, exposed to 0.3 ppm O3 or air for 6 hours (on day 7), and studied 12 hours later (day 8). Lung function, blood and bronchoalveolar lavage (BAL) fluid immune cell profile, and bronchial brushing and blood cell mRNA expression were assessed. RESULTS:O3 induced significant BAL fluid neutrophilia and eosinophilia and increased AHR and expression of IL6 and IL25 mRNA in the airway epithelium together with increased BAL fluid group 2 innate lymphoid cell (ILC2s), CD1c+ myeloid dendritic cell, and CD4+ T-cell counts and diminished surfactant protein D expression. Although LGM2605 attenuated some of the immune and inflammatory changes, it completely abolished O3-induced AHR. CONCLUSION:ILC2s, CD1c+ myeloid dendritic cells, and CD4+ T cells are selectively involved in O3-induced asthma exacerbation. The inflammatory changes were partially prevented by antioxidant pretreatment with LGM2605, which had an unexpectedly disproportionate protective effect on AHR.

Repetitive DNA sequences support the formation of structures that can interrupt replication and repair, leading to breaks and mutagenesis. One particularly stable structure is G-quadruplex (G4) DNA, which is four-stranded and formed from tandemly repetitive guanine bases. When folded within a template, G4 interferes with DNA synthesis. Similar to non-duplex structures, DNA base lesions can also halt an advancing replication fork, but the Y-family polymerases solve this problem by bypassing the damage. In order to better understand how guanine-rich DNA is replicated, we have investigated the activity of the model Y-family polymerase, Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4), on guanine-rich templates in vitro. We find that Dpo4 progression on templates containing either a single GC-rich hairpin or a G4 DNA structure is greatly reduced and synthesis stalls at the structure. Human polymerase eta (hPol eta) showed the same pattern of stalling at G4; however, and in contrast to Klenow, hPol eta and Dpo4 partially synthesise into the guanine repeat. Substitution of the nucleotide selectivity residue in Dpo4 with alanine permitted ribonucleotide incorporation on unstructured templates, but this further reduced the ability of Dpo4 to synthesise across from the guanine repeats. The advancement of Dpo4 on G4 templates was highest when the reaction was supplied with only deoxycytidine triphosphate, suggesting that high-fidelity synthesis is favoured over misincorporation. Our results are consistent with a model where the Y-family polymerases pause upon encountering G4 structures but have an ability to negotiate some synthesis through tetrad-associated guanines. This suggests that the Y-family polymerases reduce mutagenesis by catalysing the accurate replication of repetitive DNA sequences, but most likely in concert with additional replication and structure resolution activities.

The asthmatic airways are highly susceptible to inflammatory injury by air pollutants such as ozone (O-3), characterized by enhanced activation of eosinophilic granulocytes and a failure of immune protective mechanisms. Eosinophil activation during asthma exacerbation contributes to the proinflammatory oxidative stress by high levels of nitric oxide (NO) production and extracellular DNA release. Surfactant protein-D (SP-D), an epithelial cell product of the airways, is a critical immune regulatory molecule with a multimeric structure susceptible to oxidative modifications. Using recombinant proteins and confocal imaging, we demonstrate here that SP-D directly bound to the membrane and inhibited extracellular DNA trap formation by human and murine eosinophils in a concentration and carbohydrate-dependent manner. Combined allergic airway sensitization and O-3 exposure heightened eosinophilia and nos2 mRNA (iNOS) activation in the lung tissue and S-nitrosylation related de-oligomerisation of SP-D in the airways. In vitro reproduction of the iNOS action led to similar effects on SP-D. Importantly, S-nitrosylation abolished the ability of SP-D to block extracellular DNA trap formation. Thus, the homeostatic negative regulatory feedback between SP-D and eosinophils is destroyed by the NO-rich oxidative lung tissue environment in asthma exacerbations. Demonstration that specific carbohydrate-dependent inhibition of murine and human eosinophil extracellular DNA trap release is abolished by s-nitrosylation of the SP-D molecule.

Chronic exposure to air pollution can lead to altered respiratory mucosal immune function and predisposition to or exacerbation of chronic obstructive pulmonary disease (COPD) or asthma. These diseases are characterized by genetic bias, airway inflammation and remodeling, and activation of innate and adaptive respiratory immune pathways. Glucocorticoids are typically used to treat inflammation and immune cell activation in asthma and COPD, but severe cases may be resistant to treatment. T helper 17 (Th17) cells and IL-17–related pathways have been linked to both air pollution–induced airway inflammation and glucocorticoid resistance. In this issue of the Journal, Mann and colleagues (pp. 355–366) describe the effects of the air pollutant known as urban particulate matter (UPM) and vitamin D on dendritic cell (DC)-induced stimulation of Th17.1 cells (1). They used an elegant myeloid DC-memory CD4 T cell coculture system to investigate the in vitro mechanisms of UPM-driven IL-17A and IL-22 production, and to address whether glucocorticoid-resistant Th17.1 activation can be inhibited by vitamin D treatment (Figure 1). Exposure to UPM has been known to induce airway inflammation and impair lung function, and is implicated in the pathogenesis of asthma and COPD (2). In mouse models, functional changes in the airways after UPM inhalation were elicited by lymphocyte-derived cytokines (3). Indeed, T lymphocytes stimulated ex vivo with various types of UPM can produce IFN-g, IL-13, IL-17A, and IL-22, suggesting that UPM can induce activation of Th1, Th2, Th17, and Th22 cells, respectively (4, 5). However, UPM was also shown to suppress Th1 responses, indicating that the composition of UPM can drive various types of T cell responses (6). Recent evidence also suggests that the classical T cell subtype paradigm is less rigid than once thought. In fact, T cells can be programmed to produce cytokines characteristic of more than one subset based on extrinsic and intrinsic signals (7, 8). In the present study, Mann and colleagues set out to clarify precisely how UPM alters the cytokine output of T cells, because of the potential of this material to induce a variety of T cell responses (1). The authors found that UPM-primed DCs increased the proportion of memory CD4 T cells with a “Th17.1-like” (9) proinflammatory phenotype. The importance of Th17.1 cells originally emerged in the context of autoimmune diseases. It was shown that in the presence of IL-23 and IL-12, Th17 cells decreased IL-17A and (like Th1 cells) increased IFN-g production. Pathogenicity of these cells required IL-23 receptor activation and was abolished by blockade of IFN-g, but not of IL-17A (10). Indeed, Th17 cells display a remarkable developmental plasticity; share functional characteristics with inducible regulatory T, Th1, and Th22 cells; and coexpress the corresponding lineage-specific transcription factor(s) or cytokine(s) (11). Because this plasticity determines whether Th17 cells will serve immune-protective or pathogenic functions, it poses a major concern. As patients suffering from COPD or asthma are increasingly being grouped into distinct subphenotypes and endotypes based on the mechanisms that drive their disease (12–15), the ability to clarify how Th17 cells function is becoming particularly critical. In the current study, UPM-primed DCs induced CD4 T cells to produce the Th17-associated cytokines IL-17A, IL-17F, and IL-22, as well as IFN-g, granulocyte-macrophage colony-stimulating factor, and granzyme B. A large proportion of the UPM-driven IL-17A cells coexpressed these cytokines, but not IL-10 or IL-13 (1). Notably, although IL-17A is a well-described proinflammatory and host-defense cytokine, IL-22 may have either proor antiinflammatory functions depending on the presence of IL-17A. Coactivation of IL-17R and IL-22R signaling pathways was suggested to synergize in the development of inflammation (16, 17). Further, although granzyme B is known as a cytotoxic T cell granule product and a mediator of cell death in target cells, it can also stimulate proinflammatory cytokines and play a role in asthma (18). Taken together, the results of Mann and colleagues show that UPM promotes a Th17 population with a potentially pathogenic phenotype. The exact factors that control the development of Th17.1 cells in vivo are not well understood. Several cytokines, including IL-1b, IL-12, and IL-23, have been implicated in this process (19). Engagement of Th17 cells with IL-12 and IL-23 promotes the activation of transcription factor Tbet (Tbx21, T-box expressed in T cells), which in turn regulates the expression of IFN-g and related chemokine genes (CXCL9, CXCL10, and CXCL11), leading to the Th17.1 phenotype (20, 21). In addition to Th1 and Th17 cytokines, the pathogenicity of IL-23–driven Th17 cells is dependent on granulocyte-macrophage colony-stimulating factor (22). Further, although high IL-23R expression characterizes pathogenic Th17 cells in mice, human proinflammatory Th17.1 cells (identified as CCR6CXCR3CCR4CCR10CD161, and transiently c-Kit) express multidrug resistance type 1 (MDR1) (23). This is of importance because MDR1 proinflammatory Th17 cells are resistant to several glucocorticoids. In the study by Mann and colleagues, UPM pretreatment of CD1c DCs increased IL-12/23p40 and expression of IL-17a and MDR1 mRNA after 48 hours in coculture supernatants. A neutralizing antibody specific for IL-23p19 (thus inhibiting IL-23, but not IL-12) significantly reduced this effect, but not expression of IL-10. At the protein level, although anti–IL-23p19 had no effect on UPM-driven cell division, it did significantly and specifically reduce the frequency of IL-17AIFN-g cells (1). Previous studies also characterized how antigen, in combination with inhaled UPM, can alter the cytokine output of T cells in mouse models, and how the T cell response in turn affects inflammation and lung function (24, 25). Unraveling the in vivo significance of Th17.1 cells in mouse models and studies in humans should be an exciting and significant future extension of the study by Mann and colleagues. Mann and colleagues found that 1,25(OH)2D3 counteracted the UPM-driven DC maturation and inhibited the frequency of IL-17AIFN-g cells, most prominently when DCs were cotreated with the corticosteroid dexamethasone, while maintaining antiinflammatory IL-10 synthesis. These data indicate that UPM might promote an inflammatory milieu in part by

Neutrophilic inflammation and IL-17 have been implicated in the pathogenesis of severe asthma but the underlying mechanisms remain unclear. We aimed to establish the relationship between inflammatory markers of severe asthma and peripheral blood dendritic cell and lymphocyte populations and their IL-13 and IL-17 expression. Twenty-seven patients with severe asthma all requiring asthma medications and seven non-asthmatic healthy controls were recruited from the UCANTMclinic at the University of California, Davis. Complete blood count, exhaled NO and serum IgE/IgM were investigated. Peripheral blood mononuclear cells were isolated by Ficoll-Paque PLUS and T-cell subsets, dendritic cell subsets, natural killer cells and ILC2 were analyzed by multi-color flow cytometry and compared between the patients and healthy controls. Severe asthma patients had elevated blood neutrophil counts, exhaled NO, serum IgE and IgM. In comparison with healthy controls, the number of circulating CD1c dendritic cells, CD4 T cells, NK cells and ILC2 were significantly increased (p<0.05). Further, the number of CD141 DC (implicated in activation of Th2 cells) significantly correlated with peripheral blood eosinophils (r=0.70) and CD4 T cells. Intracellular IL-13 and IL-17a expression was significantly elevated in CD4 and CD8 T cells, NK cells and ILC2. We showed for the first time the presence of ILC2 producing both IL-13 and IL-17, in the peripheral blood of severe asthma patients. Our data also support that increased IL-17 expression may contribute to the elevated neutrophil count we observed in severe asthma patients.

Genome sequences that contain tandem repeats of guanine can form stable four-stranded structures known as G-quadruplex, or G4 DNA. While the molecular mechanisms are not fully defined, such guanine-rich loci are prone to mutagenesis and recombination. Various repair pathways function to reduce the potential for genome instability by correcting base damage and replication errors; however, it is not yet fully defined how well these processes function at G4 DNA. One frequent form of base damage occurs from cytidine deamination, resulting in deoxyuracil and UG mismatches. In duplex and single-stranded DNA, uracil bases are recognised and excised by uracil glycosylases. Here, we tested the efficiency of uracil glycosylase activity in vitro on uracil bases located directly adjacent to guanine repeats and G4 DNA. We show that uracil excision by bacterial UDG and human hUNG2 is reduced at uracils positioned directly 5' or 3' of a guanine tetrad. Control reactions using oligonucleotides disrupted for G4 formation or reaction conditions that do not favour G4 formation resulted in full uracil excision activity. Based on these in vitro results, we suggest that folding of guanine-rich DNA into G4 DNA results in a DNA conformation that is resistant to uracil glycosylase-initiated repair and this has the potential to increase the risk of instability at guanine repeats in the genome.

The formation of highly stable four-stranded DNA, called G-quadruplex (G4), promotes site-specific genome instability. G4 DNA structures fold from repetitive guanine sequences, and increasing experimental evidence connects G4 sequence motifs with specific gene rearrangements. The human transcription factor 3 (TCF3) gene (also termed E2A) is subject to genetic instability associated with severe disease, most notably a common translocation event t(1;19) associated with acute lymphoblastic leukemia. The sites of instability in TCF3 are not randomly distributed, but focused to certain sequences. We asked if G4 DNA formation could explain why TCF3 is prone to recombination and mutagenesis. Here we demonstrate that sequences surrounding the major t(1;19) break site and a region associated with copy number variations both contain G4 sequence motifs. The motifs identified readily adopt G4 DNA structures that are stable enough to interfere with DNA synthesis in physiological salt conditions in vitro. When introduced into the yeast genome, TCF3 G4 motifs promoted gross chromosomal rearrangements in a transcription-dependent manner. Our results provide a molecular rationale for the site-specific instability of human TCF3, suggesting that G4 DNA structures contribute to oncogenic DNA breaks and recombination.

Meiotic silencing by unpaired DNA (MSUD) is a process that detects unpaired regions between homologous chromosomes and silences them for the duration of sexual development. While the phenomenon of MSUD is well recognized, the process that detects unpaired DNA is poorly understood. In this report, we provide two lines of evidence linking unpaired DNA detection to a physical search for DNA homology. First, we have found that a putative SNF2-family protein (SAD-6) is required for efficient MSUD in Neurospora crassa. SAD-6 is closely related to Rad54, a protein known to facilitate key steps in the repair of double-strand breaks by homologous recombination. Second, we have successfully masked unpaired DNA by placing identical transgenes at slightly different locations on homologous chromosomes. This masking falls apart when the distance between the transgenes is increased. We propose a model where unpaired DNA detection during MSUD is achieved through a spatially constrained search for DNA homology. The identity of SAD-6 as a Rad54 paralog suggests that this process may be similar to the searching mechanism used during homologous recombination.