Sex Differences in the Immune System Become Evident in the Perinatal Period in the Four Core Genotypes Mouse.

We have used the four core genotypes (FCG) mouse model, which allows a distinction between effects of gonadal secretions and chromosomal complement, to determine when sex differences in the immune system first appear and what influences their development. Using splenic T cell number as a measure that could be applied to neonates with as yet immature immune responses, we found no differences among the four genotypes at postnatal day 1, but by day 7, clear sex differences were observed. These sex differences were unexpectedly independent of chromosomal complement and similar in degree to gonadectomized FCG adults: both neonatal and gonadectomized adult females (XX and XY) showed 2-fold the number of CD4+ and 7-fold the number of CD8+ T cells versus their male (XX and XY) counterparts. Appearance of this long-lived sex difference between days 1 and 7 suggested a role for the male-specific perinatal surge of testicular testosterone. Interference with the testosterone surge significantly de-masculinized the male CD4+, but not CD8+ splenic profile. Treatment of neonates demonstrated elevated testosterone limited mature cell egress from the thymus, whereas estradiol reduced splenic T cell seeding in females. Neonatal male splenic epithelium/stroma expressed aromatase mRNA, suggesting capacity for splenic conversion of perinatal testosterone into estradiol in males, which, similar to administration of estradiol in females, would result in reduced splenic T cell seeding. These sex steroid effects affected both CD4+ and CD8+ cells and yet interference with the testosterone surge only significantly de-masculinized the splenic content of CD4+ cells. For CD8+ cells, male cells in the thymus were also found to express one third the density of sphingosine-1-phosphate thymic egress receptors per cell compared to female, a male characteristic most likely an indirect result of Sry expression. Interestingly, the data also support a previously unrecognized role for non-gonadal estradiol in the promotion of intra-thymic cell proliferation in neonates of both sexes. Microarray analysis suggested the thymic epithelium/stroma as the source of this hormone. We conclude that some immune sex differences appear long before puberty and more than one mechanism contributes to differential numbers and distribution of T cells.

Inhaled corticosteroid normalizes some but not all airway vascular remodeling in COPD.

BACKGROUND: This study assessed the effects of inhaled corticosteroid (ICS) on airway vascular remodeling in chronic obstructive pulmonary disease (COPD). METHODS: Thirty-four subjects with mild-to-moderate COPD were randomly allocated 2:1 to ICS or placebo treatment in a double-blinded clinical trial over 6 months. Available tissue was compared before and after treatment for vessel density, and expression of VEGF, TGF-ß1, and TGF-ß1-related phosphorylated transcription factors p-SMAD 2/3. This clinical trial has been registered and allocated with the Australian New Zealand Clinical Trials Registry (ANZCTR) on 17/10/2012 with reference number ACTRN12612001111864. RESULTS: There were no significant baseline differences between treatment groups. With ICS, vessels and angiogenic factors did not change in hypervascular reticular basement membrane, but in the hypovascular lamina propria (LP), vessels increased and this had a proportionate effect on lung air trapping. There was modest evidence for a reduction in LP vessels staining for VEGF with ICS treatment, but a marked and significant reduction in p-SMAD 2/3 expression. CONCLUSION: Six-month high-dose ICS treatment had little effect on hypervascularity or angiogenic growth factors in the reticular basement membrane in COPD, but normalized hypovascularity in the LP, and this was physiologically relevant, though accompanied by a paradoxical reduction in growth factor expression.

A randomized controlled trial of inhaled corticosteroids (ICS) on markers of epithelial-mesenchymal transition (EMT) in large airway samples in COPD: an exploratory proof of concept study.

BACKGROUND: We recently reported that epithelial-mesenchymal transition (EMT) is active in the airways in chronic obstructive pulmonary disease (COPD), suggesting presence of an active profibrotic and promalignant stroma. With no data available on potential treatment effects, we undertook a blinded analysis of inhaled corticosteroids (ICS) effects versus placebo on EMT markers in previously obtained endobronchial biopsies in COPD patients, as a "proof of concept" study. METHODS: Assessment of the effects of inhaled fluticasone propionate (FP; 500 µg twice daily for 6 months) versus placebo in 34 COPD patients (23 on fluticasone propionate and eleven on placebo). The end points were epidermal growth factor receptor (EGFR; marker of epithelial activation) and the biomarkers of EMT: reticular basement membrane (Rbm) fragmentation ("hallmark" structural marker), matrix metalloproteinase-9 (MMP-9) cell expression, and S100A4 expression in basal epithelial and Rbm cells (mesenchymal transition markers). RESULTS: Epithelial activation, "clefts/fragmentation" in the Rbm, and changes in the other biomarkers all regressed on ICS, at or close to conventional levels of statistical significance. From these data, we have been able to nominate primary and secondary end points and develop power calculations that would be applicable to a definitive prospective study. CONCLUSION: Although only a pilot "proof of concept" study, this trial provided strong suggestive support for an anti-EMT effect of ICS in COPD airways. A larger and fully powered prospective study is now indicated as this issue is likely to be extremely important. Such studies may clarify the links between ICS use and better clinical outcomes and protection against lung cancer in COPD.

Plasticity of melanoma in vivo: murine lesions resulting from Trp53, but not Cdk4 or Arf deregulation, display neural transdifferentiation.

We previously noted that melanomas developing in Cdk4(R24C/R24C) ::Tyr-NRAS, Arf(-/-) ::Tyr-NRAS and Trp53(F/F) ::Tyr-Cre(ER)::Tyr-NRAS mice exhibited differences in behaviour in vivo. We investigated this phenomenon using global gene expression profiling of lesions from the respective genotypes. While those from the Cdk4- and Arf-mutant mice exhibited similar profiles, the Trp53(F/F) ::Tyr-Cre(ER)::Tyr-NRAS melanomas were strikingly different, showing relative down-regulation of melanocyte-related genes, and up-regulation of genes related to neural differentiation. Specifically, they highly expressed genes representative of the myelin-producing peripheral oligodendrite (Schwann cell) lineage, although histopathologically the lesions did not exhibit the classical features of schwannoma. As Schwann cell precursors can be a cellular origin of melanocytes, it is unsurprising that plasticity with respect to melanocyte-neural differentiation can occur in melanoma. What is surprising is the genotype proclivity. Comparison of gene expression signatures revealed that melanomas from the Trp53-mutant mice show significant similarities with a subset of aggressive human melanomas with relatively low levels of MITF.

Ultraviolet radiation effects on the proteome of skin cells.

Proteomic studies to date have had limited use as an investigative tool in the skin's response to UV radiation. These studies used cell lines and reconstructed skin and have shown evidence of cell injury with oxidative damage and stress induced heat shock proteins. Others changes included altered cytokeratin and cytoskeletal proteins with enhanced expression of TRIM29 as the keratinocytes regenerate. The associated DNA repair requires polη, Rad18/Rad16 and Rev1. In the whole animal these events would be associated with inflammation, remodelling of the epidermis and modulation of the immune response. Longer term changes include ageing and skin cancers such as melanoma, squamous cell carcinoma and basal cell carcinoma. In the future proteomics will be used to explore these important aspects of photobiology. Better characterisation of the proteins involved should lead to a greater understanding of the skin's response to UV radiation.

UVB-induced melanocyte proliferation in neonatal mice driven by CCR2-independent recruitment of Ly6c(low)MHCII(hi) macrophages.

Intermittent sunburns, particularly in childhood, are the strongest environmental risk factor for malignant melanoma (MM). In mice, a single neonatal UVR exposure induces MM, whereas chronic doses to adult mice do not. Neonatal UVR alters melanocyte migration dynamics by inducing their movement upward out of hair follicles into the epidermis. UVR is known to induce inflammation and recruitment of macrophages into the skin. In this study, we have used a liposomal clodronate strategy to deplete macrophages at the time of neonatal UVR, and have shown functionally that this reduces the melanocyte proliferative response. This effect was not reproduced by depletion of CD11c-expressing populations of dendritic cells. On the basis of epidermal expression array data at various time points after UVR, we selected mouse strains defective in various aspects of macrophage recruitment, activation, and effector functions, and measured their melanocyte UVR response. We identified Ly6c(low)MHCII(hi) macrophages as the major population promoting the melanocyte response across multiple strains. The activity of this subpopulation was CCR2 (C-C chemokine receptor type 2) independent and partly IL-17 dependent. By helping induce this effect, the infiltration of specific macrophage subpopulations after sunburn may be a factor in increasing the risk of subsequent neoplastic transformation of melanocytes.

Evaluation of epithelial mesenchymal transition in patients with chronic obstructive pulmonary disease.

BACKGROUND: The reticular basement membrane (Rbm) in smokers and especially smokers with COPD is fragmented with "clefts" containing cells staining for the collagenase matrix-metalloproteinase-9 (MMP-9) and fibroblast protein, S100A4. These cells are also present in the basal epithelium. Such changes are likely hallmarks of epithelial mesenchymal transition (EMT). We aimed to confirm the epithelial origin of these Rbm cells, and to exclude potential confounding by infiltrating inflammatory cells. METHODS: Endobronchial biopsy sections from 17 COPD current smokers, with documented Rbm splitting and cellularity were stained for neutrophil elastase (neutrophil marker), CD68 (macrophage/mature fibroblasts), CD4+/CD8+ T lymphocytes, CD19 (B-cells), CD11c (dendritic cells/inflammatory cells), and S100 (Langerhans cells). The number of cells in the Rbm and epithelium staining for these "inflammatory" cell markers were then compared to numbers staining for S100A4, "a documented EMT epitope". Slides were double stained for S100A4 and cytokeratin(s). RESULTS: In the basal epithelium significantly more cells stained for S100A4 compared to infiltrating macrophages, fibroblasts or immune cells: median, 26 (21.3 - 37.3) versus 0 (0 - 9.6) per mm, p < 0.003. Markedly more S100A4 staining cells were also observed in the Rbm compared to infiltrating macrophages, neutrophils, fibroblasts or immune cells or any sub-type: 58 (37.3 - 92.6) versus 0 (0 - 4.8) cells/mm Rbm, p < 0.003. Cells in the basal epithelium 26 (21.3 - 37.3) per mm) and Rbm (5.9 (2.3 - 13.8) per mm) frequently double stained for both cytokeratin and S100A4. CONCLUSIONS: These data provide additional support for active EMT in COPD airways.

Neonatal exposure to UVR alters skin immune system development, and suppresses immunity in adulthood.

Neonates have a developing immune response, with a predisposition towards induction of tolerance. As the immune system develops, immunity rather than tolerance is induced, with this development of immunity occurring in response to external factors such as the environment. As ultraviolet radiation (UVR) suppresses immunity, it is likely that the effect of UVR on the neonatal immune system would be augmentation of the suppressive response. In support, childhood exposure to UVR has been linked with an increased incidence of melanoma; consistent with an increase in suppression. To address this, phenotypic and functional immune system studies were undertaken at 8 weeks after one single exposure of solar-simulated UVR to mice, when mice had reached adulthood. Subtle changes were observed in cell populations resident in the skin-draining lymph nodes (LNs) and there also appeared to be a subtle, but not statistically significant, increase in the production of interleukin-10 and interferon-γ. Importantly, these changes also corresponded with significant suppression of the contact hypersensitivity response in irradiated mice compared with their control counterparts. This suppression was apparent when antigen sensitisation occurred during the neonatal or adult period, and thus did not appear to be analogous to UVR-induced suppression in adults. Although the percentage of T regulatory cells was increased in the skin-draining LNs, they were induced in a different manner to those induced following adult UVR exposure, with no increase in function on a per-cell basis. It therefore appears that one single neonatal exposure to UVR alters development of the immune system, leading to long-term implications for induction of immunity.

Basement membrane and vascular remodelling in smokers and chronic obstructive pulmonary disease: a cross-sectional study.

BACKGROUND: Little is known about airway remodelling in bronchial biopsies (BB) in smokers and chronic obstructive pulmonary disease (COPD). We conducted an initial pilot study comparing BB from COPD patients with nonsmoking controls. This pilot study suggested the presence of reticular basement membrane (Rbm) fragmentation and altered vessel distribution in COPD. METHODS: To determine whether Rbm fragmentation and altered vessel distribution in BB were specific for COPD we designed a cross-sectional study and stained BB from 19 current smokers and 14 ex-smokers with mild to moderate COPD and compared these to 15 current smokers with normal lung function and 17 healthy and nonsmoking subjects. RESULTS: Thickness of the Rbm was not significantly different between groups; although in COPD this parameter was quite variable. The Rbm showed fragmentation and splitting in both current smoking groups and ex-smoker COPD compared with healthy nonsmokers (p < 0.02); smoking and COPD seemed to have additive effects. Rbm fragmentation correlated with smoking history in COPD but not with age. There were more vessels in the Rbm and fewer vessels in the lamina propria in current smokers compared to healthy nonsmokers (p < 0.05). The number of vessels staining for vascular endothelial growth factor (VEGF) in the Rbm was higher in both current smoker groups and ex-smoker COPD compared to healthy nonsmokers (p < 0.004). In current smoker COPD VEGF vessel staining correlated with FEV1% predicted (r = 0.61, p < 0.02). CONCLUSIONS: Airway remodelling in smokers and mild to moderate COPD is associated with fragmentation of the Rbm and altered distribution of vessels in the airway wall. Rbm fragmentation was also present to as great an extent in ex-smokers with COPD. These characteristics may have potential physiological consequences.

Reticular basement membrane fragmentation and potential epithelial mesenchymal transition is exaggerated in the airways of smokers with chronic obstructive pulmonary disease.

BACKGROUND AND OBJECTIVE: In COPD, the airways are chronically inflamed, and we have now observed fragmentation of the reticular basement membrane (Rbm). This appears to be a hallmark of the process known as epithelial mesenchymal transition (EMT), in which epithelial cells migrate through the Rbm and differentiate into fibroblasts. The aim of this study was to confirm the extent and relevance of Rbm fragmentation in smokers and patients with COPD, and to undertake a preliminary analysis of some classical markers of EMT. METHODS: Endobronchial biopsies from current smokers (CS; n = 17) and ex-smokers with COPD (ES; n = 15), smokers with normal lung function (NS; n = 16) and never-smoking control subjects (NC; n = 15) were stained for the EMT markers, S100A4, vimentin, epidermal growth factor receptor and matrix metalloproteinase-9. RESULTS: Compared with NC, there was significant Rbm fragmentation in the CS, ES and NS groups, which was positively associated with smoking history in subjects with COPD. Staining for basal epithelial S100A4, epithelial epidermal growth factor receptor and matrix metalloproteinase-9 in cells within Rbm clefts, and for S100A4 in Rbm cells, was increased in the CS, NS and ES groups compared with the NC group. There was also increased Rbm cell S100A4 staining in the CS group compared with the ES and NS groups. Basal epithelial cell staining for S100A4 was inversely correlated with airflow limitation. Double staining for both S100A4 and vimentin further strengthened the likelihood that these changes represented active EMT. CONCLUSIONS: This is the first detailed description of fragmentation and cellularity of the Rbm in smokers, which were most marked in subjects with COPD. The data are consistent with active EMT in these subjects.

Enhancement of DNA repair using topical T4 endonuclease V does not inhibit melanoma formation in Cdk4(R24C/R24C)/Tyr-Nras(Q61K) mice following neonatal UVR.

To further investigate the use of DNA repair-enhancing agents for skin cancer prevention, we treated Cdk4(R24C/R24C)/Nras(Q61K) mice topically with the T4 endonuclease V DNA repair enzyme (known as Dimericine) immediately prior to neonatal ultraviolet radiation (UVR) exposure, which has a powerful effect in exacerbating melanoma development in the mouse model. Dimericine has been shown to reduce the incidence of basal-cell and squamous cell carcinoma. Unexpectedly, we saw no difference in penetrance or age of onset of melanoma after neonatal UVR between Dimericine-treated and control animals, although the drug reduced DNA damage and cellular proliferation in the skin. Interestingly, epidermal melanocytes removed cyclobutane pyrimidine dimers (CPDs) more efficiently than surrounding keratinocytes. Our study indicates that neonatal UVR-initiated melanomas may be driven by mechanisms other than solely that of a large CPD load and/or their inefficient repair. This is further suggestive of different mechanisms by which UVR may enhance the transformation of keratinocytes and melanocytes.

Murine neonatal melanocytes exhibit a heightened proliferative response to ultraviolet radiation and migrate to the epidermal basal layer.

Melanocytes respond to UVR not only by producing melanin, but also by proliferating. This is essentially a protective response. We have studied the melanocyte proliferative response after a single UVR exposure to neonatal mice. At 3 days post-UVR in wild-type neonates we observed a marked melanocyte activation not seen in adults. Melanocytes migrated to the epidermal basal layer, their numbers peaking at 3-5 days after UVR then diminishing. They appeared to emanate from the hair follicle, migrating to the epidermis via the outer root sheath. In melanoma-prone mice with melanocyte-specific overexpression of Hras(G12V), basal layer melanocytes were increased in size and dendricity compared to UVR-treated wild-type mice. Melanocytes in mice carrying a pRb pathway cell-cycle defect (oncogenic Cdk4(R24C)) did not show an enhanced response to UVR such as those carrying Hras(G12V). The exquisite sensitivity to UVR-induced proliferation and migration that characterizes neonatal mouse melanocytes may partly explain the utility of this form of exposure for inducing melanoma in mice that carry oncogenic mutations.

Effect of UV radiation on the neonatal skin immune system- implications for melanoma.

The neonatal immune environment and the events that occur during this time have profound effects for the adult period. While protective immune responses can develop, the neonatal immune system, particularly the skin immune system (SIS), tends to promote tolerance. With this information we undertook a number of studies to identify unique aspects of skin during the neonatal period. Proteomics revealed proteins uniquely expressed in neonatal, but not adult, skin (e.g. Stefin A, peroxiredoxins) and these may have implications in the development of SIS. Vitamin D was found to have a modulating role on SIS and this was apparent from the early neonatal period. Exposure of the neonatal skin to UV radiation altered the microenvironment resulting in the generation of regulatory T cells, which persisted in adult life. As the development of UV radiation-induced melanoma can occur following a single high dose (equivalent to burning in adults) to transgenic mice (hepatocyte growth factor/scatter factor or TPras) during the neonatal period, the early modulating events which lead to suppression may be relevant for the development of UV radiation-induced human melanoma. Any attempt to produce effective melanoma immunotherapy has to accommodate and overcome these barriers. Margaret Kripke's pioneering work on UV-induced immunosuppression still remains central to the understanding of the development of melanoma and how it frequently escapes the immune system.

Local cytokine levels associated with delayed-type hypersensitivity responses: modulation by gender, ovariectomy, and estrogen replacement.

It is well established that females mount stronger immune responses than males, but only very little is understood about the underlying mechanisms. We have analyzed local cytokine differences among intact females, those that had been ovariectomized (OVX), those receiving estrogen replacement after OVX, and males, both before and after production of delayed-type hypersensitivity (DTH) responses. We report confirmation of a much larger DTH response in females versus males. However, OVX resulted in an even larger response, while estrogen replacement resulted in a smaller response when compared with intact females. In animals exposed for the first time to an antigen (without a DTH response), OVX increased interleukin-6 (IL-6) and estrogen replacement after OVX suppressed IL-6. Of the cytokines that differed between males and females exposed for the first time to an antigen, only IL-6 was higher in females versus males when exposure to antigen occurred for the second time (when the DTH response occurs). Analysis of cytokines with OVX and estrogen replacement after a second exposure to antigen showed that IL-6 did not significantly change. Levels of IL-4; Regulated upon Activation, Normal T-cell Expressed; and Secreted; and thrombopoietin, however, correlated with the DTH response, suggesting direct or indirect positive regulation by estrogen. These results suggest an important role for both IL-6 and IL-4 in determining the degree of DTH response, with IL-6 (which appears negatively regulated by estrogen) increasing and IL-4 (which appears positively regulated by estrogen) decreasing the response. The results further suggest that IL-6 may play a role in predisposing to a larger DTH response, while IL-4 levels seem more important during an active response.

Broad tumor spectrum in a mouse model of multiple endocrine neoplasia type 1.

Multiple endocrine neoplasia type 1 (MEN1) is an inherited cancer predisposition syndrome typified by development of tumors in parathyroid, pituitary and endocrine pancreas, as well as less common sites including both endocrine and nonendocrine organs. Deletion or mutation of the tumor suppressor gene MEN1 on chromosome 11 has been identified in many cases of MEN1 as well as in sporadic tumors. The molecular biology of menin, the protein encoded by MEN1, remains poorly understood. Here we describe a mouse model of MEN1 in which tumors were seen in pancreatic islets, pituitary, thyroid and parathyroid, adrenal glands, testes and ovaries. The observed tumor spectrum therefore includes types commonly seen in MEN1 patients and additional types. Pancreatic pathology was most common, evident in over 80% of animals, while other tumor types developed with lower frequency and generally later onset. Tumors of multiple endocrine organs were observed frequently, but progression to carcinoma and metastasis were not evident. Tumors in all sites showed loss of heterozygosity at the Men1 locus, though the frequency in testicular tumors was only 36%, indicating that a different molecular mechanism of tumorigenesis occurs in those Leydig tumors that do not show loss of the normal Men1 allele. Menin expression was below the level of detection in ovary, thyroid and testis, but loss of nuclear menin immunoreactivity was observed uniformly in all pancreatic islet adenomas and in some hyperplastic islet cells, suggesting that complete loss of Men1 is a critical point in islet tumor progression in this model.

Spontaneous and UV radiation-induced multiple metastatic melanomas in Cdk4R24C/R24C/TPras mice.

Human melanoma susceptibility is often characterized by germ-line inactivating CDKN2A (INK4A/ARF) mutations, or mutations that activate CDK4 by preventing its binding to and inhibition by INK4A. We have previously shown that a single neonatal UV radiation (UVR) dose delivered to mice that carry melanocyte-specific activation of Hras (TPras) increases melanoma penetrance from 0% to 57%. Here, we report that activated Cdk4 cooperates with activated Hras to enhance susceptibility to melanoma in mice. Whereas UVR treatment failed to induce melanomas in Cdk4(R24C/R24C) mice, it greatly increased the penetrance and decreased the age of onset of melanoma development in Cdk4(R24C/R24C)/TPras animals compared with TPras alone. This increased penetrance was dependent on the threshold of Cdk4 activation as Cdk4(R24C/+)/TPras animals did not show an increase in UVR-induced melanoma penetrance compared with TPras alone. In addition, Cdk4(R24C/R24C)/TPras mice invariably developed multiple lesions, which occurred rarely in TPras mice. These results indicate that germ-line defects abrogating the pRb pathway may enhance UVR-induced melanoma. TPras and Cdk4(R24C/R24C)/TPras tumors were comparable histopathologically but the latter were larger and more aggressive and cultured cells derived from such melanomas were also larger and had higher levels of nuclear atypia. Moreover, the melanomas in Cdk4(R24C/R24C)/TPras mice, but not in TPras mice, readily metastasized to regional lymph nodes. Thus, it seems that in the mouse, Hras activation initiates UVR-induced melanoma development whereas the cell cycle defect introduced by mutant Cdk4 contributes to tumor progression, producing more aggressive, metastatic tumors.

Melanocytes in conditional Rb-/- mice are normal in vivo but exhibit proliferation and pigmentation defects in vitro.

The function of the retinoblastoma tumour suppressor (Rb1), and the pocket protein family in general, has been implicated as an important focal point for deregulation in many of the molecular pathways mutated in melanoma. We have focused on the role of Rb1 in mouse melanocyte homeostasis using gene targeting and Cre/loxP mediated tissue-specific deletion. We show that constitutive Cre-mediated ablation of Rb1 exon 2 prevents the production of Rb1 and recapitulates the phenotype encountered in other Rb1 knockout mouse models. Mice with conditional melanocyte-specific ablation of Rb1 manifest overtly normal pigmentation and are bereft of melanocytic hyperproliferative defects or apoptosis-induced depigmentation. Histologically, these mice have melanocyte morphology and distribution comparable with control littermates. In contrast, Rb1-null melanocytes removed from their in vivo micro-environment and cultured in vitro display some of the characteristics associated with a transformed phenotype. They proliferate at a heightened rate when compared with control melanocytes and have a decreased requirement for mitogens. With progressive culture the cells depigment at relatively early passage and display a gross morphology which, whilst reminiscent of early passage melanocytes, is generally different to equivalent passage control cells. These results indicate that Rb1 is dispensable for in vivo melanocyte homeostasis when its ablation is targeted from the melanoblast stage onwards, however, when cultured in vitro, Rb1 loss increases melanocyte growth but the cells are not fully transformed.