The astrocyte-produced growth factor HB-EGF limits autoimmune CNS pathology.

Central nervous system (CNS)-resident cells such as microglia, oligodendrocytes and astrocytes are gaining increasing attention in respect to their contribution to CNS pathologies including multiple sclerosis (MS). Several studies have demonstrated the involvement of pro-inflammatory glial subsets in the pathogenesis and propagation of inflammatory events in MS and its animal models. However, it has only recently become clear that the underlying heterogeneity of astrocytes and microglia can not only drive inflammation, but also lead to its resolution through direct and indirect mechanisms. Failure of these tissue-protective mechanisms may potentiate disease and increase the risk of conversion to progressive stages of MS, for which currently available therapies are limited. Using proteomic analyses of cerebrospinal fluid specimens from patients with MS in combination with experimental studies, we here identify Heparin-binding EGF-like growth factor (HB-EGF) as a central mediator of tissue-protective and anti-inflammatory effects important for the recovery from acute inflammatory lesions in CNS autoimmunity. Hypoxic conditions drive the rapid upregulation of HB-EGF by astrocytes during early CNS inflammation, while pro-inflammatory conditions suppress trophic HB-EGF signaling through epigenetic modifications. Finally, we demonstrate both anti-inflammatory and tissue-protective effects of HB-EGF in a broad variety of cell types in vitro and use intranasal administration of HB-EGF in acute and post-acute stages of autoimmune neuroinflammation to attenuate disease in a preclinical mouse model of MS. Altogether, we identify astrocyte-derived HB-EGF and its epigenetic regulation as a modulator of autoimmune CNS inflammation and potential therapeutic target in MS.

An unanticipated discourse of HB-EGF with VANGL2 signaling during embryo implantation.

Implantation is the first direct encounter between the embryo and uterus during pregnancy, and Hbegf is the earliest known molecular signaling for embryo-uterine crosstalk during implantation. The downstream effectors of heparin-binding EGF (HB-EGF) in implantation remain elusive due to the complexity of EGF receptor family. This study shows that the formation of implantation chamber (crypt) triggered by HB-EGF is disrupted by uterine deletion of Vangl2, a key planar cell polarity component (PCP). We found that HB-EGF binds to ERBB2 and ERBB3 to recruit VANGL2 for tyrosine phosphorylation. Using in vivo models, we show that uterine VAGL2 tyrosine phosphorylation is suppressed in Erbb2/Erbb3 double conditional knockout mice. In this context, severe implantation defects in these mice lend support to the critical role of HB-EGF-ERBB2/3-VANGL2 in establishing a two-way dialogue between the blastocyst and uterus. In addition, the result addresses an outstanding question how VANGL2 is activated during implantation. Taken together, these observations reveal that HB-EGF regulates the implantation process by influencing uterine epithelial cell polarity comprising VANGL2.

The chromatin-modifying enzyme Ezh2 is critical for the maintenance of regulatory T cell identity after activation.

Regulatory T cells (Treg cells) are required for immune homeostasis. Chromatin remodeling is essential for establishing diverse cellular identities, but how the epigenetic program in Treg cells is maintained throughout the dynamic activation process remains unclear. Here we have shown that CD28 co-stimulation, an extracellular cue intrinsically required for Treg cell maintenance, induced the chromatin-modifying enzyme, Ezh2. Treg-specific ablation of Ezh2 resulted in spontaneous autoimmunity with reduced Foxp3(+) cells in non-lymphoid tissues and impaired resolution of experimental autoimmune encephalomyelitis. Utilizing a model designed to selectively deplete wild-type Treg cells in adult mice co-populated with Ezh2-deficient Treg cells, Ezh2-deficient cells were destabilized and failed to prevent autoimmunity. After activation, the transcriptome of Ezh2-deficient Treg cells was disrupted, with altered expression of Treg cell lineage genes in a pattern similar to Foxp3-deficient Treg cells. These studies reveal a critical role for Ezh2 in the maintenance of Treg cell identity during cellular activation.

Altered ratio of dendritic cell subsets in skin-draining lymph nodes promotes Th2-driven contact hypersensitivity.

Plasmacytoid dendritic cells (pDCs) specialize in the production of type I IFN (IFN-I). pDCs can be depleted in vivo by injecting diphtheria toxin (DT) in a mouse in which pDCs express a diphtheria toxin receptor (DTR) transgene driven by the human CLEC4C promoter. This promoter is enriched for binding sites for TCF4, a transcription factor that promotes pDC differentiation and expression of pDC markers, including CLEC4C. Here, we found that injection of DT in CLEC4C-DTR+ mice markedly augmented Th2-dependent skin inflammation in a model of contact hypersensitivity (CHS) induced by the hapten fluorescein isothiocyanate. Unexpectedly, this biased Th2 response was independent of reduced IFN-I accompanying pDC depletion. In fact, DT treatment altered the representation of conventional dendritic cells (cDCs) in the skin-draining lymph nodes during the sensitization phase of CHS; there were fewer Th1-priming CD326+ CD103+ cDC1 and more Th2-priming CD11b+ cDC2. Single-cell RNA-sequencing of CLEC4C-DTR+ cDCs revealed that CD326+ DCs, like pDCs, expressed DTR and were depleted together with pDCs by DT treatment. Since CD326+ DCs did not express Tcf4, DTR expression might be driven by yet-undefined transcription factors activating the CLEC4C promoter. These results demonstrate that altered DC representation in the skin-draining lymph nodes during sensitization to allergens can cause Th2-driven CHS.

Profibrotic properties of C1q+ interstitial macrophages in silica-induced pulmonary fibrosis in mice.

Pulmonary fibrosis (PF) is a progressive fibrotic disease with poor prognosis and suboptimal therapeutic options. Although macrophages have been implicated in PF, the role of macrophage subsets, particularly interstitial macrophages (IMs), remains unknown. We performed a time-series single-cell RNA sequencing analysis of the silica-induced mouse PF model. Among the macrophage subsets in fibrotic lungs, Lyve1lo MHC IIhi IMs increased with fibrosis, and highly expressed profibrotic genes. Additionally, we identified C1q as an IM-specific marker. Experiments with C1q-diphtheria toxin receptor-GFP knock-in (C1qKI) mice revealed that IMs are distributed around fibrotic nodules. Depletion of C1q+ IMs in C1qKI mice decreased activated fibroblasts and epithelial cells; however, bodyweight loss and neutrophil infiltration were exacerbated in silica-induced PF. Collectively, these results suggest that IMs have profibrotic and anti-inflammatory properties and that the selective inhibition of the profibrotic function of IMs without compromising their anti-inflammatory effects is a potential novel therapeutic strategy for PF.

Combining gene expression analysis of gastric cancer cell lines and tumor specimens to identify biomarkers for anti-HER therapies-the role of HAS2, SHB and HBEGF.

BACKGROUND: The standard treatment for patients with advanced HER2-positive gastric cancer is a combination of the antibody trastuzumab and platin-fluoropyrimidine chemotherapy. As some patients do not respond to trastuzumab therapy or develop resistance during treatment, the search for alternative treatment options and biomarkers to predict therapy response is the focus of research. We compared the efficacy of trastuzumab and other HER-targeting drugs such as cetuximab and afatinib. We also hypothesized that treatment-dependent regulation of a gene indicates its importance in response and that it can therefore be used as a biomarker for patient stratification. METHODS: A selection of gastric cancer cell lines (Hs746T, MKN1, MKN7 and NCI-N87) was treated with EGF, cetuximab, trastuzumab or afatinib for a period of 4 or 24 h. The effects of treatment on gene expression were measured by RNA sequencing and the resulting biomarker candidates were tested in an available cohort of gastric cancer patients from the VARIANZ trial or functionally analyzed in vitro. RESULTS: After treatment of the cell lines with afatinib, the highest number of regulated genes was observed, followed by cetuximab and trastuzumab. Although trastuzumab showed only relatively small effects on gene expression, BMF, HAS2 and SHB could be identified as candidate biomarkers for response to trastuzumab. Subsequent studies confirmed HAS2 and SHB as potential predictive markers for response to trastuzumab therapy in clinical samples from the VARIANZ trial. AREG, EREG and HBEGF were identified as candidate biomarkers for treatment with afatinib and cetuximab. Functional analysis confirmed that HBEGF is a resistance factor for cetuximab. CONCLUSION: By confirming HAS2, SHB and HBEGF as biomarkers for anti-HER therapies, we provide evidence that the regulation of gene expression after treatment can be used for biomarker discovery. TRIAL REGISTRATION: Clinical specimens of the VARIANZ study (NCT02305043) were used to test biomarker candidates.

Elevated HB-EGF expression in neural stem cells causes middle age obesity by suppressing Hypocretin/Orexin expression.

Obesity is common in the middle aged population and it increases the risks of diabetes, cardiovascular diseases, certain cancers, and dementia. Yet, its etiology remains incompletely understood. Here, we show that ectopic expression of HB-EGF, an important regulator of neurogenesis, in Nestin+ neuroepithelial progenitors with the Cre-LoxP system leads to development of spontaneous middle age obesity in male mice accompanied by hyperglycemia and insulin resistance. The Nestin-HB-EGF mice show decreases in food uptake, energy expenditure, and physical activity, suggesting that reduced energy expenditure underlies the pathogenesis of this obesity model. However, HB-EGF expression in appetite-controlling POMC or AgRP neurons or adipocytes fails to induce obesity. Mechanistically, HB-EGF suppresses expression of Hypocretin/Orexin, an orexigenic neuropeptide hormone, in the hypothalamus of middle aged Nestin-HB-EGF mice. Hypothalamus Orexin administration alleviates the obese and hyperglycemic phenotypes in Nestin-HB-EGF mice. This study uncovers an important role for HB-EGF in regulating Orexin expression and energy expenditure and establishes a midlife obesity model whose pathogenesis involves age-dependent changes in hypothalamus neurons.

Microglial ablation in rats disrupts the circadian system.

Microglia, the key neuroimmune cells of the central nervous system, are best known for their function in defending an individual from pathogens and injury. Recent findings, including our own, suggest microglia also have several immune-independent roles, including in regulating satiety, promoting memory, and modifying pain responses. Many of these microglia-associated functions are affected by circadian rhythmicity, thus, varying substantially depending upon the time of day. To gain further insight into this link, we used a Cx3cr1-Dtr transgenic Wistar rat model to acutely deplete microglia and examined if this could lead to a disruption in diurnal temperature, metabolism, and activity measures. We also examined if differences in the physiological rhythms corresponded with changes in the expression of key circadian rhythm-regulating genes and proteins. Our data show that in the absence of microglia there is a pronounced disruption of diurnal rhythms in several domains consistent with a shift toward the inactive phase, in conjunction with changes in circadian rhythm-regulating genes and proteins. These data suggest microglia are involved in the regulation of circadian rhythms and indicate an exciting potential to manipulate these cells to improve disrupted circadian rhythms such as with shift-work or jet-lag.

Progressive Cellular Senescence Mediates Renal Dysfunction in Ischemic Nephropathy.

BACKGROUND: Peripheral vascular diseases may induce chronic ischemia and cellular injury distal to the arterial obstruction. Cellular senescence involves proliferation arrest in response to stress, which can damage neighboring cells. Renal artery stenosis (RAS) induces stenotic-kidney dysfunction and injury, but whether these arise from cellular senescenceand their temporal pattern remain unknown. METHODS: Chronic renal ischemia was induced in transgenic INK-ATTAC and wild type C57BL/6 mice by unilateral RAS, and kidney function (in vivo micro-MRI) and tissue damage were assessed. Mouse healthy and stenotic kidneys were analyzed using unbiased single-cell RNA-sequencing. To demonstrate translational relevance, cellular senescence was studied in human stenotic kidneys. RESULTS: Using intraperitoneal AP20187 injections starting 1, 2, or 4 weeks after RAS, selective clearance of cells highly expressing p16Ink4a attenuated cellular senescence and improved stenotic-kidney function; however, starting treatment immediately after RAS induction was unsuccessful. Broader clearance of senescent cells, using the oral senolytic combination dasatinib and quercetin, in C57BL/6 RAS mice was more effective in clearing cells positive for p21 (Cdkn1a) and alleviating renal dysfunction and damage. Unbiased, single-cell RNA sequencing in freshly dissociated cells from healthy and stenotic mouse kidneys identified stenotic-kidney epithelial cells undergoing both mesenchymal transition and senescence. As in mice, injured human stenotic kidneys exhibited cellular senescence, suggesting this process is conserved. CONCLUSIONS: Maladaptive tubular cell senescence, involving upregulated p16 (Cdkn2a), p19 (Cdkn2d), and p21 (Cdkn1a) expression, is associated with renal dysfunction and injury in chronic ischemia. These findings support development of senolytic strategies to delay chronic ischemic renal injury.

Geniposide alleviates choroidal neovascularization by downregulating HB-EGF release from RPE cells by downregulating the miR-145-5p/NF-κB axis.

Age-related macular degeneration (AMD), mainly wet AMD, is the major reason for nonreversible vision loss worldwide. Choroidal neovascularization (CNV) is a characteristic pathological manifestation of wet AMD. Stress or injury to the retinal pigment epithelium (RPE) induces proangiogenic factors that drive CNV. An iridoid glycoside extracted from the fruit of gardenia, geniposide (GEN) plays an antiangiogenic role. In this study, GEN inhibited the transcription and expression of heparin-binding epidermal growth factor (HB-EGF), a proangiogenic factor, in hypoxic RPE cells and a mouse laser-induced CNV model. Inhibition of glucagon-like peptide-1 receptor (GLP-1R), a GEN receptor blocker, eliminated the protective effect of GEN. Additionally, GEN decreased the transcription and expression of HB-EGF in hypoxia-exposed RPE cells by downregulating the miR-145-5p/NF-κB axis. Therefore, our research provides a promising novel strategy for wet AMD therapy.

HB-EGF Synthesized by CD4 T Cells Modulates Allergic Airway Eosinophilia by Regulating IL-5 Synthesis.

CD4 T cells express the epidermal growth factor (EGF) receptor ligand, heparin-binding EGF (HB-EGF), with no defined immuno-pathophysiological function. Therefore, we wished to elucidate the function of HB-EGF synthesized by CD4 T cells in the context of allergic pulmonary inflammation and the asthma surrogate, airway hyperresponsiveness, in a murine acute model of asthma. In this study, we show how knocking out HB-EGF expression in CD4 T cells in vivo attenuates IL-5 synthesis in the lung that is accompanied by diminished eosinophilic inflammation and airway hyperresponsiveness. HB-EGF coimmunoprecipitates with the transcriptional repressor B cell lymphoma 6 (Bcl-6) in CD4 T cells. Knocking out HB-EGF in CD4 T cells resulted in increased Bcl-6 binding to the IL-5 gene and decreased IL-5 mRNA expression. Thus, these findings suggest an immunoregulatory function for intrinsic HB-EGF expressed by CD4 T cells in TH2 inflammation and airway dysfunction by modulating IL-5 expression via binding to and inhibiting the repressive function of Bcl-6.

Failure of Diphtheria Toxin Model to Induce Parkinson-Like Behavior in Mice.

Rodent models of Parkinson's disease are based on transgenic expression of mutant synuclein, deletion of PD genes, injections of MPTP or rotenone, or seeding of synuclein fibrils. The models show histopathologic features of PD such as Lewi bodies but mostly only subtle in vivo manifestations or systemic toxicity. The models only partly mimic a predominant loss of dopaminergic neurons in the substantia nigra. We therefore generated mice that express the transgenic diphtheria toxin receptor (DTR) specifically in DA neurons by crossing DAT-Cre mice with Rosa26 loxP-STOP-loxP DTR mice. After defining a well-tolerated DTx dose, DAT-DTR and DTR-flfl controls were subjected to non-toxic DTx treatment (5 × 100 pg/g) and subsequent histology and behavioral tests. DAT protein levels were reduced in the midbrain, and tyrosine hydroxylase-positive neurons were reduced in the substantia nigra, whereas the pan-neuronal marker NeuN was not affected. Despite the promising histologic results, there was no difference in motor function tests or open field behavior. These are tests in which double mutant Pink1-/-SNCAA53T Parkinson mice show behavioral abnormalities. Higher doses of DTx were toxic in both groups. The data suggest that DTx treatment in mice with Cre/loxP-driven DAT-DTR expression leads to partial ablation of DA-neurons but without PD-reminiscent behavioral correlates.

Cytokine regulation of apoptosis-induced apoptosis and apoptosis-induced cell proliferation in vascular smooth muscle cells.

Vascular smooth muscle cells (VSMCs) are the main structural cell of blood vessels, and VSMC apoptosis occurs in vascular disease, after injury, and in vessel remodeling during development. Although VSMC apoptosis is viewed as silent, recent studies show that apoptotic cells can promote apoptosis-induced compensatory proliferation (AICP), apoptosis-induced apoptosis (AIA), and migration of both local somatic and infiltrating inflammatory cells. However, the effects of VSMC apoptosis on adjacent VSMCs, and their underlying signaling and mechanisms are unknown. We examined the consequences of VSMC apoptosis after activating extrinsic and intrinsic death pathways. VSMCs undergoing apoptosis through Fas/CD95 or the protein kinase inhibitor staurosporine transcriptionally activated interleukin 6 (IL-6) and granulocyte-macrophage colony stimulating factor (GM-CSF), leading to their secretion. Apoptosis induced activation of p38MAPK, JNK, and Akt, but neither p38 and JNK activation nor IL-6 or GM-CSF induction required caspase cleavage. IL-6 induction depended upon p38 activity, while Fas-induced GM-CSF expression required p38 and JNK. Conditioned media from apoptotic VSMCs induced VSMC apoptosis in vitro, and IL-6 and GM-CSF acted as pro-survival factors for AIA. VSMC apoptosis was studied in vivo using SM22α-DTR mice that express the diphtheria toxin receptor in VSMCs only. DT administration induced VSMC apoptosis and VSMC proliferation, and also signficantly induced IL-6 and GM-CSF. We conclude that VSMC apoptosis activates multiple caspase-independent intracellular signaling cascades, leading to release of soluble cytokines involved in regulation of both cell proliferation and apoptosis. VSMC AICP may ameliorate while AIA may amplify the effects of pro-apoptotic stimuli in vessel remodeling and disease.

Interleukin 1 beta and Matrix Metallopeptidase 3 Contribute to Development of Epidermal Growth Factor Receptor-Dependent Serrated Polyps in Mouse Cecum.

BACKGROUND & AIMS: Transgenic mice (HBUS) that express the epidermal growth factor receptor (EGFR) ligand HBEGF (heparin-binding epidermal growth factor-like growth factor) and a constitutively active G protein-coupled receptor (US28) in intestinal epithelial cells develop serrated polyps in the cecum. Development of serrated polyps depends on the composition of the gut microbiota and is associated with bacterial invasion of the lamina propria, accompanied by induction of inflammation and up-regulation of interleukin 1 beta (IL1B) and matrix metalloproteinase (MMP) 3 in the cecum. We investigated the mechanisms by which these changes contribute to development of serrated polyps. METHODS: We performed studies with C57BL/6 (control) and HBUS mice. To accelerate polyp development, we increased the exposure of the bacteria to the lamina propria by injecting HBUS mice with diphtheria toxin, which binds transgenic HBEGF expressed by the epithelial cells and causes apoptosis. Mice were given injections of IL1B-neutralizing antibody and the MMP inhibitor N-isobutyl-N-(4-methoxyphenylsulfonyl)glycyl hydroxamic acid. Intestinal tissues were collected from mice and analyzed by histology, reverse-transcription polymerase chain reaction, enzyme-linked immunosorbent assay, immunofluorescence, and flow cytometry. We examined fibroblast subsets in polyps using single-cell RNA sequencing. RESULTS: Administration of diphtheria toxin to HBUS mice accelerated development of serrated polyps (95% of treated mice developed polyps before 100 days of age, compared with 53% given vehicle). IL1B stimulated subsets of platelet-derived growth factor receptor alpha+ (PDGRFA+) fibroblasts isolated from cecum, resulting in increased expression of MMP3. Neutralizing antibodies against IL1B or administration of the MMP inhibitor reduced the number of serrated polyps that formed in the HBUS mice. Single-cell RNA sequencing analysis showed subsets of fibroblasts in serrated polyps that express genes that regulate matrix fibroblasts and inflammation. CONCLUSIONS: In studies of mice, we found that barrier breakdown and expression of inflammatory factors contribute to development of serrated polyps. Subsets of cecal PDGFRA+ fibroblasts are activated by release of IL1B from myeloid cells during the early stages of serrated polyp development. MMP3 produced by PDGFRA+ fibroblasts is important for serrated polyp development. Our findings confirm the functions of previously identified serrated polyp-associated molecules and indicate roles for immune and stromal cells in serrated polyp development.

BReDi mouse: A novel transgenic mouse strain to track and deplete B cells.

BReDi mice express a red fluorescent protein together with the diphtheria toxin receptor selectively in B cells. B cells can be effectively visualized by red fluorescence and can be efficiently depleted in a highly controlled fashion to study their functional capacity in vivo.

Underestimated Peripheral Effects Following Pharmacological and Conditional Genetic Microglial Depletion.

Microglia, predominant parenchymal resident macrophages in the central nervous system (CNS), are crucial players in neurodevelopment and CNS homeostasis. In disease conditions, pro-inflammatory microglia predominate over their regulatory counterparts, and are thus a potential immunotherapeutic target. It has been well documented that microglia can be effectively depleted using both conditional genetic Cx3cr1Cre-diphtheria toxin receptor (DTR)/diphtheria toxin subunit A (DTA) animal models and pharmacological colony-stimulating factor 1 receptor (CSF1R) inhibitors. Recent advances using these approaches have expanded our knowledge of the multitude of tasks conducted by microglia in both homeostasis and diseases. Importantly, experimental microglial depletion has been proven to exert neuroprotective effects in an increasing number of disease models, mostly explained by reduced neuroinflammation. However, the comprehensive effects of additional targets such as circulating monocytes and peripheral tissue macrophages during microglial depletion periods have not been investigated widely, and for those studies addressing the issue the conclusions are mixed. In this study, we demonstrate that experimental microglial depletion using both Cx3cr1CreER/+Rosa26DTA/+ mice and different doses of CSF1R inhibitor PLX3397 exert crucial influences on circulating monocytes and peripheral tissue macrophages. Our results suggest that effects on peripheral immunity should be considered both in interpretation of microglial depletion studies, and especially in the potential translation of microglial depletion and replacement therapies.

Eckol Alleviates Intestinal Dysfunction during Suckling-to-Weaning Transition via Modulation of PDX1 and HBEGF.

Maintaining intestinal health in livestock is critical during the weaning period. The precise mechanisms of intestinal dysfunction during this period are not fully understood, although these can be alleviated by phlorotannins, including eckol. This question was addressed by evaluating the changes in gene expression and intestinal function after eckol treatment during suckling-to-weaning transition. The biological roles of differentially expressed genes (DEGs) in intestinal development were investigated by assessing intestinal wound healing and barrier functions, as well as the associated signaling pathways and oxidative stress levels. We identified 890 DEGs in the intestine, whose expression was altered by eckol treatment, including pancreatic and duodenal homeobox (PDX)1, which directly regulate heparin-binding epidermal growth factor-like growth factor (HBEGF) expression in order to preserve intestinal barrier functions and promote wound healing through phosphoinositide 3-kinase (PI3K)/AKT and P38 signaling. Additionally, eckol alleviated H2O2-induced oxidative stress through PI3K/AKT, P38, and 5'-AMP-activated protein kinase (AMPK) signaling, improved growth, and reduced oxidative stress and intestinal permeability in pigs during the weaning period. Eckol modulates intestinal barrier functions, wound healing, and oxidative stress through PDX/HBEGF, and improves growth during the suckling-to-weaning transition. These findings suggest that eckol can be used as a feed supplement in order to preserve the intestinal functions in pigs and other livestock during this process.

Astrocyte-to-astrocyte contact and a positive feedback loop of growth factor signaling regulate astrocyte maturation.

Astrocytes are critical for the development and function of the central nervous system. In developing brains, immature astrocytes undergo morphological, molecular, cellular, and functional changes as they mature. Although the mechanisms that regulate the maturation of other major cell types in the central nervous system such as neurons and oligodendrocytes have been extensively studied, little is known about the cellular and molecular mechanisms that control astrocyte maturation. Here, we identified molecular markers of astrocyte maturation and established an in vitro assay for studying the mechanisms of astrocyte maturation. Maturing astrocytes in vitro exhibit similar molecular changes and represent multiple molecular subtypes of astrocytes found in vivo. Using this system, we found that astrocyte-to-astrocyte contact strongly promotes astrocyte maturation. In addition, secreted signals from microglia, oligodendrocyte precursor cells, or endothelial cells affect a small subset of astrocyte genes but do not consistently change astrocyte maturation. To identify molecular mechanisms underlying astrocyte maturation, we treated maturing astrocytes with molecules that affect the function of tumor-associated genes. We found that a positive feedback loop of heparin-binding epidermal growth factor-like growth factor (HBEGF) and epidermal growth factor receptor (EGFR) signaling regulates astrocytes maturation. Furthermore, HBEGF, EGFR, and tumor protein 53 (TP53) affect the expression of genes important for cilium development, the circadian clock, and synapse function. These results revealed cellular and molecular mechanisms underlying astrocytes maturation with implications for the understanding of glioblastoma.

Extraembryonic heparin-binding epidermal growth factor-like growth factor deficiency compromises placentation in mice.

Heparin-binding epidermal growth factor (EGF)-like growth factor (HBEGF) is expressed in the embryo and uterus at the implantation site, stimulating trophoblast invasive activity essential for placentation. The effect of extraembryonic HBEGF deficiency on placental development was investigated by breeding mice heterozygous for the Hbegf null mutation. On gestation day 13.5, the average placental weights of the wild-type (Hbegf+/+) and heterozygous (Hbegf+/-) mice were approximately 76 and 77 mg, respectively, as opposed to reduced average placental weights of approximately 61 mg in homozygous null (Hbgef-/-) females. In contrast, fetal weights were not significantly affected by genotype. HBEGF immunostaining in placental sections was Hbegf gene dosage-dependent, while expression of other EGF family members was comparable in Hbegf+/+ and Hbegf-/- placentas. Histological analysis revealed no apparent differences in trophoblast giant cells, but the spongiotrophoblast region was reduced compared to labyrinth (P < 0.05) in Hbegf null placentas. While no differences in cell apoptosis were noted, proliferation as assessed by nuclear Ki67 staining was elevated in the labyrinth and decreased in the spongiotrophoblast region of Hbegf-/- placentas. Labyrinth morphology appeared disrupted in Hbegf -/- placentas stained with laminin, a marker for capillary basement membrane, and the capillary density was reduced. Immunohistochemical staining revealed reduced vascular endothelial growth factor (VEGF) levels in both spongiotrophoblast and labyrinth (P < 0.01) regions of Hbegf-/- placentas. In vitro, HBEGF supplementation increases the expression of VEGF in a human trophoblast cell line. These findings suggest that trophoblast HBEGF promotes placental capillary formation by inducing VEGF in the developing placenta of mice.

Inflammatory monocytes mediate control of acute alphavirus infection in mice.

Chikungunya virus (CHIKV) and Ross River virus (RRV) are mosquito-transmitted alphaviruses that cause debilitating acute and chronic musculoskeletal disease. Monocytes are implicated in the pathogenesis of these infections; however, their specific roles are not well defined. To investigate the role of inflammatory Ly6ChiCCR2+ monocytes in alphavirus pathogenesis, we used CCR2-DTR transgenic mice, enabling depletion of these cells by administration of diptheria toxin (DT). DT-treated CCR2-DTR mice displayed more severe disease following CHIKV and RRV infection and had fewer Ly6Chi monocytes and NK cells in circulation and muscle tissue compared with DT-treated WT mice. Furthermore, depletion of CCR2+ or Gr1+ cells, but not NK cells or neutrophils alone, restored virulence and increased viral loads in mice infected with an RRV strain encoding attenuating mutations in nsP1 to levels detected in monocyte-depleted mice infected with fully virulent RRV. Disease severity and viral loads also were increased in DT-treated CCR2-DTR+;Rag1-/- mice infected with the nsP1 mutant virus, confirming that these effects are independent of adaptive immunity. Monocytes and macrophages sorted from muscle tissue of RRV-infected mice were viral RNA positive and had elevated expression of Irf7, and co-culture of Ly6Chi monocytes with RRV-infected cells resulted in induction of type I IFN gene expression in monocytes that was Irf3;Irf7 and Mavs-dependent. Consistent with these data, viral loads of the attenuated nsP1 mutant virus were equivalent to those of WT RRV in Mavs-/- mice. Finally, reconstitution of Irf3-/-;Irf7-/- mice with CCR2-DTR bone marrow rescued mice from severe infection, and this effect was reversed by depletion of CCR2+ cells, indicating that CCR2+ hematopoietic cells are capable of inducing an antiviral response. Collectively, these data suggest that MAVS-dependent production of type I IFN by monocytes is critical for control of acute alphavirus infection and that determinants in nsP1, the viral RNA capping protein, counteract this response.