Brain-borne IL-1 adjusts glucoregulation and provides fuel support to astrocytes and neurons in an autocrine/paracrine manner.

It is still controversial which mediators regulate energy provision to activated neural cells, as insulin does in peripheral tissues. Interleukin-1β (IL-1β) may mediate this effect as it can affect glucoregulation, it is overexpressed in the 'healthy' brain during increased neuronal activity, and it supports high-energy demanding processes such as long-term potentiation, memory and learning. Furthermore, the absence of sustained neuroendocrine and behavioral counterregulation suggests that brain glucose-sensing neurons do not perceive IL-1β-induced hypoglycemia. Here, we show that IL-1β adjusts glucoregulation by inducing its own production in the brain, and that IL-1β-induced hypoglycemia is myeloid differentiation primary response 88 protein (MyD88)-dependent and only partially counteracted by Kir6.2-mediated sensing signaling. Furthermore, we found that, opposite to insulin, IL-1β stimulates brain metabolism. This effect is absent in MyD88-deficient mice, which have neurobehavioral alterations associated to disorders in glucose homeostasis, as during several psychiatric diseases. IL-1β effects on brain metabolism are most likely maintained by IL-1β auto-induction and may reflect a compensatory increase in fuel supply to neural cells. We explore this possibility by directly blocking IL-1 receptors in neural cells. The results showed that, in an activity-dependent and paracrine/autocrine manner, endogenous IL-1 produced by neurons and astrocytes facilitates glucose uptake by these cells. This effect is exacerbated following glutamatergic stimulation and can be passively transferred between cell types. We conclude that the capacity of IL-1β to provide fuel to neural cells underlies its physiological effects on glucoregulation, synaptic plasticity, learning and memory. However, deregulation of IL-1β production could contribute to the alterations in brain glucose metabolism that are detected in several neurologic and psychiatric diseases.Molecular Psychiatry advance online publication, 8 December 2015; doi:10.1038/mp.2015.174.

Deficiency of MALT1 paracaspase activity results in unbalanced regulatory and effector T and B cell responses leading to multiorgan inflammation

The paracaspase MALT1 plays an important role in immune receptor-driven signaling pathways leading to NF-κB activation. MALT1 promotes signaling by acting as a scaffold, recruiting downstream signaling proteins, as well as by proteolytic cleavage of multiple substrates. However, the relative contributions of these two different activities to T and B cell function are not well understood. To investigate how MALT1 proteolytic activity contributes to overall immune cell regulation, we generated MALT1 protease-deficient mice (Malt1(PD/PD)) and compared their phenotype with that of MALT1 knockout animals (Malt1(-/-)). Malt1(PD/PD) mice displayed defects in multiple cell types including marginal zone B cells, B1 B cells, IL-10-producing B cells, regulatory T cells, and mature T and B cells. In general, immune defects were more pronounced in Malt1(-/-) animals. Both mouse lines showed abrogated B cell responses upon immunization with T-dependent and T-independent Ags. In vitro, inactivation of MALT1 protease activity caused reduced stimulation-induced T cell proliferation, impaired IL-2 and TNF-α production, as well as defective Th17 differentiation. Consequently, Malt1(PD/PD) mice were protected in a Th17-dependent experimental autoimmune encephalomyelitis model. Surprisingly, Malt1(PD/PD) animals developed a multiorgan inflammatory pathology, characterized by Th1 and Th2/0 responses and enhanced IgG1 and IgE levels, which was delayed by wild-type regulatory T cell reconstitution. We therefore propose that the pathology characterizing Malt1(PD/PD) animals arises from an immune imbalance featuring pathogenic Th1- and Th2/0-skewed effector responses and reduced immunosuppressive compartments. These data uncover a previously unappreciated key function of MALT1 protease activity in immune homeostasis and underline its relevance in human health and disease.

Growth hormone replacement therapy regulates microRNA-29a and targets involved in insulin resistance

Replacement of growth hormone (GH) in patients suffering from GH deficiency (GHD) offers clinical benefits on body composition, exercise capacity, and skeletal integrity. However, GH replacement therapy (GHRT) is also associated with insulin resistance, but the mechanisms are incompletely understood. We demonstrate that in GH-deficient mice (growth hormone-releasing hormone receptor (Ghrhr)(lit/lit)), insulin resistance after GHRT involves the upregulation of the extracellular matrix (ECM) and the downregulation of microRNA miR-29a in skeletal muscle. Based on RNA deep sequencing of skeletal muscle from GH-treated Ghrhr(lit/lit) mice, we identified several upregulated genes as predicted miR-29a targets that are negative regulators of insulin signaling or profibrotic/proinflammatory components of the ECM. Using gain- and loss-of-function studies, five of these genes were confirmed as endogenous targets of miR-29a in human myotubes (PTEN, COL3A1, FSTL1, SERPINH1, SPARC). In addition, in human myotubes, IGF1, but not GH, downregulated miR-29a expression and upregulated COL3A1. These results were confirmed in a group of GH-deficient patients after 4 months of GHRT. Serum IGF1 increased, skeletal muscle miR-29a decreased, and miR-29a targets were upregulated in patients with a reduced insulin response (homeostatic model assessment of insulin resistance (HOMA-IR)) after GHRT. We conclude that miR-29a could contribute to the metabolic response of muscle tissue to GHRT by regulating ECM components and PTEN. miR-29a and its targets might be valuable biomarkers for muscle metabolism following GH replacement. KEY MESSAGES GHRT most significantly affects the ECM cluster in skeletal muscle from mice. GHRT downregulates miR-29a and upregulates miR-29a targets in skeletal muscle from mice. PTEN, COL3A1, FSTL1, SERPINH1, and SPARC are endogenous miR-29a targets in human myotubes. IGF1 decreases miR-29a levels in human myotubes. miR-29a and its targets are regulated during GHRT in skeletal muscle from humans.

β2-microglobulin is a systemic pro-aging factor that impairs cognitive function and neurogenesis

Aging drives cognitive and regenerative impairments in the adult brain, increasing susceptibility to neurodegenerative disorders in healthy individuals. Experiments using heterochronic parabiosis, in which the circulatory systems of young and old animals are joined, indicate that circulating pro-aging factors in old blood drive aging phenotypes in the brain. Here we identify β2-microglobulin (B2M), a component of major histocompatibility complex class 1 (MHC I) molecules, as a circulating factor that negatively regulates cognitive and regenerative function in the adult hippocampus in an age-dependent manner. B2M is elevated in the blood of aging humans and mice, and it is increased within the hippocampus of aged mice and young heterochronic parabionts. Exogenous B2M injected systemically, or locally in the hippocampus, impairs hippocampal-dependent cognitive function and neurogenesis in young mice. The negative effects of B2M and heterochronic parabiosis are, in part, mitigated in the hippocampus of young transporter associated with antigen processing 1 (Tap1)-deficient mice with reduced cell surface expression of MHC I. The absence of endogenous B2M expression abrogates age-related cognitive decline and enhances neurogenesis in aged mice. Our data indicate that systemic B2M accumulation in aging blood promotes age-related cognitive dysfunction and impairs neurogenesis, in part via MHC I, suggesting that B2M may be targeted therapeutically in old age.

Adenosine mediated lung mast cell degranulation in the mouse

Adenosine has been implicated to play a role in inflammatory processes associated with asthma. Most notable is adenosine's ability to potentiate mediator release from mast cells. Mast cells are bone marrow derived inflammatory cells that can release mediators that have both immediate and chronic effects on airway constriction and inflammation. Most physiological roles of adenosine are mediated through adenosine receptors. Four subtypes of adenosine receptors have been identified, A1, A2A, A2B and A 3. The mechanisms by which adenosine can influence the release of mediators from lung tissue mast cells is not understood due to lack of in vivo models. Mice deficient in the enzyme adenosine deaminase (ADA) have been generated. ADA controls the levels of adenosine in tissues and cells, and consequently, adenosine accumulates in the lungs of ADA-deficient mice. ADA-deficient mice develop features seen in asthmatics, including lung eosinophilia and mucus hypersecretion. In addition, lung tissue mast cell degranulation was associated with elevated adenosine in ADA-deficient lungs and can be prevented by ADA enzyme therapy. We established primary murine lung mast cell cultures, and used real time RT-PCR and immunofluorescence to demonstrate that A 2A, A2B and A3 receptors are expressed on murine lung mast cells. Studies using selective adenosine receptor agonists and antagonists and A3 receptor deficient (A3−/−) mast cells suggested that activation of A3 receptors could induce mast cell mediator release in vitro. Furthermore, this mediator release was associated with increases in intracellular Ca++ that appeared to be mediated through a Gi and PI3K pathway. In addition, nebulized A3 receptor agonist directly induced lung mast cell degranulation in wild type mice while having no effect in A3−/− mice. These results demonstrate that the A3 receptor plays an important role in adenosine mediated murine lung mast cell degranulation. Therefore, the A3 adenosine receptor and its signaling pathways may represent novel therapeutic targets for the treatment and prevention of asthma. ^

The heparan sulfate-fibroblast growth factor signaling system in liver growth and function

The heparan sulfate (HS)-fibroblast growth factor (FGF) signaling system is a ubiquitous regulator that senses local environmental changes and mediates cell-to-cell communication. This system consists of three mutually interactive components. These are regulatory polypeptides (FGF), FGF receptor (FGFR) and heparan sulfate proteoglycans (FGFRHS). All four FGFR genes are expressed in the adult liver. Expression of the FGFR1–3 genes is generally associated with non-parenchymal cells while expression of the FGFR4 gene is associated with parenchymal hepatocytes. We showed that livers of mice lacking FGFR4 exhibited normal morphology and regenerated normally in response to partial hepatectomy. However, the FGFR4 (−/−) mice exhibited depleted gallbladders, an elevated bile acid pool and elevated excretion of bile acids. Cholesterol- and bile acid-controlled liver cholesterol 7α-hydroxylase (Cyp7a), the limiting enzyme for bile acid synthesis, was elevated, unresponsive to dietary cholesterol, but repressed normally by dietary cholate. These results indicated that FGFR4 was not directly involved in liver growth but exerted negative control on liver bile acid synthesis. This was confirmed in transgenic mice overexpressing the constitutively active human FGFR4 in livers. The transgenic mice exhibited decreased fecal bile acid excretion, bile acid pool size, and expression of Cyp7a. Introduction of this constitutively active human FGFR4 into FGFR4 (−/−) mice restored the inhibition of bile acid synthesis. Activation of the c-Jun N-terminal Kinase (JNK) pathway by FGFR4 correlated with the repressive effect on bile acid synthesis. ^ To determine whether FGFR4 played a broader role in liver-specific metabolic function, we examined the impact of both acute and chronic exposure to CCl 4 in FGFR4 (−/−) mice. Following acute CCl4 exposure, the FGFR4 (−/−) mice exhibited accelerated liver injury, a significant increase in liver mass and delayed hepatolobular repair, with no apparent effect on liver cell proliferation and restoration of cellularity. Chronic CCl4 exposure resulted in severe fibrosis in livers of FGFR4 (−/−) mice compared to normal mice. Analysis at both mRNA and protein levels indicated an 8 hr delay in FGFR4-deficient mice in the down-regulation of cytochrome P450 2E1 (CYP2E1) protein, the major enzyme whose products underlie CCl 4-induced injury. These results show that hepatocyte FGFR4 protects against acute and chronic insult to the liver and prevents accompanying fibrosis. ^ Of the 23 FGF polypeptides, FGF1 and FGF2 are present at significant levels in the liver. To determine whether FGF1 and FGF2 played a role in CCl 4-induced liver injury and fibrosis, we examined the impact of both acute and chronic exposure to CCl4 in both wild-type and FGF1-FGF2 double-knockout mice. Following acute CCl4 exposure, FGF1(−/−)FGF2(−/−) mice exhibited accelerated liver injury, overall normal liver growth and repair, and decreased liver collagen α1(I) induction. Liver fibrosis resulting from chronic CCl4 exposure was markedly decreased in livers of FGF1(−/−)FGF2(−/−) mice compared to wild-type mice. This study suggests a role for FGF1 and FGF2 in hepatic fibrogenesis. ^ In summary, our three part study shows that specific components of the ubiquitous HS-FGF signaling family in the liver context interfaces with metabolite- and xenobiotic-controlled networks to regulate liver function, but has no apparent direct effect on liver cell growth. ^

B cells in ADA deficiency

Deficiency of the enzyme adenosine deaminase (ADA) results in severe lymphopenia in humans. Mice with an inactivating mutation in the ADA gene also exhibit profound lymphopenia, as well as pulmonary insufficiency and ribcage abnormalities. In fact, the mouse model has a phenotype that is remarkably similar to that of the human disease, making the mice valuable tools for unraveling the mechanism of lymphocyte destruction in absence of this housekeeping gene. T cell deficiency in ADA deficiency has been extensively studied by others, revealing a block in early thymocyte development. In contrast, our studies revealed that early B cell development in the bone marrow is normal. ADA-deficient mice, however, exhibit profound defects in germinal center formation, preventing antigen-dependent B cell maturation in the spleen. ADA-deficient spleen B cells display significant defects in proliferation and activation signaling, and produce more IgM than their normal counterparts, suggesting that extrafollicular plasmablasts are overrepresented. B cells from ADA-deficient mouse spleens undergo apoptosis more readily than those from normal mouse spleens. Levels of ADA's substrates, adenosine and 2′-deoxyadenosine, are elevated in both bone marrow and spleen in ADA-deficient mice. S ′-adenosyihomoeysteine hydrolase (SAH hydrolase) activity is significantly inhibited in both locales, as well. dATP levels, though, are only elevated in spleen, where B cell development is impaired, and not in bone marrow, where B cell ontogeny is normal. This finding points to dATP as the causative agent of lymphocyte death in ADA deficiency. ADA deficiency results in inhibition of the enzyme ribonucleotide reductase, thereby depleting nucleoside pools needed for DNA repair. Another mouse model that lacks a functional gene encoding a protein involved in DNA repair and/or cell cycle checkpoint regulation, p53-binding protein 1, exhibits blocks in T and B cell development that are similar to those seen in ADA-deficient mice. Unraveling the mechanisms of lymphocyte destruction in ADA deficiency may further understanding of lymphocyte biology, facilitate better chemotherapeutic treatment for lymphoproliferative diseases, and improve gene and enzyme therapy regimens attempted for ADA deficiency. ^

The role of nitric oxide in small intestinal motility in a model of acute inflammation

Motility responses of the small intestine of iNOS deficient mice (iNOS −/−) and their wildtype littermates (iNOS+/+) to the inflammatory challenge of lipopolysaccharide (LPS) were investigated. LPS administration failed to attenuate intestinal transit in iNOS−/− mice but depressed transit in their iNOS+/+ littermates. Supporting an inhibitory role for sustained nitric oxide (NO) synthesis in the regulation of intestinal motility during inflammation, iNOS immunoreactivity was upregulated in all regions of the small intestine of iNOS+/+ mice. In contrast, neuronal NOS was barely affected. Cyclooxygenase activation was determined by prostaglandin E2 (PGE2) concentration. Following LPS challenge, PGE2 levels were elevated in all intestinal segments in both animal groups. Moreover, COX-1 and COX-2 protein levels were elevated in iNOS+/+ mice in response to LPS, while COX-2 levels were similarly increased in iNOS −/− intestine. However, no apparent relationship was observed between increased prostaglandin concentrations and attenuated intestinal transit. The presence of heme oxygenase 1 (HO-1) in the murine small intestine was also investigated. In both animal groups HO-1 immunoreactivity in the proximal intestine increased in response to treatment, while the constitutive protein levels detected in the middle and distal intestine were unresponsive to LPS administration. No apparent correlation of HO-1 to the suppression of small intestinal motility induced by LPS administration was detected. The presence of S-nitrosylated contractile proteins in the small intestine was determined. γ-smooth muscle actin was basally nitrosylated as well as in response to LPS, but myosin light chain kinase and myosin regulatory chain (MLC20) were not. In conclusion, in a model of acute intestinal inflammation, iNOS-produced NO plays a significant role in suppressing small intestinal motility while nNOS, COX-1, COX-2 and HO-1 do not participate in this event. S-nitrosylation of γ-smooth muscle actin is associated with elevated levels of nitric oxide in the smooth muscle of murine small intestine. ^

Ontogeny and regulation of interleukin-7 expressing thymic epithelial cells

T cell development is a multistage process of differentiation that depends on proper thymocyte-thymic epithelial cell (TEC) interactions. Epithelial cells in the thymus are organized in a three-dimensional network that provides support and signals for thymocyte maturation. Concurrently, proper TEC differentiation in the adult thymus relies on thymocyte-derived signals. TECs produce interleukin-7 (IL-7), a non-redundant cytokine that promotes the survival, differentiation, and proliferation of thymocytes. We have identified IL-7 expressing TECs throughout ontogeny and in the adult thymus by in situ hybridization analysis. IL-7 expression is initiated in the thymic fated domain of the thymic primordium by embryonic day 11.5, in a Foxn1 independent pathway. Marked changes occur in the localization and regulation of IL-7 expressing TECs during development. Whereas IL-7 expressing TECs are present throughout the early thymic rudiment, the majority of IL-7 producing TECs are concentrated in the adult thymic medulla. By analyzing mouse strains that sustain blocks at different stages of thymocyte development, we show that IL-7 expression is initiated independently of hematopoietic-derived signals during thymic organogenesis. However, thymocyte-derived signals play an essential role in regulating IL-7 expression in the adult TEC compartment. Furthermore, distinct thymocyte subsets regulate the expression of IL-7 and keratin 5 in adult cortical epithelium. Intraperitoneal injection of Recombination Activating Gene deficient mice (RAG-2−/−) with anti-CD3ϵ monoclonal antibody (mAb) induces CD4− 8− double negative thymocytes to undergo β-selection and differentiate into CD4+8+ cells. Analysis of the thymic stromal compartment reveals that progression through β-selection renders thymocytes competent to alter the pattern of IL-7 expression in the cortical TEC compartment. RAG-2−/− mice do not generate mature T cells and therefore the RAG-2−/− thymus is devoid of organized medullary regions. Histological examination of RAG-2−/− thymus following anti-CD3ϵ stimulation reveals the emergence of mature thymic medullary regions, as assessed by H & E staining and expression of thymic stromal medullary markers. Stromal medullary reorganization occurs in the absence of T cell receptor αβ expression, suggesting that activation of RAG-2−/− thymocytes by CD3ϵ ligation generates thymocyte-derived signals that induce thymic epithelial reorganization, generating a mature medullary compartment. This model provides a tool to assess the mechanisms underlying thymic medullary development. ^

The contribution of A3 adenosine receptor signaling to pulmonary inflammation and mucus secretion in the mouse

Adenosine has been implicated in chronic lung diseases such as asthma and COPD. Most physiological actions of adenosine are mediated through G-protein coupled adenosine receptors. Four subtypes of adenosine receptors have been identified, A1, A2A, A2B, and A 3. However, the specific roles of the various adenosine receptors in processes central to asthma and COPD are not well understood in part due to the lack of adequate animal models that examine the effect of adenosine on the development of lung disease. In this study we have investigated the expression and function of the A3 adenosine receptor in pulmonary eosinophilia and mucus production/secretion in adenosine deaminase (ADA)-deficient mice in which adenosine levels are elevated. ADA-deficient mice develop features of asthma and COPD, including lung eosinophilia and mucus hyperplasia in association with elevated lung adenosine levels. The A3 receptor was found to be expressed in eosinophils and mucus producing cells in the airways of ADA-deficient. Disruption of A3 receptor signaling in ADA-deficient mice by genetic removal of the receptor or treatment with MRS 1523, a selective A3 adenosine receptor antagonist, prevented airway eosinophilia and mucus production. Although eosinophils were decreased in the airways of ADA-deficient mice with disrupted A3 receptor signaling, elevations in circulating and lung interstitial eosinophils persisted, suggesting signaling through the A3 receptor is needed for the migration of eosinophils into the airways. Further examination of the role of the A3 receptor in mucus biology demonstrated that the A3 receptor is neither required nor is overexpression of the receptor in clara cells sufficient for mucus production in naive mice. Transgenic overexpression of the A3 receptor did elucidate a role for the A3 receptor in the secretion of mucus into the airways of ovalbumin challenged mice. These findings identify an important role for the A3 adenosine receptor in regulating lung eosinophilia and mucus secretion in inflammatory lung diseases. Therefore, the A3 adenosine receptor may represent a novel therapeutic target for the treatment and prevention of asthma. ^

Elucidation of the role of 53BP1 in DNA double strand break (DSB) repair and tumor suppression

The protein p53 binding protein one (53BP1) was discovered in a yeast two-hybrid screen that used the DNA binding domain of p53 as bait. Cloning of full-length 53BP1 showed that this protein contains several protein domains which help make up the protein, which include two tandem BRCT domains and a amino-terminal serine/glutamine cluster domain (SCD). These are two protein domains are often seen in factors that are involved in the cellular response to DNA damage and control of cell cycle checkpoints and we hypothesize that 53BP1 is involved in the cellular response to DNA damage. In support of this hypothesis we observe that 53BP1 is phosphorylated and undergoes a dramatic nuclear re-localization in response to DNA damaging agents. 53BP1 also interacts with several factors that are important in the cellular response to DNA damage, such as the BRCA1 tumor suppressor, ATM and Rad3 related (ATR), and the phosphorylated version of the histone variant H2AX. Mice deficient in 53BP1 display increased sensitivity ionizing radiation (IR), a DNA damaging agent that introduces DNA double strand breaks (DSBs). In addition, 53BP1-deficient mice do not properly undergo the process of class switch recombination (CSR). We also observe that when a defect in 53BP1 is combined with a defect in p53; the resulting mice have an increased rate of formation of spontaneous tumors, notably the formation of B and T lineage lymphomas. The T lineage tumors arise by two distinct mechanisms: one driven by defects in cell cycle regulation and a second driven by defects in the ability to repair DNA DSBs. The B lineage tumors arise by the inability to repair DNA damage and over-expression of the oncogene c-myc. ^ With these observations, we conclude that not only does 53BP1 function in the cellular response to DNA damage, but it also works in concert with p53 to suppress tumor formation. ^

The role of adenosine in pulmonary angiogenesis

Angiogenesis is a feature of chronic lung diseases such as asthma and pulmonary fibrosis; however, the pathways controlling pathological angiogenesis during lung disease are not completely understood. Adenosine is a signaling nucleoside that accumulates as a result of tissue hypoxia and damage. Adenosine has been implicated in the exacerbation of chronic lung disease and in the regulation of angiogenesis; however, the relationship between these factors has not been investigated. The work presented in this dissertation utilized adenosine deaminase (ADA)-deficient mice to determine whether chronic elevations of adenosine in vivo result in pulmonary angiogenesis, and to identify factors that could potentially mediate this process. Results demonstrate that there is substantial angiogenesis in the tracheas of ADA-deficient mice in association with adenosine elevations. Replacement enzyme therapy with pegylated ADA resulted in a lowering of adenosine levels and reversal of tracheal angiogenesis, indicating that the increases in vessel number are dependent on adenosine elevations. Levels of the ELR+ angiogenic chemokine CXCL1 were found to be elevated in an adenosine-dependent manner in the lungs of ADA-deficient mice. Neutralization of CXCL1 and its putative receptor, CXCR2, in ADA-deficient lung lysates resulted in the inhibition of angiogenic activity suggesting that CXCL1 signaling through the CXCR2 receptor is responsible for mediating the observed increases in angiogenesis. Taken together, these findings suggest that adenosine plays an important role, via CXCL1, in the induction of pulmonary angiogenesis and may therefore represent an important therapeutic target for the treatment of pathological angiogenesis. ^

The role of STAT3 in the emergency neutrophil response

Neutrophils are an essential component of innate immunity, serving to provide an immediate response to microbial invasion. In response to emergency situations such as an infection, serum levels of granulocyte colony-stimulating factor (G-CSF) are induced, causing a boost in neutrophil production and a rapid mobilization of bone marrow neutrophils to the blood, where they can circulate to clear foreign pathogens. Signal transducer and activator of transcription 3 (STAT3) is a principal downstream signaling intermediate of the G-CSF receptor. Mice null for STAT3 are embryonic lethal; therefore, to examine the role that STAT3 has in granulocytic development and function in vivo, we utilized a conditional knockout mouse that deletes functional STAT3 in the hematopoietic system (referred to herein as STAT3-deficient). Using this model, we show that STAT3 is required for G-CSF-induced expansion of granulocytic progenitor cells within the bone marrow and for acute G-CSF-dependent neutrophil mobilization into the blood. Thus, STAT3 has a critical role in the immediate G-CSF-response in vivo. Sustained G-CSF exposure causes skewed granulocytic production and mobilization in STAT3-deficient mice, suggesting an atypical granulocytic developmental pathway. To determine if STAT3-deficient neutrophils were functional, we examined neutrophil chemotaxis, since neutrophil function relies on proper chemoattractant-induced migration to infected tissue sites. STAT3-deficient neutrophils have impaired chemotaxis in response to the potent neutrophil chemoattractants MIP-2 and KC, both ligands for the chemokine receptor CXCR2. Additionally, STAT3-deficient mice have a defect in NIIP-2-induced acute neutrophil mobilization in vivo. Chemotaxis in response to fMLP and SDF-1, which utilize distinct seven-transmembrane chemokine receptors, was similar between wild type and STAT3-deficient neutrophils, suggesting that STAT3 specifically regulates CXCR2-mediated migration. MIP-2-induced activation of the Raf/MEK/ERK signaling cascade, which we show is required for MIP-2-dependent neutrophil chemotaxis, was impaired in STAT3-deficient neutrophils. Interestingly, acute G-CSF administration induced CXCR2 expression and Raf/MEK/ERK activation in neutrophils from wild type mice, whereas these responses were abrogated in neutrophils from STAT3-deficient mice. Thus, STAT3 regulation of CXCR2 functions may also contribute to STAT3's control of the acute G-CSF mobilization response. These combined results place STAT3 as a critical intermediate in neutrophil migration and G-CSF-induced neutrophil production responses required for emergency granulopoiesis. ^

The role of ATM in the cell cycle control of V(D)J recombination

Lymphocyte development requires the assembly of diversified antigen receptor complexes generated by the genetically programmed V(D)J recombination event. Because germline DNA is cut, introducing potentially dangerous double-stranded breaks (DSBs) and rearranged prior to repair, its activity is limited to the non-cycling stages of the cell cycle, G0/G1. The potential involvement of a key mediator, Ataxia Telangiectasia Mutated or ATM, in the DNA damage response (DDR) and cell cycle checkpoints has been implicated in recombination, but its role is not fully understood. Thymic lymphomas from ATM deficient mice contain clonal chromosomal translocations involving the T-cell antigen receptor (TCR). A previous report found ATM and its downstream target p53 associated with V(D)J intermediates, suggesting the DDR senses recombination. In this study, we sought to understand the role of ATM in V(D)J recombination. Developing thymocytes from ATM deficient mice were analyzed according to the cell cycle to detect V(D)J intermediates. Examination of all TCR loci in the non-cycling (G0/G1) and cycling (S/G2/M) fractions revealed the persistence of intermediates in ATM deficient thymocytes, contrary to the wild-type in which intermediates are found only during G0/G1. Further analysis found no defect in end-joining of intermediates, nor were they detected in developed T-cells. Based upon the presence of persisting intermediates, the recombination initiating nuclease Rag-2 was examined; strict regulation limits it to G 0/G1. Rag-2 regulation was not affected by an ATM deficiency as Rag-2 expression remained contained within G0/G 1, indicating recombination is not continuous. To determine if an ATM deficiency affects recognition of V(D)J breaks, sites of recombination identified by a TCR locus or Rag expression were analyzed according to co-localization with a DDR factor phosphorylated immediately after DNA damage, phosphorylated H2AX (γH2AX). No differences in co-localization were found between the wild-type and ATM deficiency, demonstrating ATM deficient lymphocytes retain the ability to recognize DSBs. Together, these results suggest ATM is necessary in the cell cycle regulation of recombination but not essential for the identification of V(D)J breaks. ATM ensures the containment of intermediates within G0/G1 and maintains genomic stability of developing lymphocytes, emphasizing its fundamental role in preventing tumorigenesis.^

Identification and characterization of mutations in SMC contractile genes involved in thoracic aortic aneurysms and dissections

Aortic aneurysms and dissections are the 15th most common cause of death in the United States. Genetic factors contribute to the pathogenesis of thoracic aortic aneurysms and dissections (TAAD). Currently, six loci and four genes have been identified for familial TAAD. Notably, mutations in smooth muscle cell (SMC) contractile genes, ACTA2 and MYH11, are responsible for 15% of familial TAAD, suggesting that proper SMC contraction is important for normal aorta function. Therefore, we hypothesize that mutations in other genes encoding SMC contractile proteins also cause familial TAAD. ^ To test this hypothesis, we used a candidate gene approach to identify causative mutations in SMC contractile genes for familial TAAD. Sequencing DNA in 80 TAAD patients from unrelated families, we identified putative mutations in eight contractile genes. We chose myosin light chain kinase (MLCK ) S1759P for further study for the following reasons: (1) Serine 1759 is conserved between vertebrates and invertebrates. (2) S1759P is predicted to be functionally deleterious by bioinformatics. (3) Low blood pressure is observed in SMC-selective MLCK-deficient mice. ^ In the presence of Ca2+/Calmodulin (CaM), MLCK containing CaM binding and kinase domains are activated to phosphorylate myosin light chain, thereby initiate SMC contraction. The CaM binding sequence of MLCK forms an α-helix structure required for CaM binding. MLCK Serine 1759 is located within the CaM binding domain. S1759P is predicted to decrease the α-helix composition in the CaM binding domain. Hence, we hypothesize that MLCK mutations cause TAAD through disturbing CaM binding and MLCK activity. ^ We further sequenced MLCK in DNA samples from additional 86 probands with familial TAAD. Two more mutations, MLCK A1754T and R1480Stop, were identified, supporting that MLCK mutations cause familial TAAD. ^ To define whether MLCK mutations disrupted CaM binding and MLCK activity, we performed co-immunoprecipitation and kinase assays. Decreased CaM binding and kinase activity was detected in A1754T and S1759P. Moreover, R1480Stop is predicted to truncate kinase and CaM binding domains. We conclude that MLCK mutations disrupt CaM binding and MLCK activity. ^ Collectively, our study is first to show mutations in genes regulating SMC contraction cause TAAD. This finding further highlights the importance of SMC contraction in maintaining aorta function. ^