CXCL14 Displays Antimicrobial Activity against Respiratory Tract Bacteria and Contributes to Clearance of Streptococcus pneumoniae Pulmonary Infection

CXCL14 is a chemokine with an atypical, yet highly conserved, primary structure characterized by a short N terminus and high sequence identity between human and mouse. Although it induces chemotaxis of monocytic cells at high concentrations, its physiological role in leukocyte trafficking remains elusive. In contrast, several studies have demonstrated that CXCL14 is a broad-spectrum antimicrobial peptide that is expressed abundantly and constitutively in epithelial tissues. In this study, we further explored the antimicrobial properties of CXCL14 against respiratory pathogens in vitro and in vivo. We found that CXCL14 potently killed Pseudomonas aeruginosa, Streptococcus mitis, and Streptococcus pneumoniae in a dose-dependent manner in part through membrane depolarization and rupture. By performing structure-activity studies, we found that the activity against Gram-negative bacteria was largely associated with the N-terminal peptide CXCL141-13. Interestingly, the central part of the molecule representing the β-sheet also maintained ∼62% killing activity and was sufficient to induce chemotaxis of THP-1 cells. The C-terminal α-helix of CXCL14 had neither antimicrobial nor chemotactic effect. To investigate a physiological function for CXCL14 in innate immunity in vivo, we infected CXCL14-deficient mice with lung pathogens and we found that CXCL14 contributed to enhanced clearance of Streptococcus pneumoniae, but not Pseudomonas aeruginosa. Our comprehensive studies reflect the complex bactericidal mechanisms of CXCL14, and we propose that different structural features are relevant for the killing of Gram-negative and Gram-positive bacteria. Taken together, our studies show that evolutionary-conserved features of CXCL14 are important for constitutive antimicrobial defenses against pneumonia.

T cell lipid peroxidation induces ferroptosis and prevents immunity to infection

The selenoenzyme glutathione peroxidase 4 (Gpx4) is a major scavenger of phospholipid hydroperoxides. Although Gpx4 represents a key component of the reactive oxygen species-scavenging network, its relevance in the immune system is yet to be defined. Here, we investigated the importance of Gpx4 for physiological T cell responses by using T cell-specific Gpx4-deficient mice. Our results revealed that, despite normal thymic T cell development, CD8(+) T cells from T(ΔGpx4/ΔGpx4) mice had an intrinsic defect in maintaining homeostatic balance in the periphery. Moreover, both antigen-specific CD8(+) and CD4(+) T cells lacking Gpx4 failed to expand and to protect from acute lymphocytic choriomeningitis virus and Leishmania major parasite infections, which were rescued with diet supplementation of high dosage of vitamin E. Notably, depletion of the Gpx4 gene in the memory phase of viral infection did not affect T cell recall responses upon secondary infection. Ex vivo, Gpx4-deficient T cells rapidly accumulated membrane lipid peroxides and concomitantly underwent cell death driven by ferroptosis but not necroptosis. These studies unveil an essential role of Gpx4 for T cell immunity.

IL-33 promotes an innate immune pathway of intestinal tissue protection dependent on amphiregulin-EGFR interactions

The barrier surfaces of the skin, lung, and intestine are constantly exposed to environmental stimuli that can result in inflammation and tissue damage. Interleukin (IL)-33-dependent group 2 innate lymphoid cells (ILC2s) are enriched at barrier surfaces and have been implicated in promoting inflammation; however, the mechanisms underlying the tissue-protective roles of IL-33 or ILC2s at surfaces such as the intestine remain poorly defined. Here we demonstrate that, following activation with IL-33, expression of the growth factor amphiregulin (AREG) is a dominant functional signature of gut-associated ILC2s. In the context of a murine model of intestinal damage and inflammation, the frequency and number of AREG-expressing ILC2s increases following intestinal injury and genetic disruption of the endogenous AREG-epidermal growth factor receptor (EGFR) pathway exacerbated disease. Administration of exogenous AREG limited intestinal inflammation and decreased disease severity in both lymphocyte-sufficient and lymphocyte-deficient mice, revealing a previously unrecognized innate immune mechanism of intestinal tissue protection. Furthermore, treatment with IL-33 or transfer of ILC2s ameliorated intestinal disease severity in an AREG-dependent manner. Collectively, these data reveal a critical feedback loop in which cytokine cues from damaged epithelia activate innate immune cells to express growth factors essential for ILC-dependent restoration of epithelial barrier function and maintenance of tissue homeostasis.

Subcellular distribution of FTY720 and FTY720-phosphate in immune cells - another aspect of Fingolimod action relevant for therapeutic application

FTY720 (Fingolimod; Gilenya®) is an immune-modulatory prodrug which, after intracellular phosphorylation by sphingosine kinase 2 (SphK2) and export, mimics effects of the endogenous lipid mediator sphingosine-1-phosphate. Fingolimod has been introduced to treat relapsing-remitting multiple sclerosis. However, little has been published about the immune cell membrane penetration and subcellular distribution of FTY720 and FTY720-P. Thus, we applied a newly established LC-MS/MS method to analyze the subcellular distribution of FTY720 and FTY720-P in subcellular compartments of spleen cells of wild type, SphK1- and SphK2-deficient mice. These studies demonstrated that, when normalized to the original cell volume and calculated on molar basis, FTY720 and FTY720-P dramatically accumulated several hundredfold within immune cells reaching micromolar concentrations. The amount and distribution of FTY720 was differentially affected by SphK1- and SphK2-deficiency. On the background of recently described relevant intracellular FTY720 effects in the nanomolar range and the prolonged application in multiple sclerosis, this data showing a substantial intracellular accumulation of FTY720, has to be considered for benefit/risk ratio estimates.

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.

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. ^

The role of UV radiation in the development of myeloid malignancy in Rag2 knockout mice

Excessive exposure to the UV radiation present in sunlight can lead to the development of skin cancer in humans. Majority of the UV-induced skin tumors in immune-competent mice are highly antigenic in nature. Additionally, they exhibit a high frequency of mutations in the p53 gene, which arise very early in the course of UV radiation and most of them disappear before the development of skin tumors. ^ Initially, this study was to determine whether UV radiation induces skin tumors much earlier in immune deficient Rag2 knockout mice than in immune-competent mice, and if so, compare their antigenic properties and p53 mutation spectra. However, chronic UV irradiation (10 kJ/m2) induced myeloproliferative disease (MPD) as early as 4 weeks in Rag2 knockout mice instead of skin tumors. Conversely, unirradiated Rag2 knockout mice developed MPD at a low frequency, but the frequency increased with the animal's age. Although the UV-irradiated wild type mice (B6129) developed MPD, its frequency was lower and the occurrence much later than the Rag2 knockout mice. ^ This observation led to our new hypothesis that UV irradiation plays a role in the development of MPD in Rag2 knockout mice. After 4 weeks of UV radiation, both histopathology (myeloid:erythroid ratio, number of blast cells) and flow cytometry (mature myeloid, granulocytes and immature cells) demonstrated an increased number of mice affected with the disease in the UV-irradiated Rag2 knockout group than the other groups. ^ We also investigated the role of cytokines and absence of T and B cells in the development of MPD in the Rag2 knockout mice. Results indicated that IL-3 and IL-3Rα chain expression was upregulated in the spleens of the UV-irradiated Rag2 knockout mice (4 weeks). Reconstitution of the Rag2 knockout mice with T and B cells abrogated the UV-accelerated development of MPD. Both histopathology and flow cytometric analysis (mature myeloid cells, granulocytes) showed a decrease in the number of mice affected with the disease in the UV-irradiated, reconstituted group rather than any other group. In summary, this study provides the first experimental evidence that exposure to UV irradiation can lead to the development of MPD in immune deficient mice. ^

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. ^

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 and mechanism of supressor of cytokine signaling 1 in brain metastasis

Brain metastasis, which occurs in 40%-60% of patients with advanced melanoma, has led directly to death in the majority of cases. Unfortunately, little is known about the biological and molecular basis of melanoma brain metastases. In our previous study, we developed a model to study human melanoma brain metastasis and found that Stat3 activity was increased in human brain metastatic melanoma cells when compared with that in cutaneous melanoma cells. The increased activation of Stat3 is also responsible for affecting melanoma angiogenesis in vivo and melanoma cell invasion in vitro and significantly affecting the expression of bFGF, VEGF, and MMP-2 in vivo and in vitro. Interestingly, a member of a new family of cytokine-inducible inhibitors of signal transduction, termed suppressors of cytokine signaling 1 (SOCS1) was found to negatively regulate the Janus kinase signal transducer and activator of transcription (Jak/STAT) signaling cascade. Here we report that restoration of SOCS1 expression by transfecting of SOCS1-expressing vector effectively inhibited melanoma brain metastasis through inhibiting Stat3 activation and further affecting melanoma angiogenesis and melanoma cell invasion in vitro, and significantly affected the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP-2) in vitro and in vivo. In addition, we used cDNA array to compare mRNA expression in the SOCS1-transfected and vector-transfected cell lines and found some genes are tightly correlated to the restoration of SOCS1. One of them is Caveolin-1 (Cav-1). Cav-1 was reported to function as a tumor suppressor gene by several groups. Finally, the Cav-1 expression is up-regulated in SOCS1-overexpressing cell line. Further study found the regulation of Cav-1 by SOCS1 occurs through inhibiting Stat3 activation. Activated Stat3 binds directly to Cav-1 promoter and the Cav-1 promoter within -575bp is essential for active Stat3 binding. My studies reveal that Stat3 activation and SOCS1 expression play important roles in melanoma metastases. Moreover, the expression between SOCS1, Stat3 and Cav-1 forms a feedback regulation loop. ^

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. ^