Authors: Drummond RA, Collar AL, Swamydas M, Rodriguez CA, Lim JK, Mendez LM, Fink DL, Hsu AP, Zhai B, Karauzum H, Mikelis CM, Rose SR, Ferre EM, Yockey L, Lemberg K, Kuehn HS, Rosenzweig SD, Lin X, Chittiboina P, Datta SK, Belhorn TH, Weimer ET, Hernandez ML, Hohl TM, Kuhns DB, Lionakis MS
Journal: PLoS Pathog. 2015 Dec 17;11(12):e1005293. doi: 10.1371/journal.ppat.1005293. eCollection 2015.
Candida is the most common human fungal pathogen and causes systemic infections that require neutrophils for effective host defense. Humans deficient in the C-type lectin pathway adaptor protein CARD9 develop spontaneous fungal disease that targets the central nervous system (CNS). However, how CARD9 promotes protective antifungal immunity in the CNS remains unclear. Here, we show that a patient with CARD9 deficiency had impaired neutrophil accumulation and induction of neutrophil-recruiting CXC chemokines in the cerebrospinal fluid despite uncontrolled CNS Candida infection. We phenocopied the human susceptibility in Card9-/- mice, which develop uncontrolled brain candidiasis with diminished neutrophil accumulation. The induction of neutrophil-recruiting CXC chemokines is significantly impaired in infected Card9-/- brains, from both myeloid and resident glial cellular sources, whereas cell-intrinsic neutrophil chemotaxis is Card9-independent. Taken together, our data highlight the critical role of CARD9-dependent neutrophil trafficking into the CNS and provide novel insight into the CNS fungal susceptibility of CARD9-deficient humans.
Authors: Jin W, Tang Q, Wan M, Cui K, Zhang Y, Ren G, Ni B, Sklar J, Przytycka TM, Childs R, Levens D, Zhao K
Journal: Nature. 2015 Dec 3;528(7580):142-6. doi: 10.1038/nature15740.
DNase I hypersensitive sites (DHSs) provide important information on the presence of transcriptional regulatory elements and the state of chromatin in mammalian cells. Conventional DNase sequencing (DNase-seq) for genome-wide DHSs profiling is limited by the requirement of millions of cells. Here we report an ultrasensitive strategy, called single-cell DNase sequencing (scDNase-seq) for detection of genome-wide DHSs in single cells. We show that DHS patterns at the single-cell level are highly reproducible among individual cells. Among different single cells, highly expressed gene promoters and enhancers associated with multiple active histone modifications display constitutive DHS whereas chromatin regions with fewer histone modifications exhibit high variation of DHS. Furthermore, the single-cell DHSs predict enhancers that regulate cell-specific gene expression programs and the cell-to-cell variations of DHS are predictive of gene expression. Finally, we apply scDNase-seq to pools of tumour cells and pools of normal cells, dissected from formalin-fixed paraffin-embedded tissue slides from patients with thyroid cancer, and detect thousands of tumour-specific DHSs. Many of these DHSs are associated with promoters and enhancers critically involved in cancer development. Analysis of the DHS sequences uncovers one mutation (chr18: 52417839G>C) in the tumour cells of a patient with follicular thyroid carcinoma, which affects the binding of the tumour suppressor protein p53 and correlates with decreased expression of its target gene TXNL1. In conclusion, scDNase-seq can reliably detect DHSs in single cells, greatly extending the range of applications of DHS analysis both for basic and for translational research, and may provide critical information for personalized medicine.
Authors: Duverger O, Beniash E, Morasso MI
Journal: Matrix Biol. 2015 Dec 17. pii: S0945-053X(15)30010-X. doi: 10.1016/j.matbio.2015.12.007.
Dental enamel is the hardest tissue in the human body, and although it starts as a tissue rich in proteins, by the time of eruption of the tooth in the oral cavity only a small fraction of the protein remains. While this organic matrix of enamel represents less than 1% by weight it plays essential roles in improving both toughness and resilience to chemical attacks. Despite the fact that the first studies of the enamel matrix began in the 19th century, its exact composition and mechanisms of its function remain poorly understood. It was proposed that keratin or a keratin-like primitive epithelial component exists in mature enamel, however due to the extreme insolubility of its organic matrix the presence of keratins there was never clearly established. We have recently identified expression of a number of hair keratins in ameloblasts, the enamel secreting cells, and demonstrated their incorporation into mature enamel. Mutation in epithelial hair keratin KRT75 leads to a skin condition called pseudofollicularis barbae. Carriers of this mutation have an altered enamel structure and mechanical properties. Importantly, these individuals have a much higher prevalence of caries. To the best of our knowledge, this is the first study showing a direct link between a mutation in a protein-coding region of a gene and increased caries rates. In this paper we present an overview of the evidence of keratin-like material in enamel that has accumulated over the last 150years. Furthermore, we propose potential mechanisms of action of KTR75 in enamel and highlight the clinical implications of the link between mutations in KRT75 and caries. Finally, we discuss the potential use of keratins for enamel repair.
Authors: Zhou Q, Wang H, Schwartz DM, Stoffels M, Park YH, Zhang Y, Yang D, Demirkaya E, Takeuchi M, Tsai WL, Layons JJ, Yu X, Ouyang C, Chen C, Chin DT, Zaal K, Chandrasekharappa SC, P Hanson E, Yu Z, Mullikin JC, Hasni SA, Wertz IE, Ombrello AK, Stone DL, Hoffmann P, Jones A, Barham BK, Leavis HL, van Royen-Kerkof A, Sibley C, Batu ED, Gül A, Siegel RM, Boehm M, Milner JD, Ozen S, Gadina M, Chae J, Laxer RM, Kastner DL, Aksentijevich I.
Journal: Nat Genet. 2015 Dec 7. doi: 10.1038/ng.3459.
Systemic autoinflammatory diseases are driven by abnormal activation of innate immunity. Herein we describe a new disease caused by high-penetrance heterozygous germline mutations in TNFAIP3, which encodes the NF-κB regulatory protein A20, in six unrelated families with early-onset systemic inflammation. The disorder resembles Behçet's disease, which is typically considered a polygenic disorder with onset in early adulthood. A20 is a potent inhibitor of the NF-κB signaling pathway. Mutant, truncated A20 proteins are likely to act through haploinsufficiency because they do not exert a dominant-negative effect in overexpression experiments. Patient-derived cells show increased degradation of IκBα and nuclear translocation of the NF-κB p65 subunit together with increased expression of NF-κB-mediated proinflammatory cytokines. A20 restricts NF-κB signals via its deubiquitinase activity. In cells expressing mutant A20 protein, there is defective removal of Lys63-linked ubiquitin from TRAF6, NEMO and RIP1 after stimulation with tumor necrosis factor (TNF). NF-κB-dependent proinflammatory cytokines are potential therapeutic targets for the patients with this disease.
Authors: Klebanoff CA, Scott CD, Leonardi AJ, Yamamoto TN, Cruz AC, Ouyang C, Ramaswamy M, Roychoudhuri R, Ji Y, Eil RL, Sukumar M, Crompton JG, Palmer DC, Borman ZA, Clever D, Thomas SK, Patel S, Yu Z, Muranski P, Liu H, Wang E, Marincola FM, Gros A, Gattinoni L, Rosenberg SA, Siegel RM, Restifo NP.
Journal: J Clin Invest. 2015 Dec 14. pii: 81217. doi: 10.1172/JCI81217.
Adoptive cell transfer (ACT) of purified naive, stem cell memory, and central memory T cell subsets results in superior persistence and antitumor immunity compared with ACT of populations containing more-differentiated effector memory and effector T cells. Despite a clear advantage of the less-differentiated populations, the majority of ACT trials utilize unfractionated T cell subsets. Here, we have challenged the notion that the mere presence of less-differentiated T cells in starting populations used to generate therapeutic T cells is sufficient to convey their desirable attributes. Using both mouse and human cells, we identified a T cell-T cell interaction whereby antigen-experienced subsets directly promote the phenotypic, functional, and metabolic differentiation of naive T cells. This process led to the loss of less-differentiated T cell subsets and resulted in impaired cellular persistence and tumor regression in mouse models following ACT. The T memory-induced conversion of naive T cells was mediated by a nonapoptotic Fas signal, resulting in Akt-driven cellular differentiation. Thus, induction of Fas signaling enhanced T cell differentiation and impaired antitumor immunity, while Fas signaling blockade preserved the antitumor efficacy of naive cells within mixed populations. These findings reveal that T cell subsets can synchronize their differentiation state in a process similar to quorum sensing in unicellular organisms and suggest that disruption of this quorum-like behavior among T cells has potential to enhance T cell-based immunotherapies.
Authors: Whirledge SD, Oakley RH, Myers PH, Lydon JP, DeMayo F, Cidlowski JA
Journal: Proc Natl Acad Sci U S A. 2015 Dec 8;112(49):15166-71. doi: 10.1073/pnas.1508056112. Epub 2015 Nov 23.
In addition to the well-characterized role of the sex steroid receptors in fertility and reproduction, organs of the female reproductive tract are also regulated by the hypothalamic-pituitary-adrenal axis. These endocrine organs are sensitive to stress-mediated actions of glucocorticoids, and the mouse uterus contains high levels of the glucocorticoid receptor (GR). Although the presence of GR in the uterus is well established, uterine glucocorticoid signaling has been largely ignored in terms of its reproductive and/or immunomodulatory functions on fertility. To define the direct in vivo function of glucocorticoid signaling in adult uterine physiology, we generated a uterine-specific GR knockout (uterine GR KO) mouse using the PR(cre) mouse model. The uterine GR KO mice display a profound subfertile phenotype, including a significant delay to first litter and decreased pups per litter. Early defects in pregnancy are evident as reduced blastocyst implantation and subsequent defects in stromal cell decidualization, including decreased proliferation, aberrant apoptosis, and altered gene expression. The deficiency in uterine GR signaling resulted in an exaggerated inflammatory response to induced decidualization, including altered immune cell recruitment. These results demonstrate that GR is required to establish the necessary cellular context for maintaining normal uterine biology and fertility through the regulation of uterine-specific actions.
Authors: Karron RA, Luongo C, Thumar B, Loehr KM, Englund JA, Collins PL, Buchholz UJ.
Journal: Sci Transl Med. 2015 Nov 4;7(312):312ra175. doi: 10.1126/scitranslmed.aac8463.
Respiratory syncytial virus (RSV) is the leading viral cause of severe pediatric respiratory illness, and a safe and effective vaccine for use in infancy and early childhood is needed. We previously showed that deletion of the coding sequence for the viral M2-2 protein (ΔM2-2) down-regulated viral RNA replication and up-regulated gene transcription and antigen synthesis, raising the possibility of development of an attenuated vaccine with enhanced immunogenicity. RSV MEDI ΔM2-2 was therefore evaluated as a live intranasal vaccine in adults, RSV-seropositive children, and RSV-seronegative children. When results in RSV-seronegative children were compared to those achieved with the previous leading live attenuated RSV candidate vaccine, vaccine virus shedding was significantly more restricted, yet the postvaccination RSV-neutralizing serum antibody achieved [geometric mean titer (GMT) = 1:97] was significantly greater. Surveillance during the subsequent RSV season showed that several seronegative RSV MEDI ΔM2-2 recipients had substantial antibody rises without reported illness, suggesting that the vaccine was protective yet primed for anamnestic responses to RSV. Rational design appears to have yielded a candidate RSV vaccine that is intrinsically superior at eliciting protective antibody in RSV-naïve children and highlights an approach for the development of live attenuated RSV vaccines.
Copyright © 2015, American Association for the Advancement of Science.
Authors: Brehm A, Liu Y, Sheikh A, Marrero B, Omoyinmi E, Zhou Q, Montealegre G, Biancotto A, Reinhardt A, Almeida de Jesus A, Pelletier M, Tsai WL, Remmers EF, Kardava L, Hill S, Kim H, Lachmann HJ, Megarbane A, Chae JJ, Brady J, Castillo RD, Brown D, Casano AV, Gao L, Chapelle D, Huang Y, Stone D, Chen Y, Sotzny F, Lee CC, Kastner DL, Torrelo A, Zlotogorski A, Moir S, Gadina M, McCoy P, Wesley R, Rother K, Hildebrand PW, Brogan P, Krüger E, Aksentijevich I, Goldbach-Mansky R.
Journal: J Clin Invest. 2015 Nov 2;125(11):4196-211. doi: 10.1172/JCI81260. Epub 2015 Oct 20.
Autosomal recessive mutations in proteasome subunit β 8 (PSMB8), which encodes the inducible proteasome subunit β5i, cause the immune-dysregulatory disease chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE), which is classified as a proteasome-associated autoinflammatory syndrome (PRAAS). Here, we identified 8 mutations in 4 proteasome genes, PSMA3 (encodes α7), PSMB4 (encodes β7), PSMB9 (encodes β1i), and proteasome maturation protein (POMP), that have not been previously associated with disease and 1 mutation in PSMB8 that has not been previously reported. One patient was compound heterozygous for PSMB4 mutations, 6 patients from 4 families were heterozygous for a missense mutation in 1 inducible proteasome subunit and a mutation in a constitutive proteasome subunit, and 1 patient was heterozygous for a POMP mutation, thus establishing a digenic and autosomal dominant inheritance pattern of PRAAS. Function evaluation revealed that these mutations variably affect transcription, protein expression, protein folding, proteasome assembly, and, ultimately, proteasome activity. Moreover, defects in proteasome formation and function were recapitulated by siRNA-mediated knockdown of the respective subunits in primary fibroblasts from healthy individuals. Patient-isolated hematopoietic and nonhematopoietic cells exhibited a strong IFN gene-expression signature, irrespective of genotype. Additionally, chemical proteasome inhibition or progressive depletion of proteasome subunit gene transcription with siRNA induced transcription of type I IFN genes in healthy control cells. Our results provide further insight into CANDLE genetics and link global proteasome dysfunction to increased type I IFN production.
Authors: Fitzhugh CD, Hsieh MM, Allen D, Coles WA, Seamon C, Ring M, Zhao X, Minniti CP, Rodgers GP, Schechter AN, Tisdale JF, Taylor JG
Journal: PLoS One. 2015 Nov 17;10(11):e0141706. doi: 10.1371/journal.pone.0141706.
BACKGROUND: Adults with sickle cell anemia (HbSS) are inconsistently treated with hydroxyurea.
OBJECTIVES: We retrospectively evaluated the effects of elevating fetal hemoglobin with hydroxyurea on organ damage and survival in patients enrolled in our screening study between 2001 and 2010.
METHODS: An electronic medical record facilitated development of a database for comparison of study parameters based on hydroxyurea exposure and dose. This study is registered with ClinicalTrials.gov, number NCT00011648.
RESULTS: Three hundred eighty-three adults with homozygous sickle cell disease were analyzed with 59 deaths during study follow-up. Cox regression analysis revealed deceased subjects had more hepatic dysfunction (elevated alkaline phosphatase, Hazard Ratio = 1.005, 95% CI 1.003-1.006, p<0.0.0001), kidney dysfunction (elevated creatinine, Hazard Ratio = 1.13, 95% CI 1.00-1.27, p = 0.043), and cardiopulmonary dysfunction (elevated tricuspid jet velocity on echocardiogram, Hazard Ratio = 2.22, 1.23-4.02, p = 0.0082). Sixty-six percent of subjects were treated with hydroxyurea, although only 66% of those received a dose within the recommended therapeutic range. Hydroxyurea use was associated with improved survival (Hazard Ratio = 0.58, 95% CI 0.34-0.97, p = 0.040). This effect was most pronounced in those taking the recommended dose of 15-35 mg/kg/day (Hazard Ratio 0.36, 95% CI 0.17-0.73, p = 0.0050). Hydroxyurea use was not associated with changes in organ function over time. Further, subjects with higher fetal hemoglobin responses to hydroxyurea were more likely to survive (p = 0.0004). While alkaline phosphatase was lowest in patients with the best fetal hemoglobin response (95.4 versus 123.6, p = 0.0065 and 96.1 versus 113.6U/L, p = 0.041 at first and last visits, respectively), other markers of organ damage were not consistently improved over time in patients with the highest fetal hemoglobin levels.
CONCLUSIONS: Our data suggest that adults should be treated with the maximum tolerated hydroxyurea dose, ideally before organ damage occurs. Prospective studies are indicated to validate these findings.
Authors: Liu Z, Gerner MY, Van Panhuys N, Levine AG, Rudensky AY, Germain RN.
Journal: Nature. 2015 Nov 25. doi: 10.1038/nature16169.
FOXP3+ regulatory T cells (Treg cells) prevent autoimmunity by limiting the effector activity of T cells that have escaped thymic negative selection or peripheral inactivation. Despite the information available about molecular factors mediating the suppressive function of Treg cells, the relevant cellular events in intact tissues remain largely unexplored, and whether Treg cells prevent activation of self-specific T cells or primarily limit damage from such cells has not been determined. Here we use multiplex, quantitative imaging in mice to show that, within secondary lymphoid tissues, highly suppressive Treg cells expressing phosphorylated STAT5 exist in discrete clusters with rare IL-2-positive T cells that are activated by self-antigens. This local IL-2 induction of STAT5 phosphorylation in Treg cells is part of a feedback circuit that limits further autoimmune responses. Inducible ablation of T cell receptor expression by Treg cells reduces their regulatory capacity and disrupts their localization in clusters, resulting in uncontrolled effector T cell responses. Our data thus reveal that autoreactive T cells are activated to cytokine production on a regular basis, with physically co-clustering T cell receptor-stimulated Treg cells responding in a negative feedback manner to suppress incipient autoimmunity and maintain immune homeostasis.
Clusters of Tregs (green) form in healthy mouse lymph nodes.