DNAAF5 is a dynein motor assembly factor associated with the autosomal heterogenic recessive condition of motile cilia, primary ciliary dyskinesia (PCD). The effects of allele heterozygosity on motile cilia function are unknown. We used CRISPR-Cas9 genome editing in mice to recreate a human missense variant identified in patients with mild PCD and a second, frameshift null deletion in Dnaaf5. Litters with Dnaaf5 heteroallelic variants showed distinct missense and null gene dosage effects. Homozygosity for the null Dnaaf5 alleles was embryonic lethal. Compound heterozygous animals with the missense and null alleles showed severe disease manifesting as hydrocephalus and early lethality. However, animals homozygous for the missense mutation had improved survival, with partial preserved cilia function and motor assembly observed by ultrastructure analysis. Notably, the same variant alleles exhibited divergent cilia function across different multiciliated tissues. Proteomic analysis of isolated airway cilia from mutant mice revealed reduction in some axonemal regulatory and structural proteins not previously reported in DNAAF5 variants. While transcriptional analysis of mouse and human mutant cells showed increased expression of genes coding for axonemal proteins. Together, these findings suggest allele-specific and tissue-specific molecular requirements for cilia motor assembly that may affect disease phenotypes and clinical trajectory in motile ciliopathies.
Amjad Horani, Deepesh Gupta, Jian Xu, Huihui Xu, Lis del C. Puga Molina, Celia M. Santi, Sruthi Ramagiri, Steven K. Brennan, Jiehong Pan, Jeffrey R. Koenitzer, Tao Huang, Rachael M. Hyland, Sean P. Gunsten, Shin-Cheng Tzeng, Jennifer M. Strahle, Pleasantine Mill, Moe R. Mahjoub, Susan K. Dutcher, Steven L. Brody
The development and progression of endometriotic lesions are poorly understood, but immune cell dysfunction and inflammation are closely associated with the pathophysiology of endometriosis. There is a need for 3D in vitro models to permit the study of interactions between cell types and the microenvironment. To address this, we developed endometriotic spheroids (ES) to explore the role of epithelial-stromal interactions and model peritoneal invasion associated with lesion development. Using a non-adherent microwell culture system, spheroids were generated with immortalized endometriotic epithelial cells(12Z) combined with endometriotic stromal (iEc-ESC) or uterine stromal (iHUF) cell lines. Transcriptomic analysis found 4,522 differentially expressed genes in ES compared to spheroids containing uterine stromal cells. The top increased gene sets were inflammation-related pathways, and an overlap with baboon endometriotic lesions was highly significant. Finally, to mimic invasion of endometrial tissue into the peritoneum, a model was developed with human peritoneal mesothelial cells in an extracellular matrix. Invasion was increased in presence of estradiol or proinflammatory macrophages and suppressed by a progestin. Taken together, our results strongly support the concept that ES are an appropriate model for dissecting mechanisms that contribute to endometriotic lesion development.
Yong Song, Gregory W. Burns, Niraj R. Joshi, Ripla Arora, Ji-Yong Julie Kim, Asgerally T. Fazleabas
Biological sex and host genetics influence HIV pathogenesis. Females have a higher likelihood of spontaneous viral control and lower setpoint viral load (spVL). No prior studies have assessed sex-specific genetics of HIV. To address this, we performed a sex stratified genome-wide association study using data from the International Collaboration for the Genomics of HIV. Although it is the largest collection of genomic data in HIV, this multi-ethnic sample of 9,705 people is 81.3% male. We sought to identify sex-specific genetic variants and genes associated with HIV spVL and control. We confirmed associations in the HLA and CCR5 regions in males, and HLA in females. Gene-based analyses detected associations between HIV spVL and PET100 (Pvalue=8.36x10-07), PCP2 (Pvalue=8.81x10-07), XAB2 (Pvalue=1.32x10-6) and STXBP2 (Pvalue=1.65x10-4) only in males. We detected variants with a significant sex-differential effect on spVL in SDC3 and PUM1 (rs10914268,Pvalue=1.93x10-08) and PSORS1C2 (rs1265159, Pvalue=3.26x10-08) and on HIV control in SUB1 (rs687659, Pvalue=1.02×10-08), AL158151.3, PTPA and IER5L (rs4387067, Pvalue=2.07×10-09). Those variants have epigenetic and genetic interactions with relevant genes with both cis and trans effects. In summary, we identified sex-shared associations at the single variant level, sex-specific associations at the gene-based level, and genetic variants with significant differential effects between the sexes.
Candelaria Vergara, Jeffrey F. Tuff, International Collaboration for the Genomics of HIV, Jacques Fellay, Priya Duggal, Eileen P. Scully, Paul J. McLaren
Patients with peripheral artery disease (PAD) and diabetes constitute a high risk population for development of critical limb ischemia (CLI) and amputation, although the underlying mechanisms remain poorly understood. Comparison of dysregulated microRNAs from diabetic human subjects with PAD and diabetic mice with limb ischemia revealed the conserved microRNA, miR-130b-3p. In vitro angiogenic assays demonstrated miR-130b rapidly promoted proliferation, migration, and sprouting in endothelial cells (ECs), whereas miR-130b inhibition exerted anti-angiogenic effects. Local delivery of miR-130b mimics into ischemic muscles of diabetic mice (db/db) following femoral artery ligation (FAL) promoted revascularization by increasing angiogenesis and markedly improved limb necrosis and amputation. RNA-sequencing, and gene set enrichment analysis from miR-130b overexpressing ECs revealed the BMP / TGF-b signaling pathway as one of the top dysregulated pathways. Accordingly, overlapping downregulated transcripts from RNA-seq and miRNA prediction algorithms identified that miR-130b directly targeted and repressed the TGF-b superfamily member inhibin-b-A (INHBA). miR-130b overexpression or siRNA-mediated knockdown of INHBA induced IL-8 expression, a potent angiogenic chemokine. Lastly, ectopic delivery of silencer RNAs (siRNA) targeting Inhba in db/db ischemic muscles following FAL improved revascularization and limb necrosis, recapitulating the phenotype of miR-130b delivery. Taken together, a miR-130b-INHBA signaling axis may provide therapeutic targets for patients with PAD and diabetes at risk of developing CLI.
Henry S. Cheng, Daniel Pérez-Cremades, Rulin Zhuang, Anurag Jamaiyar, Winona W. Wu, Jingshu Chen, Aspasia Tzani, Lauren Stone, Jorge Plutzky, Terence E. Ryan, Philip P. Goodney, Mark A. Creager, Marc S. Sabatine, Marc P. Bonaca, Mark W. Feinberg
Myelin oligodendrocyte glycoprotein (MOG) antibody-associated disease (MOGAD) is an inflammatory demyelinating central nervous system condition characterized by the presence of MOG autoantibodies. We sought to investigate whether human MOG autoantibodies are capable of mediating damage to MOG-expressing cells through multiple mechanisms. We developed high-throughput assays to measure complement activity (CA), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), and antibody-dependent cellular cytotoxicity (ADCC) of live MOG-expressing cells. MOGAD patient sera effectively mediate all of these effector functions. Our collective analyses reveal that [i] cytotoxicity is not incumbent on MOG autoantibody quantity alone, [ii] engagement of effector functions by MOGAD patient serum is bimodal, with some sera exhibiting cytotoxic capacity while others did not, [iii] the magnitude of CDC and ADCP is elevated closer to relapse, while MOG-IgG binding is not, and [iv] all IgG subclasses can damage MOG-expressing cells. Histopathology from a representative MOGAD case revealed congruence between lesion histology and serum CDC and ADCP, and we identified NK cells, mediators of ADCC, in the cerebrospinal fluid of relapsing MOGAD patients. Thus, MOGAD-derived autoantibodies are cytotoxic to MOG-expressing cells through multiple mechanisms and assays quantifying CDC and ADCP may prove to be effective tools for predicting risk of future relapses.
Soumya S. Yandamuri, Beata Filipek, Abeer H. Obaid, Nikhil Lele, Joshua M. Thurman, Naila Makhani, Richard J. Nowak, Yong Guo, Claudia F. Lucchinetti, Eoin P. Flanagan, Erin E. Longbrake, Kevin C. O'Connor
Although thymidylate synthase (TYMS) inhibitors have served as components of chemotherapy regimens, the currently available inhibitors induce TYMS overexpression or alter folate transport/metabolism feedback pathways that tumor cells exploit for drug resistance limiting overall benefit. Here we report a small molecule TYMS inhibitor that i) exhibits enhanced antitumor activity as compared to current fluoropyrimidines and antifolates without inducing TYMS overexpression, ii) is structurally distinct from classical antifolates, iii) extends survival in both pancreatic xenograft tumor models and hTS/Ink4a/Arf null genetically engineered mouse tumor model, iv) and is well tolerated with equal efficacy using either intraperitoneal or oral administration. Mechanistically, we confirm the compound is a multifunctional non-classical antifolate, and using a series of analogues, we identify structural features allowing direct TYMS inhibition while also maintaining the ability to inhibit dihydrofolate reductase (DHFR). Collectively, this work identifies new non-classical antifolate inhibitors that optimize inhibition of thymidylate biosynthesis with a favorable safety profile highlighting potential for enhanced cancer therapy.
Maria V. Guijarro, Patrick C. Kellish, Peter E. Dib, Nicholas G. Paciaroni, Akbar Nawab, Jacob Andring, Lidia Kulemina, Nicholas V. Borrero, Carlos Modenutti, Michael Feely, Elham Nasri, Robert P. Seifert, Xiaoping Luo, Richard L. Bennett, Daniil Shabashvili, Jonathan D. Licht, Robert McKenna, Adrian Roitberg, Robert W. Huigens III, Frederic J. Kaye, Maria Zajac-Kaye
The intake of dietary phosphate far exceeds recommended levels however the long-term health consequences remain relatively unknown. Here, the chronic physiological response to sustained elevated and reduced dietary phosphate consumption was investigated in mice. Although serum phosphate levels were brought into homeostatic balance, the prolonged intake of a high-phosphate diet dramatically and negatively impacted bone volume, generated a sustained increase in the phosphate responsive circulating factors, FGF23, PTH, osteopontin and osteocalcin, and produced a chronic low grade inflammatory state in the bone marrow, marked by increased numbers of T cells expressing IL-17a, RANKL, and TNFα. In contrast, a low-phosphate diet preserved trabecular bone while increasing cortical bone volume over time and reduced inflammatory T cell populations. Cell-based studies identified a direct response of T cells to elevated extracellular phosphate. Neutralizing antibodies to pro-osteoclastic cytokines RANKL, TNFα, and IL-17a blunted the high-phosphate diet induced bone loss identifying bone resorption as a regulatory mechanism. Collectively, this study illuminates that habitual consumption of a high-phosphate diet in mice induces chronic inflammation in bone even in the absence of elevated serum phosphate. Further, the study supports the concept that a reduced phosphate diet may be a simple, yet effective strategy to reduce inflammation and improve bone health during aging
Joseph L. Roberts, Mingcan Yu, Manjula Viggeswarapu, Jamie L. Arnst, Roberto Pacifici, George R. Beck
BACKGROUND. People living with HIV (PLHIV) on antiretroviral therapy (ART) exhibit persistent immune dysregulation and microbial dysbiosis, leading to the development of cardiovascular diseases (CVD). We initially compared plasma proteomic profiles between 205 PLHIV and 120 healthy controls (HCs) and validated the results in an independent cohort of 639 PLHIV and 99 HCs. Differentially expressed proteins (DEPs) were then associated to microbiome data. Finally, we assessed which proteins were linked with CVD development in PLHIV. METHOD. Proximity extension assay technology was utilized to measure 1472 plasma proteins. Markers of systemic inflammation (CRP, D-Dimer, IL6, sCD14, and sCD163) and microbial translocation (IFABP) were measured by ELISA, and gut bacterial species were identified using shotgun metagenomic sequencing. Baseline CVD data were available for all PLHIV, and 205 PLHIV were recorded for the development of CVD during a 5-year follow-up. RESULTS. PLHIV on ART displayed systemic dysregulation of protein concentrations compared to HCs. Most of the DEPs originated from the intestine and lymphoid tissues, while they enriched in immune- and lipid metabolism-related pathways. Furthermore, we observed that DEPs originating from the intestine were associated with specific gut bacterial species. Finally, we identified upregulated proteins in PLHIV (GDF15, PLAUR, RELT, NEFL, COL6A3, and EDA2R), unlike most markers of systemic inflammation, associated with the presence and risk of developing CVD in 5-year follow-up. CONCLUSIONS. Our findings suggest a systemic dysregulation of protein concentrations in PLHIV, of which some proteins were associated with CVD development. Most of DEPs originated from the gut and were related to specific gut bacterial species. TRIAL REGISTRATION. Cohorts included in this study are part of the Human Functional Genomics Project (HFGP) (www.humanfunctionalgenomics.org). The 2000HIV Human Functional Genomics Partnership Program is registered at ClinicalTrials.gov: (ID: NCT03994835). FUNDING. The 200HIV and 2000HIV studies are supported by the AIDS-fonds (#P-29001, Netherlands) and a ViiV healthcare grant (A18-1052), respectively; The ViiV healthcare grant was awarded to A.V., M.G.N., L.A.B.J., and Q.d.M; The Spinoza Prize (NWO SPI94-212) and ERC Advanced grant (no. 833247) were awarded to M.G.N; The Indonesia Endowment Fund for Education (LPDP) given by the Ministry of Finance of the Republic of Indonesia was awarded to N.V.
Nadira Vadaq, Yue Zhang, Wilhelm A.J.W. Vos, Albert L. Groenendijk, Martinus J.T. Blaauw, Louise E. van Eekeren, Maartje C.P. Jacobs-Cleophas, Lisa Van de Wijer, Jéssica Cristina dos Santos, Muhammad Hussein Gasem, Leo A.B. Joosten, Mihai G. Netea, Quirijn de Mast, Jingyuan Fu, André J.A.M. van der Ven, Vasiliki Matzaraki
Abdominal aortic aneurysm (AAA) is usually asymptomatic until life-threatening complications occur, predominantly involving aortic rupture. Currently, no drug-based treatments are available, primarily due to limited understanding of AAA pathogenesis. Transcriptional regulator PR domain-containing protein 16 (PRDM16) is highly expressed in the aorta, but its functions in the aorta are largely unknown. By RNA-seq analysis, we found that VSMCs-specific Prdm16 knockout mice (Prdm16SMKO) already showed extensive changes in the expression of genes associated with extracellular matrix (ECM) remodeling and inflammation in the abdominal aorta under normal housing conditions without any pathological stimuli. Human AAA lesions displayed lower PRDM16 expression. Periadventitial elastase application to the suprarenal region of the abdominal aorta aggravated AAA formation in Prdm16SMKO. During AAA development, VSMCs undergo apoptosis because of both intrinsic and environmental changes including inflammation and ECM remodeling. Prdm16 deficiency promoted inflammation and apoptosis in VSMCs. A disintegrin and metalloproteinase 12 (ADAM12) is a gelatinase which can degrade various ECM. We found that ADAM12 is a target of transcriptional repression by PRDM16. Adam12 knockdown reversed VSMC apoptosis induced by Prdm16 deficiency. Our study demonstrated that PRDM16 deficiency in VSMCs promoted ADAM12 expression and aggravates AAA formation, which may provide potential targets for AAA treatment.
Zhenguo Wang, Xiangjie Zhao, Guizhen Zhao, Yanhong Guo, Haocheng Lu, Wenjuan Mu, Juan Zhong, Minerva Garcia-Barrio, Jifeng Zhang, Y. Eugene Chen, Lin Chang
Muscular dystrophies are a group of genetic neuromuscular disorders that involve severe muscle wasting. Transforming growth factor β-activated kinase 1 (TAK1) is an important signaling protein that regulates cell survival, growth, and inflammation. TAK1 has been recently found to promote myofiber growth in the skeletal muscle of adult mice. However, the role of TAK1 in muscle diseases remains poorly understood. In the present study, we have investigated how TAK1 affects the progression of dystrophic phenotype in the mdx mouse model of Duchenne muscular dystrophy (DMD). TAK1 is highly activated in the dystrophic muscle of mdx mice during the peak necrotic phase. While targeted inducible inactivation of TAK1 inhibits myofiber injury in young mdx mice, it results in reduced muscle mass and contractile function. TAK1 inactivation also causes loss of muscle mass in adult mdx mice. By contrast, forced activation of TAK1 through overexpression of TAK1 and TAB1 induces myofiber growth without having any deleterious effect on muscle histopathology. Collectively, our results suggest that TAK1 is a positive regulator of skeletal muscle mass and targeted regulation of TAK1 can suppress myonecrosis and ameliorate disease progression in DMD.
Anirban Roy, Tatiana E. Koike, Aniket S. Joshi, Meiricris Tomaz da Silva, Kavya Mathukumalli, Mingfu Wu, Ashok Kumar
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