GATA2 mutation is associated with immune dysfunction and increased Mycobacterium haemophilum susceptibility in immunocompromised individuals

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Abstract

Infections with non-tuberculous mycobacterium (NTM) are on the rise. Here, we investigated an uncommon NTM infection, by M. haemophilum (Mh, n = 3), from a shared geographic location in the USA. All patients had underlying immunosuppressive conditions or treatments. We identified that all these individuals had a non-synonymous mutation in GATA2 gene, which was absent in Healthy controls (HCs, n = 4) from the same geographic area (Missouri, USA). Whole blood from these individuals had attenuated cytokine responses to Mh stimulation for IL1β, IL-6, IL-8, MIP-1α and β, but not to Phytohemagglutinin (PHA) or another NTM, M. abscessus. Impaired whole blood transcriptional responses in individuals with GATA2 mutation, included heightened Ras-homolog (Rho) guanosine triphosphate hydrolases (GTPase) and lowered Transforming growth factor (TGF)-β responses among others. Our results highlight that comparatively, M. abscessus and Mh elicit differential immune responses in humans, and we identify a 23-gene signature that distinguishes host response to Mh and M. abscessus and show that in vitro GATA2 siRNA knockdown indeed attenuates cytokine responses to Mh. Thus, we provide new evidence that links GATA2 mutation and immune dysfunction in individuals with compromised immunity to Mh infection in humans and outline host factors associated with the immune response of this clinically relevant NTM.

Authors

Ananya Gupta, Shail B. Mehta, Abhimanyu A, Bruce A. Rosa, John Martin, Mushtaq Ahmed, Shyamala Thirunavukkarasu, Farheen Fatma, Gaya Amarsinghe, Makedonka Mitreva, Thomas C. Bailey, David B. Clifford, Shabaana A. Khader

PRDM16 acts as a therapeutic downstream target of TGF-β signaling in chronic kidney disease

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Abstract

Transforming growth factor beta (TGF-β) signaling is the master modulator of renal fibrosis. However, targeting drugs have failed to prevent the progression of chronic kidney disease (CKD) in clinical trials due to the extensive biological regulation of TGF-β signaling. It is necessary to investigate the precise downstream mechanisms of TGF-β signaling that regulate renal fibrosis. In this study, we found that PR-domain containing 16 (PRDM16) expression in human renal tubular epithelial cells was markedly reduced by TGF-β. Mechanistically, activated Smad3 induced by TGF-β interacted with the cofactor H-Ras and bound to the promoter of PRDM16, downregulating its transcription. Tubular-specific knockout of PRDM16 promoted renal fibrosis in models of unilateral ureteral occlusion (UUO) and unilateral ischemia-reperfusion injury (UIRI) by exacerbating mitochondrial dysfunction. In vitro, PRDM16 blocked TGF-β-induced mitochondrial injury and lipid deposition by upregulating Peroxisome Proliferator-Activated Receptor Gamma Coactivator-1α (PGC-1α). Delivery of the exogenous PRDM16 gene preserved renal function and ameliorated the progression of renal fibrosis by protecting mitochondrial function. We report PRDM16 as a novel downstream target of TGF-β signaling that attenuates renal fibrosis by safeguarding tubular mitochondrial function.

Authors

Qian Yuan, Ben Tang, Yuting Zhu, Chao Wan, Yaru Xie, Yajuan Xie, Cheng Wan, Hua Su, Youhua Liu, Chun Zhang

Pro-inflammatory macrophages transporting gut-derived bacterial DNA drive autoimmune arthritis in spondyloarthropathy

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Abstract

Spondyloarthritis (SpA) is an inflammatory arthritis of the spine and joints associated with intestinal inflammation, in which it is hypothesized that innate immune exposure to entero-invasive species is followed by self/bacterial peptide presentation. However, the mechanisms underlying loss of tolerance to gut bacteria in genetically at-risk individuals are unclear. Curdlan (β-1,3-glucan, dectin-1 ligand)-treated ZAP-70W163C (SKG) mice develop autoimmune arthritis and ileitis associated with Gram-negative faecal dysbiosis. Using gnotobiotic mice, we show that curdlan-treated SKG mice mono-associated with Parabacteroides goldsteinii or Lactobacillus murinus developed ileitis, arthritis and enthesitis, while BALB/c mice were tolerant. Gnotobiotic SKG ileum upregulated Il23a and ER stress genes and lost goblet cells. Whereas bacterial DNA co-localised with neutrophils and inflammatory macrophages in SKG lamina propria, peri-articular bone marrow, entheses and spleen, in BALB/c bacterial DNA co-localised with resident macrophages in lamina propria and spleen. Human psoriatic-arthritis synovial tissue also contained cell-associated peri-vascular bacterial DNA. Curdlan-treated SKG spleen/bone marrow macrophages transferred severe arthritis and expanded Th17 cells in naïve SKG recipients, while BALB/c or germ free-SKG macrophages transferred mild arthritis and regulated Th17 cells. Thus, bacterial DNA and myeloid cells in the gut and their subsequent traffic regulate or enforce T cell pathogenicity in SpA.

Authors

Benjamin Cai, Rabina Giri, Amy J. Cameron, M. Arifur Rahman, Annabelle Small, Christopher Altmann, Yenkai Lim, Linda M. Rehaume, Mark Morrison, Mihir D. Wechalekar, Jakob Begun, Anne-Sophie Bergot, Ranjeny Thomas

Regulatory T cells epigenetically reprogrammed from autoreactive effector T cells mitigate established autoimmunity

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Abstract

Reprogramming autoreactive CD4⁺ effector T (Teff) cells into immunosuppressive regulatory T (Treg) cells represents a promising strategy for treating established autoimmune diseases. However, the stability and function of such reprogrammed Tregs under inflammatory conditions remain unclear. Here, we show that epigenetic activation of core Treg identity genes in Teff cells yields lineage-stable Effector T cell Reprogrammed Tregs (ER-Tregs). A single adoptive transfer of ER-Tregs not only prevents autoimmune neuroinflammation in mice when given before disease onset but also arrests its progression when administered after onset. Compared to Foxp3 overexpressing Teff cells, induced Tregs from naïve precursors, and endogenous Tregs, ER Tregs provide superior protection against autoimmune neuroinflammation. This enhanced efficacy stems from their inherited autoantigen specificity and selectively preserved effector cell transcriptional programs, which together bolster their fitness in inflammatory environments and enhance their suppressive capacity. Our results establish epigenetic reprogramming of autoreactive Teff cells as an effective approach to generate potent, stable Tregs for the treatment of refractory autoimmune conditions.

Authors

Tyler R. Colson, James J. Cameron, Hayley I. Muendlein, Mei-An Nolan, Jamie L. Leiriao, James H. Kim, Alexander N. Poltorak, Xudong Li

Efficacious genome editing in infant mice with glycogen storage disease type Ia

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Abstract

Glycogen storage disease type Ia (GSD Ia) is caused by a deficiency of glucose-6-phosphatase (G6Pase) in the liver leading to lethal hypoglycemia. Gene therapy with adeno-associated virus (AAV) vectors encoding G6Pase fails to stably treat GSD Ia early in life. We evaluated genome editing in 12 day-old infant mice with GSD Ia using two AAV vectors, one containing Cas9 from Streptococcus pyogenes and a second Donor vector that expresses a guide RNA and a G6PC transgene. Gene therapy with the Donor vector only was compared with genome editing using both Donor and CRISPR vectors. Treatment with genome editing (total vector dose 0.2 to 2E+13 vector genomes/kg) and bezafibrate (to stimulate autophagy) was efficacious as assessed by hypoglycemia prevention and the frequency of transgene integration, which correlated with improved survival. This therapy achieved 5.9% chromosomal transgene integration through homology directed repair, which surpassed a threshold to prevent long-term hepatic complications. No integration was detected in absence of the CRISPR vector. Importantly for safety, CRISPR vector genomes were depleted, and no intact, integrated CRISPR genomes were detected by long-read sequencing. Thus, genome editing warrants further development as a potentially stable treatment for human infants with GSD Ia.

Authors

Benjamin Arnson, Ekaterina Ilich, Troy von Beck, Songtao Li, Elizabeth D. Brooks, Dorothy Gheorghiu, Gordon He, Matthew Weinrub, Sze Ying Chan, Hye-Ri Kang, David Courtney, Jeffrey Everitt, Bryan R. Cullen, Dwight D. Koeberl

Low‐intensity pulsed ultrasound stimulation to treat renal fibrosis through inhibiting tubular IL-1R

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Abstract

Low-intensity pulsed ultrasound stimulation (LIPUS) has become increasingly appreciated for its therapeutic effect on kidney diseases. However, its role and biological mechanism in treating chronic kidney disease (CKD) remain poorly defined. Here, we revealed that LIPUS was applied in a safe range with an intensity of 25-315 mW/cm2. Daily LIPUS at an intensity of 315 mW/cm2 ameliorated ischemia-reperfusion (IR)-induced tubular injury and renal fibrosis, accompanied by the remarkable downregulation of IL-1R. Transcriptome sequencing showed that LIPUS significantly down-regulated IL-1R and its downstream genes in IL-1β-stimulated IR-injured mice. LIPUS effectively reversed IL-1β-induced tubular injury and reduced the production of profibrotic cytokines by down-regulating IL-1R in vivo and in vitro. Renal proximal tubule-specific Il1r1 knockout mice exhibited milder renal tubular injury and fibrosis after IR injury. However, LIPUS did not ameliorate IR injury in proximal tubule-specific Il1r1 knockout mice. Collectively, daily LIPUS at an intensity of 315 mW/cm2 relieves IR-induced tubular injury and fibrosis, potentially through down-regulating tubular IL-1R.

Authors

Zhimin Huang, Jiaxin Dong, Ziqi Fu, Li Li, Simeng liu, Lin Wu, Honglei Guo, Ao Bian, Kang Liu, Wei Sun, Changying Xing, Steven D. Crowley, Jiafa Ren, Xiangqing Kong, Huijuan Mao

Elevation of master autophagy regulator Tfeb in osteoblast lineage cells increases bone mass and strength

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Abstract

Autophagy is a recycling pathway in which damaged proteins, protein aggregates, and organelles are delivered to lysosomes for degradation. Autophagy insufficiency is thought to contribute to osteoporosis. Accordingly, autophagy elimination from the osteoblast lineage reduces bone formation and bone mass. However, whether increasing autophagy would benefit bone health is unknown. Here, we increased expression of endogenous transcription factor EB gene (Tfeb) in osteoblast lineage cells in vivo via CRISPR activation (TfebCRa mice). Elevated Tfeb stimulated autophagy and lysosomal biogenesis in osteoblasts. TfebCRa mice displayed a robust increase in femoral and vertebral cortical thickness at 4.5 months of age. Increases in cortical thickness was due to increased periosteal bone formation. Tfeb elevation also increased femoral trabecular bone volume. These changes increased bone strength of TfebCRa mice. Female TfebCRa mice displayed a progressive increase in bone mass and at 12 months of age had high cortical thickness and trabecular bone volume. Increased vertebral trabecular bone volume was due to elevated bone formation. Osteoblastic cultures showed that Tfeb elevation increased proliferation and mineral deposition. Overall, these results demonstrate TFEB-driven stimulation of autophagy in osteoblast lineage cells is associated with increased bone formation and strength and may represent an effective approach to combat osteoporosis.

Authors

Alicen James, James A. Hendrixson, Ilham Kadhim, Adriana Marques-Carvalho, Jacob Laster, Julie Crawford, Jeff Thostenson, Visanu Wanchai, Amy Y. Sato, Intawat Nookaew, Jinhu Xiong, Maria Almeida, Melda Onal

TGF-β–mediated epithelial–mesenchymal transition of keratinocytes promotes fibrosis in secondary lymphedema

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Abstract

Secondary lymphedema is characterized by fibrosis and impaired lymphatic function. Although TGF-β is a key regulator of fibrosis in this disease, the cellular mechanisms regulating this process remain unknown. Epithelial–mesenchymal transition (EMT), a mechanism by which TGF-β induces fibrosis in other skin diseases, is characterized by loss of epithelial cell markers and cellular polarity, upregulation of fibrotic gene expression, and gain of migratory capacity. Using clinical lymphedema biopsy specimens and animal models, we show that keratinocytes in the basal layer of the epidermis undergo EMT in lymphedematous skin, migrate into the dermis, and contribute to dermal fibrosis. In vitro studies using cultured primary human keratinocytes treated with lymphatic fluid from the affected limbs of patients with secondary lymphedema resulted in a TGF-β–mediated increased expression of EMT markers. We show for the first time that EMT is activated by TGF-β in secondary lymphedema and that this process plays an important role in regulating skin fibrosis in this disease.

Authors

Hyeung Ju Park, Jinyeon Shin, Ananta Sarker, Mark G. Klang, Elyn Riedel, Michelle Coriddi, Joseph H. Dayan, Sarit Pal, Babak J. Mehrara, Raghu P. Kataru

Cardiac enrichment of mutant calmodulin protein in a murine model of a human calmodulinopathy

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Abstract

Heterozygosity for missense mutations in one of 3 seemingly redundant calmodulin (CALM)-encoding genes can cause life-threatening arrhythmias, suggesting that small fractions of mutant CALM protein suffice to cause a severe phenotype. However, the exact molar ratios of wildtype to mutant CALM protein in calmodulinopathy hearts remain unknown. The aim of the present study was to quantitate mutant versus wildtype CALM transcript and protein levels in hearts of knock-in mice harboring the p.N98S mutation in the Calm1 gene. We found that the transcripts from the mutant Calm1 allele were the least abundantly expressed Calm transcripts in both hetero- and homozygous mutant hearts, while mutant hearts accumulate high levels of N98S-CALM protein in a Calm1N98S allele dosage-dependent manner, exceeding those of wildtype CALM protein. We further show that the severity of the electrophysiological phenotype incrementally increases with the graded increase in the mutant-to-wildtype CALM protein expression ratio seen in homozygous versus heterozygous mutant mice. We finally show a decrease in N98S-CALM protein degradation, suggesting that mutant CALM stabilization contributed to its enrichment in the heart. Our results support what we believe to be a novel mechanism by which a mutation in a single Calm gene can give rise to a severe phenotype.

Authors

Wen-Chin Tsai, Chiu-Fen Yang, Shu-Yu Lin, Suh-Yuen Liang, Wei-Chung Tsai, Shuai Guo, Xiaochun Li, Susan Ofner, Kai-Chien Yang, Tzu-Ching Meng, Peng-Sheng Chen, Michael Rubart

STING-adjuvanted outer membrane vesicle nanoparticle vaccine against Pseudomonas aeruginosa

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Abstract

Multidrug-resistant (MDR) bacterial pneumonias pose a critical threat to global public health. The opportunistic Gram-negative pathogen Pseudomonas aeruginosa is a leading cause of nosocomial-associated pneumonia, and an effective vaccine could protect vulnerable populations, including the elderly, immunocompromised, and those with chronic respiratory diseases. Highly heterogeneous outer membrane vesicles (OMVs), shed from Gram-negative bacteria, are studded with immunogenic lipids, proteins, and virulence factors. To overcome limitations in OMV stability and consistency, we described a believed to be novel vaccine platform that combines immunogenic OMVs with precision nanotechnology—creating a bacterial cellular nanoparticle vaccine candidate (CNP), termed Pa-STING-CNP, which incorporates an adjuvanted core that activates the STING (stimulator of interferon genes) pathway. In this design, OMVs are coated onto the surface of self-adjuvanted STING nanocores. Pa-STING CNP vaccination induced substantial antigen presenting cell recruitment and activation in draining lymph nodes, robust anti-Pseudomonas antibody responses, and provided protection against lethal challenge with the hypervirulent clinical P. aeruginosa isolate PA14. Antibody responses mediated this protection and provided passive immunity against the heterologous P. aeruginosa strain PA01. These findings provided evidence that nanotechnology can be used to create a highly efficacious vaccine platform against high priority MDR pathogens such as P. aeruginosa.

Authors

Elisabet Bjånes, Nishta Krishnan, Truman Koh, Anh T.P. Ngo, Jason Cole, Joshua Olson, Ingrid Cornax, Chih-Ho Chen, Natalie Chavarria, Samira Dahesh, Shawn M. Hannah, Alexandra Stream, Jiaqi Amber Zhang, Hervé Besançon, Daniel Sun, Siri Yendluri, Sydney Morrill, Jiarong Zhou, Animesh Mohapatra, Ronnie H. Fang, Victor Nizet