Monocytes and interstitial macrophages contribute to hypoxic pulmonary hypertension

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Abstract

Hypoxia is a major cause of pulmonary hypertension (PH) worldwide, and it is likely that interstitial pulmonary macrophages contribute to this vascular pathology. We observed in hypoxia-exposed mice an increase in resident interstitial macrophages, which expanded through proliferation and expressed the monocyte recruitment ligand CCL2. We also observed an increase in CCR2+ macrophages through recruitment, which express the protein thrombospondin-1, which functionally activates TGF-β to cause vascular disease. Blockade of monocyte recruitment with either CCL2-neutralizing antibody treatment or CCR2 deficiency in the bone marrow compartment suppressed hypoxic PH. These data were supported by analysis of plasma samples from humans who traveled from low (225 m) to high (3500 m) elevation, revealing an increase in thrombospondin-1 and TGF-β expression following ascent, which was blocked by dexamethasone prophylaxis. In the hypoxic mouse model, dexamethasone prophylaxis recapitulated these findings by mechanistically suppressing CCL2 expression and CCR2+ monocyte recruitment. These data suggest a pathologic cross talk between 2 discrete interstitial macrophage populations, which can be therapeutically targeted.

Authors

Rahul Kumar, Kevin Nolan, Biruk Kassa, Neha Chanana, Tsering Palmo, Kavita Sharma, Kanika Singh, Claudia Mickael, Dara Fonseca Balladares, Julia Nilsson, Amit Prabhakar, Aastha Mishra, Michael H. Lee, Linda Sanders, Sushil Kumar, Ari B. Molofsky, Kurt R. Stenmark, Dean Sheppard, Rubin M. Tuder, Mohit D. Gupta, Tashi Thinlas, Qadar Pasha, Brian B. Graham

KRAS mutants confer platinum resistance by regulating ALKBH5 posttranslational modifications in lung cancer

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Abstract

Constitutively active mutations of KRAS are prevalent in non–small cell lung cancer (NSCLC). However, the relationship between these mutations and resistance to platinum-based chemotherapy and the underlying mechanisms remain elusive. In this study, we demonstrate that KRAS mutants confer resistance to platinum in NSCLC. Mechanistically, KRAS mutants mediate platinum resistance in NSCLC cells by activating ERK/JNK signaling, which inhibits AlkB homolog 5 (ALKBH5) N6-methyladenosine (m6A) demethylase activity by regulating posttranslational modifications (PTMs) of ALKBH5. Consequently, the KRAS mutant leads to a global increase in m6A methylation of mRNAs, particularly damage-specific DNA-binding protein 2 (DDB2) and XPC, which are essential for nucleotide excision repair. This methylation stabilized the mRNA of these 2 genes, thus enhancing NSCLC cells’ capability to repair platinum-induced DNA damage and avoid apoptosis, thereby contributing to drug resistance. Furthermore, blocking KRAS-mutant–induced m6A methylation, either by overexpressing a SUMOylation-deficient mutant of ALKBH5 or by inhibiting methyltransferase-like 3 (METTL3) pharmacologically, significantly sensitizes KRAS-mutant NSCLC cells to platinum drugs in vitro and in vivo. Collectively, our study uncovers a mechanism that mediates KRAS-mutant–induced chemoresistance in NSCLC cells by activating DNA repair through the modulation of the ERK/JNK/ALKBH5 PTM-induced m6A modification in DNA damage repair–related genes.

Authors

Fang Yu, Shikan Zheng, Chunjie Yu, Sanhui Gao, Zuqi Shen, Rukiye Nar, Zhexin Liu, Shuang Huang, Lizi Wu, Tongjun Gu, Zhijian Qian

RNase L represses hair follicle regeneration through altered innate immune signaling

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Abstract

Mammalian injury responses are predominantly characterized by fibrosis and scarring rather than functional regeneration. This limited regenerative capacity in mammals could reflect a loss of proregeneration programs or active suppression by genes functioning akin to tumor suppressors. To uncover programs governing regeneration in mammals, we screened transcripts in human participants following laser rejuvenation treatment and compared them with mice with enhanced wound-induced hair neogenesis (WIHN), a rare example of mammalian organogenesis. We found that Rnasel–/– mice exhibit an increased regenerative capacity, with elevated WIHN through enhanced IL-36α. Consistent with RNase L’s known role to stimulate caspase-1, we found that pharmacologic inhibition of caspases promoted regeneration in an IL-36–dependent manner in multiple epithelial tissues. We identified a negative feedback loop, where RNase L–activated caspase-1 restrains the proregenerative dsRNA-TLR3 signaling cascade through the cleavage of toll-like adaptor protein TRIF. Through integrated single-cell RNA-seq and spatial transcriptomic profiling, we confirmed OAS & IL-36 genes to be highly expressed at the site of wounding and elevated in Rnasel–/– mouse wounds. This work suggests that RNase L functions as a regeneration repressor gene, in a functional trade off that tempers immune hyperactivation during viral infection at the cost of inhibiting regeneration.

Authors

Charles S. Kirby, Nasif Islam, Eric Wier, Martin P. Alphonse, Evan Sweren, Gaofeng Wang, Haiyun Liu, Dongwon Kim, Ang Li, Sam S. Lee, Andrew M. Overmiller, Yingchao Xue, Sashank Reddy, Nathan K. Archer, Lloyd S. Miller, Jianshi Yu, Weiliang Huang, Jace W. Jones, Sooah Kim, Maureen A. Kane, Robert H. Silverman, Luis A. Garza

Estrogen receptor-α ablation reverses muscle fibrosis and inguinal hernias

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Abstract

Fibrosis of the lower abdominal muscle (LAM) contributes to muscle weakening and inguinal hernia formation, an ailment that affects a noteworthy 50% of men by age 75 and necessitates surgical correction as the singular therapy. Despite its prevalence, the mechanisms driving LAM fibrosis and hernia development remain poorly understood. Using a humanized mouse model that replicates the elevated skeletal muscle tissue estrogen concentrations seen in aging men, we identified estrogen receptor-α (ESR1) as a key driver of LAM fibroblast proliferation, extracellular matrix deposition, and hernia formation. Fibroblast-specific ESR1 ablation effectively prevented muscle fibrosis and herniation, while pharmacological ESR1 inhibition with fulvestrant reversed hernias and restored normal muscle architecture. Multiomics analyses of in vitro LAM fibroblasts from humanized mice unveiled an estrogen/ESR1-mediated activation of a distinct profibrotic cistrome and gene expression signature, concordant with observations in inguinal hernia tissues in human males. Our findings hold significant promise for prospective medical interventions targeting fibrotic conditions and present non-surgical avenues for addressing inguinal hernias.

Authors

Tanvi Potluri, Tianming You, Ping Yin, John Coon V, Jonah J. Stulberg, Yang Dai, David J. Escobar, Richard L. Lieber, Hong Zhao, Serdar E. Bulun

A conserved human CD4⁺ T cell subset recognizing the mycobacterial adjuvant trehalose monomycolate

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Abstract

Mycobacterium tuberculosis causes human tuberculosis (TB). As mycobacteria are protected by a thick lipid cell wall, humans have developed immune responses against diverse mycobacterial lipids. Most of these immunostimulatory lipids are known as adjuvants acting through innate immune receptors, such as C-type lectin receptors. Although a few mycobacterial lipid antigens activate unconventional T cells, the antigenicity of most adjuvantic lipids is unknown. Here, we identified that trehalose monomycolate (TMM), an abundant mycobacterial adjuvant, activated human T cells bearing a unique αβ T cell receptor (αβTCR). This recognition was restricted by CD1b, a monomorphic antigen-presenting molecule conserved in primates but not mice. Single-cell TCR-RNA-Seq using newly established CD1b-TMM tetramers revealed that TMM-specific T cells were present as CD4+ effector memory T cells in the periphery of uninfected donors but expressed IFN-γ, TNF, and anti-mycobacterial effectors upon TMM stimulation. TMM-specific T cells were detected in cord blood and PBMCs of donors without bacillus Calmette-Guérin vaccination but were expanded in patients with active TB. A cryo-electron microscopy study of CD1b-TMM-TCR complexes revealed unique antigen recognition by conserved features of TCRs, positively charged CDR3α, and long CDR3β regions. These results indicate that humans have a commonly shared and preformed CD4+ T cell subset recognizing a typical mycobacterial adjuvant as an antigen. Furthermore, the dual role of TMM justifies reconsideration of the mechanism of action of adjuvants.

Authors

Yuki Sakai, Minori Asa, Mika Hirose, Wakana Kusuhara, Nagatoshi Fujiwara, Hiroto Tamashima, Takahiro Ikazaki, Shiori Oka, Kota Kuraba, Kentaro Tanaka, Takashi Yoshiyama, Masamichi Nagae, Yoshihiko Hoshino, Daisuke Motooka, Ildiko Van Rhijn, Xiuyuan Lu, Eri Ishikawa, D. Branch Moody, Takayuki Kato, Shinsuke Inuki, Go Hirai, Sho Yamasaki

Activating antiviral immune responses potentiates immune checkpoint inhibition in glioblastoma models

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Abstract

Viral mimicry refers to the activation of innate antiviral immune responses due to the induction of endogenous retroelements (REs). Viral mimicry augments antitumor immune responses and sensitizes solid tumors to immunotherapy. Here, we found that targeting what we believe to be a novel, master epigenetic regulator, Zinc Finger Protein 638 (ZNF638), induces viral mimicry in glioblastoma (GBM) preclinical models and potentiates immune checkpoint inhibition (ICI). ZNF638 recruits the HUSH complex, which precipitates repressive H3K9me3 marks on endogenous REs. In GBM, ZNF638 is associated with marked locoregional immunosuppressive transcriptional signatures, reduced endogenous RE expression, and poor immune cell infiltration. Targeting ZNF638 decreased H3K9 trimethylation, increased REs, and activated intracellular dsRNA signaling cascades. Furthermore, ZNF638 knockdown upregulated antiviral immune programs and significantly increased PD-L1 immune checkpoint expression in diverse GBM models. Importantly, targeting ZNF638 sensitized mice to ICI in syngeneic murine orthotopic models through innate IFN signaling. This response was recapitulated in recurrent GBM (rGBM) samples with radiographic responses to checkpoint inhibition with widely increased expression of dsRNA, PD-L1, and perivascular CD8 cell infiltration, suggesting that dsRNA signaling may mediate response to immunotherapy. Finally, low ZNF638 expression was a biomarker of clinical response to ICI and improved survival in patients with rGBM and patients with melanoma. Our findings suggest that ZNF638 could serve as a target to potentiate immunotherapy in gliomas.

Authors

Deepa Seetharam, Jay Chandar, Christian K. Ramsoomair, Jelisah F. Desgraves, Alexandra Alvarado Medina, Anna Jane Hudson, Ava Amidei, Jesus R. Castro, Vaidya Govindarajan, Sarah Wang, Yong Zhang, Adam M. Sonabend, Mynor J. Mendez Valdez, Dragan Maric, Vasundara Govindarajan, Sarah R. Rivas, Victor M. Lu, Ritika Tiwari, Nima Sharifi, Emmanuel Thomas, Marcus Alexander, Catherine DeMarino, Kory Johnson, Macarena I. De La Fuente, Ruham Alshiekh Nasany, Teresa Maria Rosaria Noviello, Michael E. Ivan, Ricardo J. Komotar, Antonio Iavarone, Avindra Nath, John Heiss, Michele Ceccarelli, Katherine B. Chiappinelli, Maria E. Figueroa, Defne Bayik, Ashish H. Shah

Deep immunophenotyping reveals circulating activated lymphocytes in individuals at risk for rheumatoid arthritis

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Abstract

Rheumatoid arthritis (RA) is a systemic autoimmune disease currently with no universally highly effective prevention strategies. Identifying pathogenic immune phenotypes in at-risk populations prior to clinical onset is crucial to establishing effective prevention strategies. Here, we applied multimodal single-cell technologies (mass cytometry and CITE-Seq) to characterize the immunophenotypes in blood from at-risk individuals (ARIs) identified through the presence of serum antibodies against citrullinated protein antigens (ACPAs) and/or first-degree relative (FDR) status, as compared with patients with established RA and people in a healthy control group. We identified significant cell expansions in ARIs compared with controls, including CCR2+CD4+ T cells, T peripheral helper (Tph) cells, type 1 T helper cells, and CXCR5+CD8+ T cells. We also found that CD15+ classical monocytes were specifically expanded in ACPA-negative FDRs, and an activated PAX5lo naive B cell population was expanded in ACPA-positive FDRs. Further, we uncovered the molecular phenotype of the CCR2+CD4+ T cells, expressing high levels of Th17- and Th22-related signature transcripts including CCR6, IL23R, KLRB1, CD96, and IL22. Our integrated study provides a promising approach to identify targets to improve prevention strategy development for RA.

Authors

Jun Inamo, Joshua Keegan, Alec Griffith, Tusharkanti Ghosh, Alice Horisberger, Kaitlyn Howard, John F. Pulford, Ekaterina Murzin, Brandon Hancock, Salina T. Dominguez, Miranda G. Gurra, Siddarth Gurajala, Anna Helena Jonsson, Jennifer A. Seifert, Marie L. Feser, Jill M. Norris, Ye Cao, William Apruzzese, S. Louis Bridges, Vivian P. Bykerk, Susan Goodman, Laura T. Donlin, Gary S. Firestein, Joan M. Bathon, Laura B. Hughes, Andrew Filer, Costantino Pitzalis, Jennifer H. Anolik, Larry Moreland, Nir Hacohen, Joel M. Guthridge, Judith A. James, Carla M. Cuda, Harris Perlman, Michael B. Brenner, Soumya Raychaudhuri, Jeffrey A. Sparks, The Accelerating Medicines Partnership RA/SLE Network, V. Michael Holers, Kevin D. Deane, James Lederer, Deepak A. Rao, Fan Zhang

High PRMT5 levels, maintained by KEAP1 inhibition, drive chemoresistance in high-grade serous ovarian cancer

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Abstract

Protein arginine methyl transferases (PRMTs) are generally upregulated in cancers. However, the mechanisms leading to this upregulation and its biological consequences are poorly understood. Here, we identify PRMT5, the main symmetric arginine methyltransferase, as a critical driver of chemoresistance in high-grade serous ovarian cancer (HGSOC). PRMT5 levels and its enzymatic activity are induced in a platinum-resistant (Pt-resistant) state at the protein level. To reveal potential regulators of high PRMT5 protein levels, we optimized intracellular immunostaining conditions and performed unbiased CRISPR screening. We identified Kelch-like ECH-associated protein 1 (KEAP1) as a top-scoring negative regulator of PRMT5. Our mechanistic studies show that KEAP1 directly interacted with PRMT5, leading to its ubiquitin-dependent degradation under normal physiological conditions. At the genomic level, ChIP studies showed that elevated PRMT5 directly interacted with the promoters of stress response genes and positively regulated their transcription. Combined PRMT5 inhibition with Pt resulted in synergistic cellular cytotoxicity in vitro and reduced tumor growth in vivo in Pt-resistant patient-derived xenograft tumors. Overall, the findings from this study identify PRMT5 as a critical therapeutic target in Pt-resistant HGSOC cells and reveal the molecular mechanisms that lead to high PRMT5 levels in Pt-treated and chemo-resistant tumors.

Authors

Harun Ozturk, Fidan Seker-Polat, Neda Abbaszadeh, Yasemin Kingham, Sandra Orsulic, Mazhar Adli

Targeting ubiquitin-independent proteasome with small molecule increases susceptibility in pan-KRAS–mutant cancer models

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Abstract

Despite advances in the development of direct KRAS inhibitors, KRAS-mutant cancers continue to exhibit resistance to the currently available therapies. Here, we identified REGγ as a mutant KRAS–associated factor that enhanced REGγ transcription through the KRAS intermediate NRF2, suggesting that the REGγ-proteasome is a potential target for pan-KRAS inhibitor development. We elucidated a mechanism involving the KRAS/NRF2/REGγ regulatory axis, which links activated KRAS to the ATP- and ubiquitin-independent proteasome. We subsequently developed RLY01, a REGγ-proteasome inhibitor that effectively suppressed tumor growth in KRAS-mutant cancer models and lung cancer organoids. Notably, the combination of RLY01 and the KRASG12C inhibitor AMG510 exhibited enhanced antitumor efficacy in KRASG12C cancer cells. Collectively, our data support the hypothesis that KRAS mutations enhance the capacity of the REGγ-proteasome by increasing REGγ expression, highlighting the potential of ubiquitin-independent proteasome inhibition as a therapeutic approach for pan-KRAS–mutant cancers.

Authors

Shihui Shen, Qiansen Zhang, Yuhan Wang, Hui Chen, Shuangming Gong, Yun Liu, Conghao Gai, Hansen Chen, Enhao Zhu, Bo Yang, Lin Liu, Siyuan Cao, Mengting Zhao, Wenjie Ren, Mengjuan Li, Zhuoya Peng, Lu Zhang, Shaoying Zhang, Juwen Shen, Bianhong Zhang, Patrick K.H. Lee, Kun Li, Lei Li, Huaiyu Yang

Identification of lysosomal lipolysis as an essential noncanonical mediator of adipocyte fasting and cold-induced lipolysis

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Abstract

Adipose tissue lipolysis is the process by which triglycerides in lipid stores are hydrolyzed into free fatty acids (FFAs), serving as fuel during fasting or cold-induced thermogenesis. Although cytosolic lipases are considered the predominant mechanism of liberating FFAs, lipolysis also occurs in lysosomes via lysosomal acid lipase (LIPA), albeit with unclear roles in lipid storage and whole-body metabolism. We found that adipocyte LIPA expression increased in adipose tissue of mice when lipolysis was stimulated during fasting, cold exposure, or β-adrenergic agonism. This was functionally important, as inhibition of LIPA genetically or pharmacologically resulted in lower plasma FFAs under lipolytic conditions. Furthermore, adipocyte LIPA deficiency impaired thermogenesis and oxygen consumption and rendered mice susceptible to diet-induced obesity. Importantly, lysosomal lipolysis was independent of adipose triglyceride lipase, the rate-limiting enzyme of cytosolic lipolysis. Our data suggest a significant role for LIPA and lysosomal lipolysis in adipocyte lipid metabolism beyond classical cytosolic lipolysis.

Authors

Yu-Sheng Yeh, Trent D. Evans, Mari Iwase, Se-Jin Jeong, Xiangyu Zhang, Ziyang Liu, Arick Park, Ali Ghasemian, Borna Dianati, Ali Javaheri, Dagmar Kratky, Satoko Kawarasaki, Tsuyoshi Goto, Hanrui Zhang, Partha Dutta, Francisco J. Schopfer, Adam C. Straub, Jaehyung Cho, Irfan J. Lodhi, Babak Razani