Sickle cell disease (SCD) is a hereditary hemoglobinopathy characterized by painful vaso-occlusive crises (VOC) and chronic hemolysis. The mononuclear phagocyte system is pivotal to SCD pathophysiology, but the mechanisms governing monocyte/macrophage differentiation remain unknown. This study examined the influence of hemolysis on circulating monocyte trajectories in SCD. We discovered that hemolysis stimulated CSF-1 production, partly by endothelial cells via Nrf2, promoting classical monocyte (CMo) differentiation into blood patrolling monocytes (PMo) in SCD mice. However, hemolysis also upregulated CCL-2 through IFN-I, inducing CMo transmigration and differentiation into tissue monocyte-derived macrophages. Blocking CMo transmigration by anti-P-selectin antibody in SCD mice increased circulating PMo, corroborating that CMo-to-tissue macrophage differentiation occurs at the expense of CMo-to-blood PMo differentiation. We observed a positive correlation between plasma CSF-1/CCL-2 ratios and blood PMo levels in SCD patients, underscoring the clinical significance of these two opposing factors in monocyte differentiation. Combined treatment with CSF-1 and anti-P-selectin antibody more effectively increased PMo numbers and reduced stasis compared to single-agent therapies in SCD mice. Altogether, these data indicate that monocyte fates are regulated by the balance between two heme pathways, Nrf2-CSF-1 and IFN-I-CCL-2, and suggest that the CSF-1/CCL-2 ratio may present a diagnostic and therapeutic target in SCD.
Yunfeng Liu, Shan Su, Sarah Shayo, Weili Bao, Mouli Pal, Kai Dou, Patricia A. Shi, Banu Aygun, Sally Campbell-Lee, Cheryl A. Lobo, Avital Mendelson, Xiuli An, Deepa Manwani, Hui Zhong, Karina Yazdanbakhsh
Glucocorticoids are steroid hormones with potent immunosuppressive properties. Their primary source is the adrenals, where they are generated via de novo synthesis from cholesterol. In addition, many tissues have a recycling pathway in which glucocorticoids are regenerated from inactive metabolites by the enzyme 11β-HSD1 (encoded by Hsd11b1). Here we find that multiple tumor types express Hsd11b1 and produce active glucocorticoids. Genetic ablation of Hsd11b1 in such cells had no effect on in vitro growth but reduced in vivo tumor progression, which corresponded with increased frequencies of tumor-infiltrating CD8+ T cells (TIL) expressing activation markers and producing effector cytokines. Tumor-derived glucocorticoids were found to promote signatures of Treg activation and suppress signatures of Tconv activation in tumor-infiltrating Treg. Indeed, CD8+ T cell activation was restored and tumor growth reduced in mice with Treg-specific glucocorticoid receptor deficiency. Importantly, pharmacologic inhibition of 11β-HSD1 reduced tumor growth to the same degree as gene knockout, and rendered immunotherapy-resistant tumors susceptible to PD-1 blockade. Given that HSD11B1 expression is upregulated in many human tumors and that inhibition of 11β-HSD1 is well-tolerated in clinical studies, these data suggest that targeting 11β-HSD1 may be a beneficial adjunct in cancer therapy.
Matthew D. Taves, Shizuka Otsuka, Michaela A. Taylor, Kaitlynn M. Donahue, Thomas J. Meyer, Margaret C. Cam, Jonathan D. Ashwell
Protease activated receptor (PAR) 4 (gene: F2RL3) harbors a functional dimorphism, rs773902 A/G (encoding Thr120/Ala120, respectively) and is associated with greater platelet aggregation. The A allele frequency is more common in Black individuals, and Black individuals have a higher incidence of ischemic stroke than White individuals. However, it is not recognized whether the A allele is responsible for worse stroke outcomes. To directly test the in vivo effect of this variant on stroke, we generated mice where F2rl3 was replaced by F2RL3, thereby expressing human PAR4 (hPAR4) with either Thr120 or Ala120. Compared to hPAR4 Ala120 mice, hPAR4 Thr120 mice had worse stroke outcomes, mediated in part by enhanced platelet activation and platelet-neutrophil interactions. Analyses of 7620 Black subjects with 487 incident ischemic strokes demonstrated the AA genotype was a risk for incident ischemic stroke and worse functional outcomes. In humanized mice, ticagrelor with or without aspirin improved stroke outcomes in hPAR4 Ala120 mice, but not in hPAR4 Thr120 mice. P-selectin blockade improved stroke outcomes and reduced platelet-neutrophil interactions in hPAR4 Thr120 mice. Our results may explain some of the racial disparity in stroke and support the need for studies of non-standard anti-platelet therapies for patients expressing PAR4 Thr120.
Frederik Denorme, Nicole D. Armstrong, Michelle L. Stoller, Irina Portier, Emilia A. Tugolukova, Rikki M. Tanner, Emilie Montenont, Seema Bhatlekar, Mark Cody, John L. Rustad, Abigail Ajanel, Neal D. Tolley, Darian C. Murray, Julie L. Boyle, Marvin T. Nieman, Steven E. McKenzie, Christian Con Yost, Leslie A. Lange, Mary Cushman, Marguerite R. Irvin, Paul F. Bray, Robert A. Campbell
RNA splicing factor SF3B1 is recurrently mutated in various cancers, particularly in hematological ma-lignancies. We previously reported that co-expression of Sf3b1 mutation and Atm deletion in B cells, but not either lesion alone, leads to the onset of chronic lymphocytic leukemia (CLL) with CLL cells harbor-ing chromosome amplification. However, the exact role of Sf3b1 mutation and Atm deletion in chromo-somal instability (CIN) remains unclear. Here, we demonstrate that SF3B1 mutation promotes centro-meric R-loop (cen-R-loop) accumulation, leading to increased chromosome oscillation, impaired chromo-some segregation, altered spindle architecture and aneuploidy, which can be alleviated by removal of cen-R-loop and exaggerated by deletion of ATM. Aberrant splicing of key genes involved in R-loop processing underlies augmentation of cen-R-loop as overexpression of the normal isoform, but not the altered form, mitigates mitotic stress in SF3B1 mutant cells. Our study underscores the critical role of novel splice variants in linking RNA splicing dysregulation and CIN, and highlights cen-R-loop augmen-tation as a key mechanism for leukemogenesis.
Martina Cusan, Haifeng Shen, Bo Zhang, Aijun Liao, Lu Yang, Meiling Jin, Mike Fernandez, Prajish Iyer, Yiming Wu, Kevyn L. Hart, Catherine Gutierrez, Sara Nik, Shondra M. Pruett-Miller, Jeremy Stark, Esther A. Obeng, Teresa V. Bowman, Catherine J. Wu, Ren-Jang Lin, Lili Wang
The liver has a high demand for phosphatidylcholine (PC) particularly in overnutrition where reduced phospholipid levels have been implicated in the development of non-alcoholic fatty liver disease (NAFLD). Whether other pathways exist in addition to de novo PC synthesis that contribute to hepatic PC pools remains unknown. Here, we identified the lysophosphatidylcholine (LPC) transporter Mfsd2a as critical for maintaining hepatic phospholipid pools. Hepatic Mfsd2a expression was induced in patients having NAFLD and in mice in response to dietary fat via glucocorticoid receptor action. Mfsd2a liver-specific deficiency in mice (L2aKO) led to a robust NASH-like phenotype within just two weeks of dietary fat challenge associated with reduced hepatic phospholipids containing linoleic acid. Reducing dietary choline intake in L2aKO mice exacerbated liver pathology and deficiency of liver phospholipids containing polyunsaturated fatty acids (PUFA). Treating hepatocytes with LPC containing oleate and linoleate, two abundant blood-derived LPCs, specifically induced lipid droplet biogenesis and contributed to phospholipid pools, while LPC containing the omega-3 fatty acid DHA promoted lipid droplet formation and suppressed lipogenesis. This study revealed that PUFA containing LPCs drive both hepatic lipid droplet formation, suppress lipogenesis and sustain hepatic phospholipid pools--processes that are critical for protecting the liver from excess dietary fat.
Cheen Fei Chin, Dwight L.A. Galam, Liang Gao, Bryan C. Tan, Bernice H. Wong, Geok-Lin Chua, Randy Y.J. Loke, Yen Ching Lim, Markus R. Wenk, Miao Shan Lim, Wei-Qiang Leow, George B.B. Goh, Federico Torta, David L. Silver
HIV-1 persists in a latent reservoir in resting CD4+ T cells despite antiretroviral therapy (ART). The reservoir decays slowly over the first seven years of ART (t1/2 = 44 months). However, whether decay continues with long-term ART is unclear. Recent integration site studies indicate gradual selection against inducible, intact proviruses, raising speculation that decades of ART might allow treatment interruption without viral rebound. Therefore, we measured the reservoir in 42 people on long-term ART (mean 22 years) using a quantitative viral outgrowth assay. After seven years of ART, there was no long-term decrease in the frequency of inducible, replication-competent proviruses but rather an increase with an estimated doubling time of 23 years. Another reservoir assay, the intact proviral DNA assay, confirmed that reservoir decay with t1/2 of 44 months did not continue with long-term ART. The lack of decay reflected infected cell proliferation. Most (79.8%) inducible, replication-competent viruses had env sequences identical to other isolates from the same sample. Thus, although integration site analysis indicates changes in reservoir composition, the proliferation of CD4+ T cells counteracts decay, maintaining the frequency of inducible, replication-competent proviruses at roughly constant levels over the long term. These results reinforce the need for lifelong ART.
Natalie F. McMyn, Joseph Varriale, Emily J. Fray, Carolin Zitzmann, Hannah J MacLeod, Jun Lai, Anushka Singhal, Milica Moskovljevic, Mauro A. Garcia, Brianna M. Lopez, Vivek Hariharan, Kyle Rhodehouse, Kenneth Lynn, Pablo Tebas, Karam Mounzer, Luis J. Montaner, Erika Benko, Colin Kovacs, Rebecca Hoh, Francesco R. Simonetti, Gregory M. Laird, Steven G. Deeks, Ruy M. Ribeiro, Alan S. Perelson, Robert Siliciano, Janet M. Siliciano
Recognition of pathogen-associated molecular patterns can trigger the IRE1α arm of the endoplasmic reticulum (ER) stress response in innate immune cells. This process maintains ER homeostasis and also coordinates diverse immunomodulatory programs during bacterial and viral infections. However, the role of innate IRE1α signaling in response to fungal pathogens remains elusive. Here, we report that systemic infection with the human opportunistic fungal pathogen Candida albicans induces proinflammatory IRE1α hyperactivation in myeloid cells that leads to fatal kidney immunopathology. Mechanistically, simultaneous activation of the TLR/IL-1R adaptor protein MyD88 and the C-type lectin receptor Dectin-1 by C. albicans induced NADPH oxidase-driven generation of reactive oxygen species that caused ER stress and IRE1α-dependent overexpression of key inflammatory mediators such as IL-1β, IL-6, CCL5, PGE2 and TNFα. Selective ablation of IRE1α in leukocytes, or treatment with an IRE1α pharmacological inhibitor, mitigated kidney inflammation and prolonged the survival of mice with systemic C. albicans infection. Therefore, controlling IRE1α hyperactivation may be useful for impeding the immunopathogenic progression of disseminated candidiasis.
Deepika Awasthi, Sahil Chopra, Byuri A. Cho, Alexander Emmanuelli, Tito A. Sandoval, Sung-Min Hwang, Chang-Suk Chae, Camilla Salvagno, Chen Tan, Liliana Vasquez-Urbina, Jose J. Fernandez Rodriguez, Sara F. Santagostino, Takao Iwawaki, E. Alfonso Romero-Sandoval, Mariano Sanchez Crespo, Diana K. Morales, Iliyan D. Iliev, Tobias M. Hohl, Juan R. Cubillos-Ruiz
Asthma is a chronic inflammatory disease associated with episodic airway narrowing. Inhaled β2-adrenergic receptor (β2AR) agonists (β2-agonists) promote bronchodilation in asthma, but have limited efficacy. All β2-agonists are canonical orthosteric ligands that bind the same site as endogenous epinephrine. We recently isolated a β2AR-selective positive allosteric modulator (PAM), compound-6 (Cmpd-6), which binds outside of the orthosteric site and modulates orthosteric ligand functions. With the emerging therapeutic potential of GPCR allosteric ligands, we investigated the impact of Cmpd-6 on β2AR-mediated bronchoprotection. Consistent with our findings using human β2ARs, Cmpd-6 allosterically potentiated β2-agonist binding to, and downstream signaling of, guinea pig β2ARs. In contrast, Cmpd-6 had no such effect on murine β2ARs which lack a crucial amino acid in the Cmpd-6 allosteric binding site. Importantly, Cmpd-6 enhanced β2-agonist-mediated bronchoprotection against methacholine-induced bronchoconstriction in guinea pig lung slices, but – in line with the binding studies – not in mice. Moreover, Cmpd-6 robustly potentiated β2-agonist-mediated bronchoprotection against allergen-induced airway constriction in lung slices obtained from a guinea pig model of allergic asthma. Cmpd-6 similarly enhanced β2-agonist-mediated bronchoprotection against methacholine-induced bronchoconstriction in human lung slices. Our results highlight the potential of β2AR-selective PAMs in the treatment of airway narrowing in asthma and other obstructive respiratory diseases.
Seungkirl Ahn, Harm Maarsingh, Julia K.L. Walker, Samuel W. Liu, Akhil Hegde, Hyeje C. Sumajit, Alem W. Kahsai, Robert J. Lefkowitz
Despite the success of KRAS G12C inhibitors in non-small cell lung cancer (NSCLC), more effective treatments are needed. One preclinical strategy has been to co-target RAS and mTOR pathways, however toxicity due to broad mTOR inhibition has limited its utility. Therefore, we sought to develop a more refined means of targeting cap-dependent translation and identify the most therapeutically important eIF4F-translated targets. Here we show that an eIF4A inhibitor, which targets a component of eIF4F, dramatically enhances the effects of KRAS G12C inhibitors in NSCLCs and together these agents induce potent tumor regression in vivo. By screening a broad panel of eIF4F targets, we show that this cooperativity is driven by effects on BCL-2 family proteins. Moreover, because multiple BCL-2 family members are concomitantly suppressed, these agents are broadly efficacious in NSCLCs, irrespective of their dependency on MCL1, BCL-xL, or BCL-2, which is known to be heterogeneous. Finally, we show that MYC overexpression confers sensitivity to this combination because it creates a dependency on eIF4A for BCL-2 family protein expression. Together, these studies identify a promising therapeutic strategy for KRAS-mutant NSCLCs, demonstrate that BCL-2 proteins are the key mediators of the therapeutic response in this tumor type, and uncover a predictive biomarker of sensitivity.
Francesca Nardi, Naiara Perurena, Amy E. Schade, Ze-Hua Li, Kenneth Ngo, Elena V. Ivanova, Aisha Saldanha, Chendi Li, Prafulla C. Gokhale, Aaron N. Hata, David A. Barbie, Cloud P. Paweletz, Pasi A. Janne, Karen Cichowski
Protein aggregation is a hallmark of many neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). Although mutations in TARDBP, encoding TDP-43, account for less than 1% of all ALS cases, TDP-43-positive aggregates are present in nearly all ALS patients, including patients with sporadic ALS (sALS) or carrying other familial ALS (fALS)-causing mutations. Interestingly, TDP-43 inclusions are also present in subsets of patients with frontotemporal dementia, Alzheimer’s disease, and Parkinson’s disease; therefore, methods of activating intracellular protein quality control machinery capable of clearing toxic cytoplasmic TDP-43 species may alleviate disease-related phenotypes. Here, we identify a novel function of Nemo-like kinase (Nlk) as a negative regulator of lysosome biogenesis. Genetic or pharmacological reduction of Nlk increased lysosome formation and improved clearance of aggregated TDP-43. Furthermore, Nlk reduction ameliorated pathological, behavioral, and lifespan deficits in two distinct mouse models of TDP-43 proteinopathy. Because many toxic proteins can be cleared along the autophagy-lysosome axis, targeted reduction of Nlk represents a potential approach to therapy development for multiple neurodegenerative disorders.
Leon Tejwani, Youngseob Jung, Hiroshi Kokubu, Sowmithra Sowmithra, Luhan Ni, Changwoo Lee, Benjamin Sanders, Paul J. Lee, Yangfei Xiang, Kimberly Luttik, Armand Soriano, Jennifer Yoon, Junhyun Park, Hannah H. Ro, Hyoungseok Ju, Clara Liao, Sofia Massaro Tieze, Frank Rigo, Paymaan Jafar-Nejad, Janghoo Lim
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