BACKGROUND. The kynurenine pathway (KP) has been identified as a potential mediator linking acute illness to cognitive dysfunction by generating neuroactive metabolites in response to inflammation. Delirium (acute confusion) is a common complication of acute illness and is associated with increased risk of dementia and mortality. However, the molecular mechanism underlying delirium, particularly in relation to the KP, remain elusive. METHODS. We undertook a multi-center observational study with 586 hospitalized patients (248 with delirium) and investigated associations between delirium and KP metabolites measured in cerebrospinal fluid (CSF) and serum by targeted metabolomics. We also explored associations between KP metabolites and markers of neuronal damage and one-year mortality. RESULTS. In delirium, we found concentrations of the neurotoxic metabolite quinolinic acid in CSF (CSF-QA, OR 2.26 [1.78, 2.87], p<0.001) to be increased, as well as increases in several other KP metabolites in serum and CSF. In addition, CSF-QA was associated with the neuronal damage marker neurofilament light chain (NfL, β 0.43, p<0.001) and was a strong predictor of one-year mortality (HR 4.35 [2.93, 6.45] for CSF-QA ≥ 100 nmol/L, p<0.001). The associations between CSF-QA and delirium, neuronal damage, and mortality remained highly significant following adjustment for confounders and multiple comparisons. CONCLUSION. Our data identified how systemic inflammation, neurotoxicity, and delirium are strongly linked via the KP, and should inform future delirium prevention and treatment clinical trials that target enzymes of the KP. FUNDING. Norwegian Health Association and the South-Eastern Norway Regional Health Authorities
Leiv Otto Watne, Christian Thomas Pollmann, Bjorn Erik Neerland, Else Quist-Paulsen, Nathalie Bodd Halaas, Ane-Victoria Idland, Bjørnar Hassel, Kristi Henjum, Anne-Brita Knapskog, Frede Frihagen, Johan Raeder, Aasmund Godø, Per Magne Ueland, Adrian McCann, Wender Figved, Geir Selbæk, Henrik Zetterberg, Evandro Fei Fang, Marius Myrstad, Lasse M. Giil
BACKGROUND. Acute febrile neutrophilic dermatosis (Sweet syndrome) is a potentially fatal multiorgan inflammatory disease characterized by fever, leukocytosis, and a rash with a neutrophilic infiltrate. Disease pathophysiology remains elusive, and current dogma suggests Sweet syndrome is a “reactive” process to an unknown antigen. Corticosteroids and steroid-sparing agents remain front-line therapies, but refractory cases pose a clinical challenge. METHODS. A 51-year-old woman with multiorgan Sweet syndrome developed serious corticosteroid-related side effects and was refractory to steroid-sparing agents. Blood counts, liver enzymes, and skin histopathology supported the diagnosis. Whole genome sequencing, transcriptomic profiling, and cellular assays of patient’s skin and neutrophils were performed. RESULTS. We identified elevated IL-1 signaling in lesional Sweet syndrome skin caused by a PIK3R1 gain-of-function mutation specifically found in neutrophils. This mutation increased neutrophil migration towards IL-1β and neutrophil respiratory burst. Targeted treatment with an IL-1R1 antagonist in the patient resulted in a dramatic therapeutic response and enabled tapering of corticosteroids. CONCLUSIONS. Dysregulated PI3K-AKT signaling is the first signaling pathway linked to Sweet syndrome and suggests Sweet syndrome may be caused by acquired mutations that modulate neutrophil function. Moreover, integration of molecular data across multiple levels identified a distinct subtype within a heterogenous disease that resulted in a rational and successful clinical intervention. Future cases will benefit from efforts to identify potential mutations. The ability to directly interrogate diseased skin allows this method to be generalizable to other inflammatory diseases and demonstrates a potential personalized medicine approach for challenging patients. FUNDING Berstein Foundation, NIH, VA, Moseley Foundation, and H.T. Leung Foundation.
Shreya Bhattacharya, Sayon Basu, Emily Sheng, Christina M. Murphy, Jenny Wei, Anna E. Kersh, Caroline A. Nelson, Joshua S. Bryer, Hovik A. Ashchyan, Katherine T. Steele, Amy K. Forrestel, John T. Seykora, Robert G. Micheletti, William D. James, Misha Rosenbach, Thomas H. Leung
Platelets and megakaryocytes are critical players in immune responses. Recent reports suggest infection and inflammation alter the megakaryocyte and platelet transcriptome to induce altered platelet reactivity. We examined if non-viral sepsis induces differential platelet gene expression and reactivity. Non-viral sepsis upregulated IFITM3, an interferon responsive gene that restricts viral replication. As IFITM3 has been linked to clathrin-mediated endocytosis, we examined if IFITM3 promoted endocytosis of alpha granule proteins. Interferon stimulation enhanced fibrinogen endocytosis in megakaryocytes and platelets from Ifitm+/+ mice, but not Ifitm-/- mice. IFITM3 overexpression or deletion in megakaryocytes demonstrated IFITM3 was necessary and sufficient to regulate fibrinogen endocytosis. Mechanistically, IFITM3 interacts with clathrin and αIIb and altered their plasma membrane localization into lipid rafts. In vivo interferon administration increased fibrinogen endocytosis, platelet reactivity, and thrombosis in an IFITM-dependent manner. In contrast, Ifitm-/- mice were completely rescued from interferon-induced platelet hyperreactivity and thrombosis. During murine sepsis, platelets from Ifitm+/+ mice demonstrated increased fibrinogen content and platelet reactivity, which was dependent on interferon-alpha and IFITMs. Platelets from patients with non-viral sepsis had increases in platelet IFITM3 expression, fibrinogen content, and hyperreactivity. These data identify IFITM3 as a regulator of platelet endocytosis, hyperreactivity, and thrombosis during inflammatory stress.
Robert A. Campbell, Bhanu Kanth Manne, Meenakshi Banerjee, Elizabeth A. Middleton, Abigail Ajanel, Hansjorg Schwertz, Frederik Denorme, Chris Stubben, Emilie Montenont, Samantha Saperstein, Lauren Page, Neal D. Tolley, Diana L. Lim, Samuel M. Brown, Colin K. Grissom, Douglas W. Sborov, Anandi Krishnan, Matthew T. Rondina
Mevalonate kinase deficiency (MKD) is characterized by recurrent fevers and flares of systemic inflammation, caused by biallelic loss-of-function mutations in MVK. The underlying disease mechanisms and triggers of inflammatory flares are poorly understood because of the lack of in vivo models. We describe genetically modified mice bearing the hypomorphic mutation p.Val377Ile (the commonest variant in patients with MKD) and amorphic, frameshift mutations in Mvk. Compound heterozygous mice recapitulated the characteristic biochemical phenotype of MKD, with increased plasma mevalonic acid and clear buildup of unprenylated GTPases in PBMCs, splenocytes, and bone marrow. The inflammatory response to LPS was enhanced in compound heterozygous mice and treatment with the NLRP3 inflammasome inhibitor MCC950 prevented the elevation of circulating IL-1β, thus identifying a potential inflammasome target for future therapeutic approaches. Furthermore, lines of mice with a range of deficiencies in mevalonate kinase and abnormal prenylation mirrored the genotype-phenotype relationship in human MKD. Importantly, these mice allowed the determination of a threshold level of residual enzyme activity, below which protein prenylation is impaired. Elevated temperature dramatically but reversibly exacerbated the deficit in the mevalonate pathway and the defective prenylation in vitro and in vivo, highlighting increased body temperature as a likely trigger of inflammatory flares.
Marcia A. Munoz, Oliver P. Skinner, Etienne Masle-Farquhar, Julie Jurczyluk, Ya Xiao, Emma K. Fletcher, Esther Kristianto, Mark P. Hodson, Seán I. O’Donoghue, Sandeep Kaur, Robert Brink, David G. Zahra, Elissa K. Deenick, Kristen A. Perry, Avril A.B. Robertson, Sam Mehr, Pravin Hissaria, Catharina M. Mulders-Manders, Anna Simon, Michael J. Rogers
Bronchiolitis obliterans syndrome (BOS) is a major impediment to lung transplant survival and is generally resistant to medical therapy. Extracorporeal photophoresis (ECP) is an immunomodulatory therapy that shows promise in stabilizing BOS patients, but its mechanisms of action are unclear. In a mouse lung transplant model, we show that ECP blunts alloimmune responses and inhibits BOS through lowering airway TGF-β bioavailability without altering its expression. Surprisingly, ECP-treated leukocytes were primarily engulfed by alveolar macrophages (AMs), which were reprogrammed to become less responsive to TGF-β and reduce TGF-β bioavailability through secretion of the TGF-β antagonist decorin. In untreated recipients, high airway TGF-β activity stimulated AMs to express CCL2, leading to CCR2+ monocyte-driven BOS development. Moreover, we found TGF-β receptor 2–dependent differentiation of CCR2+ monocytes was required for the generation of monocyte-derived AMs, which in turn promoted BOS by expanding tissue-resident memory CD8+ T cells that inflicted airway injury through Blimp-1–mediated granzyme B expression. Thus, through studying the effects of ECP, we have identified an AM functional plasticity that controls a TGF-β–dependent network that couples CCR2+ monocyte recruitment and differentiation to alloimmunity and BOS.
Zhiyi Liu, Fuyi Liao, Jihong Zhu, Dequan Zhou, Gyu Seong Heo, Hannah P. Leuhmann, Davide Scozzi, Antanisha Parks, Ramsey Hachem, Derek E. Byers, Laneshia K. Tague, Hrishikesh S. Kulkarni, Marlene Cano, Brian W. Wong, Wenjun Li, Howard J. Haung, Alexander S. Krupnick, Daniel Kreisel, Yongjian Liu, Andrew E. Gelman
Astrocytes are highly heterogenic in their phenotype and function, which contribute to CNS disease, repair and aging; however, the molecular mechanism of their functional states remains largely unknown. Here we show that activation of sirtuin 1 (SIRT1), a protein deacetylase, plays an important role in the detrimental actions of reactive astrocytes, whereas its inactivation endorsed these cells with anti-inflammatory functions that inhibited the production of proinflammatory mediators by myeloid cells/microglia and promoted the differentiation of oligodendrocyte progenitor cells. Mice with astrocyte-specific Sirt1 knockout had suppressed progression of experimental autoimmune encephalomyelitis (EAE), an animal model of CNS inflammatory demyelinating diseases. Ongoing EAE was also suppressed when Sirt1 expression in astrocytes was diminished by CRISPR/Cas vector, resulting in reduced demyelination, decreased numbers of T cells, and increased rate of IL-10-producing macrophages/microglia in the CNS, whereas peripheral immune response remained unaffected. Mechanistically, Sirt1-/- astrocytes expressed a range of nuclear factor erythroid-derived 2-like 2 (Nfe2l2) target genes, and Nfe2l2 deficiency shifted the beneficial action of Sirt1-/- astrocytes to a detrimental one. These findings identify a novel approach for switching functional state of reactive astrocytes and facilitate the development of astrocyte-targeting therapies for inflammatory neurodegenerative diseases such as multiple sclerosis.
Weifeng Zhang, Dan Xiao, Xing Li, Yuan Zhang, Javad Rasouli, Giacomo Casella, Alexandra Boehm, Daniel Hwang, Larissa L.W. Ishikawa, Rodolfo Thome, Bogoljub Ciric, Mark T. Curtis, Abdolmohamad Rostami, Guang-Xian Zhang
BACKGROUND. Anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitidies (AAV) are life-threatening systemic autoimmune conditions. ANCA directed against proteinase 3 (PR3) or myeloperoxidase (MPO) bind their cell surface-presented antigen, activate neutrophils and cause vasculitis. An imbalance between PR3 and its major inhibitor α1-antitrypsin (AAT) was proposed to underlie PR3- but not MPO-AAV. We measured AAT and PR3 in healthies and AAV patients and studied protective AAT effects pertaining to PR3- and MPO-ANCA. METHODS. Plasma and blood neutrophils were assessed for PR3 and AAT. Wild-type, mutant, and oxidation-resistant AAT species were produced to characterize AAT-PR3 interactions by flow cytometry, immunoblotting, FRET assays, and surface plasmon resonance measurements. Neutrophil activation was measured using the ferricytochrome C assay and AAT methionine-oxidation by Parallel Reaction Monitoring. RESULTS. We found significantly increased PR3 and AAT pools in both PR3- and MPO-AAV patients, however, only in PR3-AAV did the PR3 pool correlate with ANCA titer, inflammatory response and disease severity. Mechanistically, AAT prevented PR3 from binding to CD177, thereby reducing neutrophil surface antigen for ligation by PR3-ANCA. Active PR3-AAV patients showed critical methionine-oxidation in plasma AAT that was recapitulated by ANCA-activated neutrophils. The protective PR3-related AAT effects were compromised by methionine-oxidation in the AAT reactive center loop but preserved when two critical methionines were substituted by valine and leucine. CONCLUSION. Pathogenic differences between PR3- and MPO-AAV are related to AAT regulation of membrane-PR3, attenuating neutrophil activation by PR3- rather than MPO-ANCA. Oxidation-resistant AAT could serve as adjunctive therapy in PR3-AAV.
Maximilian J.P. Ebert, Uwe Jerke, Claudia Eulenberg-Gustavus, Lovis Kling, Dieter E. Jenne, Marieluise Kirchner, Philipp Mertins, Markus Bieringer, Saban Elitok, Kai-Uwe Eckardt, Adrian Schreiber, Alan D. Salama, Ralph Kettritz
The molecular mechanisms underlying obesity-induced increase in β cell mass, and the resulting β cell dysfunction need to be elucidated further. Our study revealed that GPR92, expressed in islet macrophages, is modulated by dietary interventions in metabolic tissues. Therefore, we aimed to define the role of GPR92 in islet inflammation by using high fat diet (HFD)-induced obese mouse model. GPR92 knockout mice exhibited glucose intolerance and reduced insulin level, despite the enlarged pancreatic islets, and increased islet macrophage content and inflammation level (compared to those in wild type mice). These results indicate that the lack of GPR92 in islet macrophages can cause β cell dysfunction, leading to disrupted glucose homeostasis. Alternatively, GPR92 agonist, farnesyl pyrophosphate (FPP) stimulation results in the inhibition of HFD-induced islet inflammation and increased insulin secretion in WT mice, but not in GPR92 KO mice. Thus, our study suggests that GPR92 can be a potential target to alleviate β cell dysfunction via the inhibition of islet inflammation associated with the progression of diabetes.
Camila O. de Souza, Vivian A. Paschoal, Xue-Nan Sun, Lavanya Vishvanath, Qianbin Zhang, Mengle Shao, Toshiharu Onodera, Shiuhwei Chen, Nolwenn Joffin, Lorena M.A. Bueno, Rana K. Gupta, Da Young Oh
Immunomodulation holds therapeutic promise against brain injuries, but leveraging this approach requires a precise understanding of mechanisms. We report that CD8+CD122+CD49dlo T regulatory-like cells (CD8+ TRLs) are among the earliest lymphocytes to infiltrate mouse brains after ischemic stroke and temper inflammation; they also confer neuroprotection. TRL depletion worsened stroke outcomes, an effect reversed by CD8+ TRL reconstitution. The CXCR3/CXCL10 axis served as the brain-homing mechanism for CD8+ TRLs. Upon brain entry, CD8+ TRLs were reprogrammed to upregulate leukemia inhibitory factor (LIF) receptor, epidermal growth factor–like transforming growth factor (ETGF), and interleukin 10 (IL-10). LIF/LIF receptor interactions induced ETGF and IL-10 production in CD8+ TRLs. While IL-10 induction was important for the antiinflammatory effects of CD8+ TRLs, ETGF provided direct neuroprotection. Poststroke intravenous transfer of CD8+ TRLs reduced infarction, promoting long-term neurological recovery in young males or aged mice of both sexes. Thus, these unique CD8+ TRLs serve as early responders to rally defenses against stroke, offering fresh perspectives for clinical translation.
Wei Cai, Ligen Shi, Jingyan Zhao, Fei Xu, Connor Dufort, Qing Ye, Tuo Yang, Xuejiao Dai, Junxuan Lyu, Chenghao Jin, Hongjian Pu, Fang Yu, Sulaiman Hassan, Zeyu Sun, Wenting Zhang, T. Kevin Hitchens, Yejie Shi, Angus W. Thomson, Rehana K. Leak, Xiaoming Hu, Jun Chen
Myelodysplastic syndromes (MDS) are age-related myeloid neoplasms with increased risks of progression to acute myeloid leukemia (AML). The mechanisms of MDS to AML transformation are poorly understood, especially in relation to the aging microenvironment. We previously established a mDia1/miR-146a double knockout (DKO) mouse model phenocopying MDS. These mice develop age-related pancytopenia with over-secretion of pro-inflammatory cytokines. Here, we found that most of the DKO mice underwent leukemic transformation at 12-14 months of age. These mice showed myeloblast replacement of a fibrotic bone marrow and widespread leukemic infiltration. Strikingly, depletion of IL-6 in these mice largely rescued the leukemic transformation and markedly extended the survival. Single cell RNA sequencing analyses revealed that DKO leukemic mice had increased monocytic blasts that were reduced with IL-6 knockout. We further revealed that the levels of surface and soluble IL-6 receptor (IL-6R) in the bone marrow were significantly increased in high risk MDS patients. Similarly, IL-6R was also highly expressed in older DKO mice. Blocking of IL-6 signaling significantly ameliorated AML progression in the DKO model and clonogenicity of CD34 positive cells from MDS patients. Our study establishes a mouse model of age-related MDS to AML progression and indicates the clinical significance of targeting IL-6 signaling in treating high risk MDS.
Yang Mei, Kehan Ren, Yijie Liu, Annabel Ma, Zongjun Xia, Xu Han, Ermin Li, Hamza Tariq, Haiyan Bao, Xinshu Xie, Cheng Zou, Dingxiao Zhang, Zhaofeng Li, Lili Dong, Amit Verma, Xinyan Lu, Yasmin Abaza, Jessica K. Altman, Madina Sukhanova, Jing Yang, Peng Ji
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