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BACKGROUND Leucine rich repeat kinase 2 (LRRK2) and SNCA are genetically linked to late-onset Parkinson's disease (PD). Aggregated $\alpha$-synuclein pathologically defines PD. Recent studies identified elevated LRRK2 expression in pro-inflammatory CD16+ monocytes in idiopathic PD,as well as increased phosphorylation of the LRRK2 kinase substrate Rab10 in monocytes in some LRRK2 mutation carriers. Brain-engrafting pro-inflammatory monocytes have been implicated in dopaminergic neurodegeneration in PD models. Here we examine how $\alpha$-synuclein and LRRK2 interact in monocytes and subsequent neuroinflammatory responses. METHODS Human and mouse monocytes were differentiated to distinct transcriptional states resembling macrophages,dendritic cells,or microglia,and exposed to well-characterized human or mouse $\alpha$-synuclein fibrils. LRRK2 expression and LRRK2-dependent Rab10 phosphorylation were measured with monoclonal antibodies,and myeloid cell responses to $\alpha$-synuclein fibrils in R1441C-Lrrk2 knock-in mice or G2019S-Lrrk2 BAC mice were evaluated by flow cytometry. Chemotaxis assays were performed with monocyte-derived macrophages stimulated with $\alpha$-synuclein fibrils and microglia in Boyden chambers. RESULTS $\alpha$-synuclein fibrils robustly stimulate LRRK2 and Rab10 phosphorylation in human and mouse macrophages and dendritic-like cells. In these cells,$\alpha$-synuclein fibrils stimulate LRRK2 through JAK-STAT activation and intrinsic LRRK2 kinase activity in a feed-forward pathway that upregulates phosphorylated Rab10. In contrast,LRRK2 expression and Rab10 phosphorylation are both suppressed in microglia-like cells that are otherwise highly responsive to $\alpha$-synuclein fibrils. Corroborating these results,LRRK2 expression in the brain parenchyma occurs in pro-inflammatory monocytes infiltrating from the periphery,distinct from brain-resident microglia. Mice expressing pathogenic LRRK2 mutations G2019S or R1441C have increased numbers of infiltrating pro-inflammatory monocytes in acute response to $\alpha$-synuclein fibrils. In primary cultured macrophages,LRRK2 kinase inhibition dampens $\alpha$-synuclein fibril and microglia-stimulated chemotaxis. CONCLUSIONS Pathologic $\alpha$-synuclein activates LRRK2 expression and kinase activity in monocytes and induces their recruitment to the brain. These results predict that LRRK2 kinase inhibition may attenuate damaging pro-inflammatory monocyte responses in the brain. View Publication

Fibroblast growth factor receptor 1 (FGFR1) is overexpressed in multiple types of solid tumors,including head and neck squamous cell carcinoma (HNSCC). Being associated with poor prognosis,FGFR1 is a potential therapeutic target for aggressive tumors. T cell-based cancer immunotherapy has played a central role in novel cancer treatments. However,the potential of antitumor immunotherapy targeting FGFR1 has not been investigated. Here,we showed that FGFR-tyrosine kinase inhibitors (TKIs) augmented antitumor effects of immune checkpoint inhibitors in an HNSCC mouse model and upregulated tumoral MHC class I and MHC class II expression in vivo and in vitro. This upregulation was associated with the mitogen-activated protein kinase signaling pathway,which is a crucial pathway for cancer development through FGFR signaling. Moreover,we identified an FGFR1-derived peptide epitope (FGFR1305-319) that could elicit antigen-reactive and multiple HLA-restricted CD4+ T cell responses. These T cells showed direct cytotoxicity against tumor cells that expressed FGFR1. Notably,FGFR-TKIs augmented antitumor effects of FGFR1-reactive T cells against human HNSCC cells. These results indicate that the combination of FGFR-TKIs with immunotherapy,such as an FGFR1-targeting peptide vaccine or immune checkpoint inhibitor,could be a novel and robust immunologic approach for treating patients with FGFR1-expressing cancer cells. View Publication

PURPOSE Three observations drove this study. First,2'-5'-oligoadenylate synthetase-like protein (OASL) is a negative regulator of type I interferon (IFN). Second,type I IFN plays a central role during virus infections and the pathogenesis of various diseases,including asthma. Third,influenza A virus (IAV) causes non-eosinophilic asthma. To evaluate the potential relationships between OASL,type I IFN,and pulmonary innate immune cells in IAV-induced acute airway inflammation by using Oasl1-/- mice. METHODS Asthma was induced in wild-type (WT) and Oasl1-/- mice with IAV or ovalbumin (OVA). Airway hyperreactivity (AHR) and immune cell infiltration in the bronchoalveolar lavage (BAL) fluids were measured. The immune cells in the lungs were analyzed by flow cytometry. To investigate the ability of type I IFN to shape the response of lung type 2 innate lymphoid cells (ILC2s),IFN-$\alpha$ was treated intratracheally. Plasmacytoid dendritic cells (pDCs) sorted from bone marrow and ILC2s sorted from lungs of naive mice were co-cultured with/without interferon-alpha receptor subunit 1 (IFNAR-1)-blocking antibodies. RESULTS In the IAV-induced asthma model,Oasl1-/- mice developed greater AHR and immune cell infiltration in the BAL fluids than WT mice. This was not observed in OVA-induced asthma,a standard model of allergen-induced asthma. The lungs of infected Oasl1-/- mice also had elevated DC numbers and Ifna expression and depressed IAV-induced ILC2 responses,namely,proliferation and type 2 cytokine and amphiregulin production. Intratracheal administration of type I IFN in na{\{i}}ve mice suppressed lung ILC2 production of type 2 cytokines and amphiregulin. Co-culture of ILC2s with pDCs showed that pDCs inhibit the function of ILC2s by secreting type I IFN. CONCLUSIONS OASL1 may impede the IAV-induced acute airway inflammation that drives AHR by inhibiting IAV-induced type I IFN production from lung DCs thereby preserving the functions of lung ILC2s including their amphiregulin production." View Publication

Rheumatoid arthritis (RA) is characterized by an impaired articular bone immune microenvironment,which is associated with regulatory T cells (Tregs) hypofunction and osteoclasts (OCs) hyperfunction and leads to articular bone erosion and systemic bone loss. Studies have shown that Tregs slow bone loss in RA by regulating the bone resorption function of OCs and the JAK/STAT signaling pathway can regulate the immunosuppressive function of Tregs and reduce the bone erosion function of OCs. Yi Shen Juan Bi Pill (YSJB) is a classic Chinese herbal compound for the treatment of RA. However,whether YSJB regulates bone immune microenvironment homeostasis through JAK/STAT signaling pathway remains unclear. Based on in vitro OC single culture,Treg single culture and OC-Treg coculture systems,treatments were performed using drug-containing serum,AG490 and JAK2 siRNA to explore whether YSJB-containing serum regulates the homeostasis of the bone immune microenvironment through the JAK/STAT signaling pathway. In vitro,YSJB treatment decreased the number of TRAP+ cells and the areas of bone resorption and inhibited the expression of RANK,NFATc1,c-fos,JAK2,and STAT3 in both the OC single culture system and the OC-Treg coculture system. Tregs further reduced the number of TRAP+ cells and the areas of bone resorption in the coculture system. YSJB promoted the secretion of IL-10 while inhibiting the expression of JAK2 and STAT3 in Tregs. Moreover,inhibiting the expression of JAK2 with the JAK2 inhibitor AG490 and JAK2 siRNA improved the immunosuppressive functions of Treg,inhibited OC differentiation and bone resorption. Our study demonstrates that YSJB can regulate OC-mediated bone resorption and Treg-mediated bone immunity through the JAK2/STAT3 signaling pathway. This study provides a new strategy for regulating the bone immune microenvironment in RA with traditional Chinese medicine. View Publication

Checkpoint blockade therapies targeting PD-1/PD-L1 and CTLA-4 are clinically successful but also evoke adverse events due to systemic T-cell activation. We engineered a bispecific,mAb targeting CD28 homolog (CD28H),a newly identified B7 family receptor that is constitutively expressed on T and natural killer (NK) cells,with a PD-L1 antibody to potentiate tumor-specific immune responses. The bispecific antibody led to T-cell costimulation,induced NK-cell cytotoxicity of PD-L1-expressing tumor cells,and activated tissue-resident memory CD8+ T cells. Mechanistically,the CD28H agonistic arm of the bispecific antibody reduced PD-L1/PD-1-induced SHP2 phosphorylation while simultaneously augmenting T-cell receptor signaling by activating the MAPK and AKT pathways. This bispecific approach could be used to target multiple immune cells,including CD8+ T cells,tissue-resident memory T cells,and NK cells,in a tumor-specific manner that may lead to induction of durable,therapeutic antitumor responses. View Publication

The intradermal lipopolysaccharide (LPS) challenge in healthy volunteers has proven to be a valuable tool to study local inflammation in vivo. In the current study the inhibitory effects of oral and topical corticosteroid treatment on intradermal LPS responses were evaluated to benchmark the challenge for future investigational drugs. Twenty-four healthy male volunteers received a two-and-a-half-day twice daily (b.i.d.) pretreatment with topical clobetasol propionate 0.05% and six healthy volunteers received a two-and-a-half-day b.i.d. pretreatment with oral prednisolone at 0.25 mg/kg body weight per administration. Participants received one injection regimen of either 0,2,or 4 intradermal LPS injections (5 ng LPS in 50 µL 0.9% sodium chloride solution). The LPS response was evaluated by noninvasive (perfusion,skin temperature,and erythema) and invasive assessments (cellular and cytokine responses) in suction blister exudate. Both corticosteroids significantly suppressed the clinical inflammatory response (erythema P = 0.0001 for clobetasol and P = 0.0016 for prednisolone; heat P = 0.0245 for clobetasol,perfusion P < 0.0001 for clobetasol and P = 0.0036 for prednisolone). Clobetasol also significantly reduced the number of monocytes subsets,dendritic cells,natural killer cells,and T cells in blister exudate. A similar effect was observed for prednisolone. No relevant corticosteroid effects were observed on the cytokine response to LPS. We successfully demonstrated that the anti-inflammatory effects of corticosteroids can be detected using our intradermal LPS challenge model,validating it for evaluation of future investigational drugs,as an initial assessment of the anti-inflammatory effects of such compounds in a minimally invasive manner. View Publication

BACKGROUND Monocytes are thought to be involved in venous thrombosis but the role of individual monocyte subpopulations on thrombus formation,clot inflammation,and degradation is an important unresolved issue. We investigate the role of inflammatory Ly6Chi monocytes in deep vein thrombosis and their potential therapeutic impact. METHODS Frequencies and compositions of blood monocytes were analyzed by flow cytometry in CCR2-/- (C-C chemokine receptor type 2) and wild-type mice of different ages and after treatment with the NR4A1 (nuclear receptor group 4 family A member 1,Nur77) agonist CnsB (cytosporone B). TF (tissue factor) sufficient and deficient Ly6Chi monocytes were adoptively transferred into aged CCR2-/- mice. Thrombus formation and size were followed by ultrasound over a 3-week period after surgical reduction of blood flow (stenosis) in the inferior vena cava. RESULTS Reduced numbers of peripheral monocytes in aged (>30 w) CCR2-/- mice are accompanied by reduced thrombus formation after inferior vena cava ligation. Reducing the number of inflammatory Ly6Chi monocytes in wild-type mice by CsnB treatment before ligation,similarly suspends clotting,while later treatment (d1 or d4) reduces thrombus growth and accelerates resolution. We describe how changes in inflammatory monocyte numbers affect the gradual differentiation of monocytes in thrombi and show that only tissue factor-competent Ly6Chi monocytes restore thrombosis in aged CCR2-/- mice. CONCLUSIONS We conclude that the number of inflammatory Ly6Chi monocytes controls deep vein thrombosis formation,growth,and resolution and can be therapeutically manipulated with a NR4A1 agonist at all disease stages. View Publication

The atypical cannabinoid Abn-CBD improves the inflammatory status in preclinical models of several pathologies,including autoimmune diseases. However,its potential for modulating inflammation in autoimmune type 1 diabetes (T1D) is unknown. Herein we investigate whether Abn-CBD can modulate the inflammatory response during T1D onset using a mouse model of T1D (non-obese diabetic- (NOD)-mice) and of beta cell damage (streptozotocin (STZ)-injected mice). Six-week-old female NOD mice were treated with Abn-CBD (0.1-1 mg/kg) or vehicle during 12 weeks and then euthanized. Eight-to-ten-week-old male C57Bl6/J mice were pre-treated with Abn-CBD (1 mg/kg of body weight) or vehicle for 1 week,following STZ challenge,and euthanized 1 week later. Blood,pancreas,pancreatic lymph nodes (PLNs) and T cells were collected and processed for analysis. Glycemia was also monitored. In NOD mice,treatment with Abn-CBD significantly reduced the severity of insulitis and reduced the pro-inflammatory profile of CD4+ T cells compared to vehicle. Concomitantly,Abn-CBD significantly reduced islet cell apoptosis and improved glucose tolerance. In STZ-injected mice,Abn-CBD decreased circulating proinflammatory cytokines and ameliorated islet inflammation reducing intra-islet phospho-NF-$\kappa$B and TXNIP. Abn-CBD significantly reduced 2 folds intra-islet CD8+ T cells and reduced Th1/non-Th1 ratio in PLNs of STZ-injected mice. Islet cell apoptosis and intra-islet fibrosis were also significantly reduced in Abn-CBD pre-treated mice compared to vehicle. Altogether,Abn-CBD reduces circulating and intra-islet inflammation,preserving islets,thus delaying the progression of insulitis. Hence,Abn-CBD and related compounds emerge as new candidates to develop pharmacological strategies to treat the early stages of T1D. View Publication

OBJECTIVE Long-term treatment with tyrosine kinase inhibitors (TKI) represents an effective cure for chronic myeloid leukemia (CML) patients and discontinuation of TKI therapy is now proposed to patient with deep molecular responses. However,evidence demonstrating that TKI are unable to fully eradicate dormant leukemic stem cells (LSC) indicate that new therapeutic strategies are needed to control LSC and to prevent relapse. In this study we investigated the metabolic pathways responsible for CML surviving to imatinib exposure and its potential therapeutic utility to improve the efficacy of TKI against stem-like CML cells. METHODS Using complementary cell-based techniques,metabolism was characterized in a large panel of BCR-ABL+ cell lines as well as primary CD34+ stem-like cells from CML patients exposed to TKI and L-Asparaginases. Colony forming cell (CFC) assay and flow cytometry were used to identify CML progenitor and stem like-cells. Preclinical models of leukemia dormancy were used to test the effect of treatments. RESULTS Although TKI suppressed glycolysis,compensatory glutamine-dependent mitochondrial oxidation supported ATP synthesis and CML cell survival. Glutamine metabolism was inhibited by L-asparaginases such as Kidrolase or Erwinase without inducing predominant CML cell death. However,clinically relevant concentrations of TKI render CML cells susceptible to Kidrolase. The combination of TKI with Lasparaginase reactivates the intinsic apoptotic pathway leading to efficient CML cell death. CONCLUSION Targeting glutamine metabolism with the FDA-approved drug,Kidrolase in combination with TKI that suppress glycolysis represents an effective and widely applicable therapeutic strategy for eradicating stem-like CML cells. View Publication

Oxidative stress,caused by the imbalance between reactive species generation and the dysfunctional capacity of antioxidant defenses,is one of the characteristic features of cancer. Here,we quantified hydrogen peroxide in the tumor microenvironment (TME) and demonstrated that hydrogen peroxide concentrations are elevated in tumor interstitial fluid isolated from murine breast cancers in vivo,when compared with blood or normal subcutaneous fluid. Therefore,we investigated the effects of increased hydrogen peroxide concentration on immune cell functions. NK cells were more susceptible to hydrogen peroxide than T cells or B cells,and by comparing T,B,and NK cells' sensitivities to redox stress and their antioxidant capacities,we identified peroxiredoxin-1 (PRDX1) as a lacking element of NK cells' antioxidative defense. We observed that priming with IL15 protected NK cells' functions in the presence of high hydrogen peroxide and simultaneously upregulated PRDX1 expression. However,the effect of IL15 on PRDX1 expression was transient and strictly dependent on the presence of the cytokine. Therefore,we genetically modified NK cells to stably overexpress PRDX1,which led to increased survival and NK cell activity in redox stress conditions. Finally,we generated PD-L1-CAR NK cells overexpressing PRDX1 that displayed potent antitumor activity against breast cancer cells under oxidative stress. These results demonstrate that hydrogen peroxide,at concentrations detected in the TME,suppresses NK cell function and that genetic modification strategies can improve CAR NK cells' resistance and potency against solid tumors. View Publication

Asthma and its heterogeneity change with age. Increased airspace neutrophil numbers contribute to severe steroid-resistant asthma exacerbation in the elderly,which correlates with the changes seen in adults with asthma. However,whether that resembles the same disease mechanism and pathophysiology in aged and adults is poorly understood. Here,we sought to address the underlying molecular mechanism of steroid-resistant airway inflammation development and response to corticosteroid (Dex) therapy in aged mice. To study the changes in inflammatory mechanism,we used a clinically relevant treatment model of house-dust mite (HDM)-induced allergic asthma and investigated lung adaptive immune response in adult (20-22 wk old) and aged (80-82 wk old) mice. Our result indicates an age-dependent increase in airway hyperresponsiveness (AHR),mixed granulomatous airway inflammation comprising eosinophils and neutrophils,and Th1/Th17 immune response with progressive decrease in frequencies and numbers of HDM-bearing dendritic cells (DC) accumulation in the draining lymph node (DLn) of aged mice as compared with adult mice. RNA-Seq experiments of the aged lung revealed short palate,lung,and nasal epithelial clone 1 (SPLUNC1) as one of the steroid-responsive genes,which progressively declined with age and further by HDM-induced inflammation. Moreover,we found increased glycolytic reprogramming,maturation/activation of DCs,the proliferation of OT-II cells,and Th2 cytokine secretion with recombinant SPLUNC1 (rSPLUNC1) treatment. Our results indicate a novel immunomodulatory role of SPLUNC1 regulating metabolic adaptation/maturation of DC. An age-dependent decline in the SPLUNC1 level may be involved in developing steroid-resistant airway inflammation and asthma heterogeneity. View Publication

CD8 T cell-mediated autoimmune diseases result from the breakdown of self-tolerance mechanisms in autoreactive CD8 T cells1. How autoimmune T cell populations arise and are sustained,and the molecular programmes defining the autoimmune T cell state,are unknown. In type 1 diabetes,$\beta$-cell-specific CD8 T cells destroy insulin-producing $\beta$-cells. Here we followed the fate of $\beta$-cell-specific CD8 T cells in non-obese diabetic mice throughout the course of type 1 diabetes. We identified a stem-like autoimmune progenitor population in the pancreatic draining lymph node (pLN),which self-renews and gives rise to pLN autoimmune mediators. pLN autoimmune mediators migrate to the pancreas,where they differentiate further and destroy $\beta$-cells. Whereas transplantation of as few as 20 ?»¿autoimmune progenitors induced type 1 diabetes,as many as 100,000 pancreatic autoimmune mediators did not. Pancreatic autoimmune mediators are short-lived,and stem-like ?»¿autoimmune progenitors must continuously seed the pancreas to sustain $\beta$-cell destruction. Single-cell RNA sequencing and clonal analysis revealed that autoimmune CD8 T cells represent unique T cell differentiation states and identified features driving the transition from autoimmune progenitor to autoimmune mediator. Strategies aimed at targeting the stem-like autoimmune progenitor pool could emerge as novel and powerful immunotherapeutic interventions for type 1 diabetes. View Publication