Scientific Resources

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

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

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

CD8+ memory T (TM) cells play a critical role in immune defense against infection. Two common $\gamma$-chain family cytokines,IL-2 and IL-7,although triggering the same mTORC1-S6K pathway,distinctly induce effector T (TE) cells and TM cells,respectively,but the underlying mechanism(s) remains elusive. In this study,we generated IL-7R-/and AMPK$\alpha$1-knockout (KO)/OTI mice. By using genetic and pharmaceutical tools,we demonstrate that IL-7 deficiency represses expression of FOXO1,TCF1,p-AMPK$\alpha$1 (T172),and p-ULK1 (S555) and abolishes T cell memory differentiation in IL-7R KO T cells after Listeria monocytogenesis rLmOVA infection. IL-2- and IL-7-stimulated strong and weak S6K (IL-2/S6Kstrong and IL-7/S6Kweak) signals control short-lived IL-7R-CD62L-KLRG1+ TE and long-term IL-7R+CD62L+KLRG1- TM cell formations,respectively. To assess underlying molecular pathway(s),we performed flow cytometry,Western blotting,confocal microscopy,and Seahorse assay analyses by using the IL-7/S6Kweak-stimulated TM (IL-7/TM) and the control IL-2/S6Kstrong-stimulated TE (IL-2/TE) cells. We determine that the IL-7/S6Kweak signal activates transcriptional FOXO1,TCF1,and Id3 and metabolic p-AMPK$\alpha$1,p-ULK1,and ATG7 molecules in IL-7/TM cells. IL-7/TM cells upregulate IL-7R and CD62L,promote mitochondria biogenesis and fatty acid oxidation metabolism,and show long-term cell survival and functional recall responses. Interestingly,AMPK$\alpha$1 deficiency abolishes the AMPK$\alpha$1 but maintains the FOXO1 pathway and induces a metabolic switch from fatty acid oxidation to glycolysis in AMPK$\alpha$1 KO IL-7/TM cells,leading to loss of cell survival and recall responses. Taken together,our data demonstrate that IL-7-stimulated weak strength of mTORC1-S6K signaling controls T cell memory via activation of transcriptional FOXO1-TCF1-Id3 and metabolic AMPK$\alpha$1-ULK1-ATG7 pathways. This (to our knowledge) novel finding provides a new mechanism for a distinct IL-2/IL-7 stimulation model in T cell memory and greatly impacts vaccine development. 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

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

Many solid tumors have low levels of cytotoxic CD56dim natural killer (NK) cells,suggesting that CD56dim NK-cell exclusion from the tumor microenvironment (TME) contributes to the decreased response rate of immunotherapy. Complement component 3a (C3a) is known for its tumor-promoting and immunosuppressive roles in solid tumors. Previous reports have implicated the involvement of the C3a receptor (C3aR) in immune cell trafficking into the TME. C3aR is predominantly expressed on the surface of activated cytotoxic NK cells,but a specific role for C3aR in NK-cell biology has not been investigated. Because solid tumors generate elevated C3a and have decreased NK-cell infiltration,we hypothesized that C3aR might play a role in cytotoxic NK-cell recruitment into the TME. Our results indicate that blocking C3aR signaling in NK cells increased NK-cell infiltration into the TME in mouse models and led to tumor regression. Because the critical lymphocyte trafficking integrin LFA-1 orchestrates the migration of activated NK cells,we wanted to gain insight into the interaction between C3aR signaling and LFA-1. Our results demonstrated that direct interaction between C3aR and LFA-1,which led to a high-affinity LFA-1 conformation,decreased NK-cell infiltration into the TME. We propose that approaches to enhance cytotoxic NK-cell infiltration into the TME,through either disrupting C3a and C3aR interaction or inhibiting the formation of high-affinity LFA-1,represent a new strategy to improve the efficiency of immunotherapy for cancer treatment. View Publication

New approaches beyond PD-1/PD-L1 inhibition are required to target the immunologically diverse tumor microenvironment (TME) in high-grade serous ovarian cancer (HGSOC). In this study,we explored the immunosuppressive effect of B7-H3 (CD276) via the CCL2-CCR2-M2 macrophage axis and its potential as a therapeutic target. Transcriptome analysis revealed that B7-H3 is highly expressed in PD-L1-low,nonimmunoreactive HGSOC tumors,and its expression negatively correlated with an IFN$\gamma$ signature,which reflects the tumor immune reactivity. In syngeneic mouse models,B7-H3 (Cd276) knockout (KO) in tumor cells,but not in stromal cells,suppressed tumor progression,with a reduced number of M2 macrophages and an increased number of IFN$\gamma$+CD8+ T cells. CCL2 expression was downregulated in the B7-H3 KO tumor cell lines. Inhibition of the CCL2-CCR2 axis partly negated the effects of B7-H3 suppression on M2 macrophage migration and differentiation,and tumor progression. In patients with HGSOC,B7-H3 expression positively correlated with CCL2 expression and M2 macrophage abundance,and patients with B7-H3-high tumors had fewer tumoral IFN$\gamma$+CD8+ T cells and poorer prognosis than patients with B7-H3-low tumors. Thus,B7-H3 expression in tumor cells contributes to CCL2-CCR2-M2 macrophage axis-mediated immunosuppression and tumor progression. These findings provide new insights into the immunologic TME and could aid the development of new therapeutic approaches against the unfavorable HGSOC phenotype. View Publication

Successful implantation requires the coordinated migration and invasion of trophoblast cells from out of the blastocyst and into the endometrium. This process relies on signals produced by cells in the maternal endometrium. However,the relative contribution of stroma cells remains unclear. The study of human implantation has major technical limitations,therefore the need of in vitro models to elucidate the molecular mechanisms. Using a recently described 3D in vitro models we evaluated the interaction between trophoblasts and human endometrial stroma cells (hESC),we assessed the process of trophoblast migration and invasion in the presence of stroma derived factors. We demonstrate that hESC promotes trophoblast invasion through the generation of an inflammatory environment modulated by TNF-?. We also show the role of stromal derived IL-17 as a promoter of trophoblast migration through the induction of essential genes that confer invasive capacity to cells of the trophectoderm. In conclusion,we describe the characterization of a cellular inflammatory network that may be important for blastocyst implantation. Our findings provide a new insight into the complexity of the implantation process and reveal the importance of inflammation for embryo implantation. View Publication