Airway Epithelial Cell-Derived Colony Stimulating Factor-1 Promotes Allergen Sensitization.

Airway epithelial cells (AECs) secrete innate immune cytokines that regulate adaptive immune effector cells. In allergen-sensitized humans and mice, the airway and alveolar microenvironment is enriched with colony stimulating factor-1 (CSF1) in response to allergen exposure. In this study we found that AEC-derived CSF1 had a critical role in the production of allergen reactive-IgE production. Furthermore, spatiotemporally secreted CSF1 regulated the recruitment of alveolar dendritic cells (DCs) and enhanced the migration of conventional DC2s (cDC2s) to the draining lymph node in an interferon regulatory factor 4 (IRF4)-dependent manner. CSF1 selectively upregulated the expression of the chemokine receptor CCR7 on the CSF1R+ cDC2, but not the cDC1, population in response to allergen stimuli. Our data describe the functional specification of CSF1-dependent DC subsets that link the innate and adaptive immune responses in T helper 2 (Th2) cell-mediated allergic lung inflammation.

CX3CR1+ Macrophage Facilitates the Resolution of Allergic Lung Inflammation via Interacting CCL26.

Rationale: The resolution of inflammation is an active process coordinated by mediators and immune cells to restore tissue homeostasis. However, the mechanisms for resolving eosinophilic allergic lung inflammation triggered by inhaled allergens have not been fully elucidated. Objectives: Our objectives were to investigate the cellular mechanism of tissue-resident macrophages involved in the resolution process of eosinophilic lung inflammation. Methods: For the study, we used the institutional review board-approved protocol for human subsegmental bronchoprovocation with allergen, mouse models for allergic lung inflammation, and novel transgenic mice, including a conditional CCL26 knockout. The samples were analyzed using mass cytometry, single-cell RNA sequencing, and biophysical and immunological analyses. Measurements and Main Results: We compared alveolar macrophage (AM) subsets in the BAL before and after allergen provocation. In response to provocation with inhaled allergens, the subsets of AMs are dynamically changed in humans and mice. In the steady state, the AM subset expressing CX3CR1 is a relatively small fraction in bronchoalveolar space and lung tissue but drastically increases after allergen challenges. This subset presents unique patterns of gene expression compared with classical AMs, expressing high C1q family genes. CX3CR1+ macrophages are activated by airway epithelial cell-derived CCL26 via a receptor-ligand interaction. The binding of CCL26 to the CX3CR1+ receptor induces CX3CR1+ macrophages to secrete C1q, subsequently facilitating the clearance of eosinophils. Furthermore, the depletion of CX3CR1 macrophages or CCL26 in airway epithelial cells delays the resolution of allergic lung inflammation displaying prolonged tissue eosinophilia. Conclusions: These findings indicate that the CCL26-CX3CR1 pathway is pivotal in resolving eosinophilic allergic lung inflammation.

Complement C1q essential for aeroallergen sensitization via CSF1R+ conventional dendritic cells type 2.

BACKGROUND: Dendritic cells (DCs) are heterogeneous, comprising multiple subsets with unique functional specifications. Our previous work has demonstrated that the specific conventional type 2 DC subset, CSF1R+cDC2s, plays a critical role in sensing aeroallergens. OBJECTIVE: It remains to be understood how CSF1R+cDC2s recognize inhaled allergens. We sought to elucidate the transcriptomic programs and receptor-ligand interactions essential for function of this subset in allergen sensitization. METHODS: We applied single-cell RNA sequencing to mouse lung DCs. Conventional DC-selective knockout mouse models were employed, and mice were subjected to inhaled allergen sensitization with multiple readouts of asthma pathology. Under the clinical arm of this work, human lung transcriptomic data were integrated with mouse data, and bronchoalveolar lavage (BAL) specimens were collected from subjects undergoing allergen provocation, with samples assayed for C1q. RESULTS: We found that C1q is selectively enriched in lung CSF1R+cDC2s, but not in other lung cDC2 or cDC1 subsets. Depletion of C1q in conventional DCs significantly attenuates allergen sensing and features of asthma. Additionally, we found that C1q binds directly to human dust mite allergen, and the C1q receptor CD91 (LRP1) is required for lung CSF1R+cDC2s to recognize the C1q-allergen complex and induce allergic lung inflammation. Lastly, C1q is enriched in human BAL samples following subsegmental allergen challenge, and human RNA sequencing data demonstrate close homology between lung IGSF21+DCs and mouse CSF1R+cDC2s. CONCLUSIONS: C1q is secreted from the CSF1R+cDC2 subset among conventional DCs. Our data indicate that the C1q-LRP1 axis represents a candidate for translational therapeutics in the prevention and suppression of allergic lung inflammation.

Colony-stimulating factor 1 and its receptor are new potential therapeutic targets for allergic asthma.

BACKGROUND: A new approach targeting aeroallergen sensing in the early events of mucosal immunity could have greater benefit. The CSF1-CSF1R pathway has a critical role in trafficking allergens to regional lymph nodes through activating dendritic cells. Intervention in this pathway could prevent allergen sensitization and subsequent Th2 allergic inflammation. OBJECTIVE: To examine the therapeutic effectiveness of CSF1 and CSF1R inhibition for blocking the dendritic cell function of sensing aeroallergens. METHODS: We adopted a model of chronic asthma induced by a panel of three naturally occurring allergens and novel delivery system of CSF1R inhibitor encapsulated nanoprobe. RESULTS: Selective depletion of CSF1 in airway epithelial cells abolished the production of allergen-reactive IgE, resulting in prevention of new asthma development as well as reversal of established allergic lung inflammation. CDPL-GW nanoprobe containing GW2580, a selective CSF1R inhibitor, showed favorable pharmacokinetics for inhalational treatment and intranasal insufflation delivery of CDPL-GW nanoprobe ameliorated asthma pathologies including allergen-specific serum IgE production, allergic lung and airway inflammation and airway hyper-responsiveness (AHR) with minimal pulmonary adverse reaction. CONCLUSION: The inhibition of the CSF1-CSF1R signaling pathway effectively suppresses sensitization to aeroallergens and consequent allergic lung inflammation in a murine model of chronic asthma. CSF1R inhibition is a promising new target for the treatment of allergic asthma.

IL-11 facilitates a novel connection between RA joint fibroblasts and endothelial cells.

IL-11 has been detected in inflamed joints; however, its role in the pathogenesis of arthritis is not yet clear. Studies were conducted to characterize the expression and functional significance of IL-11 and IL-11Rα in rheumatoid arthritis (RA). IL-11 levels were elevated in RA synovial fluid (SF) compared to osteoarthritis (OA) SF and plasma from RA, OA and normal individuals (NLs). Morphologic studies established that IL-11 was detected in lining fibroblasts and macrophages in addition to sublining endothelial cells and macrophages at higher levels in RA compared to NL synovial tissues. Since IL-11Rα was exclusively expressed in RA fibroblasts and endothelial cells, macrophages were not involved in IL-11 effector function. Ligation of IL-11 to IL-11Rα strongly provoked fibroblast infiltration into RA joint, while cell proliferation was unaffected by this process. Secretion of IL-8 and VEGF from IL-11 activated RA fibroblasts was responsible for the indirect effect of IL-11 on endothelial cell transmigration and tube formation. Moreover, IL-11 blockade impaired RA SF capacity to elicit endothelial cell transmigration and tube formation. We conclude that IL-11 binding to endothelial IL-11Rα can directly induce RA angiogenesis. In addition, secretion of proangiogenic factors from migrating fibroblasts potentiated by IL-11 can indirectly contribute to RA neovascularization.

Fabrication and Characterization of Finite-Size DNA 2D Ring and 3D Buckyball Structures.

In order to incorporate functionalization into synthesized DNA nanostructures, enhance their production yield, and utilize them in various applications, it is necessary to study their physical stabilities and dynamic characteristics. Although simulation-based analysis used for DNA nanostructures provides important clues to explain their self-assembly mechanism, structural function, and intrinsic dynamic characteristics, few studies have focused on the simulation of DNA supramolecular structures due to the structural complexity and high computational cost. Here, we demonstrated the feasibility of using normal mode analysis for relatively complex DNA structures with larger molecular weights, i.e., finite-size DNA 2D rings and 3D buckyball structures. The normal mode analysis was carried out using the mass-weighted chemical elastic network model (MWCENM) and the symmetry-constrained elastic network model (SCENM), both of which are precise and efficient modeling methodologies. MWCENM considers both the weight of the nucleotides and the chemical bonds between atoms, and SCENM can obtain mode shapes of a whole structure by using only a repeated unit and its connectivity with neighboring units. Our results show the intrinsic vibrational features of DNA ring structures, which experience inner/outer circle and bridge motions, as well as DNA buckyball structures having overall breathing and local breathing motions. These could be used as the fundamental basis for designing and constructing more complicated DNA nanostructures.

5 nm Silver Nanoparticles Amplify Clinical Features of Atopic Dermatitis in Mice by Activating Mast Cells.

The triggering effect of silver nanoparticles (NPs) on the induction of allergic reactions is evaluated, by studying the activation of mast cells and the clinical features of atopic dermatitis in a mouse model. Granule release is induced in RBL-2H3 mast cells by 5 nm, but not 100 nm silver NPs. Increases in the levels of reactive oxygen species (hydrogen peroxide and mitochondrial superoxide) and intracellular Ca++ in mast cells are induced by 5 nm silver NPs. In a mouse model of atopic dermatitis induced by a mite allergen, the skin lesions are more severe and appear earlier in mice treated simultaneously with 5 nm silver NPs and allergen compared with mice treated with allergen alone or 100 nm silver NPs and allergen. The histological findings reveal that number of tryptase-positive mast cells and total IgE levels in the serum increase in mice treated with 5 nm silver NPs and allergen. The results in this study indicate that cotreatment with 5 nm silver NPs stimulates mast cell degranulation and induces earlier and more severe clinical alterations in allergy-prone individuals.

Differential impact of obesity on the pathogenesis of RA or preclinical models is contingent on the disease status.

OBJECTIVE: Studies were performed to uncover the significance of obesity in rheumatoid arthritis (RA) and preclinical models. METHODS: Preclinical arthritis models were used to examine the impact of obesity on disease onset and remission. Conditioned media from RA adipose tissues were used to investigate the mechanism contributing to joint neutrophil influx and M1 macrophage differentiation observed in early and remission phases of arthritis. RESULTS: We report that mice fed with high fat diet (HFD) have an earlier onset of collagen-induced arthritis (CIA) compared with mice on regular diet. However, the differences in CIA joint swelling between the two diet groups are lost once disease is established. We found that early arthritis triggered by obesity is due to elevated joint MIP2/interleukin-8 levels detected in CIA as well as in the RA and mouse adipose tissues and the effect of this chemokine on neutrophil recruitment. Although active disease progression is similarly affected in both diet groups, arthritis resolution is accelerated in lean mice while joint inflammation is sustained in obese mice. We document that HFD can prolong toll-like receptor (TLR)4-induced arthritis by increasing joint monocyte migration and further remodelling the recruited cells into M1 macrophages. Consistently, we show that adipose condition media can transform RA and wild-type naïve myeloid cells into M1 macrophages; however, this function is impaired by TLR4 blockade or deficiency. CONCLUSIONS: We conclude that despite established disease being unaffected by obesity, the early and the resolution phases of RA are impacted by obesity through different mechanisms.

Effects of Partial Absence of Visual Feedback Information on Gait Symmetry.

The incorporation of real-time visual feedback during gait rehabilitation can improve the efficacy of training. Our prior work demonstrated that the imposed distortion of simple visual feedback information of step lengths entails an unintentional adaptive process in the subjects' spatial gait pattern, thereby suggesting the important role of implicit learning in the context of gait rehabilitation that employs visual feedback. The purpose of this study was to investigate whether the removal of a portion of visual feedback information-after it had initially been provided-had any impact on gait symmetry. Eighteen healthy subjects walked on a treadmill for 10-min periods at their preferred walking speed and at a slower walking speed (1.3 mph) during the experimental trials, in which two simple vertical bars corresponding to subject's right and left step length were displayed on a computer screen. Halfway through the trial, one of the bars was removed from the visual feedback via random selection. Subjects were instructed to continually walk normally and also look at the visual feedback until the trials were completed. The changes in step length symmetry ratio were computed and analyzed. We found that displaying only one side of visual feedback influenced subjects to spontaneously modulate gait symmetry away from the baseline, and also that the amount of modulated gait symmetry slightly increased when their walking speed decreased. The changes in gait symmetry occurred by producing either longer right steps produced than left steps or vice versa, but we were unable to find any correlation between side of removal (right or left side) and the different types of trend in response. This warrants further investigation in a study with a larger population. Nonetheless, the results of this study demonstrated the effect of partial absence of visual feedback on changes in step symmetry, and that the perturbation of visual information caused implicit (unintentional) motor processes. A gait training procedure involving a novel way of perturbing visual feedback, such as partial absence of visual feedback tested in this study, may be of value in gait rehabilitation by driving more efficient motor adaptations.

Ligation of TLR5 promotes myeloid cell infiltration and differentiation into mature osteoclasts in rheumatoid arthritis and experimental arthritis.

Our aim was to examine the impact of TLR5 ligation in rheumatoid arthritis (RA) and experimental arthritis pathology. Studies were conducted to investigate the role of TLR5 ligation on RA and mouse myeloid cell chemotaxis or osteoclast formation, and in addition, to uncover the significance of TNF-α function in TLR5-mediated pathogenesis. Next, the in vivo mechanism of action was determined in collagen-induced arthritis (CIA) and local joint TLR5 ligation models. Last, to evaluate the importance of TLR5 function in RA, we used anti-TLR5 Ab therapy in CIA mice. We show that TLR5 agonist, flagellin, can promote monocyte infiltration and osteoclast maturation directly through myeloid TLR5 ligation and indirectly via TNF-α production from RA and mouse cells. These two identified TLR5 functions are potentiated by TNF-α, because inhibition of both pathways can more strongly impair RA synovial fluid-driven monocyte migration and osteoclast differentiation compared with each factor alone. In preclinical studies, flagellin postonset treatment in CIA and local TLR5 ligation in vivo provoke homing and osteoclastic development of myeloid cells, which are associated with the TNF-α cascade. Conversely, CIA joint inflammation and bone erosion are alleviated when TLR5 function is blocked. We found that TLR5 and TNF-α pathways are interconnected, because TNF-α is produced by TLR5 ligation in RA myeloid cells, and anti-TNF-α therapy can markedly suppress TLR5 expression in RA monocytes. Our novel findings demonstrate that a direct and an indirect mechanism are involved in TLR5-driven RA inflammation and bone destruction.

Regulation of IGFBP-2 expression during fasting.

Insulin-like growth factor (IGF)-binding protein-2 (IGFBP-2), one of the most abundant circulating IGFBPs, is known to attenuate the biological action of IGF-1. Although the effect of IGFBP-2 in preventing metabolic disorders is well known, its regulatory mechanism remains unclear. In the present study, we demonstrated the transcriptional regulation of the Igfbp-2 gene by peroxisome-proliferator-activated receptor (PPAR) α in the liver. During fasting, both Igfbp-2 and PPARα expression levels were increased. Wy14643, a selective PPARα agonist, significantly induced Igfbp-2 gene expression in primary cultured hepatocytes. However, Igfbp-2 gene expression in Pparα null mice was not affected by fasting or Wy14643. In addition, through transient transfection and chromatin immunoprecipitation assay in fasted livers, we determined that PPARα bound to the putative PPAR-responsive element between -511 bp and -499 bp on the Igfbp-2 gene promoter, indicating that the Igfbp-2 gene transcription is activated directly by PPARα. To explore the role of PPARα in IGF-1 signalling, we treated primary cultured hepatocytes with Wy14643 and observed a decrease in the number of IGF-1 receptors (IGF-1Rs) and in Akt phosphorylation. No inhibition was observed in the hepatocytes isolated from Pparα null mice. These results suggest that PPARα controls IGF-1 signalling through the up-regulation of hepatic Igfbp-2 transcription during fasting and Wy14643 treatment.

Melatonin ameliorates ER stress-mediated hepatic steatosis through miR-23a in the liver.

The endoplasmic reticulum (ER) stress induces hepatic steatosis and inflammation in the liver. Although melatonin ameliorates ER stress-target genes, it remains unknown whether melatonin protects against hepatic steatosis as well as inflammation through regulation of miRNA. MicroRNAs have been identified as pivotal regulators in the field of gene regulation and their dysfunctions are a common feature in a variety of metabolic diseases. Especially, among miRNAs, miR-23a has been shown to regulate ER stress. Herein, we investigated the crucial roles of melatonin in hepatic steatosis and inflammation in vivo. Tunicamycin challenge caused increase of hepatic triglyceride and intracellular calcium levels through activation of ER stress, whereas these phenomena were partially disrupted by melatonin. We also demonstrated that expression of miR-23a stimulated with tunicamycin was rescued by melatonin treatment, resulting in reduced ER stress in primary hepatocytes. Overall, these results suggest a new function of melatonin that is involved in ameliorating ER stress-induced hepatic steatosis and inflammation by attenuating miR-23a. Melatonin may be useful as a pharmacological agent to protect against hepatic metabolic diseases due to its ability to regulate expression of miR-23a.

Characterising the expression and function of CCL28 and its corresponding receptor, CCR10, in RA pathogenesis.

OBJECTIVE: This study was conducted to determine the expression pattern, regulation and function of CCL28 and CCR10 in rheumatoid arthritis (RA) pathogenesis. METHODS: Expression of CCL28 and CCR10 was assessed in RA compared with other arthritis synovial tissues (STs) or fluids (SFs) by histology or ELISA. The factors modulating CCL28 and CCR10 expression were identified in RA myeloid and endothelial cells by ELISA, FACS and Western blotting. The mechanism by which CCL28 ligation promotes RA angiogenesis was examined in control and CCR10-knockdown endothelial cell chemotaxis and capillary formation. RESULTS: CCL28 and/or CCR10 expression levels were accentuated in STs and SFs of patients with joint disease compared with normal controls and they were predominately coexpressed in RA myeloid and endothelial cells. We show that protein expression of CCL28 and CCR10 was modulated by tumour necrosis factor (TNF)-α and toll-like receptor 4 ligation in RA monocytes and endothelial cells and by interleukin (IL)-6 stimulation in RA macrophages. Neutralisation of CCL28 in RA SF or blockade of CCR10 on human endothelial progenitor cells (EPCs) significantly reduced SF-induced endothelial migration and capillary formation, demonstrating that ligation of joint CCL28 to endothelial CCR10+ cells is involved in RA angiogenesis. We discovered that angiogenesis driven by ligation of CCL28 to CCR10 is linked to the extracellular signal regulated kinase (ERK) cascade, as CCR10-knockdown cells exhibit dysfunctional CCL28-induced ERK signalling, chemotaxis and capillary formation. CONCLUSIONS: The overexpression of CCL28 and CCR10 in RA ST and their contribution to EPC migration into RA joints support the CCL28/CCR10 cascade as a potential therapeutic target for RA.

The novel role of IL-7 ligation to IL-7 receptor in myeloid cells of rheumatoid arthritis and collagen-induced arthritis.

Although the role of IL-7 and IL-7R has been implicated in the pathogenesis of rheumatoid arthritis (RA), the majority of the studies have focused on the effect of IL-7/IL-7R in T cell development and function. Our novel data, however, document that patients with RA and greater disease activity have higher levels of IL-7, IL-7R, and TNF-α in RA monocytes, suggesting a feedback regulation between IL-7/IL-7R and TNF-α cascades in myeloid cells that is linked to chronic disease progression. Investigations into the involved mechanism showed that IL-7 is a novel and potent chemoattractant that attracts IL-7R(+) monocytes through activation of the PI3K/AKT1 and ERK pathways at similar concentrations of IL-7 detected in RA synovial fluid. To determine whether ligation of IL-7 to IL-7R is a potential target for RA treatment and to identify their mechanism of action, collagen-induced arthritis (CIA) was therapeutically treated with anti-IL-7 Ab or IgG control. Anti-IL-7 Ab treatment significantly reduces CIA monocyte recruitment and osteoclast differentiation as well as potent joint monocyte chemoattractants and bone erosion markers, suggesting that both direct and indirect pathways might contribute to the observed effect. We also demonstrate that reduction in joint MIP-2 levels is responsible for suppressed vascularization detected in mice treated with anti-IL-7 Ab compared with the control group. To our knowledge, we show for the first time that expression of IL-7/IL-7R in myeloid cells is strongly correlated with RA disease activity and that ligation of IL-7 to IL-7R contributes to monocyte homing, differentiation of osteoclasts, and vascularization in the CIA effector phase.

Angiogenesis in rheumatoid arthritis is fostered directly by toll-like receptor 5 ligation and indirectly through interleukin-17 induction.

OBJECTIVE: To examine the impact of Toll-like receptor 5 (TLR-5) on endothelial cell function in rheumatoid arthritis (RA) and vascularization in collagen-induced arthritis (CIA). METHODS: Endothelial cell migration and tube formation assays were used to demonstrate the direct role of TLR-5 ligation in angiogenesis. Mice with CIA were treated with the TLR-5 agonist flagellin to document the effect of TLR-5 ligation in RA pathology. Vascularization in CIA was determined by immunohistochemical analysis and determination of cytokine levels in ankle joints. Spleen Th17 cells and joint interleukin-17 (IL-17) were quantified by fluorescence-activated cell sorting analysis and enzyme-linked immunosorbent assay. The development of Th17 cells induced by TLR-5 ligation was validated in RA peripheral blood mononuclear cells. RESULTS: Ligation of TLR-5 to endogenous ligands expressed in RA synovial fluid contributed to endothelial cell infiltration and tube formation. Furthermore, treatment with flagellin after the onset of CIA exacerbated joint inflammation; in contrast, inflammation in control mice remained at a plateau phase. We showed that TLR-5-enhanced disease severity was attributable to Th17 cell differentiation and joint vascularization in CIA. Examination of the underlying mechanism using RA peripheral blood mononuclear cells documented that ligation of TLR-5 in myeloid cells and production of Th17-promoting cytokines were necessary for Th17 cell polarization. Additionally, we demonstrated that blockade of the IL-17 cascade markedly reduced endothelial cell migration activated by flagellin-conditioned medium, suggesting that TLR-5 ligation can mediate RA angiogenesis either directly by attracting endothelial cells or indirectly by fostering Th17 cell development. CONCLUSION: Our data demonstrate a novel role for TLR-5 in RA angiogenesis; thus, TLR-5 may be a promising new target for RA treatment.

Effect of explicit visual feedback distortion on human gait.

BACKGROUND: Gait rehabilitation often utilizes correction of stepping movements, and visual feedback is one of the interactive forms that can be used for rehabilitation. We presented a paradigm called visual feedback distortion in which we manipulated the visual representation of step length. Our previous work showed that an implicit distortion of visual feedback of step length entails unintentional modulations in the subjects' gait spatial pattern. Even in the presence of cognitive load through a distraction task, distortion of visual feedback still induced modulation of gait step length. In the current study, subjects were aware of the imposed distortion of visual feedback and they were instructed to maintain their natural gait symmetric pattern during trials. We then studied whether such an explicit "visual feedback distortion" would still influence gait spatial pattern. METHODS: Nine healthy subjects participated in the treadmill walking trial. The step length was defined as the distance between each foot. The on-line visual feedback showing right and left step length information as bar graphs was displayed on a computer screen. When distorting the visual feedback, the height of the bar for only one side was manipulated, so that subjects perceived their step length as being asymmetric. Actual step lengths were measured during trial and analyzed to see the effects of visual feedback distortion. RESULTS: Our results showed that a gradual distortion of visual feedback systematically modulated gait step length away from symmetry even at the expense of an opposing apparent task goal. It was also observed that the amount of induced gait modulation was reduced during the explicit condition compared to the implicit condition where subjects were not aware of distortion. CONCLUSIONS: Our study demonstrated that although the visual feedback display used in this study did not alter visual space or evoke illusions of motion, perturbation of visual information about subjects' movement can cause unintentional motor functions. This suggests that the effect of visual feedback distortion is spontaneous and a gait training involving the visual distortion paradigm may provide an effective way to help subjects correct gait patterns by driving implicit motor functions, thereby bringing benefits to rehabilitation.

Tumor immunogenicity regulates host immune responses, and conventional dendritic cell type 2 uptakes the majority of tumor antigens in an orthotopic lung cancer model.

Human lung cancer carries high genetic alterations, expressing high tumor-specific neoantigens. Although orthotopic murine lung cancer models recapitulate many characteristics of human lung cancers, genetically engineered mouse models have fewer somatic mutations than human lung cancer, resulting in scarce immune cell infiltration and deficient immune responses. The endogenous mouse lung cancer model driven by Kras mutation and Trp53 deletion (KP model) has minimal immune infiltration because of a scarcity of neoantigens. Fine-tuning tumor antigenicity to trigger the appropriate level of antitumor immunity would be key to investigating immune responses against human lung cancer. We engineered the KP model to express antigens of OVA peptides (minOVA) as neoantigens along with ZsGreen, a traceable fluorescent conjugate. The KP model expressing minOVA exhibited stronger immunogenicity with higher immune cell infiltration comprised of CD8+ T cells and CD11c+ dendritic cells (DCs). Consequentially, the KP model expressing minOVA exhibits suppressed tumor growth compared to its origin. We further analyzed tumor-infiltrated DCs. The majority of ZsGreen conjugated with minOVA was observed in the conventional type 2 DCs (cDC2), where cDC1 has minimal. These data indicate that tumor immunogenicity regulates host immune responses, and tumor neoantigen is mostly recognized by cDC2 cells, which may play a critical role in initiating anti-tumor immune responses in an orthotopic murine lung cancer model.

Ligation of TLR7 by rheumatoid arthritis synovial fluid single strand RNA induces transcription of TNFα in monocytes.

OBJECTIVE: The aim of the study was to characterise the expression, regulation and pathogenic role of toll-like receptor 7 (TLR7) and TLR8 in rheumatoid arthritis (RA). METHODS: Expression of TLR7 and TLR8 was demonstrated in RA, osteoarthritis (OA) and normal (NL) synovial tissues (STs) employing immunohistochemistry. The authors next examined the mechanism by which TLR7 and TLR8 ligation mediates proinflammatory response by Western blot analysis and ELISA. Expression of TLR7 and TLR8 in RA monocytes was correlated to disease activity score (DAS28) and tumour necrosis factor α (TNFα) levels. Further, the effect of TLR7 ligation in RA monocytes was determined on synovial fluid (SF)-mediated TNFα transcription. RESULTS: TLR7/8 are predominately expressed in RA ST lining and sublining macrophages. The authors show that NF-κB and/or PI3K pathways are essential for TLR7/8 induction of proinflammatory factors in RA peripheral blood (PB)-differentiated macrophages. Expression of TLR7 in RA monocytes shows a strong correlation with DAS28 and TNFα levels. By contrast, expression of TLR8 in these cells does not correlate with DAS28, TLR7 or TNFα levels. The authors further demonstrate that RNA from RA SF, but not RA or NL plasma, could modulate TNFα transcription from RA monocytes that can be downregulated by antagonising TLR7 ligation or degradation of single stand (ss) RNA. Thus, ssRNA present in RA SF may function as a potential endogenous ligand for TLR7. CONCLUSIONS: These results suggest that expression of TLR7, but not TLR8, may be a predictor for RA disease activity and anti-TNFα responsiveness, and targeting TLR7 may suppress chronic progression of RA.

The type III histone deacetylase Sirt1 protein suppresses p300-mediated histone H3 lysine 56 acetylation at Bclaf1 promoter to inhibit T cell activation.

The NAD-dependent histone deacetylase Sirt1 is a negative regulator of T cell activation. Here we report that Sirt1 inhibits T cell activation by suppressing the transcription of Bcl2-associated factor 1 (Bclaf1), a protein required for T cell activation. Sirt1-null T cells have increased acetylation of the histone 3 lysine 56 residue (H3K56) at the bclaf1 promoter, as well as increasing Bclaf1 transcription. Sirt1 binds to bclaf1 promoter upon T cell receptor (TCR)/CD28 stimulation by forming a complex with histone acetyltransferase p300 and NF-κB transcription factor Rel-A. The recruitment of Sirt1, but not p300, requires Rel-A because blocking Rel-A nuclear translocation in T cells and siRNA-mediated knockdown of Rel-A can inhibit Sirt1 binding to bclaf1 promoter. Although knockdown of either p300 or GCN5 partially suppressed global H3K56 acetylation, only p300 knockdown specifically attenuated H3K56 acetylation at the bclaf1 promoter. Lastly, knockdown of Bclaf1 suppresses the hyperactivation observed in Sirt1(-/-) T cells, indicated by less IL-2 production in CD4(+) T cells and reduced proliferation. Therefore, Sirt1 negatively regulates T cell activation via H3K56 deacetylation at the promoter region to inhibit transcription of Bclaf1.

Effects of implicit visual feedback distortion on human gait.

Gait rehabilitation after stroke often utilizes treadmill training delivered by either therapists or robotic devices. However, clinical results have shown no benefit from this modality when compared to usual care. On the contrary, results were inferior; perhaps, because in its present form it is not interactive and at least for stroke, central pattern generators at the spinal level do not appear to be the key to promote recovery. To enable gait therapy to be more effective, therapy must be interactive and visual feedback appears to be an important option to engage patients' participation. In this study, we tested healthy subjects to see whether an implicit "visual feedback distortion" influences gait spatial pattern. Subjects were not aware of the visual distortion nor did they realize changes in their gait pattern. The visual feedback of step length symmetry was distorted so that subjects perceived their step length as being asymmetric during treadmill training. We found that a gradual distortion of visual feedback, without explicit knowledge of the manipulation, systematically modulated gait step length away from symmetry and that the visual distortion effect was robust even in the presence of cognitive load. This indicates that although the visual feedback display used in this study did not create a conscious and vivid sensation of self-motion (the properties of the optical flow), experimental modifications of visual information of subjects' movement were found to cause implicit gait modulation. Nevertheless, our results indicate that modulation with visual distortion may require cognitive resources because during the distraction task, the amount of gait modulation was reduced. Our results suggest that a therapeutic program involving visual feedback distortion, in the context of gait rehabilitation, may provide an effective way to help subjects correct gait patterns, thereby improving the outcome of rehabilitation.