Intestinal overexpression of IL-18 promotes eosinophils-mediated allergic disorders.

Baseline eosinophils reside in the gastrointestinal tract; however, in several allergic disorders, excessive eosinophils accumulate in the blood as well in the tissues. Recently, we showed in vitro that interleukin (IL)-18 matures and transforms IL-5-generated eosinophils into the pathogenic eosinophils that are detected in human allergic diseases. To examine the role of local induction of IL-18 in promoting eosinophil-associated intestinal disorders, we generated enterocyte IL-18-overexpressing mice using the rat intestinal fatty acid-binding promoter (Fabpi) and analysed tissue IL-18 overexpression and eosinophilia by performing real-time polymerase chain reaction, Enzyme-Linked Immunosorbent Assay and anti-major basic protein immunostaining. Herein we show that Fabpi-IL-18 mice display highly induced IL-18 mRNA and protein in the jejunum. IL-18 overexpression in enterocytes promotes marked increases of eosinophils in the blood and jejunum. Our analysis shows IL-18 overexpression in the jejunum induces a specific population of CD101+  CD274+ tissue eosinophils. Additionally, we observed comparable tissue eosinophilia in IL-13-deficient-Fabpi-IL-18 mice, and reduced numbers of tissue eosinophils in eotaxin-deficient-Fabpi-IL-18 and IL-5-deficient-Fabpi-IL-18 mice compared with Fabpi-IL-18 transgenic mice. Notably, jejunum eosinophilia in IL-5-deficient-Fabpi-IL-18 mice is significantly induced compared with wild-type mice, which indicates the direct role of induced IL-18 in the tissue accumulation of eosinophils and mast cells. Furthermore, we also found that overexpression of IL-18 in the intestine promotes eosinophil-associated peanut-induced allergic responses in mice. Taken together, we provide direct in vivo evidence that induced expression of IL-18 in the enterocytes promotes eotaxin-1, IL-5 and IL-13 independent intestinal eosinophilia, which signifies the clinical relevance of induced IL-18 in eosinophil-associated gastrointestinal disorders (EGIDs) to food allergens.

Involvement of interleukin-18 in the pathogenesis of human eosinophilic esophagitis.

IL-18 is induced in food allergy and EoE is food allergen-induced disease. Therefore, we tested the hypothesis whether IL-18 is involved in food allergen-induced EoE pathogenesis. Accordingly, we examined normal SPT+ and SPT- EoE patient blood and biopsy samples for IL-18, IL-18Rα, ICAM and VCAM expression. Herein, we show increased IL-18 level is highly significant in food allergen SPT+ compared to SPT- EoE patients. We also report that IL-18Rα+ cells and mRNA levels are induced in the esophageal biopsies of EoE patients and blood IL-18 levels correlate with esophageal eosinophilia (P<0.01). Additionally, we report that the levels of esophageal eosinophil and mast cells correlate with ICAM expression in human EoE. Mechanistically, we show that IL-18 in vitro stimulates iNKT cells and endothelial cells and induce eosinophil active cytokines IL-5 and IL-13. We provide the evidence that IL-18 is critical cytokine involved in activation of iNKT cells and ICAM in promoting human EoE.

Allergen-induced interleukin-18 promotes experimental eosinophilic oesophagitis in mice.

Elevated levels of interleukin (IL)-18 have been reported in a number of allergic diseases. We recently reported that IL-18 in the blood and IL-18Rα mRNA in the oesophagus are induced during human eosinophilic oesophagitis (EoE). Additionally, we earlier showed that invariant natural killer T (iNKT) cells are critical to EoE pathogenesis; however, the mechanism of iNKT cell activation in EoE is not well understood. Therefore, the current study focused on the hypothesis that allergen-induced IL-18 may have an important role in iNKT cell-mediated EoE pathogenesis. We first validated the human EoE findings of IL-18 in experimental EoE by examining blood levels of IL-18 and oesophageal IL-18Rα mRNA levels in aeroallergen- and food allergen-induced experimental mouse models of EoE. We demonstrate that blood IL-18 protein and oesophageal IL-18Rα mRNA are induced in the mouse model of EoE and that IL-18Rα is expressed by iNKT cells in the oesophagus. Intranasal delivery of rIL-18 induced both mast cells and eosinophilic inflammation in the oesophagus in a time- and dose-dependent manner. To establish the significance of IL-18 in EoE pathogenesis, we examined DOX-inducible rtTA-CC10-IL-18 bitransgenic mice that induce IL-18 protein expression in the oesophagus. Our analysis indicated that induction of IL-18 in these mice resulted in the development of many of the characteristics of EoE, including oesophageal intraepithelial eosinophilia, increased mast cells, oesophageal remodelling and fibrosis. The current study provides evidence that IL-18 may induce iNKT cell activation to release the eosinophil-activating cytokine IL-5, as IL-5-deficient mice and iNKT cell-deficient (CD1d null) mice do not induce EoE in response to intranasal IL-18 challenge. Taken together, these findings provide evidence that allergen-induced IL-18 has a significant role in promoting IL-5- and iNKT-dependent EoE pathogenesis.

The Thermal Dose of Photothermal Therapy Generates Differential Immunogenicity in Human Neuroblastoma Cells.

Photothermal therapy (PTT) is an effective method for tumor eradication and has been successfully combined with immunotherapy. However, besides its cytotoxic effects, little is known about the effect of the PTT thermal dose on the immunogenicity of treated tumor cells. Therefore, we administered a range of thermal doses using Prussian blue nanoparticle-based photothermal therapy (PBNP-PTT) and assessed their effects on tumor cell death and concomitant immunogenicity correlates in two human neuroblastoma cell lines: SH-SY5Y (MYCN-non-amplified) and LAN-1 (MYCN-amplified). PBNP-PTT generated thermal dose-dependent tumor cell killing and immunogenic cell death (ICD) in both tumor lines in vitro. However, the effect of the thermal dose on ICD and the expression of costimulatory molecules, immune checkpoint molecules, major histocompatibility complexes, an NK cell-activating ligand, and a neuroblastoma-associated antigen were significantly more pronounced in SH-SY5Y cells compared with LAN-1 cells, consistent with the high-risk phenotype of LAN-1 cells. In functional co-culture studies in vitro, T cells exhibited significantly higher cytotoxicity toward SH-SY5Y cells relative to LAN-1 cells at equivalent thermal doses. This preliminary report suggests the importance of moving past the traditional focus of using PTT solely for tumor eradication to one that considers the immunogenic effects of PTT thermal dose to facilitate its success in cancer immunotherapy.

Interstitial Photothermal Therapy Generates Durable Treatment Responses in Neuroblastoma.

Photothermal therapy (PTT) represents a promising modality for tumor control typically using infrared light-responsive nanoparticles illuminated by a wavelength-matched external laser. However, due to the constraints of light penetration, PTT is generally restricted to superficially accessible tumors. With the goal of extending the benefits of PTT to all tumor settings, interstitial PTT (I-PTT) is evaluated by the photothermal activation of intratumorally administered Prussian blue nanoparticles with a laser fiber positioned interstitially within the tumor. This interstitial fiber, which is fitted with a terminal diffuser, distributes light within the tumor microenvironment from the "inside-out" as compared to from the "outside-in" traditionally observed during superficially administered PTT (S-PTT). I-PTT improves the heating efficiency and heat distribution within a target treatment area compared to S-PTT. Additionally, I-PTT generates increased cytotoxicity and thermal damage at equivalent thermal doses, and elicits immunogenic cell death at lower thermal doses in targeted neuroblastoma tumor cells compared to S-PTT. In vivo, I-PTT induces significantly higher long-term tumor regression, lower rates of tumor recurrence, and improved long-term survival in multiple syngeneic murine models of neuroblastoma. This study highlights the significantly enhanced therapeutic benefit of I-PTT compared to traditional S-PTT as a promising treatment modality for solid tumors.

An Engineered Prussian Blue Nanoparticles-based Nanoimmunotherapy Elicits Robust and Persistent Immunological Memory in a TH-MYCN Neuroblastoma Model.

A combination therapy using Prussian blue nanoparticles (PBNP) as photothermal therapy (PTT) agents coated with CpG oligodeoxynucleotides, an immunologic adjuvant, as a nanoimmunotherapy (CpG-PBNP-PTT) for neuroblastoma (NB) is described. NB driven by MYCN amplification confers high risk and correlates with a dismal prognosis, accounting for the majority of NB-related mortality. The efficacy of the CpG-PBNP-PTT nanoimmunotherapy in a clinically relevant, TH-MYCN murine NB model (9464D) overexpressing MYCN is tested. When administered to 9464D NB cells in vitro, CpG-PBNP-PTT triggers thermal dose-dependent immunogenic cell death and tumor cell priming for immune recognition in vitro, measured by the expression of specific costimulatory and antigen-presenting molecules. In vivo, intratumorally administered CpG-PBNP-PTT generates complete tumor regression and significantly higher long-term survival compared to controls. Furthermore, CpG-PBNP-PTT-treated mice reject tumor rechallenge. Ex vivo studies confirm these therapeutic responses result from the generation of robust T cell-mediated immunological memory. Consequently, in a synchronous 9464D tumor model, CpG-PBNP-PTT induces complete tumor regression on the treated flank and significantly slows tumor progression on the untreated flank, improving animal survival. These findings demonstrate that localized administration of the CpG-PBNP-PTT nanoimmunotherapy drives potent systemic T cell responses in solid tumors such as NB and therefore has therapeutic implications for NB.

CpG-coated prussian blue nanoparticles-based photothermal therapy combined with anti-CTLA-4 immune checkpoint blockade triggers a robust abscopal effect against neuroblastoma.

High-risk neuroblastoma, which is associated with regional and systemic metastasis, is a leading cause of cancer-related mortality in children. Responding to this need for novel therapies for high-risk patients, we have developed a "nanoimmunotherapy," which combines photothermal therapy (PTT) using CpG oligodeoxynucleotide-coated Prussian blue nanoparticles (CpG-PBNPs) combined with anti-CTLA-4 (aCTLA-4) immunotherapy. Our in vitro studies demonstrate that in addition to causing ablative tumor cell death, our nanoimmunotherapy alters the surface levels of co-stimulatory, antigen-presenting, and co-inhibitory molecules on neuroblastoma tumor cells. When administered in a syngeneic, murine model of neuroblastoma bearing synchronous Neuro2a tumors, the CpG-PBNP-PTT plus aCTLA-4 nanoimmunotherapy elicits complete tumor regression in both primary (CpG-PBNP-PTT-treated) and secondary tumors, and long-term survival in a significantly higher proportion (55.5%) of treated-mice compared with the controls. Furthermore, the surviving, nanoimmunotherapy-treated animals reject Neuro2a rechallenge, suggesting that the therapy generates immunological memory. Additionally, the depletion of CD4+, CD8+, and NK+ populations abrogate the observed therapeutic responses of the nanoimmunotherapy. These findings demonstrate the importance of concurrent PTT-based cytotoxicity and the antitumor immune effects of PTT, CpG, and aCTLA-4 in generating a robust abscopal effect against neuroblastoma.

Elements Involved In Promoting Eosinophilic Gastrointestinal Disorders.

Eosinophilic gastrointestinal disorders (EGID) are food allergen-induced allergic gastrointestinal disorders, characterized by accumulation of highly induced eosinophils in different segments of gastrointestinal tract along with eosinophil microabssess and extracellular eosinophilic granules in the epithelial layer. EGID are both IgE- and cell-mediated group of diseases that include eosinophilic esophagitis (EoE), eosinophilic gastritis (EG), eosinophilic gastroenteritis (EGE) and eosinophilic colitis (EC). Despite the increased incidences and considerable progress made in understanding EGID pathogenesis. The mechanism is still not well understood. It has been shown that IL-4, IL-5, IL-13, IL-15, IL-18, eotaxin-1, eotaxin-2 and eotaxin-3 play a critical role in EGID pathogenesis. Currently, the only criterion for diagnosing EoE, EGE and EC are repetitive endoscopic and histopathological evaluation of biopsies along with other clinical characteristics/manifestations. Antigen elimination and corticosteroid therapies are the most effective therapies currently in practice for the treatment of EGID. The cytokines (anti-IL-5 and anti-IL-13) therapy trials were not very successful in case of EoE. Most recently, a clinical trial using anti-IL-13 reported only 60% reduced esophageal eosinophilia without achieving primary endpoint. This clinical finding is not surprising and is in accordance with our earlier report indicating that IL-13 is not critical in the initiation of EoE. Notably, EGID still has no reliable noninvasive diagnostic biomarkers. Hence, there is a great necessity to identify novel noninvasive diagnostic biomarkers that can easily diagnose EGID and provide an effective therapy. Now, the attention is required to target cell types like iNKT cells that produce eosinophil active cytokines and is found induced in the pathogenesis of both experimental and human EoE. iNKT cell neutralization is shown to protect allergen-induced EoE in experimental model. In this review, we have discussed the key elements that are critical in the disease initiation, progression, pathogenesis and important for future diagnostic and therapeutic interventions for EGID.

Trans-blood brain barrier delivery of dopamine-loaded nanoparticles reverses functional deficits in parkinsonian rats.

Sustained and safe delivery of dopamine across the blood brain barrier (BBB) is a major hurdle for successful therapy in Parkinson's disease (PD), a neurodegenerative disorder. Therefore, in the present study we designed neurotransmitter dopamine-loaded PLGA nanoparticles (DA NPs) to deliver dopamine to the brain. These nanoparticles slowly and constantly released dopamine, showed reduced clearance of dopamine in plasma, reduced quinone adduct formation, and decreased dopamine autoxidation. DA NPs were internalized in dopaminergic SH-SY5Y cells and dopaminergic neurons in the substantia nigra and striatum, regions affected in PD. Treatment with DA NPs did not cause reduction in cell viability and morphological deterioration in SH-SY5Y, as compared to bulk dopamine-treated cells, which showed reduced viability. Herein, we report that these NPs were able to cross the BBB and capillary endothelium in the striatum and substantia nigra in a 6-hydroxydopamine (6-OHDA)-induced rat model of PD. Systemic intravenous administration of DA NPs caused significantly increased levels of dopamine and its metabolites and reduced dopamine-D2 receptor supersensitivity in the striatum of parkinsonian rats. Further, DA NPs significantly recovered neurobehavioral abnormalities in 6-OHDA-induced parkinsonian rats. Dopamine delivered through NPs did not cause additional generation of ROS, dopaminergic neuron degeneration, and ultrastructural changes in the striatum and substantia nigra as compared to 6-OHDA-lesioned rats. Interestingly, dopamine delivery through nanoformulation neither caused alterations in the heart rate and blood pressure nor showed any abrupt pathological change in the brain and other peripheral organs. These results suggest that NPs delivered dopamine into the brain, reduced dopamine autoxidation-mediated toxicity, and ultimately reversed neurochemical and neurobehavioral deficits in parkinsonian rats.