Role of IL-27 in HSV-1-Induced Herpetic Stromal Keratitis.

Herpetic stromal keratitis (HSK) is a painful and vision-impairing disease caused by recurrent HSV-1 infection of the cornea. The virus replication in the corneal epithelium and associated inflammation play a dominant role in HSK progression. Current HSK treatments targeting inflammation or virus replication are partially effective and promote HSV-1 latency, and long-term use can cause side effects. Thus, understanding molecular and cellular events that control HSV-1 replication and inflammation is crucial for developing novel HSK therapies. In this study, we report that ocular HSV-1 infection induces the expression of IL-27, a pleiotropic immunoregulatory cytokine. Our data indicate that HSV-1 infection stimulates IL-27 production by macrophages. Using a primary corneal HSV-1 infection mouse model and IL-27 receptor knockout mice, we show that IL-27 plays a critical role in controlling HSV-1 shedding from the cornea, the optimum induction of effector CD4+ T cell responses, and limiting HSK progression. Using in vitro bone marrow-derived macrophages, we show that IL-27 plays an antiviral role by regulating macrophage-mediated HSV-1 killing, IFN-ß production, and IFN-stimulated gene expression after HSV-1 infection. Furthermore, we report that IL-27 is critical for macrophage survival, Ag uptake, and the expression of costimulatory molecules involved in the optimum induction of effector T cell responses. Our results indicate that IL-27 promotes endogenous antiviral and anti-inflammatory responses and represents a promising target for suppressing HSK progression.

IFN-λ Regulates Neutrophil Biology to Suppress Inflammation in Herpes Simplex Virus-1-Induced Corneal Immunopathology.

HSV-1 infection of the cornea causes a severe immunoinflammatory and vision-impairing condition called herpetic stromal keratitis (SK). The virus replication in corneal epithelium followed by neutrophil- and CD4+ T cell-mediated inflammation plays a dominant role in SK. Although previous studies demonstrate critical functions of type I IFNs (IFN-α/ß) in HSV-1 infection, the role of recently discovered IFN-λ (type III IFN), specifically at the corneal mucosa, is poorly defined. Our study using a mouse model of SK pathogenesis shows that HSV-1 infection induces a robust IFN-λ response compared with type I IFN production at the corneal mucosal surface. However, the normal progression of SK indicates that the endogenous IFN responses are insufficient to suppress HSV-1-induced corneal pathology. Therefore, we examined the therapeutic efficacy of exogenous rIFN-λ during SK progression. Our results show that rIFN-λ therapy suppressed inflammatory cell infiltration in the cornea and significantly reduced the SK pathologic condition. Early rIFN-λ treatment significantly reduced neutrophil and macrophage infiltration, and IL-6, IL-1ß, and CXCL-1 production in the cornea. Notably, the virucidal capacity of neutrophils and macrophages measured by reactive oxygen species generation was not affected. Similarly, ex vivo rIFN-λ treatment of HSV-1-stimulated bone marrow-derived neutrophils significantly promoted IFN-stimulated genes without affecting reactive oxygen species production. Collectively, our data demonstrate that exogenous topical rIFN-λ treatment during the development and progression of SK could represent a novel therapeutic approach to control HSV-1-induced inflammation and associated vision impairment.

Short palate, lung, and nasal epithelial clone 1 (SPLUNC1) level determines steroid-resistant airway inflammation in aging.

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.

ADAMTS7 Attenuates House Dust Mite-Induced Airway Inflammation and Th2 Immune Responses.

PURPOSE: ADAMTS7 is a secreted metalloproteinase enzyme and proteoglycan associated with the early progression of coronary artery disease. However, there is limited information regarding the role of ADAMTS7 in lung adaptive immunity and inflammation. Thus, we sought to assess whether ADAMTS7 expression in the lung modulates house dust mite (HDM)-induced airway inflammation and Th2 immune response. METHODS: The role of ADAMTS7 in HDM-induced airway disease was assessed in ADAMTS7-deficient (ADAMTS7-/-) mice and compared with the wild-type control mice by flow cytometry, ELISA, and histopathology. Furthermore, the antigen priming capability of dendritic cells (DC) was determined ex vivo by employing coculture with CD4+ OT-II cells. RESULTS: ADAMTS7-/- mice develop an augmented eosinophilic airway inflammation, mucous cell metaplasia, and increased Th2 immune response to inhaled HDM. In addition, allergen uptake by lung DC and migration to draining mediastinal lymph node were significantly increased in ADAMTS7-/- mice, which shows an enhanced capacity to mount allergen-specific T-cell proliferation and effector Th2 cytokine productions. We propose that the mechanism by which ADAMTS7 negatively regulates DC function involves attenuated antigen uptake and presentation capabilities, which reduces allergic sensitization and Th2 immune responses in the lung. CONCLUSION: In aggregate, we provide compelling evidence that ADAMTS7 plays a pivotal role in allergic airway disease and Th2 immunity and would be an attractive target for asthma.

Low-density lipoprotein receptor-related protein 1 attenuates house dust mite-induced eosinophilic airway inflammation by suppressing dendritic cell-mediated adaptive immune responses.

BACKGROUND: Low-density lipoprotein receptor-related protein 1 (LRP-1) is a scavenger receptor that regulates adaptive immunity and inflammation. LRP-1 is not known to modulate the pathogenesis of allergic asthma. OBJECTIVE: We sought to assess whether LRP-1 expression by dendritic cells (DCs) modulates adaptive immune responses in patients with house dust mite (HDM)-induced airways disease. METHODS: LRP-1 expression on peripheral blood DCs was quantified by using flow cytometry. The role of LRP-1 in modulating HDM-induced airways disease was assessed in mice with deletion of LRP-1 in CD11c+ cells (Lrp1fl/fl; CD11c-Cre) and by adoptive transfer of HDM-pulsed CD11b+ DCs from Lrp1fl/fl; CD11c-Cre mice to wild-type (WT) mice. RESULTS: Human peripheral blood myeloid DC subsets from patients with eosinophilic asthma have lower LRP-1 expression than cells from healthy nonasthmatic subjects. Similarly, LRP-1 expression by CD11b+ lung DCs was significantly reduced in HDM-challenged WT mice. HDM-challenged Lrp1fl/fl; CD11c-Cre mice have a phenotype of increased eosinophilic airway inflammation, allergic sensitization, TH2 cytokine production, and mucous cell metaplasia. The adoptive transfer of HDM-pulsed LRP-1-deficient CD11b+ DCs into WT mice generated a similar phenotype of enhanced eosinophilic inflammation and allergic sensitization. Furthermore, CD11b+ DCs in the lungs of Lrp1fl/fl; CD11c-Cre mice have an increased ability to take up HDM antigen, whereas bone marrow-derived DCs display enhanced antigen presentation capabilities. CONCLUSION: This identifies a novel role for LRP-1 as a negative regulator of DC-mediated adaptive immune responses in the setting of HDM-induced eosinophilic airway inflammation. Furthermore, the reduced LRP-1 expression by circulating myeloid DCs in patients with eosinophilic asthma suggests a possible role for LRP-1 in modulating type 2-high asthma.

A Critical Role for P2X7 Receptor-Induced VCAM-1 Shedding and Neutrophil Infiltration during Acute Lung Injury.

Pulmonary neutrophils are the initial inflammatory cells that are recruited during lung injury and are crucial for innate immunity. However, pathological recruitment of neutrophils results in lung injury. The objective of this study is to determine whether the novel neutrophil chemoattractant, soluble VCAM-1 (sVCAM-1), recruits pathological levels of neutrophils to injury sites and amplifies lung inflammation during acute lung injury. The mice with P2X7 receptor deficiency, or treated with a P2X7 receptor inhibitor or anti-VCAM-1 Abs, were subjected to a clinically relevant two-hit LPS and mechanical ventilation-induced acute lung injury. Neutrophil infiltration and lung inflammation were measured. Neutrophil chemotactic activities were determined by a chemotaxis assay. VCAM-1 shedding and signaling pathways were assessed in isolated lung epithelial cells. Ab neutralization of sVCAM-1 or deficiency or antagonism of P2X7R reduced neutrophil infiltration and proinflammatory cytokine levels. The ligands for sVCAM-1 were increased during acute lung injury. sVCAM-1 had neutrophil chemotactic activities and activated alveolar macrophages. VCAM-1 is released into the alveolar airspace from alveolar epithelial type I cells through P2X7 receptor-mediated activation of the metalloproteinase ADAM-17. In conclusion, sVCAM-1 is a novel chemoattractant for neutrophils and an activator for alveolar macrophages. Targeting sVCAM-1 provides a therapeutic intervention that could block pathological neutrophil recruitment, without interfering with the physiological recruitment of neutrophils, thus avoiding the impairment of host defenses.

Platelet-derived Wnt antagonist Dickkopf-1 is implicated in ICAM-1/VCAM-1-mediated neutrophilic acute lung inflammation.

Neutrophil infiltration represents the early acute inflammatory response in acute lung injury. The recruitment of neutrophils from the peripheral blood across the endothelial-epithelial barrier into the alveolar airspace is highly regulated by the adhesion molecules on alveolar epithelial cells (AECs). Wnt/ß-catenin signaling is involved in the progression of inflammatory lung diseases including asthma, emphysema, and pulmonary fibrosis. However, the function of Wnt/ß-catenin signaling in acute lung inflammation is unknown. Here, we identified platelet-derived Dickkopf-1 (Dkk1) as the major Wnt antagonist contributing to the suppression of Wnt/ß-catenin signaling in AECs during acute lung inflammation. Intratracheal administration of Wnt3a or an antibody capable of neutralizing Dkk1 inhibited neutrophil influx into the alveolar airspace of injured lungs. Activation of Wnt/ß-catenin signaling in AECs attenuated intercellular adhesion molecule 1 (ICAM-1)/vascular cell adhesion molecule 1 (VCAM-1)-mediated adhesion of both macrophages and neutrophils to AECs. Our results suggest a role for Wnt/ß-catenin signaling in modulating the inflammatory response, and a functional communication between platelets and AECs during acute lung inflammation. Targeting Wnt/ß-catenin signaling and the communication between platelets and AECs therefore represents potential therapeutic strategies to limit the damage of acute pulmonary inflammation.

The very low density lipoprotein receptor attenuates house dust mite-induced airway inflammation by suppressing dendritic cell-mediated adaptive immune responses.

The very low density lipoprotein receptor (VLDLR) is a member of the low-density lipoprotein receptor family that binds multiple ligands and plays a key role in brain development. Although the VLDLR mediates pleiotropic biological processes, only a limited amount of information is available regarding its role in adaptive immunity. In this study, we identify an important role for the VLDLR in attenuating house dust mite (HDM)-induced airway inflammation in experimental murine asthma. We show that HDM-challenged Vldlr(-/-) mice have augmented eosinophilic and lymphocytic airway inflammation with increases in Th2 cytokines, C-C chemokines, IgE production, and mucous cell metaplasia. A genome-wide analysis of the lung transcriptome identified that mRNA levels of CD209e (DC-SIGNR4), a murine homolog of DC-SIGN, were increased in the lungs of HDM-challenged Vldlr(-/-) mice, which suggested that the VLDLR might modify dendritic cell (DC) function. Consistent with this, VLDLR expression by human monocyte-derived DCs was increased by HDM stimulation. In addition, 55% of peripheral blood CD11c(+) DCs from individuals with allergy expressed VLDLR under basal conditions. Lastly, the adoptive transfer of HDM-pulsed, CD11c(+) bone marrow-derived DCs (BMDCs) from Vldlr(-/-) mice to the airways of wild type recipient mice induced augmented eosinophilic and lymphocytic airway inflammation upon HDM challenge with increases in Th2 cytokines, C-C chemokines, IgE production, and mucous cell metaplasia, as compared with the adoptive transfer of HDM-pulsed, CD11c(+) BMDCs from wild type mice. Collectively, these results identify a novel role for the VLDLR as a negative regulator of DC-mediated adaptive immune responses in HDM-induced allergic airway inflammation.

Purinergic P2X7 receptor regulates lung surfactant secretion in a paracrine manner.

Alveolar epithelium is composed of alveolar epithelial cells of type I (AEC I) and type II (AEC II). AEC II secrete lung surfactant by means of exocytosis. P2X(7) receptor (P2X(7)R), a P2 purinergic receptor, has been implicated in the regulation of synaptic transmission and inflammation. Here, we report that P2X(7)R, which is expressed in AEC I but not AEC II, is a novel mediator for the paracrine regulation of surfactant secretion in AEC II. In primary co-cultures of AEC I and AEC II benzoyl ATP (BzATP; an agonist of P2X(7)R) increased surfactant secretion, which was blocked by the P2X(7)R antagonist Brilliant Blue G. This effect was observed in AEC II co-cultured with human embryonic kidney HEK-293 cells stably expressing rat P2X(7)R, but not when co-cultured with AEC I in which P2X(7)R was knocked down or in co-cultures of AEC I and AEC II isolated from P2X(7)R(-/-) mice. BzATP-mediated secretion involved P2Y(2) receptor signaling because it was reduced by the addition of the ATP scavengers apyrase and adenosine deaminase and the P2Y(2) receptor antagonist suramin. However, the stimulation with BzATP might also release other substances that potentially increase surfactant secretion as a greater stimulation of secretion was observed in AEC II incubated with BzATP when co-cultured with E10 or HEK-293-P2X(7)R cells than with ATP alone. P2X(7)R(-/-) mice failed to increase surfactant secretion in response to hyperventilation, pointing to the physiological relevance of P2X(7)R in maintaining surfactant homeostasis in the lung. These results suggest that the activation of P2X(7)R increases surfactant secretion by releasing ATP from AEC I and subsequently stimulating P2Y(2) receptors in AEC II.

New insights of P2X7 receptor signaling pathway in alveolar functions.

Purinergic P2X7 receptor (P2X7R), an ATP-gated cation channel, is unique among all other family members because of its ability to respond to various stimuli and to modulate pro-inflammatory signaling. The activation of P2X7R in immune cells is absolutely required for mature interleukin -1beta (IL-1beta) and IL-18 production and release. Lung alveoli are lined by the structural alveolar epithelial type I (AEC I) and alveolar epithelial type II cells (AEC II). AEC I plays important roles in alveolar barrier protection and fluid homeostasis whereas AEC II synthesizes and secrete surfactant and prevents alveoli from collapse. Earlier studies indicated that purinergic P2X7 receptors were specifically expressed in AEC I. However, their implication in alveolar functions has not been explored. This paper reviews two important signaling pathways of P2X7 receptors in surfactant homeostatsis and Acute Lung Injury (ALI). Thus, P2X7R resides at the critical nexus of alveolar pathophysiology.

Targeting ß-catenin using XAV939 nanoparticle promotes immunogenic cell death and suppresses conjunctival melanoma progression.

Many tumors dysregulate Wnt/ß-catenin pathway to promote stem-cell-like phenotype, tumorigenesis, immunosuppression, and resistance to targeted cancer immunotherapies. Therefore, targeting this pathway is a promising therapeutic approach to suppress tumor progression and elicit robust anti-tumor immunity. In this study, using a nanoparticle formulation for XAV939 (XAV-Np), a tankyrase inhibitor that promotes ß-catenin degradation, we investigated the effect of ß-catenin inhibition on melanoma cell viability, migration, and tumor progression using a mouse model of conjunctival melanoma. XAV-Nps were uniform and displayed near-spherical morphology with size stability for upto 5 days. We show that XAV-Np treatment of mouse melanoma cells significantly suppresses cell viability, tumor cell migration, and tumor spheroid formation compared to control nanoparticle (Con-Np) or free XAV939-treated groups. Further, we demonstrate that XAV-Np promotes immunogenic cell death (ICD) of tumor cells with a significant extracellular release or expression of ICD molecules, including high mobility group box 1 protein (HMGB1), calreticulin (CRT), and adenosine triphosphate (ATP). Finally, we show that local intra-tumoral delivery of XAV-Nps during conjunctival melanoma progression significantly suppresses tumor size and conjunctival melanoma progression compared to Con-Nps-treated animals. Collectively, our data suggest that selective inhibition of ß-catenin in tumor cells using nanoparticle-based targeted delivery represents a novel approach to suppress tumor progression through increased tumor cell ICD.

Targeting Wnt/ß-catenin signaling using XAV939 nanoparticles in tumor microenvironment-conditioned macrophages promote immunogenicity.

The aberrant activation of Wnt/ß-catenin signaling in tumor cells and immune cells in the tumor microenvironment (TME) promotes malignant transformation, metastasis, immune evasion, and resistance to cancer treatments. The increased Wnt ligand expression in TME activates ß-catenin signaling in antigen (Ag)-presenting cells (APCs) and regulates anti-tumor immunity. Previously, we showed that activation of Wnt/ß-catenin signaling in dendritic cells (DCs) promotes induction of regulatory T cell responses over anti-tumor CD4+ and CD8+ effector T cell responses and promotes tumor progression. In addition to DCs, tumor-associated macrophages (TAMs) also serve as APCs and regulate anti-tumor immunity. However, the role of ß-catenin activation and its effect on TAM immunogenicity in TME is largely undefined. In this study, we investigated whether inhibiting ß-catenin in TME-conditioned macrophages promotes immunogenicity. Using nanoparticle formulation of XAV939 (XAV-Np), a tankyrase inhibitor that promotes ß-catenin degradation, we performed in vitro macrophage co-culture assays with melanoma cells (MC) or melanoma cell supernatants (MCS) to investigate the effect on macrophage immunogenicity. We show that XAV-Np-treatment of macrophages conditioned with MC or MCS significantly upregulates the cell surface expression of CD80 and CD86 and suppresses the expression of PD-L1 and CD206 compared to MC or MCS-conditioned macrophages treated with control nanoparticle (Con-Np). Further, XAV-Np-treated macrophages conditioned with MC or MCS significantly increased IL-6 and TNF-α production, with reduced IL-10 production compared to Con-Np-treated macrophages. Moreover, the co-culture of MC and XAV-Np-treated macrophages with T cells resulted in increased CD8+ T cell proliferation compared to Con-Np-treated macrophages. These data suggest that targeted ß-catenin inhibition in TAMs represents a promising therapeutic approach to promote anti-tumor immunity.

Metronomic Administration of Topotecan Alone and in Combination with Docetaxel Inhibits Epithelial-mesenchymal Transition in Aggressive Variant Prostate Cancers.

Prostate cancer is the second leading cause of noncutaneous cancer-related deaths in American men. Androgen deprivation therapy (ADT), radical prostatectomy, and radiotherapy remain the primary treatment for patients with early-stage prostate cancer (castration-sensitive prostate cancer). Following ADT, many patients ultimately develop metastatic castration-resistant prostate cancer (mCRPC). Standard chemotherapy options for CRPC are docetaxel (DTX) and cabazitaxel, which increase median survival, although the development of resistance is common. Cancer stem-like cells possess mesenchymal phenotypes [epithelial-to-mesenchymal transition (EMT)] and play crucial roles in tumor initiation and progression of mCRPC. We have shown that low-dose continuous administration of topotecan (METRO-TOPO) inhibits prostate cancer growth by interfering with key cancer pathway genes. This study utilized bulk and single-cell or whole-transcriptome analysis [(RNA sequencing (RNA-seq) and single-cell RNA sequencing (scRNA-seq)], and we observed greater expression of several EMT markers, including Vimentin, hyaluronan synthase-3, S100 calcium binding protein A6, TGFB1, CD44, CD55, and CD109 in European American and African American aggressive variant prostate cancer (AVPC) subtypes-mCRPC, neuroendocrine variant (NEPC), and taxane-resistant. The taxane-resistant gene FSCN1 was also expressed highly in single-cell subclonal populations in mCRPC. Furthermore, metronomic-topotecan single agent and combinations with DTX downregulated these EMT markers as well as CD44+ and CD44+/CD133+ "stem-like" cell populations. A microfluidic chip-based cell invasion assay revealed that METRO-TOPO treatment as a single agent or in combination with DTX was potentially effective against invasive prostate cancer spread. Our RNA-seq and scRNA-seq analysis were supported by in silico and in vitro studies, suggesting METRO-TOPO combined with DTX may inhibit oncogenic progression by reducing cancer stemness in AVPC through the inhibition of EMT markers and multiple oncogenic factors/pathways. Significance: The utilization of metronomic-like dosing regimens of topotecan alone and in combination with DTX resulted in the suppression of makers associated with EMT and stem-like cell populations in AVPC models. The identification of molecular signatures and their potential to serve as novel biomarkers for monitoring treatment efficacy and disease progression response to treatment efficacy and disease progression were achieved using bulk RNA-seq and single-cell-omics methodologies.

Regulation of lung surfactant secretion by microRNA-150.

P2X7 receptor (P2X7R) is a purinergic ion-channel receptor. We have previously shown that the activation of P2X7R in alveolar type I cells stimulates surfactant secretion in alveolar type II cells. In this study, we determined whether miR-150 regulates P2X7R-mediated surfactant secretion. The miR-150 expression level in alveolar type II cells was much higher than alveolar type I cells, which was inversely correlated with the P2X7R protein level. An adenovirus expressing miR-150 significantly reduced the P2X7R protein expression in E10 cells, an alveolar type I cell line. Furthermore, pre-treatment of E10 cells with the adenovirus reduced the surfactant secretion induced by E10 cell conditioned medium. Our study demonstrates that miR-150 regulates surfactant secretion through P2X7R.

Characterization of VAMP-2 in the lung: implication in lung surfactant secretion.

Lung surfactant is crucial for reducing the surface tension of alveolar space, thus preventing the alveoli from collapse. Lung surfactant is synthesized in alveolar epithelial type II cells and stored in lamellar bodies before being released via the fusion of lamellar bodies with the apical plasma membrane. SNAREs (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptors) play an essential role in membrane fusion. We have previously demonstrated the requirement of t-SNARE (target SNARE) proteins, syntaxin 2 and SNAP-23 (N-ethylmaleimide-sensitive factor-attachment protein 23), in regulated surfactant secretion. Here, we characterized the distribution of VAMPs (vesicle-associated membrane proteins) in rat lung and alveolar type II cells. VAMP-2, -3 and -8 are shown in type II cells at both mRNA and protein levels. VAMP-2 and -8 were enriched in LB (lamellar body) fraction. Immunochemistry studies indicated that VAMP-2 was co-localized with the LB marker protein, LB-180. Functionally, the cytoplasmic domain of VAMP-2, but not VAMP-8 inhibited surfactant secretion in type II cells. We suggest that VAMP-2 is the v-SNARE (vesicle SNARE) involved in regulated surfactant secretion.

Irg1/itaconate metabolic pathway is a crucial determinant of dendritic cells immune-priming function and contributes to resolute allergen-induced airway inflammation.

Itaconate is produced from the mitochondrial TCA cycle enzyme aconitase decarboxylase (encoded by immune responsive gene1; Irg1) that exerts immunomodulatory function in myeloid cells. However, the role of the Irg1/itaconate pathway in dendritic cells (DC)-mediated airway inflammation and adaptive immunity to inhaled allergens, which are the primary antigen-presenting cells in allergic asthma, remains largely unknown. House dust mite (HDM)-challenged Irg1-/- mice displayed increases in eosinophilic airway inflammation, mucous cell metaplasia, and Th2 cytokine production with a mechanism involving impaired mite antigen presentations by DC. Adoptive transfer of HDM-pulsed DC from Irg1-deficient mice into naïve WT mice induced a similar phenotype of elevated type 2 airway inflammation and allergic sensitization. Untargeted metabolite analysis of HDM-pulsed DC revealed itaconate as one of the most abundant polar metabolites that potentially suppress mitochondrial oxidative damage. Furthermore, the immunomodulatory effect of itaconate was translated in vivo, where intranasal administration of 4-octyl itaconate 4-OI following antigen priming attenuated the manifestations of HDM-induced airway disease and Th2 immune response. Taken together, these data demonstrated for the first time a direct regulatory role of the Irg1/itaconate pathway in DC for the development of type 2 airway inflammation and suggest a possible therapeutic target in modulating allergic asthma.

Development of OX40 agonists for canine cancer immunotherapy.

Recent breakthroughs in cancer immunotherapy have provided unprecedented clinical benefits to human cancer patients. Cancer is also one of the most common causes of death in pet dogs. Thus, canine-specific immune therapies targeting similar signaling pathways can provide better treatment options for canine cancer patients. Here, we describe the development and characterization of two canine-specific anti-OX40 agonists to activate OX40 signaling. We show that canine OX40, like human OX40, is not expressed on resting T cells, and its expression is markedly increased on canine CD4 T cells and Tregs after stimulation with concanavalin A (Con-A). cOX40 is also expressed on tumor-infiltrating lymphocytes (TILs) in canine osteosarcoma patients. The canine-specific OX40 agonists strongly activates cPBMCs by increasing IFN-γ expression and do not require Fc receptor-mediated cross-linking for OX40 agonism. Together, these results suggest that cFcOX40L proteins are potent OX40 agonists and have the potential to enhance antitumor immunity in canine cancer patients.

Nanobody-based CTLA4 inhibitors for immune checkpoint blockade therapy of canine cancer patients.

Cancer is the leading cause of death in the geriatric dog population. Currently, the use of immune checkpoint inhibitors (ICIs) such as anti-CTLA4 antibodies has markedly improved the prognosis of several cancers in their advanced stages. However, ICIs targeting CTLA4 blockade to treat canine cancer patients are yet to define. In this study, we sought to develop, characterize and assess whether chimeric heavy chain only antibodies (cHcAbs) against CTLA4 are viable therapeutic candidates for the treatment of canine cancers. Anti-CTLA4 nanobodies (Nbs) were identified from a yeast nanobody (Nb) library using magnetic-assisted cell sorting (MACS) and flow cytometry. cHcAbs were engineered by genetically fusing the DNA sequences coding for anti-CTLA4 Nbs with the Fc domain of the subclass B of canine IgG. Recombinant cHcAbs were purified from ExpiCHO-S cells. Stable cell lines expressing canine CTLA4 and FcγRI were used to elucidate the binding ability and specificity of cHcAbs. PBMCs isolated from healthy dogs were used to evaluate the ability of cHcAbs to activate canine PBMCs (cPBMCs). Novel Nbs were identified using the extracellular domain of canine CTLA4 protein to screen a fully synthetic yeast nanobody library. Purified Nbs bind specifically to natïve canine CTLA4. We report that chimeric HcAbs, which were engineered by fusing the anti-CTLA4 Nbs and Fc region of subclass B of canine IgG, were half the size of a conventional mAb and formed dimers. The chimeric HcAbs specifically binds both with canine CTLA4 and Fcγ receptors. As the binding of Nbs overlapped with the MYPPPY motif of canine CTLA4, these Nbs were expected to sterically disrupt the interaction of canine CTLA4 to B-7s. Like their human counterpart, canine CTLA4 was expressed on helper T cells and a small subset of cytotoxic T cells. Canine Tregs also constitutively expressed CTLA4, and stimulation with PMA/Ionomycin dramatically increased expression of CTLA4 on the cell surface. Stimulation of cPBMCs in the presence of agonistic anti-CD3 Ab and cHcAb6 significantly increased the expression of IFN-γ as compared to the isotype control. This study identifies a novel nanobody-based CTLA4 inhibitor for the treatment of canine cancer patients.

Micro-RNA-375 inhibits lung surfactant secretion by altering cytoskeleton reorganization.

Lung surfactant is secreted via exocytosis of lamellar bodies from alveolar epithelial type II cells. Whether micro-RNAs regulate lung surfactant secretion is unknown. Micro-RNA-375 (miR-375) has been shown to be involved in insulin secretion. In this article, we report that the overexpression of miR-375 inhibited lung surfactant secretion. However, miR-125a, miR-30a, miR-1, miR-382 and miR-101 did not influence lung surfactant secretion. miR-375 had no effects on surfactant synthesis or the formation of lamellar bodies. However, miR-375 did abolish the lung surfactant secretagogue-induced disassembly and reassembly of cytoskeleton. Our results suggest that miR-375 regulates surfactant secretion via the reorganization of cytoskeleton.

Pyruvate kinase M2 in lung APCs regulates Alternaria-induced airway inflammation.

Sensitivity to allergenic fungi (Alternaria alternata) is associated with acute, severe asthma attacks. Antigen presenting cells (APCs) in the lung sense environmental perturbations that induce cellular stress and metabolic changes and are critical for allergic airway inflammation. However, the mechanisms underlying such environmental sensing by APCs in the lung remains unclear. Here we show that acute Alternaria challenge rapidly induces neutrophil accumulation in airways, and alter expressions of Pyruvate Kinase (PKM2) and hypoxia-inducible factor -1α (Hif-1α) that correlates with proinflammatory mediator release. Blockade of IL33 signaling in vivo led to reduce oxidative stress and glycolysis in lung APCs. Lung-specific ablation of CD11c+ cells abrogates Alternaria-induced neutrophil accumulation and inflammation. Furthermore, administration of Alternaria into the airways stimulated APCs and elevate the expression of Glut-1. Mechanistically, we establish that PKM2 is a critical modulator of lung APC activation in Alternaria-induced acute inflammation. Allosteric activation of PKM2 by a small molecule ML265 or siRNA-mediated knock down correlated negatively with glycolysis and activation of APCs. These results collectively demonstrates that PKM2-mediated glycolytic reprogramming by fungal allergen Alternaria influences lung APC activation, thereby promotes acute airway inflammation. Our data support a model in which Alternaria sensitization in airways induce a circuitry of glycolysis and PKM2 regulation that confers an acute activation of APCs in the lung, whose targeting might represent a strategy for asthma treatment.