Attenuated Dengue virus PV001-DV induces oncolytic tumor cell death and potent immune responses.

BACKGROUND: Viral therapies developed for cancer treatment have classically prioritized direct oncolytic effects over their immune activating properties. However, recent clinical insights have challenged this longstanding prioritization and have shifted the focus to more immune-based mechanisms. Through the potential utilization of novel, inherently immune-stimulating, oncotropic viruses there is a therapeutic opportunity to improve anti-tumor outcomes through virus-mediated immune activation. PV001-DV is an attenuated strain of Dengue virus (DEN-1 #45AZ5) with a favorable clinical safety profile that also maintains the potent immune stimulatory properties characterstic of Dengue virus infection. METHODS: In this study, we utilized in vitro tumor killing and immune multiplex assays to examine the anti-tumor effects of PV001-DV as a potential novel cancer immunotherapy. RESULTS: In vitro assays demonstrated that PV001-DV possesses the ability to directly kill human melanoma cells lines as well as patient melanoma tissue ex vivo. Importantly, further work demonstrated that, when patient peripheral blood mononuclear cells (PBMCs) were exposed to PV001-DV, a substantial induction in the production of apoptotic factors and immunostimulatory cytokines was detected. When tumor cells were cultured with the resulting soluble mediators from these PBMCs, rapid cell death of melanoma and breast cancer cell lines was observed. These soluble mediators also increased dengue virus binding ligands and immune checkpoint receptor, PD-L1 expression. CONCLUSIONS: The direct in vitro tumor-killing and immune-mediated tumor cytotoxicity facilitated by PV001-DV contributes support of its upcoming clinical evaluation in patients with advanced melanoma who have failed prior therapy.

Intratumoral injection of the seasonal flu shot converts immunologically cold tumors to hot and serves as an immunotherapy for cancer.

Reprogramming the tumor microenvironment to increase immune-mediated responses is currently of intense interest. Patients with immune-infiltrated "hot" tumors demonstrate higher treatment response rates and improved survival. However, only the minority of tumors are hot, and a limited proportion of patients benefit from immunotherapies. Innovative approaches that make tumors hot can have immediate impact particularly if they repurpose drugs with additional cancer-unrelated benefits. The seasonal influenza vaccine is recommended for all persons over 6 mo without prohibitive contraindications, including most cancer patients. Here, we report that unadjuvanted seasonal influenza vaccination via intratumoral, but not intramuscular, injection converts "cold" tumors to hot, generates systemic CD8+ T cell-mediated antitumor immunity, and sensitizes resistant tumors to checkpoint blockade. Importantly, intratumoral vaccination also provides protection against subsequent active influenza virus lung infection. Surprisingly, a squalene-based adjuvanted vaccine maintains intratumoral regulatory B cells and fails to improve antitumor responses, even while protecting against active influenza virus lung infection. Adjuvant removal, B cell depletion, or IL-10 blockade recovers its antitumor effectiveness. Our findings propose that antipathogen vaccines may be utilized for both infection prevention and repurposing as a cancer immunotherapy.

α-Gal Nanoparticles Mediated Homing of Endogenous Stem Cells for Repair and Regeneration of External and Internal Injuries by Localized Complement Activation and Macrophage Recruitment.

This review discusses a novel experimental approach for the regeneration of original tissue structure by recruitment of endogenous stem-cells to injured sites following administration of α-gal nanoparticles, which harness the natural anti-Gal antibody. Anti-Gal is produced in large amounts in all humans, and it binds the multiple α-gal epitopes (Galα1-3Galß1-4GlcNAc-R) presented on α-gal nanoparticles. In situ binding of anti-Gal to α-gal nanoparticles activates the complement system and generates complement cleavage chemotactic-peptides that rapidly recruit macrophages. Macrophages reaching anti-Gal coated α-gal nanoparticles bind them via Fc/Fc receptor interaction and polarize into M2 pro-reparative macrophages. These macrophages secrete various cytokines that orchestrate regeneration of the injured tissue, including VEGF inducing neo-vascularization and cytokines directing homing of stem-cells to injury sites. Homing of stem-cells is also directed by interaction of complement cleavage peptides with their corresponding receptors on the stem-cells. Application of α-gal nanoparticles to skin wounds of anti-Gal producing mice results in decrease in healing time by half. Furthermore, α-gal nanoparticles treated wounds restore the normal structure of the injured skin without fibrosis or scar formation. Similarly, in a mouse model of occlusion/reperfusion myocardial-infarction, near complete regeneration after intramyocardial injection of α-gal nanoparticles was demonstrated, whereas hearts injected with saline display ~20% fibrosis and scar formation of the left ventricular wall. It is suggested that recruitment of stem-cells following anti-Gal/α-gal nanoparticles interaction in injured tissues may result in induction of localized regeneration facilitated by conducive microenvironments generated by pro-reparative macrophage secretions and "cues" provided by the extracellular matrix in the injury site.

Topical α-gal nanoparticles accelerate diabetic wound healing.

An inadequate response from macrophages, key orchestrators of the wound healing process, has been implicated in the pathophysiology of impaired healing in diabetes. This study explored the utility of nanoparticles presenting the α-gal (Galα1-3Galß1-4GlcNAc-R) epitope to induce anti-Gal antibody-mediated local transient recruitment of macrophages to accelerate wound closure and healing in a diabetic murine model. α1,3galactosyltrasferase knockout mice were stimulated to produce anti-Gal antibodies and subsequently diabetes was induced by streptozotocin-induced ß-cell destruction. Six mm full-thickness skin wounds were made and α-gal nanoparticles (AGN) were topically applied on postwounding days 0 and 1. Wounds were analysed histologically for macrophage invasion and markers of wound healing, including epithelialization, vascularization and granulation tissue deposition through postoperative day 12. We found that application of AGN transiently but significantly increased macrophage recruitment into the wounds of diabetic mice. Treated wounds demonstrated more rapid closure and enhanced wound healing as demonstrated by significantly accelerated rates of epithelialization, vascularization and granulation tissue deposition. Thus, topical treatment of full-thickness wounds in diabetic mice with α-gal nanoparticles induced a transient but significant increase in macrophage recruitment resulting in an accelerated rate of wound healing. Using α-gal nanoparticles as a topical wound healing adjunct is a simple, safe and effective means of augmenting dysregulated macrophage recruitment present in the diabetic state.

IL-15 Complexes Induce Migration of Resting Memory CD8 T Cells into Mucosal Tissues.

IL-15 is an essential cytokine known to promote T cell survival and activate the effector function of memory phenotype CD8 T cells. Blocking IL-15 signals also significantly impacts tissue-specific effector and memory CD8 T cell formation. In this study, we demonstrate that IL-15 influences the generation of memory CD8 T cells by first promoting their accumulation into mucosal tissues and second by sustaining expression of Bcl-6 and T-bet. We show that the mechanism for this recruitment is largely dependent on mammalian target of rapamycin and its subsequent inactivation of FoxO1. Last, we show that IL-15 complexes delivered locally to mucosal tissues without reinfection is an effective strategy to enhance establishment of tissue resident memory CD8 T cells within mucosal tissues. This study provides mechanistic insight into how IL-15 controls the generation of memory CD8 T cells and influences their trafficking and ability to take up residence within peripheral tissues.

Pseudomonas aeruginosa ExoT Induces Atypical Anoikis Apoptosis in Target Host Cells by Transforming Crk Adaptor Protein into a Cytotoxin.

Previously, we demonstrated that Pseudomonas aeruginosa ExoT induces potent apoptosis in host epithelial cells in a manner that primarily depends on its ADP-ribosyltransferase domain (ADPRT) activity. However, the mechanism underlying ExoT/ADPRT-induced apoptosis remains undetermined. We now report that ExoT/ADPRT disrupts focal adhesion sites, activates p38ß and JNK, and interferes with integrin-mediated survival signaling; causing atypical anoikis. We show that ExoT/ADPRT-induced anoikis is mediated by the Crk adaptor protein. We found that Crk-/- knockout cells are significantly more resistant to ExoT-induced apoptosis, while Crk-/- cells complemented with Crk are rendered sensitive to ExoT-induced apoptosis. Moreover, a dominant negative (DN) mutant form of Crk phenocopies ExoT-induced apoptosis both kinetically and mechanistically. Crk is generally believed to be a component of focal adhesion (FA) and its role in cellular survival remains controversial in that it has been found to be either pro-survival or pro-apoptosis. Our data demonstrate that although Crk is recruited to FA sites, its function is likely not required for FA assembly or for survival per se. However, when modified by ExoT or by mutagenesis, it can be transformed into a cytotoxin that induces anoikis by disrupting FA sites and interfering with integrin survival signaling. To our knowledge, this is the first example whereby a bacterial toxin exerts its cytotoxicity by subverting the function of an innocuous host cellular protein and turning it against the host cell.

Pseudomonas aeruginosa ExoT Induces Mitochondrial Apoptosis in Target Host Cells in a Manner That Depends on Its GTPase-activating Protein (GAP) Domain Activity.

Pseudomonas aeruginosa is the most common cause of hospital-acquired pneumonia and a killer of immunocompromised patients. We and others have demonstrated that the type III secretion system (T3SS) effector protein ExoT plays a pivotal role in facilitating P. aeruginosa pathogenesis. ExoT possesses an N-terminal GTPase-activating protein (GAP) domain and a C-terminal ADP-ribosyltransferase (ADPRT) domain. Because it targets multiple non-overlapping cellular targets, ExoT performs several distinct virulence functions for P. aeruginosa, including induction of apoptosis in a variety of target host cells. Both the ADPRT and the GAP domain activities contribute to ExoT-induced apoptosis. The ADPRT domain of ExoT induces atypical anoikis by transforming an innocuous cellular protein, Crk, into a cytotoxin, which interferes with integrin survival signaling. However, the mechanism underlying the GAP-induced apoptosis remains unknown. In this study, we demonstrate that the GAP domain activity is both necessary and sufficient to induce mitochondrial (intrinsic) apoptosis. We show that intoxication with GAP domain results in: (i) JNK1/2 activation; (ii) substantial increases in the mitochondrial levels of activated pro-apoptotic proteins Bax and Bid, and to a lesser extent Bim; (iii) loss of mitochondrial membrane potential and cytochrome c release; and (iv) activation of initiator caspase-9 and executioner caspase-3. Further, GAP-induced apoptosis is partially mediated by JNK1/2, but it is completely dependent on caspase-9 activity. Together, the ADPRT and the GAP domains make ExoT into a highly versatile and potent cytotoxin, capable of inducing multiple forms of apoptosis in target host cells.

Pseudomonas aeruginosa uses T3SS to inhibit diabetic wound healing.

Diabetic foot ulcers are responsible for more hospitalizations than any other complication of diabetes. Bacterial infection is recognized as an important factor associated with impaired healing in diabetic ulcers. Pseudomonas aeruginosa is the most frequently detected Gram-negative pathogen in diabetic ulcers. P. aeruginosa infection has been shown to impair healing in diabetic wounds in a manner that correlates with its ability to form biofilm. While the majority of infections in diabetic ulcers are biofilm associated, 33% of infections are nonbiofilm in nature. P. aeruginosa is the most prevalent Gram-negative pathogen in all diabetic wound types, which suggests that the deleterious impact of P. aeruginosa on healing in diabetic wounds goes beyond its ability to form biofilm and likely involves other factors. The Type III Secretion System (T3SS) virulence structure is required for the pathogenesis of all P. aeruginosa clinical isolates, suggesting that it may also play a role in the inhibition of wound repair in diabetic skin ulcers. We evaluated the role of T3SS in mediating P. aeruginosa-induced tissue damage in the wounds of diabetic mice. Our data demonstrate that P. aeruginosa establishes a robust and persistent infection in diabetic wounds independent of its ability to form biofilm and causes severe wound damage in a manner that primarily depends on its T3SS.

Caloric restriction maintains OX40 agonist-mediated tumor immunity and CD4 T cell priming during aging.

Immune responses wane during aging, posing challenges to the potential effectiveness of cancer immunotherapies. We previously demonstrated that in the context of a promising immunotherapeutic, OX40 agonist (αOX40), older animals exhibited impaired anti-tumor immune responses and diminished CD4 T cell effector differentiation. In this study, we hypothesized that tumor immune responses could be maintained during aging through caloric restriction (CR) or dietary supplementation with resveratrol (RES), a CR mimetic. Mice were placed on either a calorically restricted diet or a RES-formulated diet starting between 4 and 6 months of age and continued until mice reached 12 months of age. Tumor immune responses were assessed after challenging with either sarcoma or breast tumor cells followed by αOX40 treatment. Our results show that CR, but not RES, maintained OX40-mediated anti-tumor immunity. In addition, CR fully sustained antigen-specific CD4 T cell priming in aged hosts (12 months old), whereas tumor-specific CD8 T cell priming was not fully maintained compared to young reference animals (2 months old). Thus, CR appears to maintain immunological fitness of the CD4 T cell priming environment during aging, which is critical for optimal OX40-mediated responses.

Pseudomonas aeruginosa Cytotoxins: Mechanisms of Cytotoxicity and Impact on Inflammatory Responses.

Pseudomonas aeruginosa is one of the most virulent opportunistic Gram-negative bacterial pathogens in humans. It causes many acute and chronic infections with morbidity and mortality rates as high as 40%. P. aeruginosa owes its pathogenic versatility to a large arsenal of cell-associated and secreted virulence factors which enable this pathogen to colonize various niches within hosts and protect it from host innate immune defenses. Induction of cytotoxicity in target host cells is a major virulence strategy for P. aeruginosa during the course of infection. P. aeruginosa has invested heavily in this strategy, as manifested by a plethora of cytotoxins that can induce various forms of cell death in target host cells. In this review, we provide an in-depth review of P. aeruginosa cytotoxins based on their mechanisms of cytotoxicity and the possible consequences of their cytotoxicity on host immune responses.

CrkII/Abl phosphorylation cascade is critical for NLRC4 inflammasome activity and is blocked by Pseudomonas aeruginosa ExoT.

Type 3 Secretion System (T3SS) is a highly conserved virulence structure that plays an essential role in the pathogenesis of many Gram-negative pathogenic bacteria, including Pseudomonas aeruginosa. Exotoxin T (ExoT) is the only T3SS effector protein that is expressed in all T3SS-expressing P. aeruginosa strains. Here we show that T3SS recognition leads to a rapid phosphorylation cascade involving Abl / PKCδ / NLRC4, which results in NLRC4 inflammasome activation, culminating in inflammatory responses that limit P. aeruginosa infection in wounds. We further show that ExoT functions as the main anti-inflammatory agent for P. aeruginosa in that it blocks the phosphorylation cascade through Abl / PKCδ / NLRC4 by targeting CrkII, which we further demonstrate to be important for Abl transactivation and NLRC4 inflammasome activation in response to T3SS and P. aeruginosa infection.

Near Complete Repair After Myocardial Infarction in Adult Mice by Altering the Inflammatory Response With Intramyocardial Injection of α-Gal Nanoparticles.

Background: Neonatal mice, but not older mice, can regenerate their hearts after myocardial-infarction (MI), a process mediated by pro-reparative macrophages. α-Gal nanoparticles applied to skin wounds in adult-mice bind the anti-Gal antibody, activate the complement cascade and generate complement chemotactic peptides that recruit pro-reparative macrophages which are further activated by these nanoparticles. The recruited macrophages decrease wound healing time by ~50%, restore the normal skin structure and prevent fibrosis and scar formation in mice. Objectives: The objective of this study is to determine if α-gal nanoparticles injected into the reperfused myocardium after MI in adult-mice can induce myocardial repair that restores normal structure, similar to that observed in skin injuries. Methods and Results: MI was induced by occluding the mid-portion of the left anterior descending (LAD) coronary artery for 30 min. Immediately following reperfusion, each mouse received two 10 µl injections of 100 µg α-gal nanoparticles in saline into the LAD territory (n = 20), or saline for controls (n = 10). Myocardial infarct size was measured by planimetry following Trichrome staining and macrophage recruitment by hematoxylin-eosin staining. Left ventricular (LV) function was measured by echocardiography. Control mice displayed peak macrophage infiltration at 4-days, whereas treated mice had a delayed peak macrophage infiltration at 7-days. At 28-days, control mice demonstrated large transmural infarcts with extensive scar formation and poor contractile function. In contrast, mice treated with α-gal nanoparticles demonstrated after 28-days a marked reduction in infarct size (~10-fold smaller), restoration of normal myocardium structure and contractile function. Conclusions: Intramyocardial injection of α-gal nanoparticles post-MI in anti-Gal producing adult-mice results in near complete repair of the infarcted territory, with restoration of normal LV structure and contractile function. The mechanism responsible for this benefit likely involves alteration of the usual inflammatory response post-MI, as previously observed with regeneration of injured hearts in adult zebrafish, salamanders and neonatal mice.

Pseudomonas aeruginosa ExoS Induces Intrinsic Apoptosis in Target Host Cells in a Manner That is Dependent on its GAP Domain Activity.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that causes serious infections in immunocompromised individuals and cystic fibrosis patients. ExoS and ExoT are two homologous bifunctional Type III Secretion System (T3SS) virulence factors that induce apoptosis in target host cells. They possess a GTPase Activating Protein (GAP) domain at their N-termini, which share ~76% homology, and an ADP-ribosyltransferase (ADPRT) domain at their C-termini, which target non-overlapping substrates. Both the GAP and the ADPRT domains contribute to ExoT's cytotoxicity in target epithelial cells, whereas, ExoS-induced apoptosis is reported to be primarily due to its ADPRT domain. In this report, we demonstrate that ExoS/GAP domain is both necessary and sufficient to induce mitochondrial apoptosis. Our data demonstrate that intoxication with ExoS/GAP domain leads to enrichment of Bax and Bim into the mitochondrial outer-membrane, disruption of mitochondrial membrane and release of and cytochrome c into the cytosol, which activates initiator caspase-9 and effector caspase-3, that executes cellular death. We posit that the contribution of the GAP domain in ExoS-induced apoptosis was overlooked in prior studies due to its slower kinetics of cytotoxicity as compared to ADPRT. Our data clarify the field and reveal a novel virulence function for ExoS/GAP as an inducer of apoptosis.

Apoptosis and Compensatory Proliferation Signaling Are Coupled by CrkI-Containing Microvesicles.

Apoptosis has been implicated in compensatory proliferation signaling (CPS), whereby dying cells induce proliferation in neighboring cells as a means to restore homeostasis. The nature of signaling between apoptotic cells and their neighboring cells remains largely unknown. Here we show that a fraction of apoptotic cells produce and release CrkI-containing microvesicles (distinct from exosomes and apoptotic bodies), which induce proliferation in neighboring cells upon contact. We provide visual evidence of CPS by videomicroscopy. We show that purified vesicles in vitro and in vivo are sufficient to stimulate proliferation in other cells. Our data demonstrate that CrkI inactivation by ExoT bacterial toxin or by mutagenesis blocks vesicle formation in apoptotic cells and inhibits CPS, thus uncoupling apoptosis from CPS. We further show that c-Jun amino-terminal kinase (JNK) plays a pivotal role in mediating vesicle-induced CPS in recipient cells. CPS could have important ramifications in diseases that involve apoptotic cell death.

Perioperative high inspired oxygen fraction therapy reduces surgical site infection with Pseudomonas aeruginosa in rats.

Surgical site infection (SSI) remains one of the most important causes of healthcare-associated infections, accounting for ~17 % of all hospital-acquired infections. Although short-term perioperative treatment with high fraction of inspired oxygen (FiO2) has shown clinical benefits in reducing SSI in colorectal resection surgeries, the true clinical benefits of FiO2 therapy in reducing SSI remain unclear because randomized controlled trials on this topic have yielded disparate results and inconsistent conclusions. To date, no animal study has been conducted to determine the efficacy of short-term perioperative treatments with high (FiO2>60 %) versus low (FiO2<40 %) oxygen in reducing SSI. In this report, we designed a rat model for muscle surgery to compare the effectiveness of short-term perioperative treatments with high (FiO2=80 %) versus a standard low (FiO2=30 %) oxygen in reducing SSI with Pseudomonas aeruginosa - one of the most prevalent Gram-negative pathogens, responsible for nosocomial SSIs. Our data demonstrate that 5 h perioperative treatment with 80 % FiO2 is significantly more effective in reducing SSI with P. aeruginosa compared to 30 % FiO2 treatment. We further show that whilst 80 % FiO2 treatment does not affect neutrophil infiltration into P. aeruginosa-infected muscles, neutrophils in the 80 % FiO2-treated and infected animal group are significantly more activated than neutrophils in the 30 % FiO2-treated and infected animal group, suggesting that high oxygen perioperative treatment reduces SSI with P. aeruginosa by enhancing neutrophil activation in infected wounds.

Non-oncogenic Acute Viral Infections Disrupt Anti-cancer Responses and Lead to Accelerated Cancer-Specific Host Death.

In light of increased cancer prevalence and cancer-specific deaths in patients with infections, we investigated whether infections alter anti-tumor immune responses. We report that acute influenza infection of the lung promotes distal melanoma growth in the dermis and leads to accelerated cancer-specific host death. Furthermore, we show that during influenza infection, anti-melanoma CD8+ T cells are shunted from the tumor to the infection site, where they express high levels of the inhibitory receptor programmed cell death protein 1 (PD-1). Immunotherapy to block PD-1 reverses this loss of anti-tumor CD8+ T cells from the tumor and decreases infection-induced tumor growth. Our findings show that acute non-oncogenic infection can promote cancer growth, raising concerns regarding acute viral illness sequelae. They also suggest an unexpected role for PD-1 blockade in cancer immunotherapy and provide insight into the immune response when faced with concomitant challenges.