Neutrophils and neutrophil extracellular traps in cancer: promising targets for engineered nanomaterials.

Neutrophils are the most abundant white blood cells in circulation and constitute up to 60% of circulating leukocytes. Neutrophils play a significant role in host defense against pathogens through various mechanisms, including phagocytosis, production of antimicrobial proteins, and formation of neutrophil extracellular traps (NETs). Recently, the role of neutrophils and NETs in cancer has generated significant interest, as accumulating evidence suggests that neutrophils and NETs contribute to cancer progression and are associated with adverse patient outcomes. In this review, we will first highlight the roles of neutrophils and NETs in cancer progression and metastasis and discuss new drug delivery approaches to target and modulate neutrophils and NETs for cancer therapeutics.

TNKPVI, a Putative Bioaccessible Pharmacophore of Anti-Inflammatory Potato Patatin-Derived Decapeptide DIKTNKPVIF.

Potato protein-derived decapeptide DIKTNKPVIF exerted anti-inflammatory activity in animal models when delivered via intragastric gavage and intraperitoneal injection. However, DIKTNKPVIF is susceptible to hydrolysis in the digestive tract, which will decrease its bioaccessibility and possibly bioactivity. In this study, the anti-inflammatory activity of fragments generated from in silico gastrointestinal enzymatic hydrolysis of DIKTNKPVIF was investigated using the human monocytic (THP-1) cell line. The simulated digestion by pepsin and trypsin released four fragments, DIKTNKPVI, TNKPVIF, DIK and TNKPVI. The peptides lacked the cleavage sites of chymotrypsin. All five peptides were predicted to be non-toxic, which was validated using cytotoxicity assay at 0.25-1 mM peptide concentration. However, the peptides were predicted to possess poor pharmacokinetic profiles, including low passive gastrointestinal absorption and blood-brain barrier permeability. TNKPVIF, DIK and TNKPVI significantly reduced the amount of pro-inflammatory interleukin (IL)-6, IL-8 and tumor necrosis factor in lipopolysaccharide-activated THP-1 cells. Notably, the anti-inflammatory activity of fragment TNKPVI was comparable to that of the parent decapeptide while peptide fragment DIKTNKPVI had no apparent effect on the pro-inflammatory cytokines. This highlights the important role of the C-terminal phenylalanine residue of the parent peptide in the bioactivity. Furthermore, given its activity and the absence of cleavage sites of major digestive proteases, TNKPVI could be the biostable and bioaccessible pharmacophore of potato patatin-derived anti-inflammatory decapeptide DIKTNKPVIF.

Vaccine nanodiscs plus polyICLC elicit robust CD8+ T cell responses in mice and non-human primates.

Conventional cancer vaccines based on soluble vaccines and traditional adjuvants have produced suboptimal therapeutic efficacy in clinical trials. Thus, there is an urgent need for vaccine technologies that can generate potent T cell responses with strong anti-tumor efficacy. We have previously reported the development of synthetic high-density protein (sHDL) nanodiscs for efficient lymph node (LN)-targeted co-delivery of antigen peptides and CpG oligonucleotides (a Toll-like receptor-9 agonist). Here, we performed a comparative study in mice and non-human primates (NHPs) to identify an ideal vaccine platform for induction of CD8+ T cell responses. In particular, we compared the efficacy of CpG class B, CpG class C, and polyICLC (a synthetic double-stranded RNA analog, a TLR-3 agonist), each formulated with antigen-carrying sHDL nanodiscs. Here, we report that sHDL-Ag admixed with polyICLC elicited robust Ag-specific CD8+ T cell responses in mice, and when used in combination with α-PD-1 immune checkpoint inhibitor, sHDL-Ag + polyICLC eliminated large established (~100 mm3) MC-38 tumors in mice. Moreover, sHDL-Gag + polyICLC induced robust Simian immunodeficiency virus Gag-specific, polyfunctional CD8+ T cell responses in rhesus macaques and could further amplify the efficacy of recombinant adenovirus-based vaccine. Notably, while both sHDL-Ag-CpG-B and sHDL-Ag-CpG-C generated strong Ag-specific CD8+ T cell responses in mice, their results were mixed in NHPs. Overall, sHDL combined with polyICLC offers a strong platform to induce CD8+ T cells for vaccine applications.

Cancer Immunotherapy via Targeting Cancer Stem Cells Using Vaccine Nanodiscs.

Cancer stem cells (CSCs) proliferate extensively and drive tumor metastasis and recurrence. CSCs have been identified in over 20 cancer types to date, but it remains unknown how to target and eliminate CSCs in vivo. Aldehyde dehydrogenase (ALDH) is a marker that has been used extensively for isolating CSCs. Here we present a novel approach to target and reduce the frequency of ALDHhigh CSCs by vaccination against ALDH. We have identified ALDH1-A1 and ALDH1-A3 epitopes from CSCs and developed synthetic high-density lipoprotein nanodiscs for vaccination against ALDHhigh CSCs. Nanodiscs increased antigen trafficking to lymph nodes and generated robust ALDH-specific T cell responses. Nanodisc vaccination against ALDHhigh CSCs combined with anti-PD-L1 therapy exerted potent antitumor efficacy and prolonged animal survival in multiple murine models. Overall, this is the first demonstration of a simple nanovaccine strategy against CSCs and may lead to new avenues for cancer immunotherapy against CSCs.

Inhibition of neutrophil elastase prevents neutrophil extracellular trap formation and rescues mice from endotoxic shock.

Neutrophil elastase (NE) is a serine protease stored in the azurophilic granules of neutrophils and released into the extracellular milieu during inflammatory response or formation of neutrophil extracellular traps (NETs). Neutrophils release NETs to entrap pathogens by externalizing their cellular contents in a DNA framework decorated with anti-microbials and proteases, including NE. Importantly, excess NETs in tissues are implicated in numerous pathologies, including sepsis, rheumatoid arthritis, vasculitis, and cancer. However, it remains unknown how to effectively prevent NET formation. Here, we show that NE plays a major role during NET formation and that inhibition of NE is a promising approach for decreasing NET-mediated tissue injury. NE promoted NET formation by human neutrophils. Whereas sivelestat, a small molecule inhibitor of NE, inhibited the formation of NETs in vitro , administration of free sivelestat did not have any efficacy in a murine model of lipopolysaccharide-induced endotoxic shock. To improve the efficacy of sivelestat in vivo, we have developed a nanoparticle system for delivering sivelestat. We demonstrate that nanoparticle-mediated delivery of sivelestat effectively inhibited NET formation, decreased the clinical signs of lung injury, reduced NE and other proinflammatory cytokines in serum, and rescued animals against endotoxic shock. Collectively, our data demonstrates that NE signaling can initiate NET formation and that nanoparticle-mediated inhibition of NE improves drug efficacy for preventing NET formation.

The Pivotal Role of Regulatory T Cells in the Regulation of Innate Immune Cells.

The distinction between innate and adaptive immunity is one of the basic tenets of immunology. The co-operation between these two arms of the immune system is a major determinant of the resistance or susceptibility of the host following pathogen invasion. Hence, this interactive co-operation between cells of the innate and adaptive immunity is of significant interest to immunologists. The sub-population of CD4+ T cells with regulatory phenotype (regulatory T cells; Tregs), which constitute a part of the adaptive immune system, have been widely implicated in the regulation of the immune system and maintenance of immune homeostasis. In the last two decades, there has been an explosion in research describing the role of Tregs and their relevance in several immunopathologies ranging from inflammation to cancer. The majority of these studies focus on the role of Tregs on the cells of the adaptive immune system. Recently, there is significant interest in the role of Tregs on cells of the innate immune system. In this review, we examine the literature on the role of Tregs in immunology. Specifically, we focus on the emerging knowledge of Treg interaction with dendritic cells, macrophages, neutrophils, and γδ T cells. We highlight this interaction as an important link between innate and adaptive immune systems which also indicate the far-reaching role of Tregs in the regulation of immune responses and maintenance of self-tolerance and immune homeostasis.

Regulatory T cells restrain CD4+ T cells from causing unregulated immune activation and hypersensitivity to lipopolysaccharide challenge.

Regulatory T cells (Tregs) are essential for maintenance of peripheral tolerance, and defects in Treg function have been linked to several autoimmune diseases. We previously reported that depletion of Tregs resulted in mortality to an otherwise nonlethal dose of LPS or Escherichia coli challenge. In this study, we investigated the mechanism by which Treg depletion leads to enhanced susceptibility to LPS. Using different murine lymphocyte gene knockout models, we show that the enhanced sensitivity to LPS following Treg depletion is mediated by T cells. SCID or RAG1-deficient mice, which lack T and B cells, do not show enhanced susceptibility to LPS. However, reconstitution of SCID mice with wild-type CD4(+) T cells restored Treg depletion-induced sensitivity to LPS. This CD4(+) T cell-mediated hypersensitivity to LPS challenge in the absence of Tregs was also observed upon reconstitution of SCID mice with CD4(+) T cells from CD25 knockout mice (which lack functional Tregs). Additionally, depletion of Tregs leads to increased CD4(+) T cell proliferation and proinflammatory cytokine production in response to LPS challenge. Some CD4(+) T cells express TLR4, and pretreatment of CD4(+) T cells with LPS dramatically enhanced their ability to induce inflammatory cytokine production by macrophages. Collectively, our results indicate that in the absence of functional Tregs, CD4(+) T cells are pathologic and contribute to exaggerated immune activation that is detrimental for survival in LPS-induced acute inflammation. Our data also provide evidence for direct activation of CD4(+) T cells by LPS through TLR4.