Gene expression profiling of lichenoid dermatitis immune-related adverse event from immune checkpoint inhibitors reveals increased CD14+ and CD16+ monocytes driving an innate immune response.

BACKGROUND: Cancer patients receiving antibodies abrogating immune checkpoint pathways may develop a diverse array of immune-related adverse events (irAEs), of which lichenoid dermatitis (LD) is the most common. The mechanism driving the emergence of these irAEs remain understudied, underscoring a critical need to determine the unique gene expression profiles and immune composition in LD-irAE. METHODS: LD-irAE (n = 3) and benign lichenoid keratosis (BLK) control (n = 3) were profiled with NanoString nCounter PanCancer Immune Profiling Panel interrogating the mRNA levels of 770 genes. Immunohistochemical (IHC) studies (n = 14 samples) for CD14, CD16, T-Bet, Gata-3, and FoxP3 were further evaluated using Aperio digital image analysis. RESULTS: The LD-irAE showed downregulation of 93 mRNA transcripts (P < 0.05) and upregulation of 74 mRNA transcripts (P < 0.04) including toll-like receptor (TLR) 2 and TLR4 (P < 0.05). CD14+ and CD16+ monocytes quantified by IHC (H-score) were higher in LD-irAE than in the BLK control (P < 0.05). The immune composition of LD-irAE exhibited higher numbers of T-Bet+ (Th1) cells compared with Gata-3+ (Th2) cells (P = 0.016) and lower numbers of FoxP3 (T regulatory) cells (P = 0.008). CONCLUSIONS: LD-irAE exhibited activation of CD14/TLR innate immune response with increased CD14+ and CD16+ monocytes compared with BLK control. CD14/TLR signaling may drive the development of LD-irAE.

Targeted delivery of small interfering RNA to human dendritic cells to suppress dengue virus infection and associated proinflammatory cytokine production.

Dengue is a common arthropod-borne flaviviral infection in the tropics, for which there is no vaccine or specific antiviral drug. The infection is often associated with serious complications such as dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS), in which both viral and host factors have been implicated. RNA interference (RNAi) is a potent antiviral strategy and a potential therapeutic option for dengue if a feasible strategy can be developed for delivery of small interfering RNA (siRNA) to dendritic cells (DCs) and macrophages, the major in vivo targets of the virus and also the source of proinflammatory cytokines. Here we show that a dendritic cell-targeting 12-mer peptide (DC3) fused to nona-D-arginine (9dR) residues (DC3-9dR) delivers siRNA and knocks down endogenous gene expression in heterogenous DC subsets, (monocyte-derived DCs [MDDCs], CD34(+) hematopoietic stem cell [HSC])-derived Langerhans DCs, and peripheral blood DCs). Moreover, DC3-9dR-mediated delivery of siRNA targeting a highly conserved sequence in the dengue virus envelope gene (siFvE(D)) effectively suppressed dengue virus replication in MDDCs and macrophages. In addition, DC-specific delivery of siRNA targeting the acute-phase cytokine tumor necrosis factor alpha (TNF-alpha), which plays a major role in dengue pathogenesis, either alone or in combination with an antiviral siRNA, significantly reduced virus-induced production of the cytokine in MDDCs. Finally to validate the strategy in vivo, we tested the ability of the peptide to target human DCs in the NOD/SCID/IL-2Rgamma(-/-) mouse model engrafted with human CD34(+) hematopoietic stem cells (HuHSC mice). Treatment of mice by intravenous (i.v.) injection of DC3-9dR-complexed siRNA targeting TNF-alpha effectively suppressed poly(I:C)-induced TNF-alpha production by DCs. Thus, DC3-9dR can deliver siRNA to DCs both in vitro and in vivo, and this delivery approach holds promise as a therapeutic strategy to simultaneously suppress virus replication and curb virus-induced detrimental host immune responses in dengue infection.

Targeted delivery of siRNA to macrophages for anti-inflammatory treatment.

Inflammation mediated by tumor necrosis factor-alpha (TNF-alpha) and the associated neuronal apoptosis characterizes a number of neurologic disorders. Macrophages and microglial cells are believed to be the major source of TNF-alpha in the central nervous system (CNS). Here, we show that suppression of TNF-alpha by targeted delivery of small interfering RNA (siRNA) to macrophage/microglial cells dramatically reduces lipopolysaccharide (LPS)-induced neuroinflammation and neuronal apoptosis in vivo. Because macrophage/microglia express the nicotinic acetylcholine receptor (AchR) on their surface, we used a short AchR-binding peptide derived from the rabies virus glycoprotein (RVG) as a targeting ligand. This peptide was fused to nona-D-arginine residues (RVG-9dR) to enable siRNA binding. RVG-9dR was able to deliver siRNA to induce gene silencing in macrophages and microglia cells from wild type, but not AchR-deficient mice, confirming targeting specificity. Treatment with anti-TNF-alpha siRNA complexed to RVG-9dR achieved efficient silencing of LPS-induced TNF-alpha production by primary macrophages and microglia cells in vitro. Moreover, intravenous injection with RVG-9dR-complexed siRNA in mice reduced the LPS-induced TNF-alpha levels in blood as well as in the brain, leading to a significant reduction in neuronal apoptosis. These results demonstrate that RVG-9dR provides a tool for siRNA delivery to macrophages and microglia and that suppression of TNF-alpha can potentially be used to suppress neuroinflammation in vivo.

Enhanced induction of HIV-specific cytotoxic T lymphocytes by dendritic cell-targeted delivery of SOCS-1 siRNA.

Dendritic cells (DCs) are potent antigen-presenting cells that play a critical role in the activation of T cells. RNA interference (RNAi)-mediated silencing of negative immunoregulatory molecules expressed by DCs may provide a strategy to enhance the potency of DC-based vaccines and immunotherapy. Ablation of suppressor of cytokine signaling-1 (SOCS-1) in antigen-presenting cells has been shown to enhance cellular immune response in mice. Here, we used a previously reported DC-targeting approach to deliver small interfering RNA (siRNA) against SOCS-1 to human myeloid-derived DCs (MDDCs). SOCS1-silencing in MDDCs resulted in enhanced cytokine responses to lipopolysaccharide (LPS) and a strong mixed-lymphocyte reaction. Moreover, only DCs treated with SOCS-1 siRNA, and not controls, elicited strong primary in vitro responses to well-characterized HLA-A*0201-restricted Melan-A/MART-1 and human immunodeficiency virus (HIV) Gag epitopes in naive CD8(+) T cells from healthy donors. Finally, stimulation of CD8(+) T cells from HIV-seropositive subjects with SOCS1-silenced DCs resulted in an augmented polyfunctional cytotoxic T-lymphocyte (CTL) response, suggesting that SOCS-1 silencing can restore functionally compromised T cells in HIV infection. Collectively, these results demonstrate the feasibility of DC3-9dR-mediated manipulation of DC function to enhance DC immunogenicity for potential vaccine or immunotherapeutic applications.

RNAi-mediated CCR5 silencing by LFA-1-targeted nanoparticles prevents HIV infection in BLT mice.

RNA interference (RNAi)-mediated knockdown of gene expression offers a novel treatment strategy for human immunodeficiency virus (HIV) infection. However, the major hurdle for clinical use is a practical strategy for small interfering RNA (siRNA) delivery to the multiple immune cell types important in viral pathogenesis. We have developed a novel immunoliposome method targeting the lymphocyte function-associated antigen-1 (LFA-1) integrin expressed on all leukocytes and evaluated it for systemic delivery of siRNA in a humanized mouse model. We show that in vivo administration of the LFA-1 integrin-targeted and stabilized nanoparticles (LFA-1 I-tsNPs) results in selective uptake of siRNA by T cells and macrophages, the prime targets of HIV. Further, in vivo administration of anti-CCR5 siRNA/LFA-1 I-tsNPs resulted in leukocyte-specific gene silencing that was sustained for 10 days. Finally, humanized mice challenged with HIV after anti-CCR5 siRNA treatment showed enhanced resistance to infection as assessed by the reduction in plasma viral load and disease-associated CD4 T-cell loss. This study demonstrates the potential in vivo applicability of LFA-1-directed siRNA delivery as anti-HIV prophylaxis.

Glycosylphosphatidylinositol-specific phospholipase C regulates transferrin endocytosis in the African trypanosome.

GPI-PLC (glycosylphosphatidylinositol-specific phospholipase C) is expressed in bloodstream-form Trypanosoma brucei, a protozoan that causes human African trypanosomiasis. Loss of genes encoding GPI-PLC reduces the virulence of a pleomorphic strain of the parasite, for reasons that are not clear. In the present paper, we report that GPI-PLC stimulates endocytosis of transferrin by 300-500%. Surprisingly, GPI-PLC is not detected at endosomes, suggesting that the enzyme does not interact directly with the endosomal machinery. We therefore hypothesized that a diffusible product of the GPI-PLC enzyme reaction [possibly DAG (diacylglycerol)] mediated the biological effects of the protein. Two sets of data support this assertion. First, a catalytically inactive Q81L mutant of GPI-PLC, expressed in a GPI-PLC-null background, had no effect on endocytosis, indicating that enzyme activity is essential for the protein to stimulate endocytosis. Secondly, the exogenous DAGs OAG (1-oleyl-2-acetyl-sn-glycerol) and DMG (dimyristoylglycerol) independently stimulated endocytosis of transferrin. Furthermore, the DAG mimic PMA, a phorbol ester, also activated endocytosis in T. brucei. DAG-stimulated endocytosis is a novel pathway in the trypanosome. We surmise that (i) GPI-PLC regulates transferrin endocytosis in T. brucei, (ii) GPI-PLC is a signalling enzyme, and (iii) DAG is a second messenger for GPI-PLC. We propose that regulation of endocytosis is a physiological function of GPI-PLC in bloodstream T. brucei.

Trypanosoma brucei: reduction of GPI-phospholipase C protein during differentiation is dependent on replication of newly transformed cells.

The protozoan parasite Trypanosoma brucei lives in the bloodstream of vertebrates or in a tsetse fly. Expression of a GPI-phospholipase C polypeptide (GPI-PLCp) in the parasite is restricted to the bloodstream form. Events controlling the amount of GPI-PLCp expressed during differentiation are not completely understood. Our metabolic "pulse-chase" analysis reveals that GPI-PLCp is stable in bloodstream form. However, during differentiation of bloodstream to insect stage (procyclic) T. brucei, translation GPI-PLC mRNA ceases within 8h of initiating transformation. GPI-PLCp is not lost precipitously from newly transformed procyclic trypanosomes. Nascent procyclics contain 400-fold more GPI-PLCp than established insect stage T. brucei. Reduction of GPI-PLCp in early-stage procyclics is linked to parasite replication. Sixteen cell divisions are required to reduce the amount of GPI-PLCp in newly differentiated procyclics to levels present in the established procyclic. GPI-PLCp is retained in strains of T. brucei that fail to replicate after differentiation of the bloodstream to the procyclic form. Thus, at least two factors control levels of GPI-PLCp during differentiation of bloodstream T. brucei; (i) repression of GPI-PLC mRNA translation, and (ii) sustained replication of newly transformed procyclic T. brucei. These studies illustrate the importance of repeated cell divisions in controlling the steady-state amount of GPI-PLCp during differentiation of the African trypanosome.

Safety and immunogenicity of mammalian cell derived and Modified Vaccinia Ankara vectored African swine fever subunit antigens in swine.

A reverse vaccinology system, Vaxign, was used to identify and select a subset of five African Swine Fever (ASF) antigens that were successfully purified from human embryonic kidney 293 (HEK) cells and produced in Modified vaccinia virus Ankara (MVA) viral vectors. Three HEK-purified antigens [B646L (p72), E183L (p54), and O61R (p12)], and three MVA-vectored antigens [B646L, EP153R, and EP402R (CD2v)] were evaluated using a prime-boost immunization regimen swine safety and immunogenicity study. Antibody responses were detected in pigs following prime-boost immunization four weeks apart with the HEK-293-purified p72, p54, and p12 antigens. Notably, sera from the vaccinees were positive by immunofluorescence on ASFV (Georgia 2007/1)-infected primary macrophages. Although MVA-vectored p72, CD2v, and EP153R failed to induce antibody responses, interferon-gamma (IFN-γ+) spot forming cell responses against all three antigens were detected one week post-boost. The highest IFN-γ+ spot forming cell responses were detected against p72 in pigs primed with MVA-p72 and boosted with the recombinant p72. Antigen-specific (p12, p72, CD2v, and EP153R) T-cell proliferative responses were also detected post-boost. Collectively, these results are the first demonstration that ASFV subunit antigens purified from mammalian cells or expressed in MVA vectors are safe and can induce ASFV-specific antibody and T-cell responses following a prime-boost immunization regimen in swine.

Regulated cleavage of intracellular glycosylphosphatidylinositol in a trypanosome. Peroxisome-to-endoplasmic reticulum translocation of a phospholipase C.

Cell exposure to hypo-osmolarity and alkalinity triggers a spectrum of responses including activation of phospholipases. Glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) is expressed in Trypanosoma brucei, a protozoan parasite that causes human African trypanosomiasis. We examined possible contributions of GPI-PLC to the response of T. brucei to hypo-osmotic or mildly alkaline conditions. GPIs were detected at the endoplasmic reticulum (ER). They were cleaved after exposure of T. brucei to hypo-osmolarity or mild alkalinity, which also, strikingly, caused translocation of GPI-PLC from glycosomes (peroxisomes) to the ER. A catalytically inactive Gln81Leu mutant of GPI-PLC failed to cleave GPIs despite being transported from glycosomes to the ER after hypo-osmotic or mild alkaline treatment of the parasites. In contrast, a Cys347Ser mutant of the enzyme could not exit glycosomes after treatment of cells expressing the protein with mild base or hypo-osmotic buffer. We conclude that: (a) GPI-PLC contributes to loss of GPIs in T. brucei treated with hypo-osmotic or mildly alkaline buffer; (b) access of GPI-PLC to its substrate in vivo can be regulated post-translationally; (c) translocation of GPI-PLC from glycosomes to the ER is important for in vivo cleavage of GPIs; (d) Cys347 is part of a peptide motif required for post-translational targeting of GPI-PLC to the ER. Glycosome-to-ER movement of GPI-PLC reveals a novel pathway for intracellular protein traffic. The physiological significance of GPI digestion in cells exposed to mildly alkalinity or hypo-osmolarity is discussed.

Antibody-mediated delivery of siRNAs for anti-HIV therapy.

RNA interference (RNAi) is a potent and specific gene silencing mechanism that utilizes small -double-stranded RNA intermediates (small interfering RNAs or siRNAs) to target homologous mRNA sequences for degradation. The therapeutic potential of RNAi for HIV infection has been demonstrated in many studies. However, successful clinical application of RNAi is contingent on developing practical strategies to deliver siRNA to the desired target cells and tissues. Recently, there has been significant progress towards developing reagents that selectively deliver exogenous siRNA to immune cells that are targeted by HIV or involved in viral pathogenesis, such as T cells, macrophages, and dendritic cells. Here, we describe details of two antibody-based strategies for systemic delivery of siRNA either specifically to T cells via the CD7 receptor or to multiple immune cell types via LFA-1, present on all leukocytes.

Diacylglycerol-stimulated endocytosis of transferrin in trypanosomatids is dependent on tyrosine kinase activity.

Small molecule regulation of cell function is an understudied area of trypanosomatid biology. In Trypanosoma brucei diacylglycerol (DAG) stimulates endocytosis of transferrin (Tf). However, it is not known whether other trypanosomatidae respond similarly to the lipid. Further, the biochemical pathways involved in DAG signaling to the endocytic system in T. brucei are unknown, as the parasite genome does not encode canonical DAG receptors (e.g. C1-domains). We established that DAG stimulates endocytosis of Tf in Leishmania major, and we evaluated possible effector enzymes in the pathway with multiple approaches. First, a heterologously expressed glycosylphosphatidylinositol phospholipase C (GPI-PLC) activated endocytosis of Tf 300% in L. major. Second, exogenous phorbol ester and DAGs promoted Tf endocytosis in L. major. In search of possible effectors of DAG signaling, we discovered a novel C1-like domain (i.e. C1_5) in trypanosomatids, and we identified protein Tyr kinases (PTKs) linked with C1_5 domains in T. brucei, T. cruzi, and L. major. Consequently, we hypothesized that trypanosome PTKs might be effector enzymes for DAG signaling. General uptake of Tf was reduced by inhibitors of either Ser/Thr or Tyr kinases. However, DAG-stimulated endocytosis of Tf was blocked only by an inhibitor of PTKs, in both T. brucei and L. major. We conclude that (i) DAG activates Tf endocytosis in L. major, and that (ii) PTKs are effectors of DAG-stimulated endocytosis of Tf in trypanosomatids. DAG-stimulated endocytosis of Tf may be a T. brucei adaptation to compete effectively with host cells for vertebrate Tf in blood, since DAG does not enhance endocytosis of Tf in human cells.

RNA interference-based therapeutics for human immunodeficiency virus HIV-1 treatment: synthetic siRNA or vector-based shRNA?

IMPORTANCE OF THE FIELD: Despite the clinical benefits of highly active antiretroviral therapy (HAART), the prospect of life-long antiretroviral treatment poses significant problems, which has spurred interest in developing new drugs and strategies to treat HIV infection and eliminate persistent viral reservoirs. RNAi has emerged as a therapeutic possibility for HIV. AREAS COVERED IN THIS REVIEW: We discuss progress in overcoming hurdles to translating transient and stable RNAi enabling technologies to clinical application for HIV; covering the past 2 - 3 years. WHAT THE READER WILL GAIN: HIV inhibition can be achieved by transfection of chemically or enzymatically synthesized siRNAs or by DNA-based vector systems expressing short hairpin RNAs (shRNAs) that are processed intracellularly into siRNA. We compare these approaches, focusing on technical and safety issues that will guide the choice of strategy for clinical use. TAKE HOME MESSAGE: Introduction of synthetic siRNA into cells or its stable endogenous production using vector-driven shRNA have been shown to suppress HIV replication in vitro and, in some instances, in vivo. Each method has advantages and limitations in terms of ease of delivery, duration of silencing, emergence of escape mutants and potential toxicity. Both appear to have potential as future therapeutics for HIV, once the technical and safety issues of each approach are overcome.

Lentiviral delivery of short hairpin RNAs.

In less than a decade after discovery, RNA interference-mediated gene silencing is already being tested as potential therapy in clinical trials for a number of diseases. Lentiviral vectors provide a means to express short hairpin RNA (shRNA) to induce stable and long-term gene silencing in both dividing and non-dividing cells and thus, are being intensively investigated for this purpose. However, induction of long-term shRNA expression can also cause toxicities by inducing off-target effects and interference with the endogenous micro-RNA (miRNA) pathway that regulates cellular gene expression. Recently, several advances have been made in the shRNA vector design to mimic cellular miRNA processing and to express multiplex siRNAs in a tightly regulated and reversible manner to overcome toxicities. In this review we describe some of these advances, focusing on the progress made in the development of lentiviral shRNA delivery strategies to combat viral infections.