Biological evaluation of indolactams for in vitro bryostatin 1-like activity.

Small molecule activators of protein kinase C (PKC) have traditionally been classified as either tumor promoters or suppressors. Although bryostatin 1 has well established anti-cancer activity, most natural products that target the PKC regulator domain exhibit tumor promotion properties. In this study, we examine a focused library of indolactam analogues in cell-based assays to establish the structural features of the scaffold that enhance bryostatin 1-like activity. These systematic biological assessments identified specific indole substitution patterns that impart diminished tumor promotion behavior in vitro for indolactam analogues, while still maintaining nanomolar potency for PKC.

The ε-Isozyme of Protein Kinase C (PKCε) Is Impaired in ALS Motor Cortex and Its Pulse Activation by Bryostatin-1 Produces Long Term Survival in Degenerating SOD1-G93A Motor Neuron-like Cells.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and ultimately fatal neurodegenerative disease, characterized by a progressive depletion of upper and lower motor neurons (MNs) in the brain and spinal cord. The aberrant regulation of several PKC-mediated signal transduction pathways in ALS has been characterized so far, describing either impaired expression or altered activity of single PKC isozymes (α, ß, ζ and δ). Here, we detailed the distribution and cellular localization of the ε-isozyme of protein kinase C (PKCε) in human postmortem motor cortex specimens and reported a significant decrease in both PKCε mRNA (PRKCE) and protein immunoreactivity in a subset of sporadic ALS patients. We furthermore investigated the steady-state levels of both pan and phosphorylated PKCε in doxycycline-activated NSC-34 cell lines carrying the human wild-type (WT) or mutant G93A SOD1 and the biological long-term effect of its transient agonism by Bryostatin-1. The G93A-SOD1 cells showed a significant reduction of the phosphoPKCε/panPKCε ratio compared to the WT. Moreover, a brief pulse activation of PKCε by Bryostatin-1 produced long-term survival in activated G93A-SOD1 degenerating cells in two different cell death paradigms (serum starvation and chemokines-induced toxicity). Altogether, the data support the implication of PKCε in ALS pathophysiology and suggests its pharmacological modulation as a potential neuroprotective strategy, at least in a subgroup of sporadic ALS patients.

Bryostatin-1 attenuates intestinal ischemia/reperfusion-induced intestinal barrier dysfunction, inflammation, and oxidative stress via activation of Nrf2/HO-1 signaling.

Bryostatin-1 (Bryo-1) exerts antioxidative stress effects in multiple diseases, and we confirmed that it improves intestinal barrier dysfunction in experimental colitis. Nevertheless, there are few reports on its action on intestinal ischemia/reperfusion (I/R). In this study, we mainly explored the effect of Bryo-1 on intestinal I/R injury and determined the mechanism. C57BL/6J mice underwent temporary superior mesenteric artery (SMA) obturation to induce I/R, on the contrary, Caco-2 cells suffered to oxygen and glucose deprivation/reperfusion (OGD/R) to establish the in vitro model. RAW264.7 cells were stimulated with LPS to induce macrophage inflammation. The drug gradient experiment was used to demonstrate in vivo and in vitro models. Bryo-1 ameliorated the intestinal I/R-induced injury of multiple organs and epithelial cells. It also alleviated intestinal I/R-induced barrier disruption of intestines according to the histology, intestinal permeability, intestinal bacterial translocation rates, and tight junction protein expression results. Bryo-1 significantly inhibited oxidative stress damages and inflammation, which may contribute to the restoration of intestinal barrier function. Further, Bryo-1 significantly activated Nrf2/HO-1 signaling in vivo. However, the deletion of Nrf2 in Caco-2 and RAW264.7 cells attenuated the protective functions of Bryo-1 and significantly abolished the anti-inflammatory effect of Bryo-1 on LPS-induced macrophage inflammation. Bryo-1 protects intestines against I/R-induced injury. It is associated with intestinal barrier protection, as well as inhibition of inflammation and oxidative stress partly through Nrf2/HO-1 signaling.

Effects of exercise and bryostatin-1 on functional recovery and posttranslational modification in the perilesional cortex after cerebral infarction.

Strokes can cause a variety of sequelae, such as paralysis, particularly in the early stages after stroke onset. Rehabilitation therapy atthis time often provides some degree of paralysis recovery. Neuroplasticity in the peri-infarcted cerebral cortex induced by exercise training may contribute to recovery of paralysis after cerebral infarction. However, the molecular mechanism of this process remains unclear. This study focused on brain protein kinase C (PKC), which is speculated to be involved in neuroplasticity. We evaluated the functional recovery of cerebral infarction model rats, by using rotarod test after running wheel training and with/without administration of bryostatin, a PKC activator. In addition, the expression of phosphorylated and unphosphorylated PKC subtypes, glycogen synthase kinase 3ß (GSK3ß), and collapsin response-mediator proteins 2 (CRMP2) were analyzed by Western blotting. In the rotarod test, bryostatin administration alone had no effect on gait duration, but the combination of training and this drug significantly prolonged gait duration compared with training alone. In protein expression analysis, the combination of training and bryostatin significantly increased phosphorylation of PKCα and PKCε isoforms, increased phosphorylation of GSK3ß, which acts downstream of PKC, and decreased phosphorylation of CRMP2. The effect of bryostatin in combination with training appears to be mediated via PKC phosphorylation, with effects on functional recovery occurring through the downstream regulation of GSK3ß and CRMP2 phosphorylation.

Secreted factors induced by PKC modulators do not indirectly cause HIV latency reversal.

HIV can establish a long-lived latent infection in cells harboring integrated non-expressing proviruses. Latency reversing agents (LRAs), including protein kinase C (PKC) modulators, can induce expression of latent HIV, thereby reducing the latent reservoir in animal models. However, PKC modulators such as bryostatin-1 also cause cytokine upregulation in peripheral blood mononuclear cells (PBMCs), including cytokines that might independently reverse HIV latency. To determine whether cytokines induced by PKC modulators contribute to latency reversal, primary human PBMCs were treated with bryostatin-1 or the bryostatin analog SUW133, a superior LRA, and supernatant was collected. As anticipated, LRA-treated cell supernatant contained increased levels of cytokines compared to untreated cell supernatant. However, exposure of latently-infected cells with this supernatant did not result in latency reactivation. These results indicate that PKC modulators do not have significant indirect effects on HIV latency reversal in vitro and thus are targeted in their latency reversing ability.

Computational insight into the binding of bryostatin 1 with ferritin: implication of natural compounds in Alzheimer's disease therapeutics.

Neuronal damage in iron-sensitive brain regions occurs as a result of iron dyshomeostasis. Increased iron levels and iron-related pathogenic triggers are associated with neurodegenerative diseases, including Alzheimer's disease (AD). Ferritin is a key player involved in iron homeostasis. Major pathological hallmarks of AD are amyloid plaques, neurofibrillary tangles (NFTs) and synaptic loss that lead to cognitive dysfunction and memory loss. Natural compounds persist in being the most excellent molecules in the area of drug discovery because of their different range of therapeutic applications. Bryostatins are naturally occurring macrocyclic lactones that can be implicated in AD therapeutics. Among them, Bryostatin 1 regulates protein kinase C, a crucial player in AD pathophysiology, thus highlighting the importance of bryostatin 1 in AD management. Thus, this study explores the binding mechanism of Bryotstain 1 with ferritin. In this work, the molecular docking calculations revealed that bryostatin 1 has an appreciable binding potential towards ferritin by forming stable hydrogen bonds (H-bonds). Molecular dynamics simulation studies deciphered the binding mechanism and conformational dynamics of ferrritin-bryostatin 1 system. The analyses of root mean square deviation, root mean square fluctuations, Rg, solvent accessible surface area, H-bonds and principal component analysis revealed the stability of the ferritin-bryostatin 1 docked complex throughout the trajectory of 100 ns. Moreover, the free energy landscape analysis advocated that the ferritin-bryostatin 1 complex stabilized to the global minimum. Altogether, the present work delineated the binding of bryostatin 1 with ferritin that can be implicated in the management of AD.Communicated by Ramaswamy H. Sarma.

Impact of latency-reversing agents on human macrophage physiology.

INTRODUCTION: HIV-1 eradication is hindered by the presence of inducible long-lived reservoirs of latently infected cells which rapidly disseminate viral particles upon treatment interruption. Eliminating these reservoirs by the so-called shock and kill strategy represents a crucial concept toward an HIV-1 cure. Several molecules called latency-reversing agents (LRAs) are under intensive investigations to reactivate virus gene expression. These studies are mainly conducted on CD4+ T cells where LRAs are well tolerated and did not induce global cellular activation. However, despite their broad spectrum, the putative impact of LRAs on other cellular reservoirs such as macrophages is still ill-defined. METHODS: We investigated the impact of the protein kinase C (PKC) activator bryostatin-1, bromodomain inhibitor JQ1 and histone deacetylase inhibitor romidepsin used either alone or in combination on human primary monocyte-derived macrophages (MDMs). RESULTS: We demonstrate that bryostatin-1, JQ1, and romidepsin or their combinations are not toxic at nanomolar concentrations but induce metabolic and morphologic alterations of MDMs. Bryostatin-1 triggered the secretion of pro-inflammatory cytokines, while JQ-1 decreased it. Phagocytosis and endocytosis were modestly impaired upon bryostatin-1 treatment whereas efferocytosis was markedly downregulated by romidepsin. Despite its pro-inflammatory profile, bryostatin-1 did not induce classically activated macrophage markers. Finally, we reveal that conditioned medium from bryostatin-1-treated macrophages did not potentiate its reactivation feature. CONCLUSIONS: Our study reveals that LRAs can diversely impact basic physiologic features of human primary macrophages and could potentially decrease reactivation of nearby CD4+ T cells latently infected with HIV-1. Our observations further stress the need to include different cell populations when assessing HIV-1 cure strategies.

Off-Target Effect of Activation of NF-κB by HIV Latency Reversal Agents on Transposable Elements Expression.

Many drugs have been evaluated to reactivate HIV-1 from cellular reservoirs, but the off-target effects of these latency reversal agents (LRA) remain poorly defined. Transposable elements (TEs) are reactivated during HIV-1 infection, but studies of potential off-target drug effects on TE expression have been limited. We analyzed the differential expression of TEs induced by canonical and non-canonical NF-κB signaling. We evaluated the effect of PKC agonists (Bryostatin and Ingenol B) on the expression of TEs in memory CD4+ T cells. Ingenol B induced 38 differentially expressed TEs (17 HERV (45%) and 21 L1 (55%)). Interestingly, TE expression in effector memory CD4+ T cells was more affected by Bryostatin compared to other memory T-cell subsets, with 121 (107 upregulated and 14 downregulated) differentially expressed (DE) TEs. Of these, 31% (n = 37) were HERVs, and 69% (n = 84) were LINE-1 (L1). AZD5582 induced 753 DE TEs (406 HERV (54%) and 347 L1 (46%)). Together, our findings show that canonical and non-canonical NF-κB signaling activation leads to retroelement expressions as an off-target effect. Furthermore, our data highlights the importance of exploring the interaction between LRAs and the expression of retroelements in the context of HIV-1 eradication strategies.

Bryostatin-1 Attenuates Ischemia-Elicited Neutrophil Transmigration and Ameliorates Graft Injury after Kidney Transplantation.

Ischemia reperfusion injury (IRI) is a form of sterile inflammation whose severity determines short- and long-term graft fates in kidney transplantation. Neutrophils are now recognized as a key cell type mediating early graft injury, which activates further innate immune responses and intensifies acquired immunity and alloimmunity. Since the macrolide Bryostatin-1 has been shown to block neutrophil transmigration, we aimed to determine whether these findings could be translated to the field of kidney transplantation. To study the effects of Bryostatin-1 on ischemia-elicited neutrophil transmigration, an in vitro model of hypoxia and normoxia was equipped with human endothelial cells and neutrophils. To translate these findings, a porcine renal autotransplantation model with eight hours of reperfusion was used to study neutrophil infiltration in vivo. Graft-specific treatment using Bryostatin-1 (100 nM) was applied during static cold storage. Bryostatin-1 dose-dependently blocked neutrophil activation and transmigration over ischemically challenged endothelial cell monolayers. When applied to porcine renal autografts, Bryostatin-1 reduced neutrophil graft infiltration, attenuated histological and ultrastructural damage, and improved renal function. Our novel findings demonstrate that Bryostatin-1 is a promising pharmacological candidate for graft-specific treatment in kidney transplantation, as it provides protection by blocking neutrophil infiltration and attenuating functional graft injury.

Bryostatin Activates CAR T-Cell Antigen-Non-Specific Killing (CTAK), and CAR-T NK-Like Killing for Pre-B ALL, While Blocking Cytolysis of a Burkitt Lymphoma Cell Line.

The advent of CAR-T cell therapy has changed the face of clinical care for relapsed and refractory pre-B-acute lymphocytic leukemia (B-ALL) and lymphoma. Although curative responses are reported, long-term cures remain below 50%. Different CAR T-cell leukemia targets appear to have different mechanisms of CAR-T escape. For CD22, therapeutic evasion is linked to down-modulation of the number CD22 proteins expressed on the extracellular aspect of the leukemia cell plasma membrane. Recently, pharmacologic agents known to induce cellular differentiation or epigenetic modification of leukemia have been shown to impact CD22 and CD19 expression levels on B-ALL, and thereby increase sensitivity to CAR-T mediated cytolysis. We explored the impact of epigenetic modifiers and differentiation agents on leukemia cell lines of B cell origin, as well as normal B cells. We confirmed the activity of bryostatin to increase CD22 expression on model cell lines. However, bryostatin does not change CD22 levels on normal B cells. Furthermore, bryostatin inhibited CAR-T mediated cytolysis of the Raji Burkitt lymphoma cell line. Bryostatin increased the cytolysis by CD22 CAR-T for B-ALL cell lines by at least three mechanisms: 1) the previously reported increase in CD22 target cell numbers on the cell surface, 2) the induction of NK ligands, and 3) the induction of ligands that sensitize leukemia cells to activated T cell antigen-non-specific killing. The opposite effect was seen for Burkitt lymphoma, which arises from a more mature B cell lineage. These findings should caution investigators against a universal application of agents shown to increase killing of leukemia target cells by CAR-T in a specific disease class, and highlights that activation of non-CAR-mediated killing by activated T cells may play a significant role in the control of disease. We have termed the killing of leukemia targets, by a set of cell-surface receptors that does not overlap with NK-like killing "CTAK," CAR-T Cell antigen-non-specific killing.

Bryostatin Placebo-Controlled Trials Indicate Cognitive Restoration Above Baseline for Advanced Alzheimer's Disease in the Absence of Memantine1.

BACKGROUND: In pre-clinical studies of Alzheimer's disease (AD) transgenic mice, bryostatin restored synaptic connections, prevented neuronal death, reduced amyloid plaques, and reduced neurofibrillary tangles. OBJECTIVE: Within pre-specified cohorts of advanced AD patients in two double-blind placebo-controlled bryostatin Phase II trials, to conduct exploratory statistical analyses of patients with identical conditions of enrollment and treatment. METHODS: Severe Impairment Battery (SIB) scores above baseline at 5, 9, and 13 weeks were analyzed initially in the complete cases, with multiple imputation methods based on an iterative Markov chain Monte Carlo algorithm used for missing SIB scores. To mitigate confounding by a chance imbalance of 4.9 SIB baseline scores (Study #203), each patient was used as their own control with differences in 13-week SIB from baseline in single trial and pooled analyses to measure benefit at 13 weeks using general estimating equations (GEE) modeling. RESULTS: Patients treated with bryostatin pre-specified at Mini-Mental State Examination scores 10-14, without memantine, showed baseline balance, complete safety, and SIB improvements at 13 weeks with multiple imputation analysis: Study #203 = 4.1 SIB points above baseline (p = 0.005), and Study #202 = 4.2 SIB points above baseline (p = 0.016). An increased power (N = 95) "pooled analysis" showed an increased SIB over time and a higher mean SIB at 13 weeks in the bryostatin treatment group (p < 0.001) but not significant (NS) for the placebo patients. CONCLUSION: Pre-specified exploratory analyses for the individual trials and the pooled trials confirmed significant bryostatin-induced improvement over baseline (treatment p < 0.001, placebo NS).

Engineered extracellular vesicles encapsulated Bryostatin-1 as therapy for neuroinflammation.

Targeted and effective drug delivery to central nervous system (CNS) lesions is a major challenge in the treatment of multiple sclerosis (MS). Extracellular vesicles (EVs) have great promise as a drug delivery nanosystem given their unique characteristics, including a strong cargo-loading capacity, low immunogenicity, high biocompatibility, inherent stability, high delivery efficiency, ease of manipulation, and blood-brain barrier (BBB) penetration. Clinical applications are, however, limited by their insufficient targeting capability and "dilution effects" upon systemic administration. Neural stem cells (NSCs) provide an abundant source of EVs because of their remarkable capacity for self-renewal. Here, we developed a novel therapeutic strategy for local delivery and treatment using EVPs, which are derived from NSCs with the expression of the CNS lesion targeting ligand-PDGFRα. Furthermore, we used EVPs as a targeting carrier for encapsulating Bryostatin-1 (Bryo-1), a natural compound with remarkable anti-inflammation ability. Our data showed that Bryo-1 delivered by EVPs was more stable and concentrated in the CNS than native Bryo-1. Systemic injection of a low dosage (1 × 108 particles) of EVPs + Bryo-1, versus only EVPs or Bryo-1 administration, significantly ameliorated clinical disease development, decreased the infiltration of pro-inflammatory cells, blocked myelin loss and astrogliosis, protected BBB integrity, and altered microglia pro-inflammatory phenotype in the CNS of EAE mice. Taken as a whole, our study showed that engineered EVs have a CNS targeting capacity, and it provides potentially powerful therapeutic effects for the treatment of various neuroinflammatory diseases.

Encapsulation of bryostatin-1 by targeted exosomes enhances remyelination and neuroprotection effects in the cuprizone-induced demyelinating animal model of multiple sclerosis.

Demyelination is a critical neurological disease, and there is still a lack of effective treatment methods. In the past two decades, stem cells have emerged as a novel therapeutic effector for neural regeneration. However, owing to the existence of the blood-brain barrier (BBB) and the complex microenvironment, targeted therapy still faces multiple challenges. Targeted exosome carriers for drug delivery may be considered a promising therapeutic method. Exosomes were isolated from mice neural stem cells. To develop targeting exosomes, we generated a lentivirus armed PDGFRα ligand that could anchor the membrane. Exosome targeting tests were carried out in vitro and in vivo. The modified exosomes showed an apparent ability to target OPCs in the lesion area. Next, the exosomes were loaded with Bryostatin-1 (Bryo), and the cuprizone-fed mice were administered with the targeting exosomes. The data show that Bryo exhibits a powerful therapeutic effect compared with Bryo alone after exosome encapsulation. Specifically, this novel exosome-based targeting delivery of Bryo significantly improves the protection ability of the myelin sheath and promotes remyelination. Moreover, it blocks astrogliosis and axon damage, and also has an inhibitory effect on pro-inflammatory microglia. The results of this investigation provide a straightforward strategy to produce targeting exosomes and indicate a potential therapeutic approach for demyelinating disease.

Bryostatin-1 Decreases HIV-1 Infection and Viral Production in Human Primary Macrophages.

While combination antiretroviral therapy maintains undetectable viremia in people living with HIV (PLWH), a lifelong treatment is necessary to prevent viremic rebound after therapy cessation. This rebound seemed mainly caused by long-lived HIV-1 latently infected cells reverting to a viral productive status. Reversing latency and elimination of these cells by the so-called shock-and-kill strategy is one of the main investigated leads to achieve an HIV-1 cure. Small molecules referred to as latency reversal agents (LRAs) proved to efficiently reactivate latent CD4+ T cells. However, the LRA impact on de novo infection or HIV-1 production in productively infected macrophages remains elusive. Nontoxic doses of bryostatin-1, JQ1, and romidepsin were investigated in human monocyte-derived macrophages (MDMs). Treatment with bryostatin-1 or romidepsin resulted in a downregulation of CD4 and CCR5 receptors, respectively, accompanied by a reduction of R5 tropic virus infection. HIV-1 replication was mainly regulated by receptor modulation for bryostatin-1, while romidepsin effects rely on upregulation of SAMHD1 activity. LRA stimulation of chronically infected cells did not enhance HIV-1 production or gene expression. Surprisingly, bryostatin-1 caused a major decrease in viral production. This effect was not viral strain specific but appears to occur only in myeloid cells. Bryostatin-1 treatment of infected MDMs led to decreased amounts of capsid and matrix mature proteins with little to no modulation of precursors. Our observations revealed that bryostatin-1-treated myeloid and CD4+ T cells respond differently upon HIV-1 infection. Therefore, additional studies are warranted to more fully assess the efficiency of HIV-1 eradicating strategies. IMPORTANCE HIV-1 persists in a cellular latent form despite therapy that quickly propagates infection upon treatment interruption. Reversing latency would contribute to eradicate these cells, closing the gap to a cure. Macrophages are an acknowledged HIV-1 reservoir during therapy and are suspected to harbor latency establishment in vivo. However, the impact of latency reversal agents (LRAs) on HIV-1 infection and viral production in human macrophages is poorly known but nonetheless crucial to probe the safety of this strategy. In this in vitro study, we discovered encouraging antireplicative features of distinct LRAs in human macrophages. We also described a new viral production inhibition mechanism by protein kinase C agonists that is specific to myeloid cells. This study provides new insights into HIV-1 propagation restriction potentials by LRAs in human macrophages and underline the importance of assessing latency reversal strategy on all HIV-1-targeted cells.

Spatiotemporal distribution of PKCα, Cdc42, and Rac1 before directed cell migration.

Cdc42 is a key factor in directed cell migration and accumulates at the leading edge of migrating cells. However, what kind of proteins control Cdc42 and when is unclear. After mechanical wounding, protein kinase C α (PKCα), a conventional PKC isozyme, begins to accumulate at the edges of cells adjacent to the wounded cells (WCs). In this study, we hypothesized that PKCα may be implicated in directed cell migration at an early stage before Cdc42 controls the migration. We focused on the spatiotemporal distribution of PKCα, Cdc42, and Rac1 before cell migration. After wounding, at the edges of cells adjacent to the WCs, PKCα accumulation, Cdc42 accumulation, Rac1 accumulation, and filopodia formation occurred in that order. The PKCα inhibitor suppressed Cdc42 accumulation at the cell edges. These results suggest that inhibition of PKCα activity inhibits cell migration. In addition, it is not Cdc42 but PKCα that may decide the direction of cell migration.

The Novel PKC Activator 10-Methyl-Aplog-1 Combined with JQ1 Induced Strong and Synergistic HIV Reactivation with Tolerable Global T Cell Activation.

The presence of latent human immunodeficiency virus (HIV) reservoirs is a major obstacle to a cure. The "shock and kill" therapy is based on the concept that latent reservoirs in HIV carriers with antiretroviral therapy are reactivated by latency-reversing agents (LRAs), followed by elimination due to HIV-associated cell death or killing by virus-specific cytotoxic T lymphocytes. Protein kinase C (PKC) activators are considered robust LRAs as they efficiently reactivate latently infected HIV. However, various adverse events hamper the intervention trial of PKC activators as LRAs. We found in this study that a novel PKC activator, 10-Methyl-aplog-1 (10MA-1), combined with an inhibitor of bromodomain and extra-terminal domain motifs, JQ1, strongly and synergistically reactivated latently infected HIV. Notably, higher concentrations of 10MA-1 alone induced the predominant side effect, i.e., global T cell activation as defined by CD25 expression and pro-inflammatory cytokine production in primary CD4+ T lymphocytes; however, JQ1 efficiently suppressed the 10MA-1-induced side effect in a dose-dependent manner. Considering the reasonable accessibility and availability of 10MA-1 since the chemical synthesis of 10MA-1 requires fewer processes than that of bryostatin 1 or prostratin, our results suggest that the combination of 10MA-1 with JQ1 may be a promising pair of LRAs for the clinical application of the "shock and kill" therapy.

Development of a Novel In Vitro Primary Human Monocyte-Derived Macrophage Model To Study Reactivation of HIV-1 Transcription.

Latent HIV reservoirs persist in people living with HIV despite effective antiretroviral therapy and contribute to rebound viremia upon treatment interruption. Macrophages are an important reservoir cell type, but analysis of agents that modulate latency in macrophages is limited by lack of appropriate in vitro models. We therefore generated an experimental system to investigate this by purifying nonproductively infected human monocyte-derived macrophages (MDM) following in vitro infection with an M-tropic enhanced green fluorescent protein reporter HIV clone and quantified activation of HIV transcription using live-cell fluorescence microscopy. The proportion of HIV-infected MDM was quantified by qPCR detection of HIV DNA, and GFP expression was validated as a marker of productive HIV infection by colabeling of HIV Gag protein. HIV transcription spontaneously reactivated in latently infected MDM at a rate of 0.22% ± 0.04% cells per day (mean ± the standard error of the mean, n = 10 independent donors), producing infectious virions able to infect heterologous T cells in coculture experiments, and both T cells and TZM-bl cells in a cell-free infection system using MDM culture supernatants. Polarization to an M1 phenotype with gamma interferon plus tumor necrosis factor resulted in a 2.3-fold decrease in initial HIV infection of MDM (P < 0.001, n = 8) and a 1.4-fold decrease in spontaneous reactivation (P = 0.025, n = 6), whereas M2 polarization using interleukin-4 prior to infection led to a 1.6-fold decrease in HIV infectivity (P = 0.028, n = 8) but a 2.0-fold increase in the rate of HIV reactivation in latently infected MDM (P = 0.023, n = 6). The latency reversing agents bryostatin and vorinostat, but not panobinostat, significantly induced HIV reactivation in latently infected MDM (P = 0.031 and P = 0.038, respectively, n = 6). IMPORTANCE Agents which modulate latent HIV reservoirs in infected cells are of considerable interest to HIV cure strategies. The present study characterizes a robust, reproducible model enabling quantification of HIV reactivation in primary HIV-infected human MDM which is relatively insensitive to the monocyte donor source and hence suitable for evaluating latency modifiers in MDM. The rate of initial viral infection was greater than the rate of HIV reactivation, suggesting that different mechanisms regulate these processes. HIV reactivation was sensitive to macrophage polarization, suggesting that cellular and tissue environments influence HIV reactivation in different macrophage populations. Importantly, latently infected MDM showed different susceptibilities to certain latency-reversing agents known to be effective in T cells, indicating that dedicated strategies may be required to target latently infected macrophage populations in vivo.

Selective BCL-XL Antagonists Eliminate Infected Cells from a Primary-Cell Model of HIV Latency but Not from Ex Vivo Reservoirs.

HIV persists, despite immune responses and antiretroviral therapy, in viral reservoirs that seed rebound viremia if therapy is interrupted. Previously, we showed that the BCL-2 protein contributes to HIV persistence by conferring a survival advantage to reservoir-harboring cells. Here, we demonstrate that many of the BCL-2 family members are overexpressed in HIV-infected CD4+ T cells, indicating increased tension between proapoptotic and prosurvival family members-and suggesting that inhibition of prosurvival members may disproportionately affect the survival of HIV-infected cells. Based on these results, we chose to study BCL-XL due to its consistent overexpression and the availability of selective antagonists. Infection of primary CD4+ T cells with HIV resulted in increased BCL-XL protein expression, and treatment with two selective BCL-XL antagonists, A-1155463 and A-1551852, led to selective death of productively infected CD4+ T cells. In a primary cell model of latency, both BCL-XL antagonists drove reductions in HIV DNA and in infectious cell frequencies both alone and in combination with the latency reversing agent bryostatin-1, with little off-target cytotoxicity. However, these antagonists, with or without bryostatin-1 or in combination with the highly potent latency reversing agent combination phorbol myristate acetate (PMA) + ionomycin, failed to reduce total HIV DNA and infectious reservoirs in ex vivo CD4+ T cells from antiretroviral therapy (ART)-suppressed donors. Our results add to growing evidence that bona fide reservoir-harboring cells are resistant to multiple "kick and kill" modalities-relative to latency models. We also interpret our results as encouraging further exploration of BCL-XL antagonists for cure, where combination approaches, including with immune effectors, may unlock the ability to eliminate ex vivo reservoirs. IMPORTANCE Although antiretroviral therapy (ART) has transformed HIV infection into a manageable chronic condition, there is no safe or scalable cure. HIV persists in "reservoirs" of infected cells that reinitiate disease progression if ART is interrupted. Whereas most efforts to eliminate this reservoir have focused on exposing these cells to immune-mediated clearance by reversing viral latency, recent work shows that these cells also resist being killed. Here, we identify a "prosurvival" factor, BCL-XL, that is overexpressed in HIV-infected cells, and demonstrate selective toxicity to these cells by BCL-XL antagonists. These antagonists also reduced reservoirs in a primary-cell latency model but were insufficient to reduce "natural" reservoirs in ex vivo CD4+ T cells-adding to growing evidence that the latter are resilient in a way that is not reflected in models. We nonetheless suggest that the selective toxicity of BCL-XL antagonists to HIV-infected cells supports their prioritization for testing in combinations aimed at reducing ex vivo reservoirs.

Rebound increases in chemokines by CXCR2 antagonist in breast cancer can be prevented by PKCδ and PKCε activators.

Activation of CXCR2 by chemokines such as CXCL1 and CXCL2 increases aggressiveness of breast cancer, inducing chemoresistance, hence CXCR2 antagonists are in clinical trials. We previously reported that inhibition of CXCR2 increases MIP-2 (CXCL2), which may inhibit anti-tumoral effects of CXCR2 antagonists. This seems to be due to inhibition of protein kinase C (PKC) by CXCR2 antagonist since specific inhibitor of PKC also enhances MIP-2 secretion. We here examined whether CXCR2 inhibitor also increases KC (CXCL1) secretion, ligand for CXCR2 involved in metastasis and PKC activators can prevent increases in chemokine secretion. We used SB 225002, which is a specific CXCR2 antagonist. The effects of PKC activators that have documented anti-tumoral effects and activates multiple isozymes of PKC such as Ingenol-3-angelate (I3A) and bryostatin-1 were examined here. In addition, FR236924, PKCε selective and 7α-acetoxy-6ß-benzoyloxy-12-O-benzoylroyleanone (Roy-Bz), PKCδ selective activators were also tested. The effects of activators were determined using brain metastatic (4TBM) and heart metastatic (4THM) subset of 4T1 breast carcinoma cells because these aggressive carcinoma cells with cancer stem cell features secrete high levels of KC and MIP-2. Inhibition of CXCR-2 activity increased KC (CXCL1) secretion. PKC activators prevented SB225002-induced increases in KC and MIP-2 secretion. Different activators/modulators induce differential changes in basal and SB225002-induced chemokine secretion as well as cell proliferation and the activators that act on PKCδ and/or PKCε such as bryostatin 1, FR236924 and Roy-Bz are the most effective. These activators alone also decrease cell proliferation or chemokine secretion or both. Given the role of KC and MIP-2 in drug resistance including chemotherapeutics, activators of PKCε and PKCδ may prevent emerging of resistance to CXCR2 inhibitors as well as other chemotherapeutics.

Designed PKC-targeting bryostatin analogs modulate innate immunity and neuroinflammation.

Neuroinflammation characterizes multiple neurologic diseases, including primary inflammatory conditions such as multiple sclerosis and classical neurodegenerative diseases. Aberrant activation of the innate immune system contributes to disease progression, but drugs modulating innate immunity, particularly within the central nervous system (CNS), are lacking. The CNS-penetrant natural product bryostatin-1 attenuates neuroinflammation by targeting innate myeloid cells. Supplies of natural bryostatin-1 are limited, but a recent scalable good manufacturing practice (GMP) synthesis has enabled access to it and its analogs (bryologs), the latter providing a path to more efficacious, better tolerated, and more accessible agents. Here, we show that multiple synthetically accessible bryologs replicate the anti-inflammatory effects of bryostatin-1 on innate immune cells in vitro, and a lead bryolog attenuates neuroinflammation in vivo, actions mechanistically dependent on protein kinase C (PKC) binding. Our findings identify bryologs as promising drug candidates for targeting innate immunity in neuroinflammation and create a platform for evaluation of synthetic PKC modulators in neuroinflammatory diseases.