Mitigation of doxorubicin-induced cardiotoxicity with an H2O2-Activated, H2S-Donating hybrid prodrug.

Doxorubicin (DOX) is one of the most effective anticancer agents in clinical oncology. Its continued use, however, is severely limited by its dose-dependent cardiotoxicity which stems, in part, from its overproduction of reactive oxygen species (ROS) and often manifests itself as full-blown cardiomyopathy in patients, years after the cessation of treatment. Therefore, identifying DOX analogs, or prodrugs, with a diminished cardiotoxic profile is highly desirable. Herein, we describe a novel, H2O2-responsive DOX hybrid codrug (mutual prodrug) that has been rationally designed to concurrently liberate hydrogen sulfide (H2S), a purported cardioprotectant with anticancer activity, in an effort to maintain the antitumor effects of DOX while simultaneously reducing its cardiotoxic side effects. Experiments with cardiomyoblast cells in culture demonstrated a rapid accumulation of prodrug into the cells, but diminished apoptotic effects compared with DOX, dependent upon its release of H2S. Cells treated with the prodrug exhibited significantly higher Nrf2 activation relative to DOX-treated cells. Preliminary indications, using a mouse triple-negative breast cancer cell line sensitive to DOX treatment, are that the prodrug maintains considerable toxicity against the tumor-inducing cell line, suggesting significant promise for this prodrug as a cardioprotective chemotherapeutic to replace DOX.

PD-L2 Regulates B-1 Cell Antibody Production against Phosphorylcholine through an IL-5-Dependent Mechanism.

B-1 cells produce natural Abs which provide an integral first line of defense against pathogens while also performing important homeostatic housekeeping functions. In this study, we demonstrate that programmed cell death 1 ligand 2 (PD-L2) regulates the production of natural Abs against phosphorylcholine (PC). Naive PD-L2-deficient (PD-L2-/-) mice produced significantly more PC-reactive IgM and IgA. This afforded PD-L2-/- mice with selectively enhanced protection against PC-expressing nontypeable Haemophilus influenzae, but not PC-negative nontypeable Haemophilus influenzae, relative to wild-type mice. PD-L2-/- mice had significantly increased PC-specific CD138+ splenic plasmablasts bearing a B-1a phenotype, and produced PC-reactive Abs largely of the T15 Id. Importantly, PC-reactive B-1 cells expressed PD-L2 and irradiated chimeras demonstrated that B cell-intrinsic PD-L2 expression regulated PC-specific Ab production. In addition to increased PC-specific IgM, naive PD-L2-/- mice and irradiated chimeras reconstituted with PD-L2-/- B cells had significantly higher levels of IL-5, a potent stimulator of B-1 cell Ab production. PD-L2 mAb blockade of wild-type B-1 cells in culture significantly increased CD138 and Blimp1 expression and PC-specific IgM, but did not affect proliferation. PD-L2 mAb blockade significantly increased IL-5+ T cells in culture. Both IL-5 neutralization and STAT5 inhibition blunted the effects of PD-L2 mAb blockade on B-1 cells. Thus, B-1 cell-intrinsic PD-L2 expression inhibits IL-5 production by T cells and thereby limits natural Ab production by B-1 cells. These findings have broad implications for the development of therapeutic strategies aimed at altering natural Ab levels critical for protection against infectious disease, autoimmunity, allergy, cancer, and atherosclerosis.

Endogenous, regulatory cysteine sulfenylation of ERK kinases in response to proliferative signals.

ERK-dependent signaling is key to many pathways through which extracellular signals are transduced into cell-fate decisions. One conundrum is the way in which disparate signals induce specific responses through a common, ERK-dependent kinase cascade. While studies have revealed intricate ways of controlling ERK signaling through spatiotemporal localization and phosphorylation dynamics, additional modes of ERK regulation undoubtedly remain to be discovered. We hypothesized that fine-tuning of ERK signaling could occur by cysteine oxidation. We report that ERK is actively and directly oxidized by signal-generated H2O2 during proliferative signaling, and that ERK oxidation occurs downstream of a variety of receptor classes tested in four cell lines. Furthermore, within the tested cell lines and proliferative signals, we observed that both activation loop-phosphorylated and non-phosphorylated ERK undergo sulfenylation in cells and that dynamics of ERK sulfenylation is dependent on the cell growth conditions prior to stimulation. We also tested the effect of endogenous ERK oxidation on kinase activity and report that phosphotransfer reactions are reversibly inhibited by oxidation by as much as 80-90%, underscoring the importance of considering this additional modification when assessing ERK activation in response to extracellular signals.

Aging promotes B-1b cell responses to native, but not protein-conjugated, pneumococcal polysaccharides: implications for vaccine protection in older adults.

The efficacy of different vaccines in protecting elderly individuals against Streptococcus pneumoniae infections is not clear. In the current study, aged mice (22-25 months old) exhibited significantly increased susceptibility to respiratory infection with serotype 3 S. pneumoniae relative to younger adult mice, regardless of whether mice were naive or immunized with native pneumococcal polysaccharide (PPS; Pneumovax23) or protein-PPS conjugate (Prevnar-13) vaccines. Nonetheless, Pneumovax-immunized aged mice developed limited bacteremia following respiratory challenge and exhibited significantly increased survival following systemic challenge relative to Prevnar-immune aged mice and young mice that had received either vaccine. This was explained by >10-fold increases in PPS-specific immunoglobulin G (IgG) levels in Pneumovax-immunized aged mice relative to other groups. Remarkably, PPS3-specific B-cell expansion, IgG switching, plasmablast differentiation, and spleen and bone marrow antibody-secreting cell frequencies were 10-fold higher in aged mice following Pneumovax immunization relative to young mice, due to significantly increased B-1b cell participation. In summary, this study highlights (1) the need to devise strategies to enhance respiratory immunity in aged populations, (2) the diverse responses young and aged populations generate to Pneumovax vs Prevnar vaccines, and (3) the potential value of exploiting B-1b cell responses in aged individuals for increased vaccine efficacy.

In vivo modulation of avidity in highly sensitive CD8(+) effector T cells following viral infection.

Numerous studies have demonstrated a critical role for T cell avidity in predicting in vivo efficacy. Even though the measurement of avidity is now a routine assessment for the analysis of effector and memory T cell populations, our understanding of how this property is controlled in vivo at both the population and individual cell levels is limited. Our previous studies have identified high avidity as a property of the initial effector population generated in mice following respiratory virus infection. As the response progresses, lower avidity cells appear in the effector pool. The studies described here investigate the mechanistic basis of this in vivo regulation of avidity. We present data supporting in vivo avidity modulation within the early high avidity responders that results in a population of lower avidity effector cells. Changes in avidity were correlated with decreased lck expression and increased sensitivity to lck inhibitors in effector cells present at late versus early times postinfection. The possibility of tuning within select individual effectors is a previously unappreciated mechanism for the control of avidity in vivo.

High viral burden restricts short-lived effector cell number at late times postinfection through increased natural regulatory T cell expansion.

Generating and maintaining a robust CD8(+) T cell response in the face of high viral burden is vital for host survival. Further, balancing the differentiation of effectors along the memory precursor effector cell pathway versus the short-lived effector cell (SLEC) pathway may be critical in controlling the outcome of virus infection with regard to clearance and establishing protection. Although recent studies have identified several factors that have the capacity to regulate effector CD8(+) T cell differentiation-for example, inflammatory cytokines-we are far from a complete understanding of how cells choose the memory precursor effector cell versus SLEC fate following infection. In this study, we have modulated the infectious dose of the poxvirus vaccinia virus as an approach to modulate the environment present during activation and expansion of virus-specific effector cells. Surprisingly, in the face of a high virus burden, the number of SLECs was decreased. This decrease was the result of increased natural regulatory T cells (Tregs) generated by high viral burden, as depletion of these cells restored SLECs. Our data suggest Treg modulation of differentiation occurs via competition for IL-2 during the late expansion period, as opposed to the time of T cell priming. These findings support a novel model wherein modulation of the Treg response as a result of high viral burden regulates late-stage SLEC number.

Primate B-1 cells generate antigen-specific B cell responses to T cell-independent type 2 antigens.

Ab responses to T cell-independent type 2 (TI-2) Ags, such as bacterial capsular polysaccharides, are critical for host defense. In mice, B-1b cells expressing a CD11b(+)FSC(hi)CD21(lo/-)CD19(hi) phenotype play a key role in producing Abs against TI-2 Ags. In primates, a distinct IgM(+)CD27(+) "memory" B cell population is thought to generate TI-2 Ab responses, and evidence for a B-1b-like cell population participating in these responses is lacking. In this article, we demonstrate that nonhuman primates (NHPs; African green monkeys and cynomolgus macaques) harbor serosal B cells expressing a CD11b(+)FSC(hi)CD21(lo/-)CD80(+/-)CD19(hi) phenotype, constitutively active Stat3, and increased reactivity with phosphorylcholine, similar to murine peritoneal B-1a and B-1b cell populations. Like what is observed for murine B-1b cells, NHP CD11b(+)FSC(hi)CD21(lo/-)CD19(hi) B cells dominate the Ag-specific B cell response and Ab production against the TI-2 Ag trinitrophenyl-Ficoll. Although Ag-specific IgM(+) B cells expressing CD27 were not detected prior to immunization, Ag-specific CD11b(+)CD19(hi) B cells expressed and maintained an IgM(+)IgD(lo)CD27(+)CD80(+) phenotype following immunization. Thus, the murine and NHP B cell populations responding to trinitrophenyl-Ficoll are highly similar, with the main exception being that Ag-specific NHP B-1-like cells express CD27 following TI-2 Ag encounter. Therefore, murine B-1b and primate IgM(+)CD27(+) "memory" B cell subsets proposed to produce TI-2 Ab responses may be highly related, if not identical. Overall, these data not only support that B-1-like cells are present in NHPs but also provide evidence that these cells perform the same functions attributed to murine B-1b cells.