Caspase-8 activity mediates TNFα production and restricts Coxiella burnetii replication during murine macrophage infection.

Coxiella burnetii is an obligate intracellular bacteria that causes the global zoonotic disease Q Fever. Treatment options for chronic infection are limited, and the development of novel therapeutic strategies requires a greater understanding of how C. burnetii interacts with immune signaling. Cell death responses are known to be manipulated by C. burnetii, but the role of caspase-8, a central regulator of multiple cell death pathways, has not been investigated. In this research, we studied bacterial manipulation of caspase-8 signaling and the significance of caspase-8 to C. burnetii infection, examining bacterial replication, cell death induction, and cytokine signaling. We measured caspase, RIPK, and MLKL activation in C. burnetii-infected tumor necrosis factor alpha (TNFα)/cycloheximide-treated THP-1 macrophage-like cells and TNFα/ZVAD-treated L929 cells to assess apoptosis and necroptosis signaling. Additionally, we measured C. burnetii replication, cell death, and TNFα induction over 12 days in RIPK1-kinase-dead, RIPK3-kinase-dead, or RIPK3-kinase-dead-caspase-8-/- bone marrow-derived macrophages (BMDMs) to understand the significance of caspase-8 and RIPK1/3 during infection. We found that caspase-8 is inhibited by C. burnetii, coinciding with inhibition of apoptosis and increased susceptibility to necroptosis. Furthermore, C. burnetii replication was increased in BMDMs lacking caspase-8, but not in those lacking RIPK1/3 kinase activity, corresponding with decreased TNFα production and reduced cell death. As TNFα is associated with the control of C. burnetii, this lack of a TNFα response may allow for the unchecked bacterial growth we saw in caspase-8-/- BMDMs. This research identifies and explores caspase-8 as a key regulator of C. burnetii infection, opening novel therapeutic doors.

Caspase-8 activity mediates TNFα production and restricts Coxiella burnetii replication during murine macrophage infection.

Coxiella burnetii is an obligate intracellular bacteria which causes the global zoonotic disease Q Fever. Treatment options for infection are limited, and development of novel therapeutic strategies requires a greater understanding of how C. burnetii interacts with immune signaling. Cell death responses are known to be manipulated by C. burnetii, but the role of caspase-8, a central regulator of multiple cell death pathways, has not been investigated. In this research, we studied bacterial manipulation of caspase-8 signaling and the significance of caspase-8 to C. burnetii infection, examining bacterial replication, cell death induction, and cytokine signaling. We measured caspase, RIPK, and MLKL activation in C. burnetii-infected TNFα/CHX-treated THP-1 macrophage-like cells and TNFα/ZVAD-treated L929 cells to assess apoptosis and necroptosis signaling. Additionally, we measured C. burnetii replication, cell death, and TNFα induction over 12 days in RIPK1-kinase-dead, RIPK3-kinase-dead, or RIPK3-kinase-dead-caspase-8-/- BMDMs to understand the significance of caspase-8 and RIPK1/3 during infection. We found that caspase-8 is inhibited by C. burnetii, coinciding with inhibition of apoptosis and increased susceptibility to necroptosis. Furthermore, C. burnetii replication was increased in BMDMs lacking caspase-8, but not in those lacking RIPK1/3 kinase activity, corresponding with decreased TNFα production and reduced cell death. As TNFα is associated with the control of C. burnetii, this lack of a TNFα response may allow for the unchecked bacterial growth we saw in caspase-8-/- BMDMs. This research identifies and explores caspase-8 as a key regulator of C. burnetii infection, opening novel therapeutic doors.

Fostering Diversity, Equity, Inclusion, and Belonging Through the Lens of Gratitude.

Diversity, equity, inclusion, and belonging is the work of leaders. The opportunity to embrace individuality and grow collectively is something to be appreciated. The work requires leadership at all levels and starts from within. Having the courage to lean into discomfort that comes with the work reaps great rewards. Assessing your organization and applying learnings is the start to culture change. This is only the start, this work is ongoing, and it is with gratitude we should embrace to opportunity for inclusion. We as individuals and teams will benefit as well as those we serve.

Cell-type dependence of necroptosis pathways triggered by viral infection.

Necroptosis, a potent host defense mechanism, limits viral replication and pathogenesis through three distinct initiation pathways. Toll-like receptor 3 (TLR3) via TIR-domain-containing adapter-inducing interferon-ß (TRIF), Z-DNA-binding protein 1 (ZBP1) and tumor necrosis factor (TNF)α mediate necroptosis, with ZBP1 and TNF playing pivotal roles in controlling viral infections, with the role of TLR3-TRIF being less clear. ZBP1-mediated necroptosis is initiated when host ZBP1 senses viral Z-form double stranded RNA and recruits receptor-interacting serine/threonine-protein kinase 3 (RIPK3), driving a mixed lineage kinase domain-like pseudokinase (MLKL)-dependent necroptosis pathway, whereas TNF-mediated necroptosis is initiated by TNF signaling, which drives a RIPK1-RIPK3-MLKL pathway, resulting in necroptosis. Certain viruses (cytomegalovirus, herpes simplex virus and vaccinia) have evolved to produce proteins that compete with host defense systems, preventing programmed cell death pathways from being initiated. Two engineered viruses deficient of active forms of these proteins, murine cytomegalovirus M45mutRHIM and vaccinia virus E3∆Zα, trigger ZBP1-dependent necroptosis in mouse embryonic fibroblasts. By contrast, when bone-marrow-derived macrophages are infected with the viruses, necroptosis is initiated predominantly through the TNF-mediated pathway. However, when the TNF pathway is blocked by RIPK1 inhibitors or a TNF blockade, ZBP1-mediated necroptosis becomes the prominent pathway in bone-marrow-derived macrophages. Overall, these data implicate a cell-type preference for either TNF-mediated or ZBP1-mediated necroptosis pathways in host responses to viral infections. These preferences are important to consider when evaluating disease models that incorporate necroptosis because they may contribute to tissue-specific reactions that could alter the balance of inflammation versus control of virus, impacting the organism as a whole.

Approaches to Evaluating Necroptosis in Virus-Infected Cells.

The immune system functions to protect the host from pathogens. To counter host defense mechanisms, pathogens have developed unique strategies to evade detection or restrict host immune responses. Programmed cell death is a major contributor to the multiple host responses that help to eliminate infected cells for obligate intracellular pathogens like viruses. Initiation of programmed cell death pathways during the early stages of viral infections is critical for organismal survival as it restricts the virus from replicating and serves to drive antiviral inflammation immune recruitment through the release of damage-associated molecular patterns (DAMPs) from the dying cell. Necroptosis has been implicated as a critical programmed cell death pathway in a diverse set of diseases and pathological conditions including acute viral infections. This cell death pathway occurs when certain host sensors are triggered leading to the downstream induction of mixed-lineage kinase domain-like protein (MLKL). MLKL induction leads to cytoplasmic membrane disruption and subsequent cellular destruction with the release of DAMPs. As the role of this cell death pathway in human disease becomes apparent, methods identifying necroptosis patterns and outcomes will need to be further developed. Here, we discuss advances in our understanding of how viruses counteract necroptosis, methods to quantify the pathway, its effects on viral pathogenesis, and its impact on cellular signaling.

Strengthening New Nurse Manager Leadership Skills Through a Transition-to-Practice Program.

The nurse manager role is increasingly complex, and oftentimes, leadership competencies are learned through experience rather than an effective role transition. The need for a transition-to-practice program for new nurse managers is frequently cited in the literature. This evidence-based practice project resulted in significant improvement in leadership competencies and was used in the development of an American Organization for Nursing Leadership nurse manager transition-to-practice program to be offered in 2021.

Chemical Contaminants from Plastics in the Animal Environment.

Accidental exposure of our mice to bisphenol A (BPA) from damaged polycarbonate cages 20 y ago provided some of the first evidence of the harmful effects of exposure to this common chemical. Recently we found that housing mice in damaged polysulfone cages resulted in similar harmful effects due to exposure to bisphenol S (BPS). This problem was unexpected for 2 reasons. First, polysulfone is a far more chemically resistant polymer than polycarbonate. Second, BPS is not a component in the manufacture of polysulfone. We report here our efforts to verify the source of the BPS and eliminate the exposure. Our analysis of new polysulfone caging materials confirmed that BPS is a breakdown product of damaged polysulfone plastic. Furthermore, we found that BPS can cross-contaminate new or undamaged cages in facilities that process damaged caging materials. Neither the use of disposable cages nor replacement of caging materials used solely for our colony was sufficient to eliminate exposure effects. Only the replacement of all cages and water bottles in the facility corrected the problem and allowed us to resume our studies. Taken together, our previous and current findings underscore the concern that chemicals from plastics are harmful environmental contaminants for both humans and animals. Furthermore, our results provide strong evidence that the presence of damaged plastic in a facility may be sufficient to affect research results and, by exten- sion, animal health.

Replacement Bisphenols Adversely Affect Mouse Gametogenesis with Consequences for Subsequent Generations.

20 years ago, accidental bisphenol A (BPA) exposure caused a sudden increase in chromosomally abnormal eggs from our control mice [1]. Subsequent rodent studies demonstrated developmental effects of exposure with repercussions on adult health and fertility (e.g., [2-9]; reviewed in [10-17]). Studies in monkeys, humans, fish, and worms suggest BPA effects extend across species (e.g., [18-30]; reviewed in [31-33]). Widespread use has resulted in ubiquitous environmental contamination and human BPA exposure. Consumer concern resulted in "BPA-free" products produced using structurally similar bisphenols that are now detectable environmental and human contaminants (e.g., [34-41]). We report here studies initiated by meiotic changes mirroring our previous BPA experience and implicating exposure to BPS (a common BPA replacement) from damaged polysulfone cages. Like with BPA [1, 2, 5], our data show that exposure to common replacement bisphenols induces germline effects in both sexes that may affect multiple generations. These findings add to growing evidence of the biological risks posed by this class of chemicals. Rapid production of structural variants of BPA and other EDCs circumvents efforts to eliminate dangerous chemicals, exacerbates the regulatory burden of safety assessment, and increases environmental contamination. Our experience suggests that these environmental contaminants pose a risk not only to reproductive health but also to the integrity of the research environment. EDCs, like endogenous hormones, can affect diverse processes. The sensitivity of the germline allows us to detect effects that, although not immediately apparent in other systems, may induce variability that undermines experimental reproducibility and impedes scientific advancement.

Correction: Germline and reproductive tract effects intensify in male mice with successive generations of estrogenic exposure.

[This corrects the article DOI: 10.1371/journal.pgen.1006885.].

Germline and reproductive tract effects intensify in male mice with successive generations of estrogenic exposure.

The hypothesis that developmental estrogenic exposure induces a constellation of male reproductive tract abnormalities is supported by experimental and human evidence. Experimental data also suggest that some induced effects persist in descendants of exposed males. These multi- and transgenerational effects are assumed to result from epigenetic changes to the germline, but few studies have directly analyzed germ cells. Typically, studies of transgenerational effects have involved exposing one generation and monitoring effects in subsequent unexposed generations. This approach, however, has limited human relevance, since both the number and volume of estrogenic contaminants has increased steadily over time, intensifying rather than reducing or eliminating exposure. Using an outbred CD-1 mouse model, and a sensitive and quantitative marker of germline development, meiotic recombination, we tested the effect of successive generations of exposure on the testis. We targeted the germline during a narrow, perinatal window using oral exposure to the synthetic estrogen, ethinyl estradiol. A complex three generation exposure protocol allowed us to compare the effects of individual, paternal, and grandpaternal (ancestral) exposure. Our data indicate that multiple generations of exposure not only exacerbate germ cell exposure effects, but also increase the incidence and severity of reproductive tract abnormalities. Taken together, our data suggest that male sensitivity to environmental estrogens is increased by successive generations of exposure.

Control of meiotic pairing and recombination by chromosomally tethered 26S proteasome.

During meiosis, paired homologous chromosomes (homologs) become linked via the synaptonemal complex (SC) and crossovers. Crossovers mediate homolog segregation and arise from self-inflicted double-strand breaks (DSBs). Here, we identified a role for the proteasome, the multisubunit protease that degrades proteins in the nucleus and cytoplasm, in homolog juxtaposition and crossing over. Without proteasome function, homologs failed to pair and instead remained associated with nonhomologous chromosomes. Although dispensable for noncrossover formation, a functional proteasome was required for a coordinated transition that entails SC assembly between longitudinally organized chromosome axes and stable strand exchange of crossover-designated DSBs. Notably, proteolytic core and regulatory proteasome particles were recruited to chromosomes by Zip3, the ortholog of mammalian E3 ligase RNF212, and SC protein Zip1 . We conclude that proteasome functions along meiotic chromosomes are evolutionarily conserved.

Bisphenol A alters early oogenesis and follicle formation in the fetal ovary of the rhesus monkey.

Widespread use of the endocrine disrupting chemical bisphenol A (BPA) in consumer products has resulted in nearly continuous human exposure. In rodents, low-dose exposures have been reported to adversely affect two distinct stages of oogenesis in the developing ovary: the events of prophase at the onset of meiosis in the fetal ovary and the formation of follicles in the perinatal ovary. Because these effects could influence the reproductive longevity and success of the exposed individual, we conducted studies in the rhesus monkey to determine whether BPA induces similar disturbances in the developing primate ovary. The routes and levels of human exposure are unclear; hence, two different exposure protocols were used: single daily oral doses and continuous exposure via subdermal implant. Our analyses of second trimester fetuses exposed at the time of meiotic onset suggest that, as in mice, BPA induces subtle disturbances in the prophase events that set the stage for chromosome segregation at the first meiotic division. Our analyses of third-trimester fetuses exposed to single daily oral doses during the time of follicle formation revealed an increase in multioocyte follicles analogous to that reported in rodents. However, two unique phenotypes were evident in continuously exposed animals: persistent unenclosed oocytes in the medullary region and small, nongrowing oocytes in secondary and antral follicles. Because effects on both stages of oogenesis were elicited using doses that yield circulating levels of BPA analogous to those reported in humans, these findings raise concerns for human reproductive health.

Gene expression in the fetal mouse ovary is altered by exposure to low doses of bisphenol A.

Evidence from experimental studies suggests that fetal exposure to the endocrine-disrupting chemical bisphenol A (BPA) has adverse reproductive effects in both males and females. Studies from our laboratory suggest that exposure to the developing female fetus produces a unique, multigenerational effect. Specifically, maternal exposure affects the earliest stages of oogenesis in the developing fetal ovary, and the resulting subtle meiotic defects increase the likelihood that embryos produced by the exposed female in adulthood (i.e., the grandchildren) will be chromosomally abnormal. To understand the impact of BPA on the developing ovary, we conducted expression studies to characterize gene expression changes in the fetal ovary that result from BPA exposure. We first tested the validity of the approach, asking whether we could reliably detect temporal changes in expression levels of meiotic genes in controls. As anticipated, we were able to identify appropriate increases in expression in meiotic, but in few other, genes. Intriguingly, this analysis provided data on a small set of genes for which timing and expression changes suggest that they may have important and heretofore unrecognized meiotic roles. After verifying the utility of our approach, we focused our analysis on BPA-exposed animals. We found modest, but significant, changes in gene expression in the fetal ovaries from exposed fetuses. The first changes were evident within 24 h of exposure, and the most extensive changes correlated with the onset of meiosis. Furthermore, gene ontology analysis suggested that BPA acts to down-regulate mitotic cell-cycle genes, raising the possibility that fetal BPA exposure may act to limit expansion of the primordial germ cell population.

The mouse A/HeJ Y chromosome: another good Y gone bad.

In both humans and mice there are numerous reports of Y chromosome abnormalities that interfere with sex determination. Recent studies in the mouse of one such mutation have identified Y chromosome nondisjunction during preimplantation development as the cause of abnormal testis determination that results in a high frequency of true hermaphroditism. We report here that the mouse Y chromosome from the A/HeJ inbred strain induces similar aberrations in sex determination. Our analyses provide evidence, however, that the mechanism underlying these aberrations is not Y chromosome nondisjunction. On the basis of our findings, we postulate that a mutation at or near the centromere affects both the segregation and sex-determining properties of the A/HeJ Y chromosome. This Y chromosome adds to the growing list of Y chromosome aberrations in humans and mice. In both species, the centromere of the Y is structurally and morphologically distinct from the centromeres of all other chromosomes. We conclude that these centromeric features make the human and mouse Y chromosomes extremely sensitive to minor structural alterations, and that our studies provide yet another example of a good Y chromosome gone 'bad.'

Immunolocalization and regulation of cystatin 12 in mouse testis and epididymis.

In previous studies, we identified a new member of the male reproductive tract subgroup within family 2 cystatins, termed cystatin 12 (Cst12, previously known as Cst TE-1 or Cres3). The mouse Cst12 mRNA was primarily localized to the Sertoli cells in the testis and to the epithelial cells of the proximal caput region of the epididymis. In this report, studies were carried out to characterize the cystatin 12 (CST12) protein in mouse testis and epididymis. A recombinant His-CST12 fusion protein was expressed in E. coli and purified to generate an anti-CST12 polyclonal antibody. Western blot analysis showed little or no cross-reaction between the anti-CST12 antibody and several other known male reproductive tract cystatins. Immunohistochemistry revealed that CST12 protein was predominantly localized to the cytoplasm of Sertoli cells in the seminiferous epithelium in a stage-dependent manner. All stages showed high levels of expression except stages VII and VIII, in which very limited expression of CST12 was observed. In the epididymis, CST12 was highly expressed in the cytoplasm of the epithelial cells in the proximal caput and secreted into the lumen. The mouse CST12 protein was also detected in other regions of the epididymis; however, the localization varied greatly along the epididymal tubules. Indirect immunofluorescence showed that CST12 protein was localized to the cytoplasmic droplets in both testicular and epididymal spermatozoa. These observations suggest that CST12 protein may play a specialized role during spermatogenesis and sperm maturation. Northern blot analyses demonstrated that Cst12 transcript levels in the epididymis decreased after castration, and testosterone propionate (T) treatment further repressed the expression of this gene. However, 17-beta estradiol (E) administration maintained the expression of Cst12 mRNA after castration, whereas treatment with both T and E failed to maintain Cst12 mRNA levels in epididymis. These results suggest that androgen and estrogen, probably with other testicular factors, are involved in the regulation of this gene.