A necroptosis-independent function of RIPK3 promotes immune dysfunction and prevents control of chronic LCMV infection.

Necroptosis is a lytic and inflammatory form of cell death that is highly constrained to mitigate detrimental collateral tissue damage and impaired immunity. These constraints make it difficult to define the relevance of necroptosis in diseases such as chronic and persistent viral infections and within individual organ systems. The role of necroptotic signalling is further complicated because proteins essential to this pathway, such as receptor interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL), have been implicated in roles outside of necroptotic signalling. We sought to address this issue by individually defining the role of RIPK3 and MLKL in chronic lymphocytic choriomeningitis virus (LCMV) infection. We investigated if necroptosis contributes to the death of LCMV-specific CD8+ T cells or virally infected target cells during infection. We provide evidence showing that necroptosis was redundant in the pathogenesis of acute forms of LCMV (Armstrong strain) and the early stages of chronic (Docile strain) LCMV infection in vivo. The number of immune cells, their specificity and reactivity towards viral antigens and viral loads are not altered in the absence of either MLKL or RIPK3 during acute and during the early stages of chronic LCMV infection. However, we identified that RIPK3 promotes immune dysfunction and prevents control of infection at later stages of chronic LCMV disease. This was not phenocopied by the loss of MLKL indicating that the phenotype was driven by a necroptosis-independent function of RIPK3. We provide evidence that RIPK3 signaling evoked a dysregulated type 1 interferone response which we linked to an impaired antiviral immune response and abrogated clearance of chronic LCMV infection.

Endothelial Caspase-8 prevents fatal necroptotic hemorrhage caused by commensal bacteria.

Caspase-8 transduces signals from death receptor ligands, such as tumor necrosis factor, to drive potent responses including inflammation, cell proliferation or cell death. This is a developmentally essential function because in utero deletion of endothelial Caspase-8 causes systemic circulatory collapse during embryogenesis. Whether endothelial Caspase-8 is also required for cardiovascular patency during adulthood was unknown. To address this question, we used an inducible Cre recombinase system to delete endothelial Casp8 in 6-week-old conditionally gene-targeted mice. Extensive whole body vascular gene targeting was confirmed, yet the dominant phenotype was fatal hemorrhagic lesions exclusively within the small intestine. The emergence of these intestinal lesions was not a maladaptive immune response to endothelial Caspase-8-deficiency, but instead relied upon aberrant Toll-like receptor sensing of microbial commensals and tumor necrosis factor receptor signaling. This lethal phenotype was prevented in compound mutant mice that lacked the necroptotic cell death effector, MLKL. Thus, distinct from its systemic role during embryogenesis, our data show that dysregulated microbial- and death receptor-signaling uniquely culminate in the adult mouse small intestine to unleash MLKL-dependent necroptotic hemorrhage after loss of endothelial Caspase-8. These data support a critical role for Caspase-8 in preserving gut vascular integrity in the face of microbial commensals.

Killing in self-defense: proapoptotic drugs to eliminate intracellular pathogens.

Intracellular infections rely on host cell survival for replication and have evolved several mechanisms to prevent infected cells from dying. Drugs that promote apoptosis, a noninflammatory form of cell death, can dysregulate these survival mechanisms to kill infected cells via a mechanism that resists the evolution of drug resistance. Two such drug classes, known as SMAC mimetics and BH3 mimetics, have shown preclinical efficacy at mediating clearance of liver-stage malaria and chronic infections such as hepatitis-B virus and Mycobacterium tuberculosis. Emerging toxicity and efficacy data have reinforced the broad applicability of these drugs and form the foundations for preclinical and clinical studies into their various usage cases.

A clinicopathological study of episomal papillomavirus infection of the human placenta and pregnancy complications.

Viral infections are known to adversely affect pregnancy, but scant attention has been given to human papilloma virus (HPV) infection. We aimed to determine the molecular and histopathological features of placental HPV infection, in association with pregnancy complications including fetal growth restriction, pre-maturity, pre-eclampsia, and diabetes. Three hundred and thirty-nine placentae were selected based on the presence or absence of pregnancy complications. Five independent methods were used to identify HPV in the placenta, namely, immunohistochemistry for L1 viral capsid, in situ hybridization to high-risk HPV DNA, PCR, western blotting, and transmission electron microscopy. Pregnancy complications and uterine cervical smear screening results were correlated with placental HPV histopathology. In this study, which was deliberately biased towards complications, HPV was found in the decidua of 75% of placentae (253/339) and was statistically associated with histological acute chorioamnionitis (P<0.05). In 14% (35/253) of the HPV positive cases, HPV L1 immunoreactivity also occurred in the villous trophoblast where it was associated with a lymphohistiocytic villitis (HPV-LHV), and was exclusively of high-risk HPV type. HPV-LHV significantly associated with fetal growth restriction, preterm delivery, and pre-eclampsia (all P<0.05). All cases of pre-eclampsia (20/20) in our cohort had high-risk placental HPV. A further 55 cases (22%, 55/253) of HPV positive placentae had minimal villous trophoblast HPV L1 immunoreactivity, but a sclerosing pauci-immune villitis, statistically associated with diabetes (49.1%, 27/55, P<0.05). For women with placental HPV, 33% (69/207) had an HPV-related positive smear result before pregnancy compared with (9.4% 8/85) of women with HPV-negative placentae (P=0.0001). Our findings support further investigations to determine if vaccination of women and men will improve pregnancy outcomes.

Loss of ATRX and DAXX expression identifies poor prognosis for smooth muscle tumours of uncertain malignant potential and early stage uterine leiomyosarcoma.

Uterine smooth muscle tumours of uncertain malignant potential (STUMP) are diagnostically and clinically challenging. The alternative lengthening of telomeres (ALT) telomere maintenance mechanism is associated with poor survival in soft tissue leiomyosarcoma. Time to first recurrence and survival were known for 18 STUMP and 43 leiomyosarcomata (LMS). These were screened for ALT telomere maintenance by the presence of ALT-associated PML bodies (APBs) and for changes associated with the ALT phenotype, namely aberrant p53 expression, isocitrate dehydrogenase 1 mutation (R132H substitution) expression, mutant ATRX (αthalassemia/mental retardation syndrome X-linked) expression and mutant DAXX (death-domain-associated protein) expression by immunohistochemistry (IHC). Overexpression of p16(INK4A) was examined immunohistologically in a subset of cases. Many of the tumours associated with death or recurrence demonstrated APBs commensurate with ALT telomere maintenance. However, all uterine STUMP (4/4), and vaginal STUMP (2/2) patients, and almost all LMS patients (88.4%, 23/26, including 90% (9/10) of stage 1 LMS cases), who had died of disease or who had recurrent disease, displayed loss of ATRX or DAXX expression. Loss of ATRX or DAXX expression identified poor prognosis (95% CI 2.1 to 40.8, p < 0.003), in the LMS group. Thus, loss of ATRX or DAXX expression in uterine smooth muscle tumours identifies a clinically aggressive molecular subtype of early stage LMS and when histopathological features are problematic such as in STUMP. As ATRX and DAXX IHC is readily performed in diagnostic laboratories these are potentially useful for routine histopathological classification and management.

Smoking during pregnancy causes double-strand DNA break damage to the placenta.

Despite the adverse effects of smoking, many pregnancies are exposed to tobacco smoke. Recent studies have investigated whether smoking damages placental DNA by measuring DNA adducts. This study investigated whether a more severe lesion, double-strand DNA breaks, was also present in the tobacco smoking-exposed placenta. Term placentae from women who smoked during their entire pregnancies (n = 52), from those who had ceased smoking for at least 4 weeks before delivery (previous smokers, n = 34), and from nonsmoking women (n = 150) were examined using the DNA double-strand break marker phosphorylated γ H2AX. The extent of DNA damage was assessed according to cell type and additional markers were applied for cell fate (apoptosis and DNA repair), and function (human chorionic gonadotropin, human placental lactogen, and glucose transporter 1), to characterize the effect of the DNA damage on placental integrity. Marked phosphorylated γ H2AX-positive cells occurred in the villous syncytiotrophoblast and syncytial knot nuclei in placentae from smokers (P < .001). Phosphorylated γ H2AX foci did not colocalize with the DNA repair protein 53BP1, and damaged nuclei had a marked reduction in expression of human chorionic gonadotropin, human placental lactogen, and glucose transporter 1. Minimal DNA damage, similar to nonsmokers, was present in previous smokers including those that had ceased smoking for just over 4 weeks before delivery. In summary, smoking during pregnancy was associated with marked double-strand DNA break damage to the syncytiotrophoblast. We suggest that smoking cessation is important to prevent additional DNA damage and to facilitate DNA repair.