Seminal fluid and cytokine control of regulatory T-cells in murine pregnancy.

For successful pregnancy, the maternal immune system must tolerate the presence of a fetus that expresses alloantigens. The appropriate and timely acquisition of this state of tolerance is critical and emerging evidence suggests that it needs to be present from the time the embryo implants into the uterus. Recently it has been demonstrated that a subpopulation of lymphocytes termed CD4⁺CD25⁺ regulatory T cells (Treg cells) are required for immune tolerance of the fetus during pregnancy. Despite their importance the factors that control regulatory T cells during pregnancy, and in particular in the peri-implantation period, are poorly understood. Using mouse models we have assessed the role of the ejaculate and its components (sperm and seminal plasma) in coordinating Treg cells in the period prior to embryo implantation. We have also used mice with a null mutation in the interleukin 10 (IL-10) gene to assess the role of this cytokine in coordination of Treg cell populations in later pregnancy. Experiments in the peri-implantation period just prior to implantation (day 3.5 postcoitum) showed that there was a significant increase (approximately 2-fold; p<0.05) in the total number of (CD4⁺Foxp3⁺) Treg cells in the iliac lymph nodes (LNs) that drain the uterus, but not in the distal inguinal LNs. This appeared not to be the result of a selective expansion in Treg cells but due to expansion of the entire CD4⁺ cell pool, since the percent of CD4+ cells expressing Foxp3 in any of the lymphoid tissues studied did not increase in response to mating. In addition, there was a similar increase in the density of these cells in the uterus just prior to implantation at day 3.5pc (p<0.05). By using males deficient in the sperm or seminal plasma components of the ejaculate we could show that the increase in both the lymph node and uterine Treg cell populations occurred in response to seminal plasma. The role of seminal plasma in regulating expression of mRNAs encoding migratory molecules in the peri-implantation uterus, and the involvement of these genes in recruiting Treg cells following mating, was then assessed. We analysed the mRNAs for the chemokines Ccl4, Ccl5, Ccl19, Ccl22, the chemokine receptors Ccr4, Ccr5, Ccr7 and the integrin Cd103 using qRT-PCR. We showed a significant elevation in Ccl19 and Ccr5 mRNA at day 3.5pc following mating to intact males. However the increase in mRNA was independent of factors associated with seminal fluid and might instead be regulated by ovarian steroid hormones. Using IL-10 null mutant (IL-10-/-) mice it was then shown that the cytokine IL-10 is involved in controlling Treg cell numbers in mid gestation. At gestational day (gd) 9.5, in IL-10-/- mice, there was an approximate 40% elevation in the proportion of CD4⁺ cells expressing Foxp3 compared with wild‐type control mice (p<0.01). This was seen in both the iliac LNs and inguinal LNs. In addition, there was a greater than 10-fold increase (p<0.0001) in the total number of Treg cells in the uterine-draining iliac LNs of IL-10-/- mice compared to wild-type mice. This was not seen in the inguinal LNs. Experiments comparing allogeneic and syngeneic mated mice showed that the proportional changes seen in the CD4⁺ cell population was dependent on fetal alloantigens, although the elevation in total numbers still occurred in the absence of fetal alloantigens. This study begins to unravel the process by which Treg cell populations are expanded and recruited into the uterus prior to embryo implantation and later in gestation. A greater understanding of this process may aid in the diagnosis and prevention of a range of pregnancy pathologies associated with immune dysregulation, such as preeclampsia and recurrent spontaneous abortion.Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 201

Whole-genome sequencing reveals host factors underlying critical COVID-19

Critical COVID-19 is caused by immune-mediated inflammatory lung injury. Host genetic variation influences the development of illness requiring critical care1 or hospitalization2–4 after infection with SARS-CoV-2. The GenOMICC (Genetics of Mortality in Critical Care) study enables the comparison of genomes from individuals who are critically ill with those of population controls to find underlying disease mechanisms. Here we use whole-genome sequencing in 7,491 critically ill individuals compared with 48,400 controls to discover and replicate 23 independent variants that significantly predispose to critical COVID-19. We identify 16 new independent associations, including variants within genes that are involved in interferon signalling (IL10RB and PLSCR1), leucocyte differentiation (BCL11A) and blood-type antigen secretor status (FUT2). Using transcriptome-wide association and colocalization to infer the effect of gene expression on disease severity, we find evidence that implicates multiple genes—including reduced expression of a membrane flippase (ATP11A), and increased expression of a mucin (MUC1)—in critical disease. Mendelian randomization provides evidence in support of causal roles for myeloid cell adhesion molecules (SELE, ICAM5 and CD209) and the coagulation factor F8, all of which are potentially druggable targets. Our results are broadly consistent with a multi-component model of COVID-19 pathophysiology, in which at least two distinct mechanisms can predispose to life-threatening disease: failure to control viral replication; or an enhanced tendency towards pulmonary inflammation and intravascular coagulation. We show that comparison between cases of critical illness and population controls is highly efficient for the detection of therapeutically relevant mechanisms of disease

AusTraits, a curated plant trait database for the Australian flora

We introduce the AusTraits database - a compilation of values of plant traits for taxa in the Australian flora (hereafter AusTraits). AusTraits synthesises data on 448 traits across 28,640 taxa from field campaigns, published literature, taxonomic monographs, and individual taxon descriptions. Traits vary in scope from physiological measures of performance (e.g. photosynthetic gas exchange, water-use efficiency) to morphological attributes (e.g. leaf area, seed mass, plant height) which link to aspects of ecological variation. AusTraits contains curated and harmonised individual- and species-level measurements coupled to, where available, contextual information on site properties and experimental conditions. This article provides information on version 3.0.2 of AusTraits which contains data for 997,808 trait-by-taxon combinations. We envision AusTraits as an ongoing collaborative initiative for easily archiving and sharing trait data, which also provides a template for other national or regional initiatives globally to fill persistent gaps in trait knowledge

Whole-genome sequencing reveals host factors underlying critical COVID-19

Critical COVID-19 is caused by immune-mediated inflammatory lung injury. Host genetic variation influences the development of illness requiring critical care1 or hospitalization2,3,4 after infection with SARS-CoV-2. The GenOMICC (Genetics of Mortality in Critical Care) study enables the comparison of genomes from individuals who are critically ill with those of population controls to find underlying disease mechanisms. Here we use whole-genome sequencing in 7,491 critically ill individuals compared with 48,400 controls to discover and replicate 23 independent variants that significantly predispose to critical COVID-19. We identify 16 new independent associations, including variants within genes that are involved in interferon signalling (IL10RB and PLSCR1), leucocyte differentiation (BCL11A) and blood-type antigen secretor status (FUT2). Using transcriptome-wide association and colocalization to infer the effect of gene expression on disease severity, we find evidence that implicates multiple genes—including reduced expression of a membrane flippase (ATP11A), and increased expression of a mucin (MUC1)—in critical disease. Mendelian randomization provides evidence in support of causal roles for myeloid cell adhesion molecules (SELE, ICAM5 and CD209) and the coagulation factor F8, all of which are potentially druggable targets. Our results are broadly consistent with a multi-component model of COVID-19 pathophysiology, in which at least two distinct mechanisms can predispose to life-threatening disease: failure to control viral replication; or an enhanced tendency towards pulmonary inflammation and intravascular coagulation. We show that comparison between cases of critical illness and population controls is highly efficient for the detection of therapeutically relevant mechanisms of disease

Unstable Foxp3(+) Regulatory T Cells and Altered Dendritic Cells Are Associated with Lipopolysaccharide-Induced Fetal Loss in Pregnant Interleukin 10-Deficient Mice

Maternal interleukin (IL) 10 deficiency elevates susceptibility to fetal loss induced by the model Toll-like receptor agonist lipopolysaccharide, but the mechanisms are not well elucidated. Here, we show that Il10 null mutant (Il10(-/-)) mice exhibit altered local T cell responses in pregnancy, exhibiting pronounced hyperplasia in para-aortic lymph nodes draining the uterus with >6-fold increased CD4(+) and CD8(+) T cells compared with wild-type controls. Among these CD4(+) cells, Foxp3(+) T regulatory (Treg) cells were substantially enriched, with 11-fold higher numbers at Day 9.5 postcoitum. Lymph node hypertrophy in Il10(-/-) mice was associated with more activated phenotypes in dendritic cells and macrophages, with elevated expression of MHCII, scavenger receptor, and CD80. Affymetrix microarray revealed an altered transcriptional profile in Treg cells from pregnant Il10(-/-) mice, with elevated expression of Ctse (cathepsin E), Il1r1, Il12rb2, and Ifng. In vitro, Il10(-/-) Treg cells showed reduced steady-state Foxp3 expression, and polyclonal stimulation caused greater loss of Foxp3 and reduced capacity to suppress IL17 in CD4(+) Foxp3(+) T cells. We conclude that despite a substantially expanded Treg cell pool, the diminished stability of Treg cells, increased numbers of effector T cells, and altered phenotypes in dendritic cells and macrophages in pregnancy all potentially confer vulnerability to inflammation-induced fetal loss in Il10(-/-) mice. These findings suggest that IL10 has a pivotal role in facilitating robust immune protection of the fetus from inflammatory challenge and that IL10 deficiency could contribute to human gestational disorders in which altered T cell responses are implicated

Seminal Fluid Drives Expansion of the CD4+CD25+ T Regulatory Cell Pool and Induces Tolerance to Paternal Alloantigens in Mice1

T regulatory (Treg) cells are implicated in maternal immune tolerance of the conceptus at implantation; however, the antigenic and regulatory signals controlling Treg cells in early pregnancy are undefined. To examine the role of male seminal fluid in tolerance induction, the effect of exposure to seminal fluid at mating on responsiveness to paternal alloantigens was examined using paternal tumor cell grafts and by delayed-type hypersensitivity (DTH) challenge on Day 3.5 postcoitum. Exposure to seminal fluid inhibited rejection of paternal tumor cells, independently of fertilization and embryo development, while seminal fluid from major histocompatability complex (MHC)-dissimilar males was less effective. Similarly, mating with intact males suppressed the DTH response to paternal alloantigens in an MHC-specific fashion. Excision of the seminal vesicle glands diminished the tolerance-inducing activity of seminal fluid. Mating with intact males caused an increase in CD4+CD25+ cells expressing FOXP3 in the para-aortic lymph nodes draining the uterus, beyond the estrus-associated peak in cycling mice. The increase in CD4+CD25+ cells was abrogated when males were vasectomized or seminal vesicles were excised. Collectively, these data provide evidence that exposure to seminal fluid at mating promotes a state of functional tolerance to paternal alloantigens that may facilitate maternal acceptance of the conceptus at implantation, and the effects of seminal fluid are likely to be mediated by expansion of the Treg cell pool. Both seminal plasma and sperm components of the seminal fluid are necessary to confer full tolerance and elicit the Treg cell response, potentially through provision of immune-deviating cytokines and antigens, respectively

Seminal fluid regulates accumulation of FOXP3(+) regulatory T cells in the preimplantation mouse uterus through expanding the FOXP3(+) cell pool and CCL19-mediated recruitment

Regulatory T (Treg) cells facilitate maternal immune tolerance of the semiallogeneic conceptus in early pregnancy, but the origin and regulation of these cells at embryo implantation is unclear. During the preimplantation period, factors in the seminal fluid delivered at coitus cause expansion of a CD4+CD25+ putative Treg cell population in the para-aortic lymph nodes draining the uterus. Using flow cytometry, immunohistochemistry, and real-time quantitative PCR (qPCR) for the signature Treg cell transcription factor FOXP3, we confirmed the identity of the expanded lymph node population as FOXP3+ Treg cells and showed that this is accompanied by a comparable increase in the uterus of FOXP3+ Treg cells and expression of Foxp3 mRNA by Day 3.5 postcoitum. Seminal plasma was necessary for uterine Treg cell accumulation, as mating with seminal vesicle-deficient males failed to elicit an increase in uterine Treg cells. Furthermore seminal fluid induced expression of mRNA encoding the Treg chemokine CCL19 (MIP3beta), which acts through the CCR7 receptor to regulate Treg cell recruitment and retention in peripheral tissues. Glandular and luminal epithelial cells were identified as the major cellular origins of uterine CCL19, and exposure to both seminal plasma and sperm was required for maximum expression. Together, these results indicate that Treg cells accumulate in the uterus prior to embryo implantation and that seminal fluid is a key regulator of the uterine Treg cell population, operating by both increasing the pool of available Treg cells and promoting their CCL19-mediated recruitment from the circulation into the implantation site.Leigh R. Guerin, Lachlan M. Moldenhauer, Jelmer R. Prins, John J. Bromfield, John D. Hayball and Sarah A. Robertso

Dasatinib alters the metastatic phenotype of B16-OVA melanoma in vivo

The Src/Abl tyrosine kinase inhibitor dasatinib is an approved chronic myeloid leukemia treatment and is under investigation for solid tumor therapy. Members of the Src family of kinases (SFKs) are involved in the process of metastasis and dasatinib inhibits the migration and invasiveness of human melanoma cell lines in vitro. SFKs are also involved in immune function and angiogenesis, which both contribute to As active and passive immunotherapies continue to be investigated in metastatic melanoma, we investigated possible interactions between kinase inhibitors and immunotherapies. A murine syngenic model of metastatic melanoma in which B16F10 cells expressed ovalbumin (B16-OVA) was employed and the active immunotherapy comprised immunization with an OVA-expressing recombinant fowlpox virus (FPVOVA).Dasatinib did not affect B16-OVA viability, proliferation, migration or soft agar colony formation. However, depending on drug dose and schedule, differences in the metastatic behavior of B16-OVA were observed in vivo after dasatinib therapy. At a dose of 5 mg/kg/day given before tumor challenge, dasatinib therapy reduced the number of pulmonary metastases. Conversely, a higher dose (25 mg/kg/day), did not affect the number of pulmonary metastases and increased the number of extra-pulmonary metastases. Finally, immunization of B16-OVA-bearing mice with FPVOVA reduced the number of lung metastases. Prior treatment of these mice with dasatinib 5 mg/kg/day did not affect the incidence of lung metastases. Although the mechanisms by which dasatinib alters the metastatic behavior of B16-OVA cells in vivo remain to be determined, we hypothesize that dasatinib acts via multiple tumor-extrinsic processes that include immune function and neoangiogenesis.