Using machine learning to dissect host kinases required for Leishmania internalization and development.

The Leishmania life cycle alternates between promastigotes, found in the sandfly, and amastigotes, found in mammals. When an infected sandfly bites a host, promastigotes are engulfed by phagocytes (i.e., neutrophils, dendritic cells, and macrophages) to establish infection. When these phagocytes die or break down, amastigotes must be re-internalized to survive within the acidic phagolysosome and establish disease. To define host kinase regulators of Leishmania promastigote and amastigote uptake and survival within macrophages, we performed an image-based kinase regression screen using a panel of 38 kinase inhibitors with unique and overlapping kinase targets. We also targeted inert beads to complement receptor 3 (CR3) or Fcγ receptors (FcR) as controls by coating them with complement/C3bi or IgG respectively. Through this approach, we identified several host kinases that regulate receptor-mediated phagocytosis and/or the uptake of L. amazonensis. Findings included kinases previously implicated in Leishmania uptake (such as SRC family kinases (SFK), Abl family kinases (ABL1/c-Abl, ABL2/Arg), and spleen tyrosine kinase (SYK)); we also uncovered many novel kinases. These methods also predicted kinases necessary for promastigotes to convert to amastigotes or for amastigotes to survive within macrophages. Overall, our results suggest that the concerted action of multiple interconnected networks of host kinases are needed over the course of Leishmania infection, and that the kinases required for the parasite's life cycle substantially differ depending on which receptors are bound and the life cycle stage that is internalized. In addition, using our screen, we identified kinases that preferentially regulate the uptake of parasites over beads, indicating that the methods required for Leishmania to be internalized by macrophages differ significantly from generalized phagocytic mechanisms. Our findings are intended to be used as a hypothesis generation resource for the broader scientific community studying the roles of kinases in host-pathogen interactions.

Pediatric human nose organoids demonstrate greater susceptibility, epithelial responses, and cytotoxicity than adults during RSV infection.

Respiratory syncytial virus (RSV) is a common cause of respiratory infections, causing significant morbidity and mortality, especially in young children. Why RSV infection in children is more severe as compared to healthy adults is not fully understood. In the present study, we infect both pediatric and adult human nose organoid-air liquid interface (HNO-ALIs) cell lines with two contemporary RSV isolates and demonstrate how they differ in virus replication, induction of the epithelial cytokine response, cell injury, and remodeling. Pediatric HNO-ALIs were more susceptible to early RSV replication, elicited a greater overall cytokine response, demonstrated enhanced mucous production, and manifested greater cellular damage compared to their adult counterparts. Adult HNO-ALIs displayed enhanced mucus production and robust cytokine response that was well controlled by superior regulatory cytokine response and possibly resulted in lower cellular damage than in pediatric lines. Taken together, our data suggest substantial differences in how pediatric and adult upper respiratory tract epithelium responds to RSV infection. These differences in epithelial cellular response can lead to poor mucociliary clearance and predispose infants to a worse respiratory outcome of RSV infection.

Visualization and Lineage Tracing of Pax7+ Spermatogonial Stem Cells in the Mouse.

The precise identity of spermatogonial stem cells-the germline stem cell of the adult testis-remains a controversial topic. Technical limitations have included the lack of specific markers and methods for lineage tracing of Asingle spermatogonia and their subsets. Immunolocalization of proteins in tissue sections has been a standard tool for the in situ identification and visualization of rare cellular subsets. However, these studies are limited by the need for faithful and reliable protein markers to define these cell types, as well as the availability of specific antibodies to these markers. Here we describe the use of a monoclonal antibody to Pax7 as a means to detect spermatogonial stem cells (SSCs) both in tissue sections and in intact seminiferous tubules. Furthermore, we describe methods for lineage tracing as an alternative method to visualize Pax7+ spermatogonial stem cells and their progeny.

Regulation of FOXO3 subcellular localization by Kit ligand in the neonatal mouse ovary.

PURPOSE: Foxo3 protein is required in the oocyte nucleus for the maintenance of primordial follicles in a dormant state. PI3K/AKT-dependent phosphorylation of Foxo3 leads to its relocalization to the cytoplasm and subsequent follicular activation. However, the nature of the upstream signals controlling Foxo3 activity and subcellular localization remains unknown. We aimed to study the in vitro effects of Kit ligand (stem cell factor) on the subcellular localization of Foxo3 in primordial follicles within the postnatal mouse ovary. METHODS: This was an in vitro study using explants of intact neonatal mouse ovaries. The study was performed in laboratory animal facility and basic science research laboratory at a University Hospital. The animals used for this study were FVB mice. Neonatal FVB mice ovaries at postnatal day 7 (PD7) were harvested and incubated in culture medium (DMEM) at 37 °C and 5 % CO(2) for 60-90 min with (n = 3) or without (n = 3) Kit ligand at 150 ng/mL (8 nM). Similar experimental conditions were used to establish a dose-response curve for the effects of Kit ligand and assess the effects of imatinib (small molecule inhibitor of the Kit receptor). Immunofluorescence was used to identify the subcellular location of Foxo3 in oocytes. Proportions of cytoplasmic versus nuclear Foxo3 in primordial follicles were determined. RESULTS: Kit ligand treatment increased the cytoplasmic localization of Foxo3 from 40 % in the untreated ovaries to 74 % in the treated group (p = 0.007 in paired samples and p = 0.03 in unpaired samples). Furthermore, this effect was reversible with imatinib (p = 0.005). A dose-response curve for Kit ligand treatment showed that maximum effect was seen at 150 ng/mL. CONCLUSION: Kit ligand treatment in vitro increases the proportion of cytoplasmic Foxo3 in primordial follicles at PD7, lending support to the idea that Kit receptor/ligand controls Foxo3 activity in the context of primordial follicle activation.

LKB1 loss promotes endometrial cancer progression via CCL2-dependent macrophage recruitment.

Endometrial cancer is the most common gynecologic malignancy and the fourth most common malignancy in women. For most patients in whom the disease is confined to the uterus, treatment results in successful remission; however, there are no curative treatments for tumors that have progressed beyond the uterus. The serine/threonine kinase LKB1 has been identified as a potent suppressor of uterine cancer, but the biological modes of action of LKB1 in this context remain incompletely understood. Here, we have shown that LKB1 suppresses tumor progression by altering gene expression in the tumor microenvironment. We determined that LKB1 inactivation results in abnormal, cell-autonomous production of the inflammatory cytokine chemokine (C-C motif) ligand 2 (CCL2) within tumors, which leads to increased recruitment of macrophages with prominent tumor-promoting activities. Inactivation of Ccl2 in an Lkb1-driven mouse model of endometrial cancer slowed tumor progression and increased survival. In human primary endometrial cancers, loss of LKB1 protein was strongly associated with increased CCL2 expression by tumor cells as well as increased macrophage density in the tumor microenvironment. These data demonstrate that CCL2 is a potent effector of LKB1 loss in endometrial cancer, creating potential avenues for therapeutic opportunities.

PAX7 expression defines germline stem cells in the adult testis.

Spermatogenesis is a complex, multistep process that maintains male fertility and is sustained by rare germline stem cells. Spermatogenic progression begins with spermatogonia, populations of which express distinct markers. The identity of the spermatogonial stem cell population in the undisturbed testis is controversial due to a lack of reliable and specific markers. Here we identified the transcription factor PAX7 as a specific marker of a rare subpopulation of A(single) spermatogonia in mice. PAX7+ cells were present in the testis at birth. Compared with the adult testis, PAX7+ cells constituted a much higher percentage of neonatal germ cells. Lineage tracing in healthy adult mice revealed that PAX7+ spermatogonia self-maintained and produced expanding clones that gave rise to mature spermatozoa. Interestingly, in mice subjected to chemotherapy and radiotherapy, both of which damage the vast majority of germ cells and can result in sterility, PAX7+ spermatogonia selectively survived, and their subsequent expansion contributed to the recovery of spermatogenesis. Finally, PAX7+ spermatogonia were present in the testes of a diverse set of mammals. Our data indicate that the PAX7+ subset of A(single) spermatogonia functions as robust testis stem cells that maintain fertility in normal spermatogenesis in healthy mice and mediate recovery after severe germline injury, such as occurs after cancer therapy.

Gonadal expression of Foxo1, but not Foxo3, is conserved in diverse Mammalian species.

The Foxos are key effectors of the PI3K/Akt signaling pathway and regulate diverse physiologic processes. Two of these factors, Foxo1 and Foxo3, serve specific roles in reproduction in the mouse. Foxo3 is required for suppression of primordial follicle activation in females, while Foxo1 regulates spermatogonial stem cell maintenance in males. In the mouse ovary, Foxo1 is highly expressed in somatic cells (but not in oocytes), suggesting an important functional role for Foxo1 in these cells. Given that invertebrate model species such as Caenorhabditis elegans and Drosophila melanogaster harbor a single ancestral Foxo homolog, these observations suggest that gene duplication conferred a selective advantage by permitting the Foxos to adopt distinct roles in oogenesis and spermatogenesis. Our objective was to determine if the remarkably specific expression patterns of Foxo1 and Foxo3 in mouse gonads (and, by inference, Foxo function) are conserved in diverse mammalian species. Western blotting was used to validate isoform-specific antibodies in rodents, companion animals, farm animals, nonhuman primates, and humans. Following validation of each antibody, immunohistochemistry was performed to ascertain Foxo1 and Foxo3 gonadal expression patterns. While Foxo1 expression in spermatogonia and granulosa cells was conserved in each species evaluated, Foxo3 expression in oocytes was not. Our findings suggest that Foxo3 is not uniquely required for primordial follicle maintenance in nonrodent species and that other Foxos, particularly Foxo1, may contribute to oocyte maintenance in a functionally redundant manner.