Adrian Erlebacher

Toward a molecular understanding of skeletal development

Much of our knowledge about cartilage and bone has come from descriptive anatomy, endocrinology, and cellular studies of bone turnover. Recent approaches have led to the identification of local factors that regulate skeletal morphogenesis. Molecular and biochemical studies of bone and cartilage cells in vitro, gene inactivation in mice, and the identification of genes responsible for mouse and human skeletal abnormalities have documented the importance of specific growth and differentiation factors, extracellular matrix proteins, signaling mediators, and transcription factors in bone and cartilage development. The successful convergence of mouse and human genetics in skeletal biology is illustrated in this issue of Cell with two papers that show that mutations in collagen type Xl cause chondrodysplasia both in cho/cho mice as well as in patients with Stickler syndrome (Li et al., 1995; Vikkula et al., 1995). In general, recent results emphasize the need to view skeletal development at various integrated levels of organization and illustrate how single gene products affect development at these different levels. Pattern information determines not only the body plan of the early skeleton but also the shape of each individual skeletal element. In addition, the sequence of events during bone growth and development must be temporally and spatially controlled to ensure correct proportions of bony elements. Positional information must also regulate the establishment of bone internal structure throughout growth, while local homeostatic mechanisms must maintain bone integrity throughout adult life. Lastly, a complex extracellular matrix must generate skeletal tissues with specific biomechanical properties. Ultimately, the morphogenesis of the skeleton derives from the regulated differentiation, function, and interactions of its component cell types. Three major cell types contribute to the skeleton: chondrocytes, which form cartilage; osteoblasts, which deposit bone matrix; and osteoclasts, which resorb bone. Chondrocytes and osteoblasts are of mesenchymal origin, whereas osteoclasts derive from the hematopoietic system. Once embedded in bone matrix, osteoblasts mature into terminally differentiated osteocytes. The activity and differentiation of osteoblasts and osteoclasts are closely coordinated during development as bone is formed and during growth and adult life as bone undergoes continuous remodeling. More specifically, the formation of internal bone structures and bone remodeling result from coupling bone resorption by activated osteoclasts with subsequent deposition of new matrix by osteoblasts (Figure 1). Bone remodeling also links bone turnover to the endocrine homeostasis of calcium and phosphorus, since the mineralized bone matrix serves as the major repository for these ions in the body. Descriptive embryology and anatomy distinguish two types of bone development: intramembranous and endochondral. Intramembranous ossification occurs when mesenchymal precursor cells differentiate directly into bone-forming osteoblasts, a process employed in generating the flat bones of the skull as well as in adding new bone to the outer surfaces of long bones. In contrast, endochondral bone formation entails the conversion of an initial cartilage template into bone and is responsible for generating most bones of the skeleton. Cartilage templates originally form during embryogenesis when mesenchymal cells condense and then differentiate into chondrocytes. These cells subsequently undergo a program of hypertrophy, calcification, and cell death. Concomitant neovascularization occurs, and osteoclasts and osteoblasts are recruited to replace the cartilage scaffold gradually with bone matrix and to excavate the bone marrow cavity. Longitudinal bone growth takes place through a similar pattern of endochondral ossification in the growth plates located at the epiphyses (ends) of long bones. In these epiphyseal plates, the calcified, hypertrophic cartilage provides a scaffold for the formation of new trabecular bone. Ultimately, all remaining cartilage is replaced by bone except at the articular surfaces of the joints (Figure 2). Skeletal Patterning Classical embryology has shown that three distinct embryonic lineages contribute to the early skeleton. The neural crest gives rise to the branchial arch derivatives of the craniofacial skeleton, the sclerotome generates most of the axial skeleton, and the lateral plate mesoderm forms the appendicular skeleton. Transplantation studies have indicated that information regarding the number and ana-

IMMUNOLOGY OF THE MATERNAL-FETAL INTERFACE

The immune cells that reside at the interface between the placenta and uterus are thought to play many important roles in pregnancy. Recent work has revealed that the composition and function of these cells are locally controlled by the specialized uterine stroma (the decidua) that surrounds the implanted conceptus. Here, I discuss how key immune cell types (natural killer cells, macrophages, dendritic cells, and T cells) are either enriched or excluded from the decidua, how their function is regulated within the decidua, and how they variously contribute to pregnancy success or failure. The discussion emphasizes the relationship between human and mouse studies. Deeper understanding of the immunology of the maternal-fetal interface promises to yield significant insight into the pathogenesis of many human pregnancy complications, including preeclampsia, intrauterine growth restriction, spontaneous abortion, preterm birth, and congenital infection.

MiR-30b/30d regulation of GalNAc transferases enhances invasion and immunosuppression during metastasis

To metastasize, a tumor cell must acquire abilities such as the capacity to colonize new tissue and evade immune surveillance. Recent evidence suggests that microRNAs can promote the evolution of malignant behaviors by regulating multiple targets. We performed a microRNA analysis of human melanoma, a highly invasive cancer, and found that miR-30b/30d upregulation correlates with stage, metastatic potential, shorter time to recurrence, and reduced overall survival. Ectopic expression of miR-30b/30d promoted the metastatic behavior of melanoma cells by directly targeting the GalNAc transferase GALNT7, resulted in increased synthesis of the immunosuppressive cytokine IL-10, and reduced immune cell activation and recruitment. These data support a key role of miR-30b/30d and GalNAc transferases in metastasis, by simultaneously promoting cellular invasion and immunosuppression.

Chemokine Gene Silencing in Decidual Stromal Cells Limits T Cell Access to the Maternal-Fetal Interface

Keeping Baby Safe Because half the genes from a developing fetus are inherited from the father, from the mothers perspective, a fetus is “foreign.” How, then, does the maternal immune system tolerate the fetus? Nancy et al. (p. 1317) found that careful regulation of cellular recruitment signals allowed for fetal tolerance in mice. Although high numbers of T cells localized to the myometrium layer of the uterine wall in pregnant mice, very few T cells were found in the decidua, the uterine tissue that encapsulates the fetus and placenta. Thus, in pregnancy, regulation of immune cell localization may allow for organ-specific immune tolerance. Turning off the expression in the placenta of T cell attractants allows the mother to tolerate the fetus. The chemokine-mediated recruitment of effector T cells to sites of inflammation is a central feature of the immune response. The extent to which chemokine expression levels are limited by the intrinsic developmental characteristics of a tissue has remained unexplored. We show in mice that effector T cells cannot accumulate within the decidua, the specialized stromal tissue encapsulating the fetus and placenta. Impaired accumulation was in part attributable to the epigenetic silencing of key T cell–attracting inflammatory chemokine genes in decidual stromal cells, as evidenced by promoter accrual of repressive histone marks. These findings give insight into mechanisms of fetomaternal immune tolerance, as well as reveal the epigenetic modification of tissue stromal cells as a modality for limiting effector T cell trafficking.

Dendritic cell entrapment within the pregnant uterus inhibits immune surveillance of the maternal/fetal interface in mice.

Embryo implantation induces formation of the decidua, a stromal cell-derived structure that encases the fetus and placenta. Using the mouse as a model organism, we have found that this tissue reaction prevents DCs stationed at the maternal/fetal interface from migrating to the lymphatic vessels of the uterus and thus reaching the draining lymph nodes. Strikingly, decidual DCs remained immobile even after being stimulated with LPS and exhibiting responsiveness to CCL21, the chemokine that drives DC entry into lymphatic vessels. An analysis of maternal T cell reactivity toward a surrogate fetal/placental antigen furthermore revealed that regional T cell responses toward the fetus and placenta were driven by passive antigen transport and thus the tolerogenic mode of antigen presentation that predominates when there is negligible input from tissue-resident DCs. Indeed, the lack of involvement of tissue-resident DCs in the T cell response to the fetal allograft starkly contrasts with their prominent role in organ transplant rejection. Our results suggest that DC entrapment within the decidua minimizes immunogenic T cell exposure to fetal/placental antigens and raise the possibility that impaired development or function of the human decidua, which unlike that of the mouse contains lymphatic vessels, might lead to pathological T cell activation during pregnancy.

Ovarian insufficiency and early pregnancy loss induced by activation of the innate immune system

We describe a murine model of early pregnancy failure induced by systemic activation of the CD40 immune costimulatory pathway. Although fetal loss involved an NK cell intermediate, it was not due to lymphocyte-mediated destruction of the fetus and placenta. Rather, pregnancy failure resulted from impaired progesterone synthesis by the corpus luteum of the ovary, an endocrine defect in turn associated with ovarian resistance to the gonadotropic effects of prolactin. Pregnancy failure also required the proinflammatory cytokine TNF-alpha and correlated with the luteal induction of the prolactin receptor signaling inhibitors suppressor of cytokine signaling 1 (Socs1) and Socs3. Such links between immune activation and reproductive endocrine dysfunction may be relevant to pregnancy loss and other clinical disorders of reproduction.

Coordinate regulation of tissue macrophage and dendritic cell population dynamics by CSF-1

CSF-1 drives the homeostatic expansion of macrophages within the growing myometrium of pregnant mice by stimulating in situ proliferation and inducing monocyte precursor recruitment from the blood.

Immune mechanisms at the maternal-fetal interface: perspectives and challenges

Leaders gathered at the US National Institutes of Health in November 2014 to discuss recent advances and emerging research areas in aspects of maternal-fetal immunity that may affect fetal development and pregnancy success.

Why isn't the fetus rejected?

The long-standing question of how the fetal allograft avoids immune rejection during pregnancy has lately been generating renewed interest. Recent insights have emerged from studies in mice on uterine NK cells, NKT cells, complement inhibition and the reproductive effects of 1-methyl-tryptophan.

Differential transcription of Eomes and T-bet during maturation of mouse uterine natural killer cells

During human and rodent uterine decidualization, transient but abundant numbers of uterine natural killer (uNK) cells appear, proliferate, and differentiate. uNK cells share features with peripheral NK cells but are specialized to promote interferon‐γ (IFN‐γ)‐mediated, pregnancy‐associated, structural changes in maternal placental arteries. In CD8+ T cells and NK cells, the transcription factors T‐bet and eomesodermin (Eomes) regulate maturation and effector functions, including IFN‐γ production. No studies are reported for uNK cells. Implantation sites in T‐bet null mice, which have a defect in NK cell maturation, had uNK cells normal in morphology and number and normally modified spiral arteries. As Eomes null mice are not viable, real‐time polymerase chain reaction comparisons between C57Bl/6J (B6) and alymphoid (Rag20/0γc0/0) mice were used to assess uNK cell expression of T‐bet, Eomes, and the target genes IFN‐γ, granzyme A, and perforin. Gestation dated (gd) uterine tissues (mixed cell composition) and 200 morphologically homogeneous, laser‐capture, microdissected uNK cells of different maturation stages were used. In uterus, Eomes transcripts greatly outnumbered those of T‐bet, whether donors were nonpregnant or pregnant, and increased to gd10. In uNK cells, transcripts for T‐bet, Eomes, and IFN‐γ were most abundant in mature stage cells, and transcripts for granzyme A and perforin were lower at this stage than in immature or senescent cells. Thus, Eomes dominance to T‐bet discriminates regulation of the uNK cell subset from that observed for peripheral NK cells.