Kelly M. McNagny

Infiltrating monocytes trigger EAE progression, but do not contribute to the resident microglia pool

In multiple sclerosis and the experimental autoimmune encephalitis (EAE) mouse model, two pools of morphologically indistinguishable phagocytic cells, microglia and inflammatory macrophages, accrue from proliferating resident precursors and recruitment of blood-borne progenitors, respectively. Whether these cell types are functionally equivalent is hotly debated, but is challenging to address experimentally. Using a combination of parabiosis and myeloablation to replace circulating progenitors without affecting CNS-resident microglia, we found a strong correlation between monocyte infiltration and progression to the paralytic stage of EAE. Inhibition of chemokine receptor–dependent recruitment of monocytes to the CNS blocked EAE progression, suggesting that these infiltrating cells are essential for pathogenesis. Finally, we found that, although microglia can enter the cell cycle and return to quiescence following remission, recruited monocytes vanish, and therefore do not ultimately contribute to the resident microglial pool. In conclusion, we identified two distinct subsets of myelomonocytic cells with distinct roles in neuroinflammation and disease progression.

Group 2 Innate Lymphoid Cells Are Critical for the Initiation of Adaptive T Helper 2 Cell-Mediated Allergic Lung Inflammation

Summary Naive CD4+ T cell differentiation into distinct subsets of T helper (Th) cells is a pivotal process in the initiation of the adaptive immune response. Allergens predominantly stimulate Th2 cells, causing allergic inflammation. However, why allergens induce Th2 cell differentiation is not well understood. Here we show that group 2 innate lymphoid cells (ILC2s) are required to mount a robust Th2 cell response to the protease-allergen papain. Intranasal administration of papain stimulated ILC2s and Th2 cells, causing allergic lung inflammation and elevated immunoglobulin E titers. This process was severely impaired in ILC2-deficient mice. Whereas interleukin-4 (IL-4) was dispensable for papain-induced Th2 cell differentiation, ILC2-derived IL-13 was critical as it promoted migration of activated lung dendritic cells into the draining lymph node where they primed naive T cells to differentiate into Th2 cells. Papain-induced ILC2 activation and Th2 cell differentiation was IL-33-dependent, suggesting a common pathway in the initiation of Th2 cell responses to allergen.

Early life antibiotic-driven changes in microbiota enhance susceptibility to allergic asthma

Allergic asthma rates have increased steadily in developed countries, arguing for an environmental aetiology. To assess the influence of gut microbiota on experimental murine allergic asthma, we treated neonatal mice with clinical doses of two widely used antibiotics—streptomycin and vancomycin—and evaluated resulting shifts in resident flora and subsequent susceptibility to allergic asthma. Streptomycin treatment had little effect on the microbiota and on disease, whereas vancomycin reduced microbial diversity, shifted the composition of the bacterial population and enhanced disease severity. Neither antibiotic had a significant effect when administered to adult mice. Consistent with the ‘hygiene hypothesis’, our data support a neonatal, microbiota‐driven, specific increase in susceptibility to experimental murine allergic asthma.

The Molecular Basis of Vascular Lumen Formation in the Developing Mouse Aorta

In vertebrates, endothelial cells (ECs) form blood vessels in every tissue. Here, we investigated vascular lumen formation in the developing aorta, the first and largest arterial blood vessel in all vertebrates. Comprehensive imaging, pharmacological manipulation, and genetic approaches reveal that, in mouse embryos, the aortic lumen develops extracellularly between adjacent ECs. We show that ECs adhere to each other, and that CD34-sialomucins, Moesin, F-actin, and non-muscle Myosin II localize at the endothelial cell-cell contact to define the luminal cell surface. Resultant changes in EC shape lead to lumen formation. Importantly, VE-Cadherin and VEGF-A act at different steps. VE-Cadherin is required for localizing CD34-sialomucins to the endothelial cell-cell contact, a prerequisite to Moesin and F-actin recruitment. In contrast, VEGF-A is required for F-actin-nm-Myosin II interactions and EC shape change. Based on these data, we propose a molecular mechanism of in vivo vascular lumen formation in developing blood vessels.

Retinoic-Acid-Receptor-Related Orphan Nuclear Receptor Alpha Is Required for Natural Helper Cell Development and Allergic Inflammation

Natural helper (NH) cells are innate lymphoid cells (ILCs) that produce T helper-2 (Th2)-cell-type cytokines in the lung- and gut-associated lymphoid tissues. Currently, the lineage relationship between NH cells in different tissues and between NH cells and interleukin-22 (IL-22)-producing retinoic-acid-receptor-related orphan receptor (ROR)γt-positive ILCs is unclear. Here, we report that NH cells express RORα, but not RORγt. RORα-deficient, but not RORγt-deficient, mice lacked NH cells in all tissues, whereas all other lymphocytes, including RORγt(+) ILCs, were unaffected. NH-cell-deficient mice generated by RORα-deficient bone-marrow transplantation had normal Th2 cell responses but failed to develop acute lung inflammation in response to protease allergen, thus confirming the essential role of NH cells in allergic lung inflammation. We have also identified RORα-dependent NH cell progenitors in the bone marrow. Thus, all NH cells belong to a unique RORα-dependent cell lineage separate from other lymphoid cell lineages.

Novel functions of the CD34 family

For almost 30 years, the cell-surface protein CD34 has been widely used as a marker to assist in the identification and isolation of hematopoietic stem cells (HSCs) and progenitors in preparation for bone-marrow transplantation. In addition, it has increasingly been used as a marker to help identify other tissue-specific stem cells, including muscle satellite cells and epidermal precursors. Despite its utility as a stem-cell marker, however, the function of CD34 has remained remarkably elusive. This is probably because: (1) it is subject to a range of tissue-specific post-transcriptional and post-translational modifications that are expected to alter its function dramatically; (2) the simple interpretation of CD34 gain- and loss-of-function experiments has been confounded by the overlapping expression of the two recently discovered CD34-related proteins podocalyxin and endoglycan; and (3) there has been a glaring lack of robust in vitro and in vivo functional assays that permit the structural and functional analysis of CD34 and its relatives. Here, we provide a brief review of the domain structure, genomic organization, and tissue distribution of the CD34 family. We also describe recent insights from gain- and loss-of-function experiments and improved assays, which are elucidating a fascinating role for these molecules in cell morphogenesis and migration.

Mast cells are an essential hematopoietic component for polyp development

It is generally agreed that most colon cancers develop from adenomatous polyps, and it is this fact on which screening strategies are based. Although there is overwhelming evidence to link intrinsic genetic lesions with the formation of these preneoplastic lesions, recent data suggest that the tumor stromal environment also plays an essential role in this disease. In particular, it has been suggested that CD34+ immature myeloid precursor cells are required for tumor development and invasion. Here we have used mice conditional for the stabilization of β-catenin or defective for the adenomatous polyposis coli (APC) gene to reinvestigated the identity and importance of tumor-infiltrating hematopoietic cells in polyposis. We show that, from the onset, polyps are infiltrated with proinflammatory mast cells (MC) and their precursors. Depletion of MC either pharmacologically or through the generation of chimeric mice with genetic lesions in MC development leads to a profound remission of existing polyps. Our data suggest that MC are an essential hematopoietic component for preneoplastic polyp development and are a novel target for therapeutic intervention.

Mast cells in tumor growth: angiogenesis, tissue remodelling and immune-modulation.

There is a growing acceptance that tumor-infiltrating myeloid cells play an active role in tumor growth and mast cells are one of the earliest cell types to infiltrate developing tumors. Mast cells accumulate at the boundary between healthy tissues and malignancies and are often found in close association with blood vessels within the tumor microenvironment. They express many pro-angiogenic compounds, and may play an early role in angiogenesis within developing tumors. Mast cells also remodel extracellular matrix during wound healing, and this function is subverted in tumor growth, promoting tumor spread and metastasis. In addition, mast cells modulate immune responses by dampening immune rejection or directing immune cell recruitment, depending on local stimuli. In this review, we focus on key roles for mast cells in angiogenesis, tissue remodelling and immune modulation and highlight recent findings on the integral role that mast cells play in tumor growth. New findings suggest that mast cells may serve as a novel therapeutic target for cancer treatment and that inhibiting mast cell function may lead to tumor regression.

Overexpression of the Anti-Adhesin Podocalyxin Is an Independent Predictor of Breast Cancer Progression

Podocalyxin is a CD34-related cell surface molecule with anti-adhesive qualities. We probed a tissue microarray (n = 272) linked to long-term outcome data and found that podocalyxin was highly overexpressed in a distinct subset of invasive breast carcinomas (n = 15; 6%). Univariate disease-specific (P < 0.01) and multivariate regression (P < 0.0005) analyses indicated that this overexpression is an independent indicator of poor outcome. Forced podocalyxin expression perturbed cell junctions between MCF-7 breast carcinoma cells, and it caused cell shedding from confluent monolayers. Therefore, podocalyxin overexpression is a novel predictor of breast cancer progression that may contribute to the process by perturbing tumor cell adhesion.

Modelling kidney disease with CRISPR-mutant kidney organoids derived from human pluripotent epiblast spheroids.

Human-pluripotent-stem-cell-derived kidney cells (hPSC-KCs) have important potential for disease modelling and regeneration. Whether the hPSC-KCs can reconstitute tissue-specific phenotypes is currently unknown. Here we show that hPSC-KCs self-organize into kidney organoids that functionally recapitulate tissue-specific epithelial physiology, including disease phenotypes after genome editing. In three-dimensional cultures, epiblast-stage hPSCs form spheroids surrounding hollow, amniotic-like cavities. GSK3β inhibition differentiates spheroids into segmented, nephron-like kidney organoids containing cell populations with characteristics of proximal tubules, podocytes and endothelium. Tubules accumulate dextran and methotrexate transport cargoes, and express kidney injury molecule-1 after nephrotoxic chemical injury. CRISPR/Cas9 knockout of podocalyxin causes junctional organization defects in podocyte-like cells. Knockout of the polycystic kidney disease genes PKD1 or PKD2 induces cyst formation from kidney tubules. All of these functional phenotypes are distinct from effects in epiblast spheroids, indicating that they are tissue specific. Our findings establish a reproducible, versatile three-dimensional framework for human epithelial disease modelling and regenerative medicine applications.