Jami Bennett

Local self-renewal can sustain CNS microglia maintenance and function throughout adult life

Microgliosis is a common response to multiple types of damage in the CNS. However, the origin of the cells involved in this process is still controversial and the relative importance of local expansion versus recruitment of microglia progenitors from the bloodstream is unclear. Here, we investigated the origin of microglia using chimeric animals obtained by parabiosis. We found no evidence of microglia progenitor recruitment from the circulation in denervation or CNS neurodegenerative disease, suggesting that maintenance and local expansion of microglia are solely dependent on the self-renewal of CNS resident cells in these models.

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.

Blood-brain barrier disruption and enhanced vascular permeability in the multiple sclerosis model EAE

Multiple sclerosis (MS) is a demyelinating disease characterized by the breakdown of the blood-brain barrier (BBB), and accumulation of inflammatory infiltrates in the central nervous system. Tight junctions are specialized cell-cell adhesion structures and critical components of the BBB that have previously been shown to be abnormally distributed in MS tissue. To evaluate whether experimental autoimmune encephalomyelitis (EAE) provides a suitable model for this aspect of MS disease, we examined the expression and distribution of ZO-1 over the course of disease in EAE. We observed a dramatic relocalization of ZO-1 which precedes overt clinical disease and correlates with the sites of inflammatory cell accumulation. Treatment of in vitro cultures of murine brain endothelial cells with components of EAE induction provided similar findings, with relocalization of ZO-1 and increased permeability of endothelial monolayers. BBB disruption in the EAE model appears to parallel disease progression in MS, with direct effects on the cerebrovascular endothelium, making it an ideal tool for future evaluation of tight junction breakdown and repair in MS-like pathology.

Bone Marrow-Derived Mast Cells Accumulate in the Central Nervous System During Inflammation but Are Dispensable for Experimental Autoimmune Encephalomyelitis Pathogenesis

Reports showing that W/Wv mice are protected from experimental autoimmune encephalomyelitis (EAE, a murine model of multiple sclerosis), have implicated mast cells as an essential component in disease susceptibility, but the role of mast cell trafficking has not been addressed. In this study, we have used both mast cell transplantation and genetic mutations (Cd34−/−, W/Wv, Wsh/Wsh) to investigate the role of mast cell trafficking in EAE in detail. We show, for the first time, that bone marrow-derived mast cells are actively recruited to the CNS during EAE. Unexpectedly, however, we found that EAE develops unabated in two independent genetic backgrounds in the complete absence of mast cells or bone marrow-derived mast cell reconstitution. We conclude that although mast cells do accumulate in the brain and CNS during demyelinating disease via peripheral mast cell trafficking, they are completely dispensable for development of disease.

iPS Cells: Mapping the Policy Issues

Given the explosion of research on induced pluripotent stem (iPS) cells, it is timely to consider the various ethical, legal, and social issues engaged by this fast-moving field. Here, we review issues associated with the procurement, basic research, and clinical translation of iPS cells.

CD34 Is Required for Dendritic Cell Trafficking and Pathology in Murine Hypersensitivity Pneumonitis

RATIONALE Although recent work has shown that CD34 plays an important role in the trafficking of inflammatory cells during Th2-biased inflammatory responses, its role in Th1/Th17-biased disease as well as dendritic cell (DC) trafficking is unknown. OBJECTIVES We used CD34-deficient mice (Cd34(-/-)) to investigate the role of CD34 in the Th1/Th17-biased lung inflammatory disease, hypersensitivity pneumonitis (HP). METHODS HP was induced in wild-type (wt) and Cd34(-/-) mice by repeated intranasal administration of Saccharopolyspora rectivirgula antigen. Lung inflammation was assessed by histology and analysis of bronchoalveolar lavage cells. Primary and secondary immune responses were evaluated by cytokine recall responses of pulmonary inflammatory cells as well as draining lymph node cells. MEASUREMENTS AND MAIN RESULTS Cd34(-/-) mice were highly resistant to the development of HP and exhibited an inflammatory pattern more reflective of a primary response to S. rectivirgula rather than the chronic lymphocytosis that is typical of this disease. Cytokine recall responses from Cd34(-/-) lymph node cells were dampened and consistent with a failure of antigen-loaded Cd34(-/-) DCs to deliver antigen and prime T cells in the draining lymph nodes. In agreement with this interpretation, adoptive transfer of wt DCs into Cd34(-/-) mice was sufficient to restore normal sensitivity to HP. CD34 was found to be expressed by wt DCs, and Cd34(-/-) DCs exhibited an impaired ability to chemotax toward a subset of chemokines in vitro. Finally, expression of human CD34 in Cd34(-/-) mice restored normal susceptibility to HP. CONCLUSIONS We conclude that CD34 is expressed by mucosal DCs and plays an important role in their trafficking through the lung and to the lymph nodes. Our data also suggest that CD34 may play a selective role in the efficient migration of these cells to a subset of chemokines.

Loss of CD34 Leads To Exacerbated Autoimmune Arthritis through Increased Vascular Permeability

CD34 is a cell surface sialomucin expressed by hematopoietic precursors, eosinophils, mast cells, and vascular endothelia and is suggested to play an integral role in mucosal inflammatory responses. Although Cd34−/− mice have normal hematopoietic cell subsets in peripheral tissues at steady state, they exhibit a cell recruitment defect when challenged, offering a unique opportunity to distinguish between local inflammatory cell proliferation and peripheral recruitment in disease. Autoimmune arthritis is an inflammatory disease dependent on hematopoietic infiltration, and in this study, we have examined the role of CD34 in disease development and progression. Using an autoimmune serum transfer model, arthritis was induced in C57BL/6 wild-type and Cd34−/− mice. Surprisingly, we found that Cd34−/− mice were more susceptible to arthritis than wild-type mice. We examined mast cell-transplanted, eosinophil-deficient, and bone marrow-chimeric mice to determine the role of CD34 expression on disease progression. These experiments excluded CD34-deficient mast cells, eosinophils, or hematopoietic cells as the cause of the exacerbated disease. Further study demonstrated that Cd34−/− mice exhibit increased vascular leakage at onset of disease and in response to TNF, which correlated with a subsequent increase in disease severity. We conclude that loss of CD34 expression leads to increased vascular permeability in the joints at onset of disease, leading to exacerbated arthritic disease in Cd34−/− mice.

NUP98-HOXA10hd-Expanded Hematopoietic Stem Cells Efficiently Reconstitute Bone Marrow of Mismatched Recipients and Induce Tolerance:

Gene therapy as well as methods capable of returning cells to a pluripotent state (iPS) have enabled the correction of genetic deficiencies in syngenic adult progenitors, reducing the need for immunosuppression in cell therapy approaches. However, in diseases involving mutations that lead to the complete lack of a protein, such as Duchenne muscular dystrophy, the main immunogens leading to rejection of transplanted cells are the therapeutic proteins themselves. In these cases even iPS cells would not circumvent the need for immunosuppression, and alternative strategies must be developed. One such potential strategy seeks to induce immune tolerance using hematopoietic stem cells originated from the same donor or iPS line from which the therapeutic progenitors are derived. However, donor hematopoietic stem cells (HSCs) are available in limiting numbers and embryonic stem (ES) cell-derived HSCs engraft poorly in adults. While these limitations have been circumvented by ectopic expression of HOXB4, overexpression of this protein is associated with inefficient lymphoid reconstitution. Here we show that adult HSCs expanded with a NUP98-HOXA10hd fusion protein sustain long-term engraftment in immunologically mismatched recipients and generate normal numbers of lymphoid cells. In addition, NUP98-HOXA10hd-expanded cells induce functional immune tolerance to a subsequent transplant of myogenic progenitors immunologically matched with the transplanted HSCs.

IL-7Rα and L-selectin, but not CD103 or CD34, are required for murine peanut-induced anaphylaxis

BackgroundAllergy to peanuts results in severe anaphylactic responses in affected individuals, and has dramatic effects on society and public policy. Despite the health impacts of peanut-induced anaphylaxis (PIA), relatively little is known about immune mechanisms underlying the disease. Using a mouse model of PIA, we evaluated mice with deletions in four distinct immune molecules (IL7Rα, L-selectin, CD34, CD103), for perturbed responses.MethodsPIA was induced by intragastric sensitization with peanut antigen and cholera toxin adjuvant, followed by intraperitoneal challenge with crude peanut extract (CPE). Disease outcome was assessed by monitoring body temperature, clinical symptoms, and serum histamine levels. Resistant mice were evaluated for total and antigen specific serum IgE, as well as susceptibility to passive systemic anaphylaxis.ResultsPIA responses were dramatically reduced in IL7Rα−/− and L-selectin−/− mice, despite normal peanut-specific IgE production and susceptibility to passive systemic anaphylaxis. In contrast, CD34−/− and CD103−/− mice exhibited robust PIA responses, indistinguishable from wild type controls.ConclusionsLoss of L-selectin or IL7Rα function is sufficient to impair PIA, while CD34 or CD103 ablation has no effect on disease severity. More broadly, our findings suggest that future food allergy interventions should focus on disrupting sensitization to food allergens and limiting antigen-specific late-phase responses. Conversely, therapies targeting immune cell migration following antigen challenge are unlikely to have significant benefits, particularly considering the rapid kinetics of PIA.

The changing landscape of human-animal chimera research: A Canadian regulatory perspective.

Human stem cell research is at the forefront of ethical debate, perhaps because of the potential of these cells to effect biological change. In addition to the well-known controversy concerning the permissibility of deriving stem cells from human embryos, the question of whether experiments involving manipulation of cells in this domain should be constrained by species boundaries between the human and the nonhuman animals has arisen. Progressive accomplishments in scientific discovery and technology have initiated distinct lines of discussion regarding hybrid and chimera regulations: (1) If embryonic stem cell research is legal and ethically acceptable, how will the materials for study or therapy be obtained? (2) Does inducible pluripotency of differentiated cells circumvent the ethical challenge of obtaining embryonic material? (3) Is it ethically acceptable to utilize hybrids and chimeras of human and