Activity-Induced DNA Breaks Govern the Expression of Neuronal Early-Response Genes.

Neuronal activity causes the rapid expression of immediate early genes that are crucial for experience-driven changes to synapses, learning, and memory. Here, using both molecular and genome-wide next-generation sequencing methods, we report that neuronal activity stimulation triggers the formation of DNA double strand breaks (DSBs) in the promoters of a subset of early-response genes, including Fos, Npas4, and Egr1. Generation of targeted DNA DSBs within Fos and Npas4 promoters is sufficient to induce their expression even in the absence of an external stimulus. Activity-dependent DSB formation is likely mediated by the type II topoisomerase, Topoisomerase IIß (Topo IIß), and knockdown of Topo IIß attenuates both DSB formation and early-response gene expression following neuronal stimulation. Our results suggest that DSB formation is a physiological event that rapidly resolves topological constraints to early-response gene expression in neurons.

TGF-ß-producing regulatory B cells induce regulatory T cells and promote transplantation tolerance.

Regulatory B (Breg) cells have been shown to play a critical role in immune homeostasis and in autoimmunity models. We have recently demonstrated that combined anti-T cell immunoglobulin domain and mucin domain-1 and anti-CD45RB antibody treatment results in tolerance to full MHC-mismatched islet allografts in mice by generating Breg cells that are necessary for tolerance. Breg cells are antigen-specific and are capable of transferring tolerance to untreated, transplanted animals. Here, we demonstrate that adoptively transferred Breg cells require the presence of regulatory T (Treg) cells to establish tolerance, and that adoptive transfer of Breg cells increases the number of Treg cells. Interaction with Breg cells in vivo induces significantly more Foxp3 expression in CD4(+) CD25(-) T cells than with naive B cells. We also show that Breg cells express the TGF-ß associated latency-associated peptide and that Breg-cell mediated graft prolongation post-adoptive transfer is abrogated by neutralization of TGF-ß activity. Breg cells, like Treg cells, demonstrate preferential expression of both C-C chemokine receptor 6 and CXCR3. Collectively, these findings suggest that in this model of antibody-induced transplantation tolerance, Breg cells promote graft survival by promoting Treg-cell development, possibly via TGF-ß production.

Profiling DNA break sites and transcriptional changes in response to contextual fear learning.

Neuronal activity generates DNA double-strand breaks (DSBs) at specific loci in vitro and this facilitates the rapid transcriptional induction of early response genes (ERGs). Physiological neuronal activity, including exposure of mice to learning behaviors, also cause the formation of DSBs, yet the distribution of these breaks and their relation to brain function remains unclear. Here, following contextual fear conditioning (CFC) in mice, we profiled the locations of DSBs genome-wide in the medial prefrontal cortex and hippocampus using γH2AX ChIP-Seq. Remarkably, we found that DSB formation is widespread in the brain compared to cultured primary neurons and they are predominately involved in synaptic processes. We observed increased DNA breaks at genes induced by CFC in neuronal and non-neuronal nuclei. Activity-regulated and proteostasis-related transcription factors appear to govern some of these gene expression changes across cell types. Finally, we find that glia but not neurons have a robust transcriptional response to glucocorticoids, and many of these genes are sites of DSBs. Our results indicate that learning behaviors cause widespread DSB formation in the brain that are associated with experience-driven transcriptional changes across both neuronal and glial cells.

B-cell depletion improves islet allograft survival with anti-CD45RB.

A short course of anti-CD45RB leads to long-term islet allograft survival and donor-specific tolerance in approximately half of immunocompetent mice. We have previously demonstrated that anti-CD45RB antibody-mediated tolerance requires B-cells for cardiac allograft survival. We therefore asked whether B-cells were also required for anti-CD45RB antibody-mediated survival of islets. Unexpectedly, we found that nearly 100% of islet allografts survive long term in B-cell-deficient mice. Similarly, B-cell depletion by anti-CD22/cal augmented anti-CD45RB-mediated tolerance when administered pretransplant, although it had no effect on tolerance induction when administered posttransplant. Our results demonstrate that the role of B-cells in promoting tolerance with anti-CD45RB is graft specific, promoting tolerance in cardiac grafts but resisting tolerance in islet transplantation. These findings may help elucidate the varied action of B-cells in promoting tolerance versus rejection.

Site of origin of and sex differences in the vasopressin innervation of the mouse (Mus musculus) brain.

Defining how arginine vasopressin (AVP) acts centrally to regulate homeostasis and behavior is problematic, as AVP is made in multiple nuclei in the hypothalamus (i.e., paraventricular [PVN], supraoptic [SON], and suprachiasmatic [SCN]) and extended amygdala (i.e., bed nucleus of the stria terminalis [BNST] and medial amygdala [MeA]), and these groups of neurons have extensive projections throughout the brain. To understand the function of AVP, it is essential to know the site of origin of various projections. In mice, we used gonadectomy to eliminate gonadal steroid hormone-dependent expression of AVP in the BNST and MeA and electrolytic lesions to eliminate the SCN, effectively eliminating those AVP-immunoreactive projections; we also quantified AVP-immunoreactive fiber density in gonadectomized and sham-operated male and female mice to examine sex differences in AVP innervation. Our results suggest that the BNST/MeA AVP system innervates regions containing major modulatory neurotransmitters (e.g., serotonin and dopamine) and thus may be involved in regulating behavioral state. Furthermore, this system may be biased toward the regulation of male behavior, given the numerous regions in which males have a denser AVP-immunoreactive innervation than females. AVP from the SCN is found in regions important for the regulation of hormone output and behavior. Innervation from the PVN and SON is found in brain regions that likely work in concert with the well-known peripheral AVP actions of controlling homeostasis and stress response; female-biased sex differences in this system may be related to the heightened stress response observed in females.

Elevated levels of interferon-γ production by memory T cells do not promote transplant tolerance resistance in aged recipients.

Immunosenescence predisposes the elderly to infectious and autoimmune diseases and impairs the response to vaccination. We recently demonstrated that ageing also impedes development of transplantation tolerance. Unlike their young counterparts (8-12 weeks of age) aged male recipients (greater than 12 months of age) transplanted with a full MHC-mismatched heart are resistant to tolerance mediated by anti-CD45RB antibody. Surprisingly, either chemical or surgical castration restored tolerance induction to levels observed using young recipients. Based on the strong impact of endocrine modulation on transplant tolerance, we explored the impact of ageing and castration on the immune system. Here we report a significant increase in the percentage of T cells that produce interferon-γ (IFN-γ) in aged male versus young male animals and that the overall increase in IFN-γ production was due to an expansion of IFN-γ-producing memory T cells in aged animals. In contrast to IFN-γ production, we did not observe differences in IL-10 expression in young versus old male mice. We hypothesized that endocrine modulation would diminish the elevated levels of IFN-γ production in aged recipients, however, we observed no significant reduction in the percentage of IFN-γ+ T cells upon castration. Furthermore, we neutralized interferon-γ by antibody and did not observe an effect on graft survival. We conclude that while elevated levels of interferon-γ serves as a marker of tolerance resistance in aged mice, other as yet to be identified factors are responsible for its cause. Defining these factors may be relevant to design of tolerogenic strategies for aged recipients.