Indiana University-Purdue University Indianapolis (IUPUI) / Autonomic innervation of the heart begins in utero and continues during the neonatal phase of life. A balance between the sympathetic and parasympathetic arms of the autonomic nervous system is required to regulate heart rate as well as the force of each contraction. Our lab studies the development of sympathetic innervation of the early postnatal heart in a conditional knockout (cKO) of Src homology protein tyrosine phosphatase 2 (Shp2). Shp2 is a ubiquitously expressed non-receptor phosphatase involved in a variety of cellular functions including survival, proliferation, and differentiation. We targeted Shp2 in post-migratory neural crest (NC) lineages using our novel Periostin-Cre. This resulted in a fully penetrant mouse model of diminished cardiac sympathetic innervation and concomitant bradycardia that progressively worsen. Shp2 is thought to mediate its basic cellular functions through a plethora of signaling cascades including extracellular signal-regulated kinases (ERK) 1 and 2. We hypothesize that abrogation of downstream ERK1/2 signaling in NC lineages is primarily responsible for the failed sympathetic innervation phenotype observed in our mouse model. Shp2 cKOs are indistinguishable from control littermates at birth and exhibit no gross structural cardiac anomalies; however, in vivo electrocardiogram (ECG) characterization revealed sinus bradycardia that develops as the Shp2 cKO ages. Significantly, 100% of Shp2 cKOs die within 3 weeks after birth. Characterization of the expression pattern of the sympathetic nerve marker tyrosine hydroxylase (TH) revealed a loss of functional sympathetic ganglionic neurons and reduction of cardiac sympathetic axon density in Shp2 cKOs. Shp2 cKOs exhibit lineage-specific suppression of activated pERK1/2 signaling, but not of other downstream targets of Shp2 such as pAKT (phosphorylated-Protein kinase B). Interestingly, restoration of pERK signaling via lineage-specific expression of constitutively active MEK1 (Mitogen-activated protein kinase kinase1) rescued TH-positive cardiac innervation as well as heart rate. These data suggest that the diminished sympathetic cardiac innervation and the resulting ECG abnormalities are a result of decreased pERK signaling in post-migratory NC lineages.

Indiana University-Purdue University Indianapolis (IUPUI) / Neurofibromatosis Type 1 (NF1) is a genetic disorder resulting from mutations in the NF1 tumor suppressor gene. Neurofibromin is the protein product of NF1 and functions as a negative regulator of Ras activity in both hematopoietic and vascular wall cells, which are critical for maintaining blood vessel homeostasis. NF1 patients are predisposed to chronic inflammation and premature cardiovascular disease, including development of large arterial aneurysms, which may result in sudden death secondary to their rupture. However, the molecular pathogenesis of NF1 aneurysm formation is completely unknown. Utilizing a novel model of Nf1 murine aneurysm formation, we demonstrate that heterozygous inactivation of Nf1 (Nf1+/-) results in enhanced aneurysm formation with myeloid cell infiltration and increased reactive oxygen species in the vessel wall. Using cell lineage-restricted transgenic mice, we show that loss of a single Nf1 allele in myeloid cells is sufficient to recapitulate the Nf1+/- aneurysm phenotype in vivo. Additionally, oral administration of simvastatin, a statin with antioxidant and anti-inflammatory effects, significantly reduced aneurysm formation in Nf1+/- mice. Finally, the antioxidant apocynin was administered orally and also resulted in a significant reduction of Nf1+/- aneurysms. These data provide genetic and pharmacologic evidence that neurofibromin-deficient myeloid cells are the central cellular triggers for aneurysm formation in a novel model of NF1 vascular disease, implicated oxidative stress as the key biochemical mechanisms of NF1 aneurysm formation and provide a potential therapeutic target for NF1 vasculopathy.

Indiana University-Purdue University Indianapolis (IUPUI) / The transcriptional repressor BCL6 has been shown to be essential for the differentiation of germinal center (GC) B cells and follicular T helper (TFH) cells. The interaction of TFH and GC B cells is necessary for the development of high affinity antibodies specific for an invading pathogen. Germline BCL6-deficient mouse models limit our ability to study BCL6 function in T cells due to the strong inflammatory responses seen in these mice. To overcome this, our lab has developed a new BCL6 conditional knockout (cKO) mouse using the cre/lox system, wherein the zinc finger region of the BCL6 gene is flanked by loxP sites. Mating to a CD4-Cre mouse allowed us to study the effects of BCL6 loss specifically in T cells, without the confounding effects seen in germline knockout models. Using this cKO model, we have reaffirmed the necessity of BCL6 for TFH differentiation, including its role in sustained CXCR5 surface expression, a signature marker for TFH cells. This model also allowed us to recognize the role of BCL6 in promoting the expression of PD-1, another key surface marker for TFH cells. Without BCL6, CD4+ T cells cannot express PD-1 at the high levels seen on TFH cells. Our discovery of DNMT3b as a target for BCL6 suggests BCL6-deficient T cells have increased DNA methyltransferase activity at the PD-1 promoter. This data establishes a novel pathway for explaining how BCL6, a transcriptional repressor, can activate genes. Experiments with the BCL6 cKO model have also established a role for BCL6 in naïve CD4+ T cell activation. Furthermore, we did not observe increased differentiation of other helper T cell subsets, in contrast to what has been reported elsewhere with germline BCL6-deficient models. Unexpectedly, we found decreased T helper type 2 (Th2) cells, whereas mouse models with a germline mutation of BCL6 have increased Th2 cells. These results indicate that BCL6 activity in non-T cells is critical for controlling T cell differentiation. Finally, using an HIV-1 gp120 immunization model, we have, for the first time, shown BCL6-dependent GCs to be limiting for antibody development and affinity maturation in a prime-boost vaccine scheme.

Indiana University-Purdue University Indianapolis (IUPUI) / Regulatory T (Treg) cells represent an important layer of immune-regulation indispensible for curtailing exuberant inflammatory responses and maintaining self-tolerance. Treg cells have translational potential for autoimmunity, inflammation, transplantation and cancer. Therefore, delineating the molecular underpinnings underlying the development, suppressor function and stability of Tregs is particularly warranted. The transcriptional repressor Bcl6 is a critical arbiter of helper T cell fate, promoting the follicular helper (Tfh) lineage while repressing Th1, Th2 and Th17 differentiation. Bcl6-deficient mice develop a spontaneous and severe Th2-type inflammatory disease including myocarditis and pulmonary vasculitis, suggesting a potential role for Bcl6 in Treg cell function. Bcl6-deficient Treg cells are competent in controlling Th1 responses, but fail to control Th2 inflammation in an airway allergen model. Importantly, mice with Bcl6 deleted specifically in the Treg lineage develop severe myocarditis, thus highlighting a critical role for Bcl6 in Treg-mediated control of Th2 inflammation. Bcl6-deficient Tregs display an intrinsic increase in Th2 genes and microRNA-21 (miR-21) expression. MiR-21 is a novel Bcl6 gene target in T cells and ectopic expression of miR-21 directs Th2 differentiation in non-polarized T cells. MiR-21 is up-regulated in mouse models of airway inflammation and also in human patients with eosinophilic esophagitis and asthma. Thus, miR-21 is a clinically relevant biomarker for Th2-type pathologies. Our results define a key function for Bcl6 in repressing Gata3 function and miR-21 expression in Tregs, and provide greater understanding of the control of Th2 inflammatory responses by Treg cells.