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Sphingosine-1-phosphate (S1P) is a potent lipid signaling molecule that regulates pleiotropic biological functions including cell migration, survival, angiogenesis, immune cell trafficking, inflammation, and carcinogenesis. It acts as a ligand for a family of cell surface receptors. S1P concentrations are high in blood and lymph but low in tissues, especially the thymus and lymphoid organs. S1P chemotactic gradients are essential for lymphocyte egress and other aspects of physiological cell trafficking. S1P is irreversibly degraded by S1P lyase (SPL). SPL regulates lymphocyte trafficking, inflammation and other physiological and pathological processes. For example, SPL located in thymic dendritic cells acts as a metabolic gatekeeper that controls the normal egress of mature T lymphocytes from the thymus into the circulation, whereas SPL deficiency in gut epithelial cells promotes colitis and colitis-associated carcinogenesis (CAC). Recently, we identified a complex syndrome comprised of nephrosis, adrenal insufficiency, and immunological defects caused by inherited mutations in human SGPL1, the gene encoding SPL. In the present article, we review current evidence supporting the role of SPL in thymic egress, inflammation, and cancer. Lastly, we summarize recent progress in understanding other SPL functions, its role in inherited disease, and SPL targeting for therapeutic purposes.

Consumption of probiotics for health purposes has increased vastly in the past few decades, and yet the scientific evidence to support health benefits from probiotics is only beginning to emerge. As more probiotics are studied, we are beginning to understand the mechanisms of action by which they benefit human health, as well as to identify the bacterial molecules responsible for these benefits. A new era of therapeutics is on the horizon in which purified molecules from probiotics will be used to prevent and treat diseases. In this review, we summarize the active molecules from probiotic bacteria that have been shown to affect innate and adaptive immunity and have health benefits in experimental settings. We focus particularly on the cellular and molecular mechanisms of the probiotic Bacillus subtilis and its active molecule, exopolysaccharide (ESPBs).

Mutations underlying disease in tuberous sclerosis complex (TSC) give rise to tumors with biallelic mutations in TSC1 or TSC2 and hyperactive mammalian target of rapamycin complex 1 (mTORC1). Benign tumors might exhibit de novo expression of immunogens, targetable by immunotherapy. As tumors may rely on ganglioside D3 (GD3) expression for mTORC1 activation and growth, we compared GD3 expression in tissues from patients with TSC and controls. GD3 was overexpressed in affected tissues from patients with TSC and also in aging Tsc2+/ – mice. As GD3 overexpression was not accompanied by marked natural immune responses to the target molecule, we performed preclinical studies with GD3 chimeric antigen receptor (CAR) T cells. Polyfunctional CAR T cells were cytotoxic toward GD3-overexpressing targets. In mice challenged with Tsc2 –/– tumor cells, CAR T cells substantially and durably reduced the tumor burden, correlating with increased T cell infiltration. We also treated aged Tsc2+/ – heterozygous (>60 weeks) mice that carry spontaneous Tsc2 –/– tumors with GD3 CAR or untransduced T cells and evaluated them at endpoint. Following CAR T cell treatment, the majority of mice were tumor free while all control animals carried tumors. The outcomes demonstrate a strong treatment effect and suggest that targeting GD3 can be successful in TSC.

Probiotics are a class of microorganisms can offer a health benefit to the host. How these microorganisms alter the host remains a mystery. The scientific group of Dr. Katherine L. Knight, Ph.D. studies the probiotic bacterium Bacillus subtilis. Previous reports from the Knight Laboratory showed that B. subtilis can prevent disease caused by the colonic pathogen Citrobacter rodentium. This protection is mediated by the production of exopolysaccharide (EPS). Intraperitoneal administration of purified EPS is sufficient to protect mice from colitis disease. Furthermore, EPS is protective in disease models of sepsis, allergic eosinophilia, and graft versus host disease, indicating that EPS from B. subtilis has anti-inflammatory properties. In this dissertation, we determined the chemical and structural characteristics of EPS and how EPS can generate an anti-inflammatory response. Our results showed that EPS is a complex carbohydrate with a composition of arabinose, mannose, glucose, galactose, N-acetyl glucosamine, and N-acetyl galactosamine. The backbone sugar is decorated with arabinosyl oligosaccharides that by themselves have lymphocyte inhibitory activity. EPS can generate inhibitory bone marrow-derived dendritic cells that inhibit pan- T cell activation. This inhibition is mediated by the activation and cross-linking of the Toll-like receptor 4 (TLR4), which initiates the activation of the canonical NF-kB pathway. The transcription factors in this pathway transcribe the immune-suppressive enzyme indolamine 2,3-dioxygenase (IDO), and by using ido1-/- mice and IDO inhibitors, we showed that IDO was required for EPS inhibition of T cell proliferation. IDO metabolizes the essential amino acid tryptophan into kynurenine metabolites, and depletion of tryptophan in the microenvironment and the formation of kynurenines lead to the inhibition of T cell activation. Furthermore, IDO expression induces the formation of the IDO-Kynurenine-AhR loop which further extends the anti-inflammatory response of EPS. By examining how EPS induces an anti-inflammatory response through TLR4, we found that EPS does not internalize CD14, a TLR4 co-receptor of LPS, but instead EPS internalizes the receptors CD11b and Dectin-1. Both CD11b and Dectin-1can shape TLR4 signaling into an anti-inflammatory response while blocking the pro-inflammatory branch. We hypothesize that EPS acts through these receptors in conjunction with TLR4. Data in this thesis demonstrate that carbohydrate from a gram-positive bacterium activates TLR4 on dendritic cells and induces expression of IDO, which inhibits T cell proliferation. By identifying and understanding active molecules from probiotics, a new class of therapeutics to treat and prevent diseases will likely become available.

Duchenne muscular dystrophy (DMD) is a congenital myopathy caused by mutations in the dystrophin gene. DMD pathology is marked by myositis, muscle fiber degeneration, and eventual muscle replacement by fibrosis and adipose tissue. Satellite cells (SC) are muscle stem cells critical for muscle regeneration. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that promotes SC proliferation, regulates lymphocyte trafficking, and is irreversibly degraded by sphingosine phosphate lyase (SPL). Here, we show that SPL is virtually absent in normal human and murine skeletal muscle but highly expressed in inflammatory infiltrates and degenerating fibers of dystrophic DMD muscle. In mdx mice that model DMD, high SPL expression is correlated with dysregulated S1P metabolism. Perinatal delivery of the SPL inhibitor LX2931 to mdx mice augmented muscle S1P and SC numbers, reduced leukocytes in peripheral blood and skeletal muscle, and attenuated muscle inflammation and degeneration. The effect on SC was also observed in SCID/mdx mice that lack mature T and B lymphocytes. Transcriptional profiling in the skeletal muscles of LX2931-treated vs. control mdx mice demonstrated changes in innate and adaptive immune functions, plasma membrane interactions with the extracellular matrix (ECM), and axon guidance, a known function of SC. Our cumulative findings suggest that by raising muscle S1P and simultaneously disrupting the chemotactic gradient required for lymphocyte egress, SPL inhibition exerts a combination of muscle-intrinsic and systemic effects that are beneficial in the context of muscular dystrophy.