Carlos Zárate-Bladés

Microbiota-Dependent Activation of an Autoreactive T Cell Receptor Provokes Autoimmunity in an Immunologically Privileged Site

Activated retina-specific T cells that have acquired the ability to break through the blood-retinal barrier are thought to be causally involved in autoimmune uveitis, a major cause of human blindness. It is unclear where these autoreactive T cells first become activated, given that their cognate antigens are sequestered within the immune-privileged eye. We demonstrate in a novel mouse model of spontaneous uveitis that activation of retina-specific T cells is dependent on gut commensal microbiota. Retina-specific T cell activation involved signaling through the autoreactive T cell receptor (TCR) in response to non-cognate antigen in the intestine and was independent of the endogenous retinal autoantigen. Our findings not only have implications for the etiology of human uveitis, but also raise the possibility that activation of autoreactive TCRs by commensal microbes might be a more common trigger of autoimmune diseases than is currently appreciated.

The Living Eye “Disarms” Uncommitted Autoreactive T Cells by Converting Them to Foxp3+ Regulatory Cells following Local Antigen Recognition

Immune privilege is used by the eye, brain, reproductive organs, and gut to preserve structural and functional integrity in the face of inflammation. The eye is arguably the most vulnerable and, therefore, also the most “privileged” of tissues; paradoxically, it remains subject to destructive autoimmunity. It has been proposed, although never proven in vivo, that the eye can induce T regulatory cells (Tregs) locally. Using Foxp3-GFP reporter mice expressing a retina-specific TCR, we now show that uncommitted T cells rapidly convert in the living eye to Foxp3+ Tregs in a process involving retinal Ag recognition, de novo Foxp3 induction, and proliferation. This takes place within the ocular tissue and is supported by retinoic acid, which is normally present in the eye because of its function in the chemistry of vision. Nonconverted T cells showed evidence of priming but appeared restricted from expressing effector function in the eye. Pre-existing ocular inflammation impeded conversion of uncommitted T cells into Tregs. Importantly, retina-specific T cells primed in vivo before introduction into the eye were resistant to Treg conversion in the ocular environment and, instead, caused severe uveitis. Thus, uncommitted T cells can be disarmed, but immune privilege is unable to protect from uveitogenic T cells that have acquired effector function prior to entering the eye. These findings shed new light on the phenomenon of immune privilege and on its role, as well as its limitations, in actively controlling immune responses in the tissue.

SDF-1/CXCL12 induces directional cell migration and spontaneous metastasis via a CXCR4/Gαi/mTORC1 axis

Multiple human malignancies rely on C‐X‐C motif chemokine receptor type 4 (CXCR4) and its ligand, SDF‐1/CXCL12 (stroma cell‐derived factor 1/C‐X‐C motif chemokine 12), to metastasize. CXCR4 inhibitors promote the mobilization of bone marrow stem cells, limiting their clinical application for metastasis prevention. We investigated the CXCR4‐initiated signaling circuitry to identify new potential therapeutic targets. We used HeLa human cancer cells expressing high levels of CXCR4 endogenously. We found that CXCL12 promotes their migration in Boyden chamber assays and single cell tracking. CXCL12 activated mTOR (mechanistic target of rapamycin) potently in a pertussis‐sensitive fashion. Inhibition of mTOR complex 1 (mTORC1) by rapamycin [drug concentration causing 50% inhibition (lC50) = 5 nM] and mTORC1/mTORC2 by Torin2 (IC50 = 6 nM), or by knocking down key mTORC1/2 components, Raptor and Rictor, respectively, decreased directional cell migration toward CXCL12. We developed a CXCR4‐mediated spontaneous metastasis model by implanting HeLa cells in the tongue of SCIDNOD mice, in which 80% of the animals develop lymph node metastasis. It is surprising that mTORC1 disruption by Raptor knockdown was sufficient to reduce tumor growth by 60% and spontaneous metastasis by 72%, which were nearly abolished by rapamycin. In contrast, disrupting mTORC2 had no effect in tumor growth or metastasis compared with control short hairpin RNAs. These data suggest that mTORC 1 may represent a suitable therapeutic target in human malignancies using CXCR4 for their meta‐static spread.—Dillenburg‐Pilla, P., Patel, V., Mikelis, C. M., Zárate‐Bladés, C. R., Doçi, C. L., Amornphimoltham, P., Wang, Z., Martin, D., Leelahavanichkul, K., Dorsam, R. T., Masedunskas, A., Weigert, R., Molinolo, A. A., Gutkind, J. S., SDF‐1/CXCL12 induces directional cell migration and spontaneous metastasis via a CXCR4/Gαi/mTORC1 axis. FASEB J. 29, 1056–1068 (2015). www.fasebj.org

Complete Genome Sequence of Turicibacter sp. Strain H121, Isolated from the Feces of a Contaminated Germ-Free Mouse

ABSTRACT Turicibacter bacteria are commonly detected in the gastrointestinal tracts and feces of humans and animals, but their phylogeny, ecological role, and pathogenic potential remain unclear. We present here the first complete genome sequence of Turicibacter sp. strain H121, which was isolated from the feces of a mouse line contaminated following germ-free derivation.

Gut microbiota as a source of a surrogate antigen that triggers autoimmunity in an immune privileged site

ABSTRACT Recent discoveries on the role of commensal microbiota have significantly changed our understanding of human physiology. The host-microbiota interplay is now an important aspect to take into account to understand immune responses and immunological diseases. Autoimmune uveitis is a sight-threatening disease that arises without a known infectious etiology. It is unknown where and how autoreactive T cells become primed to trigger disease in the eye, which is an immune privileged site. We recently reported data supporting the notion that retina-specific T cells receive a signal in the gut from commensal microbiota-derived cross-reactive antigen(s) and trigger autoimmune uveitis in the R161H mouse model. Here we discuss our published findings, as well as our recent attempts to identify the responsible microbe(s) by using different antibiotic treatments, 16S rDNA sequencing and homology searches for candidate antigenic mimic(s) of the retinal antigen.

Abstract 4050: A central role for mTORC1 in CXCR4-mediated directional migration and metastasis

Tumor cells can co-opt the promigratory activity of chemokines and their cognate G protein-coupled receptors to disseminate to regional lymph nodes and distant organs. Indeed, the migration to SDF-1 of tumor cells expressing its receptor, CXCR4, is implicated in lymphatic and organ-specific metastasis in multiple human malignancies. However, the critical role of CXCR4 in bone marrow stem cell retention has hampered the development of CXCR4 inhibitors for metastasis prevention. Therefore the study of CXCR4 downstream signaling circuitry may provide new opportunities for the treatment of many human malignancies that depend on CXCR4 for their metastatic spread. The PI3K/mTOR pathway represents a major player in normal and aberrant cell growth. Consistently, mTOR inhibitors targeting its complex 1 (mTORC1) alone or mTORC1/mTORC2 have already shown promising therapeutic responses in many tumor types. Our data show that mTOR is activated upon SDF-1 stimulation of CXCR4 on epithelial-derived cancer cells, and that the disruption of mTORC1 or mTORC2 abrogates CXCR4-mediated directional migration. To study the CXCR4/mTOR axis in vivo we developed a simple and robust model for CXCR4-mediated spontaneous metastasis. Surprisingly, disruption of mTORC1 alone was sufficient to decrease tumor cell proliferation, angiogenesis, lymphangiogenesis, and CXCR4-mediated metastasis, whereas mTORC2 impairment had no effect on tumor dissemination or growth in vivo. Taken together, our data suggest that mTORC1 blockade could inhibit the migration of CXCR4 expressing cancer cells to their secondary sites, while disrupting the establishment of a permissive tumor microenvironment thereby halting the spread of highly aggressive tumors that require CXCR4 to metastasize. Citation Format: Patricia Dillenburg-Pilla, Vyomesh Patel, Constantinos M. Mikelis, Carlos Rodrigo Zarate-Blades, Panomwat Amornphimoltham, Zhiyong Wang, Daniel Martin, Kantima Leelahavanichkul, Colleen L. Doci, Robert T. Dorsam, Andrius Masedunskas, Nijiro Nohata , Roberto Weigert, Alfredo A. Molinolo, J. Silvio Gutkind . A central role for mTORC1 in CXCR4-mediated directional migration and metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4050. doi:10.1158/1538-7445.AM2014-4050