Lysosomal amino acid transporters as key players in inflammatory diseases.

Controlling inflammation can alleviate immune-mediated, lifestyle-related and neurodegenerative diseases. The endolysosome system plays critical roles in inflammatory responses. Endolysosomes function as signal transduction hubs to convert various environmental danger signals into gene expression, enabling metabolic adaptation of immune cells and efficient orchestration of inflammation. Solute carrier family 15 member A3 (SLC15A3) and member A4 (SLC15A4) are endolysosome-resident amino acid transporters that are preferentially expressed in immune cells. These transporters play essential roles in signal transduction through endolysosomes, and the loss of either transporter can alleviate multiple inflammatory diseases because of perturbed endolysosome-dependent signaling events, including inflammatory and metabolic signaling. Here, we summarize the findings leading to a proof-of-concept for anti-inflammatory strategies based on targeting SLC15 transporters.

Glutamine metabolism in Th17/Treg cell fate: applications in Th17 cell-associated diseases.

Alteration in the Th17/Treg cell balance is implicated in various autoimmune diseases and these disease-associated pathologies. Increasing investigations have shown that glutamine metabolism regulates the differentiation of Th17 and Treg cells. Here we summarize the mechanisms by which glutamine metabolism regulates Th17/Treg cell fate. Some examples of a glutamine metabolism-dependent modulation of the development and progression of several Th17 Treg cell-associated diseases are provided afterward. This review will provide a comprehensive understanding of the importance of glutamine metabolism in the fate of Th17 Treg cell differentiation.

Nutrient mTORC1 signaling underpins regulatory T cell control of immune tolerance.

Foxp3+ regulatory T (T reg) cells are pivotal regulators of immune tolerance, with T cell receptor (TCR)-driven activated T reg (aT reg) cells playing a central role; yet how TCR signaling propagates to control aT reg cell responses remains poorly understood. Here we show that TCR signaling induces expression of amino acid transporters, and renders amino acid-induced activation of mTORC1 in aT reg cells. T reg cell-specific ablation of the Rag family small GTPases RagA and RagB impairs amino acid-induced mTORC1 signaling, causing defective amino acid anabolism, reduced T reg cell proliferation, and a rampant autoimmune disorder similar in severity to that triggered by T reg cell-specific TCR deficiency. Notably, T reg cells in peripheral tissues, including tumors, are more sensitive to Rag GTPase-dependent nutrient sensing. Ablation of RagA alone impairs T reg cell accumulation in the tumor, resulting in enhanced antitumor immunity. Thus, nutrient mTORC1 signaling is an essential component of TCR-initiated T reg cell reprogramming, and Rag GTPase activities may be titrated to break tumor immune tolerance.

Detrimental Effects of Induced Antibodies on Aedes aegypti Reproduction.

Aedes aegypti (Linnaeus) (Diptera: Culicidae) is the main vector of viruses causing dengue, chikungunya, Zika, and yellow fever, worldwide. This report focuses on immuno-blocking four critical proteins in the female mosquito when fed on blood containing antibodies against ferritin, transferrin, one amino acid transporter (NAAT1), and acetylcholinesterase (AchE). Peptides from these proteins were selected, synthetized, conjugated to carrier proteins, and used as antigens to immunize New Zealand rabbits. After rabbits were immunized, a minimum of 20 female mosquitos were fed on each rabbit, per replicate. No effect in their viability was observed after blood-feeding; however, the number of infertile females was 20% higher than the control when fed on AchE-immunized rabbits. The oviposition period was significantly longer in females fed on immunized rabbits than those fed on control (non-immunized) rabbits. Fecundity (eggs/female) of treated mosquitoes was significantly reduced (about 50%) in all four treatments, as compared with the control. Fertility (hatched larvae) was also significantly reduced in all four treatments, as compared with the control, being the effect on AchE and transferrin the highest, by reducing hatching between 70 and 80%. Survival to the adult stage of the hatched larvae showed no significant effect, as more than 95% survival was observed in all treatments, including the control. In conclusion, immuno-blocking of these four proteins caused detrimental effects on the mosquito reproduction, being the effect on AchE the most significant.

Immunotargeting of Antigen xCT Attenuates Stem-like Cell Behavior and Metastatic Progression in Breast Cancer.

Resistance to therapy and lack of curative treatments for metastatic breast cancer suggest that current therapies may be missing the subpopulation of chemoresistant and radioresistant cancer stem cells (CSC). The ultimate success of any treatment may well rest on CSC eradication, but specific anti-CSC therapies are still limited. A comparison of the transcriptional profiles of murine Her2(+) breast tumor TUBO cells and their derived CSC-enriched tumorspheres has identified xCT, the functional subunit of the cystine/glutamate antiporter system xc(-), as a surface protein that is upregulated specifically in tumorspheres. We validated this finding by cytofluorimetric analysis and immunofluorescence in TUBO-derived tumorspheres and in a panel of mouse and human triple negative breast cancer cell-derived tumorspheres. We further show that downregulation of xCT impaired tumorsphere generation and altered CSC intracellular redox balance in vitro, suggesting that xCT plays a functional role in CSC biology. DNA vaccination based immunotargeting of xCT in mice challenged with syngeneic tumorsphere-derived cells delayed established subcutaneous tumor growth and strongly impaired pulmonary metastasis formation by generating anti-xCT antibodies able to alter CSC self-renewal and redox balance. Finally, anti-xCT vaccination increased CSC chemosensitivity to doxorubicin in vivo, indicating that xCT immunotargeting may be an effective adjuvant to chemotherapy.

Toxoplasma gondii is dependent on glutamine and alters migratory profile of infected host bone marrow derived immune cells through SNAT2 and CXCR4 pathways.

The obligate intracellular parasite, Toxoplasma gondii, disseminates through its host inside infected immune cells. We hypothesize that parasite nutrient requirements lead to manipulation of migratory properties of the immune cell. We demonstrate that 1) T. gondii relies on glutamine for optimal infection, replication and viability, and 2) T. gondii-infected bone marrow-derived dendritic cells (DCs) display both "hypermotility" and "enhanced migration" to an elevated glutamine gradient in vitro. We show that glutamine uptake by the sodium-dependent neutral amino acid transporter 2 (SNAT2) is required for this enhanced migration. SNAT2 transport of glutamine is also a significant factor in the induction of migration by the small cytokine stromal cell-derived factor-1 (SDF-1) in uninfected DCs. Blocking both SNAT2 and C-X-C chemokine receptor 4 (CXCR4; the unique receptor for SDF-1) blocks hypermotility and the enhanced migration in T. gondii-infected DCs. Changes in host cell protein expression following T. gondii infection may explain the altered migratory phenotype; we observed an increase of CD80 and unchanged protein level of CXCR4 in both T. gondii-infected and lipopolysaccharide (LPS)-stimulated DCs. However, unlike activated DCs, SNAT2 expression in the cytosol of infected cells was also unchanged. Thus, our results suggest an important role of glutamine transport via SNAT2 in immune cell migration and a possible interaction between SNAT2 and CXCR4, by which T. gondii manipulates host cell motility.

[An efficient method for producing monoclonal antibodies against multi-pass membrane proteins].

Antibodies have greatly contributed to the development of medical science and pharmacology, because of their high specificity. The cell fusion method has developed monoclonal antibodies (mAb) technology, such that massive amounts of mAb with a uniform structure can be produced. Although mAb have been produced against many proteins so far, the production of mAb against multi-pass transmembrane proteins, such as G-protein coupled receptor (GPCR) and various transporter proteins has been extremely difficult. The complicated structures, poorly extracellular regions, and high hydrophobicity of multiple-transmembrane proteins make it difficult to produce mAb against them. Production of mAb that recognize the extracellular region of living cells is thought to be important in determining the ability of a protein. Based on these findings, we tried to produce mAb against a multi-pass transmembrane transporter using green fluorescent protein (GFP)-fused full-length target proteins as immunogens. Furthermore, the immunizing method has proved to be important in generating functional mAb. We succeeded in producing functional mAb that react against the extracellular region of a 12-pass transmembrane transporter in a living cell. Based on this success, we began to produce mAb against seven-transmembrane GPCR. In this symposium, we report on the results of producing mAb against S1P receptors, a type of GPCR.

Immunohistochemical localization of the amino acid transporter SNAT2 in the rat brain.

SNAT2 is a neutral amino acid carrier that belongs to the system A family. Since its function in the nervous system remains unclear, we have analyzed its distribution in the rat CNS using specific antisera. Although SNAT2 is expressed widely in the CNS, it is enriched in the spinal cord and the brainstem nuclei, especially those of the auditory system. At the cellular level, SNAT2 was preferentially located in neuronal cell bodies and processes, although it was also strongly expressed in the meninges and ependyma. In astrocytes, the localization of SNAT2 was more restricted since it was intensely expressed in the perivascular end-feet, glia limitans, cerebellar astrocytes and Bergmann glia, but it was less intense in astrocytes of the cerebral parenchyma. Among neurons, the primary sensory neurons of the mesencephalic trigeminal nucleus appeared to be those that most strongly express SNAT2, but many other neurons, including cortical pyramidal cells and their dendrites were also intensely stained. In several regions the transporter was detected in axons, especially in the brainstem, and its presence in both dendrites and axons was confirmed by confocal microscopy and ultrastructural studies. However, while SNAT2 was observed in the large principal dendrites and the small distal dendrites, it was only found in axonal shafts and was excluded from terminals. Some glutamatergic neurons were among the more intensely labeled cells whereas SNAT2 was not detected on GABAergic neurons. The expression of SNAT2 partially coincides with that reported for SNAT1, especially in glutamatergic neurons. Hence, both proteins could fulfill complementary roles in replenishing glutamate pools and be differentially regulated under different physiological conditions. They also seem to co-localize in non-neuronal cells probably contributing to amino acid fluxes through the blood-brain barrier.