Collaboration between a cis-interacting natural killer cell receptor and membrane sphingolipid is critical for the phagocyte function.

Inhibitory natural killer (NK) cell receptors recognize MHC class I (MHC-I) in trans on target cells and suppress cytotoxicity. Some NK cell receptors recognize MHC-I in cis, but the role of this interaction is uncertain. Ly49Q, an atypical Ly49 receptor expressed in non-NK cells, binds MHC-I in cis and mediates chemotaxis of neutrophils and type I interferon production by plasmacytoid dendritic cells. We identified a lipid-binding motif in the juxtamembrane region of Ly49Q and found that Ly49Q organized functional membrane domains comprising sphingolipids via sulfatide binding. Ly49Q recruited actin-remodeling molecules to an immunoreceptor tyrosine-based inhibitory motif, which enabled the sphingolipid-enriched membrane domain to mediate complicated actin remodeling at the lamellipodia and phagosome membranes during phagocytosis. Thus, Ly49Q facilitates integrative regulation of proteins and lipid species to construct a cell type-specific membrane platform. Other Ly49 members possess lipid binding motifs; therefore, membrane platform organization may be a primary role of some NK cell receptors.

SLC15A4 mediates M1-prone metabolic shifts in macrophages and guards immune cells from metabolic stress.

The amino acid and oligopeptide transporter Solute carrier family 15 member A4 (SLC15A4), which resides in lysosomes and is preferentially expressed in immune cells, plays critical roles in the pathogenesis of lupus and colitis in murine models. Toll-like receptor (TLR)7/9- and nucleotide-binding oligomerization domain-containing protein 1 (NOD1)-mediated inflammatory responses require SLC15A4 function for regulating the mechanistic target of rapamycin complex 1 (mTORC1) or transporting L-Ala-γ-D-Glu-meso-diaminopimelic acid, IL-12: interleukin-12 (Tri-DAP), respectively. Here, we further investigated the mechanism of how SLC15A4 directs inflammatory responses. Proximity-dependent biotin identification revealed glycolysis as highly enriched gene ontology terms. Fluxome analyses in macrophages indicated that SLC15A4 loss causes insufficient biotransformation of pyruvate to the tricarboxylic acid cycle, while increasing glutaminolysis to the cycle. Furthermore, SLC15A4 was required for M1-prone metabolic change and inflammatory IL-12 cytokine productions after TLR9 stimulation. SLC15A4 could be in close proximity to AMP-activated protein kinase (AMPK) and mTOR, and SLC15A4 deficiency impaired TLR-mediated AMPK activation. Interestingly, SLC15A4-intact but not SLC15A4-deficient macrophages became resistant to fluctuations in environmental nutrient levels by limiting the use of the glutamine source; thus, SLC15A4 was critical for macrophage's respiratory homeostasis. Our findings reveal a mechanism of metabolic regulation in which an amino acid transporter acts as a gatekeeper that protects immune cells' ability to acquire an M1-prone metabolic phenotype in inflammatory tissues by mitigating metabolic stress.

The inhibitory NK receptor Ly49Q protects plasmacytoid dendritic cells from pyroptotic cell death.

Ly49Q is an ITIM-bearing MHC class I receptor that is highly expressed in plasmacytoid dendritic cells (pDCs). Ly49Q is required for the TLR9-mediated IFN-I production in pDCs, although the mechanism is not fully understood. We here demonstrate that Ly49Q protects pDCs from pyroptotic cell death induced by CpG oligodeoxynucleotides (CpG). In the Ly49Q-deficient (Klra17-/-) mouse spleen, the number of ssDNA-positive pDCs increased significantly after CpG treatment, strongly suggesting that Klra17-/- pDCs were susceptible to CpG-induced cell death. In Klra17-/- bone-marrow-derived dendritic cells (BMDCs), CpG-induced cell death was accompanied by increased cathepsin B leakage from the vesicular compartments into the cytoplasm. Concurrently, IL-1ß secretion increased in the CpG-treated Klra17-/- BMDCs, strongly suggesting that the CpG-induced cell death in these cells is pyroptotic in nature. Consistent with these observations, inhibiting cathepsin B or caspase 1 in CpG-stimulated Klra17-/- BMDCs reversed the increase in cell death. Pyroptotic cell death and IL-1ß secretion were also observed in BMDCs derived from transgenic mice expressing an ITIM-less Ly49Q (Ly49Q-YF Tg). CpG also increased the IL-1ß production and cell death in B2m-/- BMDCs. These results suggest that Ly49Q and MHC class I play important roles for protecting pyroptosis-like cell death of DCs by influencing lysosome state.

Human SLC15A4 is crucial for TLR-mediated type I interferon production and mitochondrial integrity.

Solute carrier family 15 member 4 (SLC15A4) is an endolysosome-resident amino acid transporter that regulates innate immune responses, and is genetically associated with inflammatory diseases such as systemic lupus erythematosus (SLE) and colitis. SLC15A4-deficient mice showed the amelioration of symptoms of these model diseases, and thus SLC15A4 is a promising therapeutic target of SLE and colitis. For developing a SLC15A4-based therapeutic strategy, understanding human SLC15A4's properties is essential. Here, we characterized human SLC15A4 and demonstrated that human SLC15A4 possessed pH- and temperature-dependent activity for the transportation of dipeptides or tripeptides. Human SLC15A4 localized in LAMP1+ compartments and constitutively associated with Raptor and LAMTORs. We also investigated SLC15A4's role in inflammatory responses using the human plasmacytoid dendritic cell line, CAL-1. Knock down (KD) of the SLC15A4 gene in CAL-1 (SLC15A4-KD CAL-1) impaired Toll-like receptor (TLR) 7/8 or TLR9-triggered type I interferon (IFN-I) production and mTORC1 activity, indicating that human SLC15A4 is critical for TLR7/8/9-mediated inflammatory signaling. We also examined SLC15A4's role in the autophagy response since SLC15A4 loss caused the decrease of mTORC1 activity, which greatly influences autophagy. We found that SLC15A4 was not required for autophagy induction, but was critical for autophagy sustainability. Notably, SLC15A4-KD CAL-1 severely decreased mitochondrial membrane potential in starvation conditions. Our findings revealed that SLC15A4 plays a key role in mitochondrial integrity in human cells, which might benefit immune cells in fulfilling their functions in an inflammatory milieu.

Lysosome biogenesis regulated by the amino-acid transporter SLC15A4 is critical for functional integrity of mast cells.

Mast cells possess specialized lysosomes, so-called secretory granules, which play a key role not only in allergic responses but also in various immune disorders. The molecular mechanisms that control secretory-granule formation are not fully understood. Solute carrier family member 15A4 (SLC15A4) is a lysosome-resident amino-acid/oligopeptide transporter that is preferentially expressed in hematopoietic lineage cells. Here, we demonstrated that SLC15A4 is required for mast-cell secretory-granule homeostasis, and limits mast-cell functions and inflammatory responses by controlling the mTORC1-TFEB signaling axis. In mouse Slc15a4-/- mast cells, diminished mTORC1 activity increased the expression and nuclear translocation of TFEB, a transcription factor, which caused secretory granules to degranulate more potently. This alteration of TFEB function in mast cells strongly affected the FcεRI-mediated responses and IL-33-triggered inflammatory responses both in vitro and in vivo. Our results reveal a close relationship between SLC15A4 and secretory-granule biogenesis that is critical for the functional integrity of mast cells.

The histidine transporter SLC15A4 coordinates mTOR-dependent inflammatory responses and pathogenic antibody production.

SLC15A4 is a lysosome-resident, proton-coupled amino-acid transporter that moves histidine and oligopeptides from inside the lysosome to the cytosol of eukaryotic cells. SLC15A4 is required for Toll-like receptor 7 (TLR7)- and TLR9-mediated type I interferon (IFN-I) productions in plasmacytoid dendritic cells (pDCs) and is involved in the pathogenesis of certain diseases including lupus-like autoimmunity. How SLC15A4 contributes to diseases is largely unknown. Here we have shown that B cell SLC15A4 was crucial for TLR7-triggered IFN-I and autoantibody productions in a mouse lupus model. SLC15A4 loss disturbed the endolysosomal pH regulation and probably the v-ATPase integrity, and these changes were associated with disruption of the mTOR pathway, leading to failure of the IFN regulatory factor 7 (IRF7)-IFN-I regulatory circuit. Importantly, SLC15A4's transporter activity was necessary for the TLR-triggered cytokine production. Our findings revealed that SLC15A4-mediated optimization of the endolysosomal state is integral to a TLR7-triggered, mTOR-dependent IRF7-IFN-I circuit that leads to autoantibody production.