The role of extracellular ATP in homeostatic immune cell migration.

Antigen stimulation induces adenosine triphosphate (ATP) release from naïve lymphocytes in lymphoid tissues. However, previous studies indicated that the non-lytic release of ATP also occurs in most tissues and cell types under physiological conditions. Here, we show that extracellular ATP (eATP) is indeed constitutively produced by naïve T cells in response to lymphoid chemokines in uninflamed lymph nodes and is involved in the regulation of immune cell migration. In this review, we briefly summarize the homeostatic role of extracellular ATP in immune cell migration in vivo.

The mechanosensitive ion channel PIEZO1 promotes satellite cell function in muscle regeneration.

Muscle satellite cells (MuSCs), myogenic stem cells in skeletal muscles, play an essential role in muscle regeneration. After skeletal muscle injury, quiescent MuSCs are activated to enter the cell cycle and proliferate, thereby initiating regeneration; however, the mechanisms that ensure successful MuSC division, including chromosome segregation, remain unclear. Here, we show that PIEZO1, a calcium ion (Ca2+)-permeable cation channel activated by membrane tension, mediates spontaneous Ca2+ influx to control the regenerative function of MuSCs. Our genetic engineering approach in mice revealed that PIEZO1 is functionally expressed in MuSCs and that Piezo1 deletion in these cells delays myofibre regeneration after injury. These results are, at least in part, due to a mitotic defect in MuSCs. Mechanistically, this phenotype is caused by impaired PIEZO1-Rho signalling during myogenesis. Thus, we provide the first concrete evidence that PIEZO1, a bona fide mechanosensitive ion channel, promotes proliferation and regenerative functions of MuSCs through precise control of cell division.

Prevention of Shiga toxin 1-caused colon injury by plant-derived recombinant IgA.

Immunoglobulin A (IgA) is a candidate antibody for oral passive immunization against mucosal pathogens like Shiga toxin-producing Escherichia coli (STEC). We previously established a mouse IgG monoclonal antibody (mAb) neutralizing Shiga toxin 1 (Stx1), a bacterial toxin secreted by STEC. We designed cDNA encoding an anti-Stx1 antibody, in which variable regions were from the IgG mAb and all domains of the heavy chain constant region from a mouse IgA mAb. Considering oral administration, we expressed the cDNA in a plant expression system aiming at the production of enough IgA at low cost. The recombinant-IgA expressed in Arabidopsis thaliana formed the dimeric IgA, bound to the B subunit of Stx1, and neutralized Stx1 toxicity to Vero cells. Colon injury was examined by exposing BALB/c mice to Stx1 via the intrarectal route. Epithelial cell death, loss of crypt and goblet cells from the distal colon were observed by electron microscopy. A loss of secretory granules containing MUC2 mucin and activation of caspase-3 were observed by immunohistochemical methods. Pretreatment of Stx1 with the plant-based recombinant IgA completely suppressed caspase-3 activation and loss of secretory granules. The results indicate that a plant-based recombinant IgA prevented colon damage caused by Stx1 in vivo.

Pyruvate enhances oral tolerance via GPR31.

CX3CR1high myeloid cells in the small intestine mediate the induction of oral tolerance by driving regulatory T (Treg) cells. Bacterial metabolites, e.g. pyruvate and lactate, induce a dendrite extension of CX3CR1high myeloid cells into the intestinal lumen via GPR31. However, it remains unclear whether the pyruvate-GPR31 axis is involved in the induction of oral tolerance. Here, we show that pyruvate enhances oral tolerance in a GPR31-dependent manner. In ovalbumin (OVA)-fed Gpr31-deficient mice, an OVA-induced delayed-type hypersensitivity response was substantially induced, demonstrating the defective induction of oral tolerance in Gpr31-deficient mice. The percentage of RORγt+ Treg cells in the small intestine was reduced in Gpr31-deficient mice. In pyruvate-treated wild-type mice, a low dose of OVA efficiently induced oral tolerance. IL-10 production from intestinal CX3CR1high myeloid cells was increased by OVA ingestion in wild-type mice, but not in Gpr31-deficient mice. CX3CR1high myeloid cell-specific IL-10-deficient mice showed a defective induction of oral tolerance to OVA and a decreased accumulation of OVA-specific Treg cells in the small intestine. These findings demonstrate that pyruvate enhances oral tolerance through a GPR31-dependent effect on intestinal CX3CR1high myeloid cells.

Extracellular ATP Limits Homeostatic T Cell Migration Within Lymph Nodes.

Whereas adenosine 5'-triphosphate (ATP) is the major energy source in cells, extracellular ATP (eATP) released from activated/damaged cells is widely thought to represent a potent damage-associated molecular pattern that promotes inflammatory responses. Here, we provide suggestive evidence that eATP is constitutively produced in the uninflamed lymph node (LN) paracortex by naïve T cells responding to C-C chemokine receptor type 7 (CCR7) ligand chemokines. Consistently, eATP was markedly reduced in naïve T cell-depleted LNs, including those of nude mice, CCR7-deficient mice, and mice subjected to the interruption of the afferent lymphatics in local LNs. Stimulation with a CCR7 ligand chemokine, CCL19, induced ATP release from LN cells, which inhibited CCR7-dependent lymphocyte migration in vitro by a mechanism dependent on the purinoreceptor P2X7 (P2X7R), and P2X7R inhibition enhanced T cell retention in LNs in vivo. These results collectively indicate that paracortical eATP is produced by naïve T cells in response to constitutively expressed chemokines, and that eATP negatively regulates CCR7-mediated lymphocyte migration within LNs via a specific subtype of ATP receptor, demonstrating its fine-tuning role in homeostatic cell migration within LNs.

Microbiota-derived butyrate limits the autoimmune response by promoting the differentiation of follicular regulatory T cells.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic debilitating autoimmune disorder with a high prevalence, especially in industrialized countries. Dysbiosis of the intestinal microbiota has been observed in RA patients. For instance, new-onset untreated RA (NORA) is associated with the underrepresentation of the Clostridium cluster XIVa, including Lachnospiraceae, which are major butyrate producers, although the pathological relevance has remained obscure. Follicular regulatory T (TFR) cells play critical regulatory roles in the pathogenesis of autoimmune diseases, including RA. Reduced number of circulating TFR cells has been associated with the elevation of autoantibodies and disease severity in RA. However, the contribution of commensal microbe-derived butyrate in controlling TFR cell differentiation remains unknown. METHODS: We examined the contribution of microbe-derived butyrate in controlling autoimmune arthritis using collagen-induced arthritis (CIA) and SKG arthritis models. We phenotyped autoimmune responses in the gut-associated lymphoid tissues (GALT) in the colon and joint-draining lymph nodes in the CIA model. We developed an in vitro CXCR5+Bcl-6+Foxp3+ TFR (iTFR) cell culture system and examined whether butyrate promotes the differentiation of iTFR cells. FINDINGS: Microbe-derived butyrate suppressed the development of autoimmune arthritis. The immunization of type II collagen (CII) caused hypertrophy of the GALT in the colon by amplifying the GC reaction prior to the onset of the CIA. Butyrate mitigated these pathological events by promoting TFR cell differentiation. Butyrate directly induced the differentiation of functional TFR cells in vitro by enhancing histone acetylation in TFR cell marker genes. This effect was attributed to histone deacetylase (HDAC) inhibition by butyrate, leading to histone hyperacetylation in the promoter region of the TFR-cell marker genes. The adoptive transfer of the butyrate-treated iTFR cells reduced CII-specific autoantibody production and thus ameliorated the symptoms of arthritis. INTERPRETATION: Accordingly, microbiota-derived butyrate serves as an environmental cue to enhance TFR cells, which suppress autoantibody production in the systemic lymphoid tissue, eventually ameliorating RA. Our findings provide mechanistic insights into the link between the gut environment and RA risk. FUNDING: This work was supported by AMED-Crest (16gm1010004h0101, 17gm1010004h0102, 18gm1010004h0103, and 19gm1010004s0104 to KH), the Japan Society for the Promotion of Science (JP17KT0055, JP16H01369, and JP18H04680 to KH; JP17K15734 to DT), Keio University Special Grant-in-Aid for Innovative Collaborative Research Projects (KH), Keio Gijuku Fukuzawa Memorial Fund for the Advancement of Education and Research (DT), the SECOM Science and Technology Foundation (KH), the Cell Science Research Foundation (KH), the Mochida Memorial Foundation for Medical and Pharmaceutical Research (DT), the Suzuken Memorial Foundation (KH and DT), the Takeda Science Foundation (KH and DT), The Science Research Promotion Fund, and The Promotion and Mutual Aid Corporation for Private Schools of Japan (KH).

TRPM5 Negatively Regulates Calcium-Dependent Responses in Lipopolysaccharide-Stimulated B Lymphocytes.

B cells produce high amounts of cytokines and immunoglobulins in response to lipopolysaccharide (LPS) stimulation. Calcium signaling cascades are critically involved in cytokine production of T cells, and the cytosolic calcium concentration is regulated by calcium-activated monovalent cation channels (CAMs). Calcium signaling is also implicated in B cell activation; however, its involvement in the cytokine production of LPS-stimulated B cells remains less well characterized. Here, we show that the transient receptor potential melastatin 5 channel (TRPM5), which is one of the CAMs, negatively modulates calcium signaling, thereby regulating LPS-induced proliferative and inflammatory responses by B cells. LPS-stimulated B cells of Trpm5-deficient mice exhibit an increased cytosolic calcium concentration, leading to enhanced proliferation and the production of the inflammatory cytokines interleukin-6 and CXCL10. Furthermore, Trpm5-deficient mice show an exacerbation of endotoxic shock with high mortality. Our findings demonstrate the importance of TRPM5-dependent regulatory mechanisms in LPS-induced calcium signaling of splenic B cells.

Hv1/VSOP regulates neutrophil directional migration and ERK activity by tuning ROS production.

High-level reactive oxygen species (ROS) production in neutrophils is tightly regulated, as it can damage host cells. Neutrophils also undergo low-level ROS production when stimulated by cytokines or chemoattractants, but its biologic significance remains largely unknown. Voltage-gated proton channels (Hv1/VSOP) activity reportedly supports ROS production in neutrophils; however, we show here that Hv1/VSOP balances ROS production to suppress neutrophil directional migration in the presence of low concentrations of N-formyl-Met-Leu-Phe (fMLF). Neutrophils derived from Hvcn1 gene knockout mice produced more ROS than neutrophils from wild-type mice in the stimulation with fMLF at concentration of 1 µM and nonstimulus condition. They also exhibited stronger chemotactic responses to low concentrations of fMLF than did wild-type neutrophils. Receptor sensitivity to fMLF and evoked Ca2+ responses did not differ between Hv1/VSOP-deficient and wild-type neutrophils. Activation of ERK, but not p38, was enhanced and prolonged during the increased ROS production seen after fMLF stimulation in Hv1/VSOP-deficient neutrophils. Inhibiting ROS production suppressed the enhanced ERK activation in Hv1/VSOP-deficient neutrophils and their directional migration. These results indicate that Hv1/VSOP balances ROS production to reduce ERK signaling and suppress excessive neutrophil migration in response to fMLF. Our findings thus reveal a novel role for ROS in the directional migration of neutrophils.

High-endothelial cell-derived S1P regulates dendritic cell localization and vascular integrity in the lymph node.

While the sphingosine-1-phosphate (S1P)/sphingosine-1-phosphate receptor-1 (S1PR1) axis is critically important for lymphocyte egress from lymphoid organs, S1PR1-activation also occurs in vascular endothelial cells (ECs), including those of the high-endothelial venules (HEVs) that mediate lymphocyte immigration into lymph nodes (LNs). To understand the functional significance of the S1P/S1PR1-Gi axis in HEVs, we generated Lyve1;Spns2Δ/Δ conditional knockout mice for the S1P-transporter Spinster-homologue-2 (SPNS2), as HEVs express LYVE1 during development. In these mice HEVs appeared apoptotic and were severely impaired in function, morphology and size; leading to markedly hypotrophic peripheral LNs. Dendritic cells (DCs) were unable to interact with HEVs, which was also observed in Cdh5CRE-ERT2;S1pr1Δ/Δ mice and wildtype mice treated with S1PR1-antagonists. Wildtype HEVs treated with S1PR1-antagonists in vitro and Lyve1-deficient HEVs show severely reduced release of the DC-chemoattractant CCL21 in vivo. Together, our results reveal that EC-derived S1P warrants HEV-integrity through autocrine control of S1PR1-Gi signaling, and facilitates concomitant HEV-DC interactions.

GPR31-dependent dendrite protrusion of intestinal CX3CR1+ cells by bacterial metabolites.

Small intestinal mononuclear cells that express CX3CR1 (CX3CR1+ cells) regulate immune responses1-5. CX3CR1+ cells take up luminal antigens by protruding their dendrites into the lumen1-4,6. However, it remains unclear how dendrite protrusion by CX3CR1+ cells is induced in the intestine. Here we show in mice that the bacterial metabolites pyruvic acid and lactic acid induce dendrite protrusion via GPR31 in CX3CR1+ cells. Mice that lack GPR31, which was highly and selectively expressed in intestinal CX3CR1+ cells, showed defective dendrite protrusions of CX3CR1+ cells in the small intestine. A methanol-soluble fraction of the small intestinal contents of specific-pathogen-free mice, but not germ-free mice, induced dendrite extension of intestinal CX3CR1+ cells in vitro. We purified a GPR31-activating fraction, and identified lactic acid. Both lactic acid and pyruvic acid induced dendrite extension of CX3CR1+ cells of wild-type mice, but not of Gpr31b-/- mice. Oral administration of lactate and pyruvate enhanced dendrite protrusion of CX3CR1+ cells in the small intestine of wild-type mice, but not in that of Gpr31b-/- mice. Furthermore, wild-type mice treated with lactate or pyruvate showed an enhanced immune response and high resistance to intestinal Salmonella infection. These findings demonstrate that lactate and pyruvate, which are produced in the intestinal lumen in a bacteria-dependent manner, contribute to enhanced immune responses by inducing GPR31-mediated dendrite protrusion of intestinal CX3CR1+ cells.

A Distinct Subset of Fibroblastic Stromal Cells Constitutes the Cortex-Medulla Boundary Subcompartment of the Lymph Node.

The spatiotemporal regulation of immune responses in the lymph node (LN) depends on its sophisticated tissue architecture, consisting of several subcompartments supported by distinct fibroblastic stromal cells (FSCs). However, the intricate details of stromal structures and associated FSC subsets are not fully understood. Using several gene reporter mice, we sought to discover unrecognized stromal structures and FSCs in the LN. The four previously identified FSC subsets in the cortex are clearly distinguished by the expression pattern of reporters including PDGFRß, CCL21-ser, and CXCL12. Herein, we identified a unique FSC subset expressing both CCL21-ser and CXCL12 in the deep cortex periphery (DCP) that is characterized by preferential B cell localization. This subset was clearly different from CXCL12highLepRhigh FSCs in the medullary cord, which harbors plasma cells. B cell localization in the DCP was controlled chiefly by CCL21-ser and, to a lesser extent, CXCL12. Moreover, the optimal development of the DCP as well as medulla requires B cells. Together, our findings suggest the presence of a unique microenvironment in the cortex-medulla boundary and offer an advanced view of the multi-layered stromal framework constructed by distinct FSC subsets in the LN.

Two-Photon Imaging of T-Cell Motility in Lymph Nodes: In Vivo and Ex Vivo Approaches.

T-cell motility is essential for the T cells' ability to scan antigens within lymph nodes and initiate contact with antigen-presenting cells. While T-cell migration has been extensively studied using in vitro migration assays, accumulating evidence indicates that the T-cell migration within lymph nodes is modulated by the surrounding cells and extracellular matrix, which form the confined architecture of the lymph nodes. Therefore, to understand the mechanisms of T-cell motility in vivo, their cell migration must be analyzed under physiological conditions. To this end, two-photon microscopy is extremely useful; this technique enables the tracking of fluorescently labeled cells in vivo and ex vivo, with high spatial and temporal resolutions. Here we describe the experimental procedures for applying two-photon microscopy to the in vivo and ex vivo imaging of T-cell migration in mouse lymph nodes. These approaches provide physiological insight into the mechanisms of T-cell behavior at a single-cell level in the three-dimensional lymph node environment.

Hydrogen-Rich Saline Regulates Intestinal Barrier Dysfunction, Dysbiosis, and Bacterial Translocation in a Murine Model of Sepsis.

Bacterial translocation is a major cause of multiple organ dysfunction syndrome in critical illness, and its management is an important therapeutic strategy. In this study, we focused on the key factors responsible for bacterial translocation including the intestinal microbiome and investigated the impact of molecular hydrogen therapy as a countermeasure against bacterial translocation in a murine model of sepsis. The experimental protocols were divided into the sham, saline treatment (control), and hydrogen treatment (H2) groups. In the H2 group, 15 mL/kg of hydrogen-rich saline (7 ppm) was gavaged daily for 7 days following cecal ligation and puncture (CLP). In the control group, normal saline was gavaged in the same way. In the results, the 7-day survival rate was significantly improved in the H2 group versus the control group (69% vs. 31%, P < 0.05). The incidence of bacterial translocation at 24 h after CLP as assessed by cultivation of mesenteric lymph nodes and blood was significantly decreased in the H2 group versus the control group. Administration of hydrogen-rich saline also prevented the expansion of facultative anaerobic Enterobacteriaceae and ameliorated intestinal hyperpermeability at 24 h after CLP. Intestinal tissue levels of inflammatory mediators such as inducible nitric oxide synthases, tumor necrosis factor α, interleukin (IL)-1ß, IL-6, and oxidative stress marker malondialdehyde at 6 h after CLP were down-regulated in the H2 group. These results suggest luminal administration of hydrogen-rich saline, which prevents intestinal dysbiosis, hyperpermeability, and bacterial translocation, could potentially be a new therapeutic strategy in critical illness.

Slc3a2 Mediates Branched-Chain Amino-Acid-Dependent Maintenance of Regulatory T Cells.

Foxp3+ regulatory T (Treg) cells, which suppress immune responses, are highly proliferative in vivo. However, it remains unclear how the active replication of Treg cells is maintained in vivo. Here, we show that branched-chain amino acids (BCAAs), including isoleucine, are required for maintenance of the proliferative state of Treg cells via the amino acid transporter Slc3a2-dependent metabolic reprogramming. Mice fed BCAA-reduced diets showed decreased numbers of Foxp3+ Treg cells with defective in vivo proliferative capacity. Mice lacking Slc3a2 specifically in Foxp3+ Treg cells showed impaired in vivo replication and decreased numbers of Treg cells. Slc3a2-deficient Treg cells showed impaired isoleucine-induced activation of the mTORC1 pathway and an altered metabolic state. Slc3a2 mutant mice did not show an isoleucine-induced increase of Treg cells in vivo and exhibited multi-organ inflammation. Taken together, these findings demonstrate that BCAA controls Treg cell maintenance via Slc3a2-dependent metabolic regulation.

E-NPP3 controls plasmacytoid dendritic cell numbers in the small intestine.

Extracellular adenosine 5'-triphosphate (ATP) performs multiple functions including activation and induction of apoptosis of many cell types. The ATP-hydrolyzing ectoenzyme ecto-nucleotide pyrophosphatase/phosphodiesterase 3 (E-NPP3) regulates ATP-dependent chronic allergic responses by mast cells and basophils. However, E-NPP3 is also highly expressed on epithelial cells of the small intestine. In this study, we showed that E-NPP3 controls plasmacytoid dendritic cell (pDC) numbers in the intestine through regulation of intestinal extracellular ATP. In Enpp3-/- mice, ATP concentrations were increased in the intestinal lumen. pDC numbers were remarkably decreased in the small intestinal lamina propria and Peyer's patches. Intestinal pDCs of Enpp3-/- mice showed enhanced cell death as characterized by increases in annexin V binding and expression of cleaved caspase-3. pDCs were highly sensitive to ATP-induced cell death compared with conventional DCs. ATP-induced cell death was abrogated in P2rx7-/- pDCs. Accordingly, the number of intestinal pDCs was restored in Enpp3-/- P2rx7-/- mice. These findings demonstrate that E-NPP3 regulates ATP concentration and thereby prevents the decrease of pDCs in the small intestine.

Dysbiosis Contributes to Arthritis Development via Activation of Autoreactive T Cells in the Intestine.

OBJECTIVE: The intestinal microbiota is involved in the pathogenesis of arthritis. Altered microbiota composition has been demonstrated in patients with rheumatoid arthritis (RA). However, it remains unclear how dysbiosis contributes to the development of arthritis. The aim of this study was to investigate whether altered composition of human intestinal microbiota in RA patients contributes to the development of arthritis. METHODS: We analyzed the fecal microbiota of patients with early RA and healthy controls, using 16S ribosomal RNA-based deep sequencing. We inoculated fecal samples from RA patients and healthy controls into germ-free arthritis-prone SKG mice and evaluated the immune responses. We also analyzed whether the lymphocytes of SKG mice harboring microbiota from RA patients react with the arthritis-related autoantigen 60S ribosomal protein L23a (RPL23A). RESULTS: A subpopulation of patients with early RA harbored intestinal microbiota dominated by Prevotella copri; SKG mice harboring microbiota from RA patients had an increased number of intestinal Th17 cells and developed severe arthritis when treated with zymosan. Lymphocytes in regional lymph nodes and the colon, but not the spleen, of these mice showed enhanced interleukin-17 (IL-17) responses to RPL23A. Naive SKG mouse T cells cocultured with P copri-stimulated dendritic cells produced IL-17 in response to RPL23A and rapidly induced arthritis. CONCLUSION: We demonstrated that dysbiosis increases sensitivity to arthritis via activation of autoreactive T cells in the intestine. Autoreactive SKG mouse T cells are activated by dysbiotic microbiota in the intestine, causing joint inflammation. Dysbiosis is an environmental factor that triggers arthritis development in genetically susceptible mice.

Lypd8 promotes the segregation of flagellated microbiota and colonic epithelia.

Colonic epithelial cells are covered by thick inner and outer mucus layers. The inner mucus layer is free of commensal microbiota, which contributes to the maintenance of gut homeostasis. In the small intestine, molecules critical for prevention of bacterial invasion into epithelia such as Paneth-cell-derived anti-microbial peptides and regenerating islet-derived 3 (RegIII) family proteins have been identified. Although there are mucus layers providing physical barriers against the large number of microbiota present in the large intestine, the mechanisms that separate bacteria and colonic epithelia are not fully elucidated. Here we show that Ly6/PLAUR domain containing 8 (Lypd8) protein prevents flagellated microbiota invading the colonic epithelia in mice. Lypd8, selectively expressed in epithelial cells at the uppermost layer of the large intestinal gland, was secreted into the lumen and bound flagellated bacteria including Proteus mirabilis. In the absence of Lypd8, bacteria were present in the inner mucus layer and many flagellated bacteria invaded epithelia. Lypd8(-/-) mice were highly sensitive to intestinal inflammation induced by dextran sulfate sodium (DSS). Antibiotic elimination of Gram-negative flagellated bacteria restored the bacterial-free state of the inner mucus layer and ameliorated DSS-induced intestinal inflammation in Lypd8(-/-) mice. Lypd8 bound to flagella and suppressed motility of flagellated bacteria. Thus, Lypd8 mediates segregation of intestinal bacteria and epithelial cells in the colon to preserve intestinal homeostasis.

IL-10-producing lung interstitial macrophages prevent neutrophilic asthma.

Inflammatory responses contribute to host defense against harmful organisms and allergens, whereas a failure of immune tolerance can cause chronic inflammation including asthma. The lung has several innate myeloid cell subsets. Among these subsets, there are two types of macrophages: alveolar macrophages (AMs) and interstitial macrophages (IMs). However, compared with AMs, the role of IMs in lung homeostasis remains poorly understood. In this study, we characterized AMs and IMs in healthy and inflammatory conditions. Pulmonary IMs constitutively produce the anti-inflammatory cytokine IL-10 through activation of the TLR4/MyD88 pathway in a microbiota-independent manner. In addition to IMs, Foxp3+ Treg cells show persistent IL-10 expression in the lung, with IL-10-producing IMs more prevalent than Foxp3+ Treg cells. IMs, but not Foxp3+ Treg cells, increased IL-10 production in house dust mite (HDM)-challenged mice, a model of human asthma. HDM-challenged Il10 -/- mice exhibited severe lung pathology characterized by neutrophilia compared with that of wild-type mice. In addition, transplantation of wild-type IMs reduced neutrophilic inflammation, goblet cell mucus production and decreased expression of lung IL-13 and Th17-related neutrophil-activating cytokines such as IL-17, GM-CSF, and TNF-α. Together these results demonstrate that IL-10-producing IMs negatively regulate Th2- and Th17-mediated inflammatory responses, helping prevent neutrophilic asthma.

Fibroblastic reticular cell-derived lysophosphatidic acid regulates confined intranodal T-cell motility.

Lymph nodes (LNs) are highly confined environments with a cell-dense three-dimensional meshwork, in which lymphocyte migration is regulated by intracellular contractile proteins. However, the molecular cues directing intranodal cell migration remain poorly characterized. Here we demonstrate that lysophosphatidic acid (LPA) produced by LN fibroblastic reticular cells (FRCs) acts locally to LPA2 to induce T-cell motility. In vivo, either specific ablation of LPA-producing ectoenzyme autotaxin in FRCs or LPA2 deficiency in T cells markedly decreased intranodal T cell motility, and FRC-derived LPA critically affected the LPA2-dependent T-cell motility. In vitro, LPA activated the small GTPase RhoA in T cells and limited T-cell adhesion to the underlying substrate via LPA2. The LPA-LPA2 axis also enhanced T-cell migration through narrow pores in a three-dimensional environment, in a ROCK-myosin II-dependent manner. These results strongly suggest that FRC-derived LPA serves as a cell-extrinsic factor that optimizes T-cell movement through the densely packed LN reticular network.

Lysophosphatidic acid receptors LPA4 and LPA6 differentially promote lymphocyte transmigration across high endothelial venules in lymph nodes.

Naive lymphocytes continuously migrate from the blood into lymph nodes (LNs) via high endothelial venules (HEVs). To extravasate from the HEVs, lymphocytes undergo multiple adhesion steps, including tethering, rolling, firm adhesion and transmigration. We previously showed that autotaxin (ATX), an enzyme that generates lysophosphatidic acid (LPA), is highly expressed in HEVs, and that the ATX/LPA axis plays an important role in the lymphocyte transmigration across HEVs. However, the detailed mechanism underlying this axis's involvement in lymphocyte transmigration has remained ill-defined. Here, we show that two LPA receptors, LPA4 and LPA6, are selectively expressed on HEV endothelial cells (ECs) and that LPA4 plays a major role in the lymphocyte transmigration across HEVs in mice. In the absence of LPA4 expression, lymphocytes accumulated heavily within the HEV EC layer, compared to wild-type (WT) mice. This accumulation was also observed in the absence of LPA6 expression, but it was less pronounced. Adoptive transfer experiments using WT lymphocytes revealed that the LPA4 deficiency in ECs specifically compromised the lymphocyte transmigration process, whereas the effect of LPA6 deficiency was not significant. These results indicate that the signals evoked in HEV ECs via the LPA4 and LPA6 differentially regulate lymphocyte extravasation from HEVs in the peripheral LNs.