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CD8 T lymphocytes are able to eliminate nascent tumor cells through a process referred to as immunosurveillance. However, multiple inhibitory mechanisms within the tumor microenvironment have been described that impede tumor rejection by CD8 T cells, including increased signaling by inhibitory receptors. Lysophosphatidic acid (LPA) is a bioactive lysophospholipid that has been shown repeatedly to promote diverse cellular processes benefiting tumorigenesis. Accordingly, the exaggerated expression of LPA and LPA receptors is a common feature of diverse tumor cell lineages and can result in elevated systemic LPA levels. LPA is recognized by at least six distinct G protein–coupled receptors, several of which are expressed by T cells, although the precise function of LPA signaling in CD8 T-cell activation and function has not been defined. Here, we show that LPA signaling via the LPA5 receptor expressed by CD8 T cells suppresses antigen receptor signaling, cell activation, and proliferation in vitro and in vivo. Importantly, in a mouse melanoma model tumor-specific CD8 T cells that are LPA5-deficient are able to control tumor growth significantly better than wild-type tumor-specific CD8 T cells. Together, these data suggest that the production of LPA by tumors serves not only in an autocrine manner to promote tumorigenesis, but also as a mechanism to suppress adaptive immunity and highlights a potential novel target for cancer treatment. Cancer Immunol Res; 1(4); 245–55. ©2013 AACR.

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Supplementary Figure 5. ATX and LPA receptor profiling in isolated rat alveolar type I-like (A) or type II (B) epithelial cells. Note that expression levels in type II alveolar cells are approximately 10-fold lower than in type I-like cells. Data presented are representative of an experiment performed three times in quadruplicates and are expressed as mean {plus minus} SD.

Supplementary Figure 3. (A) ATX and LPA receptor profiling in B16F10 cells transduced with GFP lentivirus using quantitative real-time PCR. Data are expressed as mean {plus minus} SD of an experiment performed twice in quadruplicates. (B) Representative images of lungs harvested at day 21 from mice that were injected with PBS (left panel, control) and mice that were injected with 1 x 105 GFP-expressing B16F10 cells (right panel). Arrow denotes a single fluorescent tumor nodule on lung surface. Scale bar = 200 microns.

Autotaxin (ENPP2/ATX) and lysophosphatidic acid (LPA) receptors represent two key players in regulating cancer progression. The present study sought to understand the mechanistic role of LPA G protein–coupled receptors (GPCR), not only in the tumor cells but also in stromal cells of the tumor microenvironment. B16F10 melanoma cells predominantly express LPA5 and LPA2 receptors but lack LPA1. LPA dose dependently inhibited invasion of cells across a Matrigel layer. RNAi-mediated knockdown of LPA5 relieved the inhibitory effect of LPA on invasion without affecting basal invasion. This suggests that LPA5 exerts an anti-invasive action in melanoma cells in response to LPA. In addition, both siRNA-mediated knockdown and pharmacologic inhibition of LPA2 reduced the basal rate invasion. Unexpectedly, when probing the role of this GPCR in host tissues, it was found that the incidence of melanoma-derived lung metastasis was greatly reduced in LPA5 knockout (KO) mice compared with wild-type (WT) mice. LPA1-KO but not LPA2-KO mice also showed diminished melanoma-derived lung metastasis, suggesting that host LPA1 and LPA5 receptors play critical roles in the seeding of metastasis. The decrease in tumor cell residence in the lungs of LPA1-KO and LPA5-KO animals was apparent 24 hours after injection. However, KO of LPA1, LPA2, or LPA5 did not affect the subcutaneous growth of melanoma tumors.Implications: These findings suggest that tumor and stromal LPA receptors, in particular LPA1 and LPA5, play different roles in invasion and the seeding of metastasis. Mol Cancer Res; 13(1); 174–85. ©2014 AACR.

Supplementary Figure 2. LPA1-5 receptor profiling in whole lung tissue isolated from WT, LPA1-, LPA2- and LPA5KO mice, respectively using quantitative real-time PCR. Data are expressed as mean {plus minus} SEM (n = 3 mice).

Supplementary Figure 1. Time course of ATX activity measured in the conditioned media (CM) of (A) MM1 rat hepatocarcinoma, (B) HUVEC, (C) RLMVEC, and (D) isolated murine mesothelial cells. Control represents media that were not exposed to cells. Data are expressed as mean {plus minus} SD of an experiment performed three times in triplicates.

Supplementary Figure 4. Effect of BMP22 on the basal invasion of B16F10 cells across a matrigel layer. Data are representative of an experiment performed twice in quadruplicates and are expressed as mean {plus minus} SD. *Denotes p value < 0.05 using one-way ANOVA followed by a Bonferroni post-test.

Supplementary Table 1. Primer sequences used in quantitative Real-Time PCR analysis.

As an anti-inflammatory strategy, MAPK-activated protein kinase-2 (MK2) inhibition can potentially avoid the clinical failures seen for direct p38 inhibitors, especially tachyphylaxis. CC-99677, a selective targeted covalent MK2 inhibitor, employs a rare chloropyrimidine that bonds to the sulfur of cysteine 140 in the ATP binding site via a nucleophilic aromatic substitutions (S

The bioactive sphingolipid sphingosine-1-phosphate (S1P) and the kinase that produces it have been implicated in inflammatory bowel diseases in mice and humans; however, little is known about the role of the 2 S1P-specific phosphohydrolase isoforms, SGPP1 and SGPP2, which catalyze dephosphorylation of S1P to sphingosine. To elucidate their functions, we generated specific knockout mice. Deletion of Sgpp2, which is mainly expressed in the gastrointestinal tract, significantly reduced dextran sodium sulfate (DSS)-induced colitis severity, whereas deletion of ubiquitously expressed Sgpp1 slightly worsened colitis. Moreover, Sgpp1 deletion enhanced expression of multifunctional proinflammatory cytokines, IL-6, TNF-α, and IL-1β, activation of the transcription factor signal transducer and activator of transcription 3, and immune cell infiltration into the colon. Conversely, Sgpp2-null mice failed to mount a DSS-induced systemic inflammatory response. Of interest, Sgpp2 deficiency suppressed DSS-induced intestinal epithelial cell apoptosis and improved mucosal barrier integrity. Furthermore, down-regulation of Sgpp2 attenuated LPS-induced paracellular permeability in cultured cells and enhanced expression of the adherens junction protein E-cadherin. Finally, in patients with ulcerative colitis, SGPP2 expression was elevated in colitis tissues relative to that in uninvolved tissues. These results indicate that induction of SGPP2 expression contributes to the pathogenesis of colitis by promoting disruption of the mucosal barrier function. SGPP2 may represent a novel therapeutic target in inflammatory bowel disease.-Huang, W.-C., Liang, J., Nagahashi, M., Avni, D., Yamada, A., Maceyka, M., Wolen, A. R., Kordula, T., Milstien, S., Takabe, K., Oravecz, T., Spiegel, S. Sphingosine-1-phosphate phosphatase 2 promotes disruption of mucosal integrity, and contributes to ulcerative colitis in mice and humans.

Expressed strongly by myeloid cells, damage-associated molecular pattern (DAMP) proteins S100A8 and S100A9 are found in the serum of patients with infectious and autoimmune diseases. Compared to S100A9, the role of S100A8 is controversial. We investigated its biological activity in collagen-induced arthritis using the first known viable and fertile S100a8-deficient (S100a8-/-) mouse. Although comparable to the wild type (WT) in terms of lymphocyte distribution in blood and in the primary and secondary lymphoid organs, S100a8-/- mice had increased numbers of neutrophils, monocytes and dendritic cells in the blood and bone marrow, and these all expressed myeloid markers such as CD11b, Ly6G and CD86 more strongly. Granulocyte-macrophage common precursors were increased in S100a8-/- bone marrow and yielded greater numbers of macrophages and dendritic cells in culture. The animals also developed more severe arthritic disease leading to aggravated osteoclast activity and bone destruction. These findings were correlated with increased inflammatory cell infiltration and cytokine secretion in the paws. This study suggests that S100A8 is an anti-inflammatory DAMP that regulates myeloid cell differentiation, thereby mitigating the development of experimental arthritis.

Autotaxin (ENPP2/ATX) and lysophosphatidic acid (LPA) receptors represent two key players in regulating cancer progression. The present study sought to understand the mechanistic role of LPA G protein–coupled receptors (GPCR), not only in the tumor cells but also in stromal cells of the tumor microenvironment. B16F10 melanoma cells predominantly express LPA5 and LPA2 receptors but lack LPA1. LPA dose dependently inhibited invasion of cells across a Matrigel layer. RNAi-mediated knockdown of LPA5 relieved the inhibitory effect of LPA on invasion without affecting basal invasion. This suggests that LPA5 exerts an anti-invasive action in melanoma cells in response to LPA. In addition, both siRNA-mediated knockdown and pharmacologic inhibition of LPA2 reduced the basal rate invasion. Unexpectedly, when probing the role of this GPCR in host tissues, it was found that the incidence of melanoma-derived lung metastasis was greatly reduced in LPA5 knockout (KO) mice compared with wild-type (WT) mice. LPA1-KO but not LPA2-KO mice also showed diminished melanoma-derived lung metastasis, suggesting that host LPA1 and LPA5 receptors play critical roles in the seeding of metastasis. The decrease in tumor cell residence in the lungs of LPA1-KO and LPA5-KO animals was apparent 24 hours after injection. However, KO of LPA1, LPA2, or LPA5 did not affect the subcutaneous growth of melanoma tumors. Implications: These findings suggest that tumor and stromal LPA receptors, in particular LPA1 and LPA5, play different roles in invasion and the seeding of metastasis. Mol Cancer Res; 13(1); 174–85. ©2014 AACR.

Lysophospholipids have emerged as biologically important chemoattractants capable of directing lymphocyte development, trafficking, and localization. Lysophosphatidic acid (LPA) is a major lysophospholipid found systemically, and its levels are elevated in certain pathological settings, such as cancer and infections. In this study, we demonstrate that BCR signal transduction by mature murine B cells is inhibited upon LPA engagement of the LPA5 (GPR92) receptor via a Gα12/13-Arhgef1 pathway. The inhibition of BCR signaling by LPA5 manifests by impaired intracellular calcium store release and most likely by interfering with inositol 1,4,5-triphosphate receptor activity. We further show that LPA5 also limits Ag-specific induction of CD69 and CD86 expression and that LPA5-deficient B cells display enhanced Ab responses. Thus, these data show that LPA5 negatively regulates BCR signaling, B cell activation, and immune response. Our findings extend the influence of lysophospholipids on immune function and suggest that alterations in LPA levels likely influence adaptive humoral immunity.

Progressive lung disease with early onset is the main cause of mortality and morbidity in cystic fibrosis patients. Here we report a reduction of sphingosine-1-phosphate (S1P) in the lung of unchallenged Cftr tm1EUR F508del CFTR mutant mice. This correlates with enhanced infiltration by inducible nitric oxide synthase (iNOS)-expressing granulocytes, B cells, and T cells. Furthermore, the ratio of macrophage-derived dendritic cells (MoDC) to conventional dendritic cells (cDC) is higher in mutant mouse lung, consistent with unprovoked inflammation. Oral application of a S1P lyase inhibitor (LX2931) increases S1P levels in mutant mouse tissues. This normalizes the lung MoDC/cDC ratio and reduces B and T cell counts. Lung granulocytes are enhanced, but iNOS expression is reduced in this population. Although lung LyC6+ monocytes are enhanced by LX2931, they apparently do not differentiate to MoDC and macrophages. After challenge with bacterial toxins (LPS-fMLP) we observe enhanced levels of proinflammatory cytokines TNF-α, KC, IFNγ, and IL-12 and the inducible mucin MUC5AC in mutant mouse lung, evidence of deficient resolution of inflammation. LX2931 does not prevent transient inflammation or goblet cell hyperplasia after challenge, but it reduces MUC5AC and proinflammatory cytokine levels toward normal values. We conclude that lung pathology in homozygous mice expressing murine F508del CFTR, which represents the most frequent mutation in CF patients, is characterized by abnormal behavior of infiltrating myeloid cells and delayed resolution of induced inflammation. This phenotype can be partially corrected by a S1P lyase inhibitor, providing a rationale for therapeutic targeting of the S1P signaling pathway in CF patients.

Cerebral malaria (CM) is associated with excessive inflammatory responses and endothelial activation. Sphingosine 1-phosphate (S1P) is a signaling sphingolipid implicated in regulating vascular integrity, inflammation and T-cell migration. We hypothesized that altered S1P signaling during malaria contributes to endothelial activation and inflammation, and show that plasma S1P levels were decreased in Ugandan children with CM compared with children with uncomplicated malaria. Using the Plasmodium berghei ANKA (PbA) model of experimental CM (ECM), we demonstrate that humanized S1P lyase (hS1PL)(-/-) mice with reduced S1P lyase activity (resulting in increased bio-available S1P) had improved survival compared with wild-type littermates. Prophylactic and therapeutic treatment of infected mice with compounds that modulate the S1P pathway and are in human trials for other conditions (FTY720 or LX2931) significantly improved survival in ECM. FTY720 treatment improved vascular integrity as indicated by reduced levels of soluble intercellular adhesion molecule (sICAM), increased angiopoietin 1 (Ang1) (regulator of endothelial quiescence) levels, and decreased Evans blue dye leakage into brain parenchyma. Furthermore, treatment with FTY720 decreased IFNγ levels in plasma as well as CD4(+) and CD8(+) T-cell infiltration into the brain. Finally, when administered during infection in combination with artesunate, FTY720 treatment resulted in increased survival to ECM. These findings implicate dysregulation of the S1P pathway in the pathogenesis of human and murine CM and suggest a novel therapeutic strategy to improve clinical outcome in severe malaria.

An early lesion in many kidney diseases is damage to podocytes, which are critical components of the glomerular filtration barrier. A number of proteins are essential for podocyte filtration function, but the signaling events contributing to development of nephrotic syndrome are not well defined. Here we show that class II phosphoinositide 3-kinase C2α (PI3KC2α) is expressed in podocytes and plays a critical role in maintaining normal renal homeostasis. PI3KC2α-deficient mice developed chronic renal failure and exhibited a range of kidney lesions, including glomerular crescent formation and renal tubule defects in early disease, which progressed to diffuse mesangial sclerosis, with reduced podocytes, widespread effacement of foot processes, and modest proteinuria. These findings were associated with altered expression of nephrin, synaptopodin, WT-1, and desmin, indicating that PI3KC2α deficiency specifically impacts podocyte morphology and function. Deposition of glomerular IgA was observed in knockout mice; importantly, however, the development of severe glomerulonephropathy preceded IgA production, indicating that nephropathy was not directly IgA mediated. PI3KC2α deficiency did not affect immune responses, and bone marrow transplantation studies also indicated that the glomerulonephropathy was not the direct consequence of an immune-mediated disease. Thus, PI3KC2α is critical for maintenance of normal glomerular structure and function by supporting normal podocyte function.

Sphingosine 1-phosphate lyase (S1PL) has been characterized as a novel target for the treatment of autoimmune disorders using genetic and pharmacological methods. Medicinal chemistry efforts targeting S1PL by direct in vivo evaluation of synthetic analogues of 2-acetyl-4(5)-(1(R),2(S),3(R),4-tetrahydroxybutyl)-imidazole (THI, 1) led to the discovery of 2 (LX2931) and 4 (LX2932). The immunological phenotypes observed in S1PL deficient mice were recapitulated by oral administration of 2 or 4. Oral dosing of 2 or 4 yielded a dose-dependent decrease in circulating lymphocyte numbers in multiple species and showed a therapeutic effect in rodent models of rheumatoid arthritis (RA). Phase I clinical trials indicated that 2, the first clinically studied inhibitor of S1PL, produced a dose-dependent and reversible reduction of circulating lymphocytes and was well tolerated at dose levels of up to 180 mg daily. Phase II evaluation of 2 in patients with active rheumatoid arthritis is currently underway.

Sh2d3c is an adaptor protein that has been implicated in T cell activation and shown to associate with different components of the integrin signaling pathway ex vivo. However, the in vivo significance of Sh2d3c expression in the regulation of the immune response and/or hematopoietic cell lineage development is not known. In this study, we show that expression of Sh2d3c is more critical for development and function of marginal zone B (MZB) cells than for T cell maturation. Mice deficient in Sh2d3c expression (Sh2d3c−/−) had a reduced number of MZB cells, and the residual MZB cells failed to properly capture polysaccharide Ags. Activation-induced proliferation, cytokine production, and migration of Sh2d3c−/− splenic B cells were also significantly reduced in vitro compared with wild-type (Sh2d3c+/+) cells. In contrast, T cell development and function were largely normal in Sh2d3c−/− mice. The thymi of Sh2d3c−/− mice showed no maturational abnormalities, the number of splenic T cells was only modestly reduced, and the T cells responded normally to in vitro polyclonal activation. The observed B cell deficiency in the Sh2d3c−/− mice led to diminished humoral immune response against thymus-independent type 2, but not thymus-dependent Ags, which highlights the primary in vivo role of Sh2d3c in regulating B cell development and function.

Large collections of knockout organisms facilitate the elucidation of gene functions. Here we used retroviral insertion or homologous recombination to disrupt 472 genes encoding secreted and membrane proteins in mice, providing a resource for studying a large fraction of this important class of drug target. The knockout mice were subjected to a systematic phenotypic screen designed to uncover alterations in embryonic development, metabolism, the immune system, the nervous system and the cardiovascular system. The majority of knockout lines exhibited altered phenotypes in at least one of these therapeutic areas. To our knowledge, a comprehensive phenotypic assessment of a large number of mouse mutants generated by a gene-specific approach has not been described previously.

2-Acetyl-4(5)-tetrahydroxybutyl imidazole (THI) has been shown to reduce rodent peripheral blood lymphocytes through increasing lymphoid sphingosine 1-phosphate (S1P) by inhibiting S1P lyase. The objective of this study was to characterize the relationship between systemic THI exposure, splenic S1P concentrations, and lymphopenia in rats. Following the oral administration of 10 and 100 mg kg(-1) THI to male rats, THI was rapidly absorbed and reached a plasma peak level at 1 h post-dosing. Splenic S1P increased and reached the peak level at 24 h. Blood lymphocyte count decreased as the splenic S1P level increased. THI plasma concentration was linked to splenic S1P concentration using an indirect model incorporated with a four-step signal transduction model. In turn, the S1P level was directly coupled with blood lymphocyte number. The integrated model simultaneously captured the splenic S1P and blood lymphocyte responses. This pharmacokinetic-biomarker-pharmacodynamic model resolved the remarkable discrepancy between plasma THI concentration and the pharmacological response and quantitatively described the relationship of THI exposure, S1P, and lymphopenic response.

During nearly a decade of research dedicated to the study of sphingosine signaling pathways, we identified sphingosine-1-phosphate lyase (S1PL) as a drug target for the treatment of autoimmune disorders. S1PL catalyzes the irreversible decomposition of sphingosine-1-phosphate (S1P) by a retro-aldol fragmentation that yields hexadecanaldehyde and phosphoethanolamine. Genetic models demonstrated that mice expressing reduced S1PL activity had decreased numbers of circulating lymphocytes due to altered lymphocyte trafficking, which prevented disease development in multiple models of autoimmune disease. Mechanistic studies of lymphoid tissue following oral administration of 2-acetyl-4(5)-(1(R),2(S),3(R),4-tetrahydroxybutyl)-imidazole (THI) 3 showed a clear relationship between reduced lyase activity, elevated S1P levels, and lower levels of circulating lymphocytes. Our internal medicinal chemistry efforts discovered potent analogues of 3 bearing heterocycles as chemical equivalents of the pendant carbonyl present in the parent structure. Reduction of S1PL activity by oral administration of these analogues recapitulated the phenotype of mice with genetically reduced S1PL expression.

Sphingosine 1-phosphate (S1P) is a pleiotropic bioactive sphingolipid metabolite that regulates numerous processes important for immune responses. S1P is made within cells and must be transported out of cells to exert its effects through activation of 5 specific cell surface GPCRs in an autocrine or paracrine fashion. Spinster 2 (Spns2) transports S1P out of cells, and its deletion in mice reduces circulating levels of S1P, alters immune cell trafficking, and induces lymphopenia. Here we examined the effects of Spns2 deletion on adaptive immune responses and in autoimmune disease models. Airway inflammation and hypersensitivity as well as delayed-type contact hypersensitivity were attenuated in Spns2−/− mice. Similarly, Spns2 deletion reduced dextran sodium sulfate– and oxazolone-induced colitis. Intriguingly, Spns2−/− mice were protected from the development of experimental autoimmune encephalopathy, a model of the autoimmune disease multiple sclerosis. Deletion of Spns2 also strongly alleviated disease development in collagen-induced arthritis. These results point to a broad role for Spns2-mediated S1P transport in the initiation and development of adaptive immune related disorders.—Donoviel, M. S., Hait, N. C., Ramachandran, S., Maceyka, M., Takabe, K., Milstien, S., Oravecz, T., Spiegel, S. Spinster 2, a sphingosine-1-phosphate transporter, plays a critical role in inflammatory and autoimmune diseases.

It is known that β1 integrins mediate the migratory response of cells to chemokine stimulation. Also, both β1 integrins and chemokines have roles in tumor development. In the present study, the β1 integrin-chemokine axis is assessed using human osteosarcoma (HOS) transfectant cells expressing the CXCR4 receptor for chemokine SDF-1 (CXCL12). We first identified in vitro the specific β1 integrins that mediated the migratory response to SDF-1 stimulation. Results showed that on collagen type I and laminin, the chemotactic response to SDF-1 was predominantly mediated by α2β1 integrin. On fibronectin, SDF-1-stimulated chemotaxis involved both α4β1 and α5β1 integrins. A comparison of the transfectant clones expressing CXCR4 at low, intermediate, and high levels and the control transfectant revealed that the transfectant clones migratory response in vitro and their ability to form tumors in vivo was related to their levels of CXCR4 expression. In addition, treatment by injection with mAbs to CXCR4, integrin α2β1, or integrin α5β1 effectively inhibited the growth of HOS-CXCR4 transfectant cells in vivo. Therefore, our results show that the β1 integrins that mediated the migratory response were also functionally linked to the enhanced tumor growth of CXCR4-expressing HOS transfectant cells.Key words: integrins, chemokines, chemotaxis, osteosarcoma, tumorigenesis.

ObjectivesTo investigate the role of liver X receptors (LXRs) in experimental skin fibrosis and evaluate their potential as novel antifibrotic targets.MethodsWe studied the role of LXRs in bleomycin-induced skin fibrosis, in the model of sclerodermatous graft-versus-host disease (sclGvHD) and in tight skin-1 (Tsk-1) mice, reflecting different subtypes of fibrotic disease. We examined both LXR isoforms using LXRα-, LXRβ- and LXR-α/β-double-knockout mice. Finally, we investigated the effects of LXRs on fibroblasts and macrophages to establish the antifibrotic mode of action of LXRs.ResultsLXR activation by the agonist T0901317 had antifibrotic effects in bleomycin-induced skin fibrosis, in the sclGvHD model and in Tsk-1 mice. The antifibrotic activity of LXRs was particularly prominent in the inflammation-driven bleomycin and sclGvHD models. LXRα-, LXRβ- and LXRα/β-double-knockout mice showed a similar response to bleomycin as wildtype animals. Low levels of the LXR target gene ABCA-1 in the skin of bleomycin-challenged and control mice suggested a low baseline activation of the antifibrotic LXR signalling, which, however, could be specifically activated by T0901317. Fibroblasts were not the direct target cells of LXRs agonists, but LXR activation inhibited fibrosis by interfering with infiltration of macrophages and their release of the pro-fibrotic interleukin-6.ConclusionsWe identified LXRs as novel targets for antifibrotic therapies, a yet unknown aspect of these nuclear receptors. Our data suggest that LXR activation might be particularly effective in patients with inflammatory disease subtypes. Activation of LXRs interfered with the release of interleukin-6 from macrophages and, thus, inhibited fibroblast activation and collagen release.

It is well established that chemokines have a major role in the stimulation of cell movement on extracellular matrix (ECM) substrates. However, it is also clear that ECM substrates may influence the ability of cells to undergo migration. Using the migration chamber method, we assessed the migratory response of human embryonic kidney-293 (HEK) transfectant cells expressing the CC chemokine receptor 5 (CCR5) (HEK-CCR5) to stimulation by chemokines (macrophage inflamatory protein (MIP)-1α, MIP-1β, and regulated on activation normal-T cell expressed and secreted (RANTES)) on ECM substrates (collagen type I and fibronectin). Using filters coated with collagen (20 µg/mL), results showed that the chemokines differed in their ability to elicit cell movement according to the order MIP-1β > RANTES [Formula: see text] MIP-1α. In contrast, using filters coated with fibronectin (20 µg/mL), all three chemokines were similar in their ability to stimulate migration of HEK-CCR5 cells. In addition, the migratory response with respect to the concentrations of ECM substrates appeared biphasic; thus, chemokine-stimulated cell movement was inhibited at high ECM concentrations (100 µg/mL). To determine the involvement of β1 integrins, results showed that the migratory response to chemokine stimulation on collagen was largely inhibited by monoclonal antibody (mAb) to α2β1; however, complete inhibition required a combination of mAbs to α1β1 and α2β1. In comparison, migration on fibronectin was inhibited by mAb to α3β1 and α5β1. Our results suggest that the migratory response to CCR5 stimulation may vary quantitatively with both the CCR5 ligand (MIP-1α, MIP-1β, and RANTES), as well as the nature and concentration of the ECM substrate involved.Key words: chemokines, integrins, cell movement, extracellular matrix proteins, CCR5.

Mammalian sterile 20-like kinase 1 (Mst1) is a MAPK kinase kinase kinase which is involved in a wide range of cellular responses, including apoptosis, lymphocyte adhesion and trafficking. The contribution of Mst1 to Ag-specific immune responses and autoimmunity has not been well defined. In this study, we provide evidence for the essential role of Mst1 in T cell differentiation and autoimmunity, using both genetic and pharmacologic approaches. Absence of Mst1 in mice reduced T cell proliferation and IL-2 production in vitro, blocked cell cycle progression, and elevated activation-induced cell death in Th1 cells. Mst1 deficiency led to a CD4+ T cell development path that was biased toward Th2 and immunoregulatory cytokine production with suppressed Th1 responses. In addition, Mst1−/− B cells showed decreased stimulation to B cell mitogens in vitro and deficient Ag-specific Ig production in vivo. Consistent with altered lymphocyte function, deletion of Mst1 reduced the severity of experimental autoimmune encephalomyelitis (EAE) and protected against collagen-induced arthritis development. Mst1−/− CD4+ T cells displayed an intrinsic defect in their ability to respond to encephalitogenic antigens and deletion of Mst1 in the CD4+ T cell compartment was sufficient to alleviate CNS inflammation during EAE. These findings have prompted the discovery of novel compounds that are potent inhibitors of Mst1 and exhibit desirable pharmacokinetic properties. In conclusion, this report implicates Mst1 as a critical regulator of adaptive immune responses, Th1/Th2-dependent cytokine production, and as a potential therapeutic target for immune disorders.

CD8 T lymphocytes are able to eliminate nascent tumor cells through a process referred to as immunosurveillance. However, multiple inhibitory mechanisms within the tumor microenvironment have been described that impede tumor rejection by CD8 T cells, including increased signaling by inhibitory receptors. Lysophosphatidic acid (LPA) is a bioactive lysophospholipid that has been shown repeatedly to promote diverse cellular processes benefiting tumorigenesis. Accordingly, the exaggerated expression of LPA and LPA receptors is a common feature of diverse tumor cell lineages and can result in elevated systemic LPA levels. LPA is recognized by at least six distinct G protein–coupled receptors, several of which are expressed by T cells, although the precise function of LPA signaling in CD8 T-cell activation and function has not been defined. Here, we show that LPA signaling via the LPA5 receptor expressed by CD8 T cells suppresses antigen receptor signaling, cell activation, and proliferation in vitro and in vivo. Importantly, in a mouse melanoma model tumor-specific CD8 T cells that are LPA5-deficient are able to control tumor growth significantly better than wild-type tumor-specific CD8 T cells. Together, these data suggest that the production of LPA by tumors serves not only in an autocrine manner to promote tumorigenesis, but also as a mechanism to suppress adaptive immunity and highlights a potential novel target for cancer treatment. Cancer Immunol Res; 1(4); 245–55. ©2013 AACR.

The phenylalanyl-glycyl–glycyl-alanyl-prolyl (FG-GAP) domain plays an important role in protein–protein interactions, including interaction of integrins with their ligands. Integrin-α FG-GAP repeat-containing protein 2 (Itfg2) is a highly conserved protein in vertebrates that carries two FG-GAP domains, but its role in mammalian physiology is unknown. In this article, we show that Itfg2 is an intracellular protein and it plays a critical role in B cell differentiation and development of autoimmunity. Itfg2-deficient mice displayed a phenotype consistent with retention of B cells in the spleen and had a lower concentration of IgG in the blood when compared with wild-type littermates. Itfg2-deficient splenocytes also showed a defect in cell migration in vitro. After immunization with a thymus-dependent Ag, the absence of Itfg2 caused a shift in B cell maturation from the germinal centers to the extrafollicular regions of the spleen and blocked deposition of Ag-specific plasma cells in the bone marrow. In support of hematopoietic cell intrinsic activity of Itfg2, bone marrow transplantation of Itfg2-deficient cells was sufficient to impair germinal center development in wild-type mice. Furthermore, Itfg2 deficiency exacerbated development of autoimmune disease in MRL/lpr lupus-prone mice. These results identify Itfg2 as a novel contributor to B cell differentiation and a negative regulator of the autoimmune response during lupus.

Objective Enterochromaffin cell-derived serotonin (5-HT) promotes intestinal inflammation. We tested hypotheses that peripheral tryptophan hydroxylase (TPH) inhibitors, administered orally, block 5-HT biosynthesis and deplete 5-HT from enterochromaffin cells sufficiently to ameliorate intestinal inflammation; moreover, peripheral TPH inhibitors fail to enter the murine enteric nervous system (ENS) or central nervous systems and thus do not affect constitutive gastrointestinal motility. Design Two peripheral TPH inhibitors, LP-920540 and telotristat etiprate (LX1032; LX1606) were given orally to mice. Effects were measured on 5-HT levels in the gut, blood and brain, 5-HT immunoreactivity in the ENS, gastrointestinal motility and severity of trinitrobenzene sulfonic acid (TNBS)-induced colitis. Quantitation of clinical scores, histological damage and intestinal expression of inflammation-associated cytokines and chemokines with focused microarrays and real-time reverse transcriptase PCR were employed to evaluate the severity of intestinal inflammation. Results LP-920540 and LX1032 reduced 5-HT significantly in the gut and blood but not in the brain. Neither LP-920540 nor LX1032 decreased 5-HT immunoreactive neurons or fibres in the myenteric plexus and neither altered total gastrointestinal transit time, colonic motility or gastric emptying in mice. In contrast, oral LP-920540 and LX1032 reduced the severity of TNBS-induced colitis; the expression of 24% of 84 genes encoding inflammation-related cytokines and chemokines was lowered at least fourfold and the reduced expression of 17% was statistically significant. Conclusions Observations suggest that that peripheral TPH inhibitors uncouple the positive linkage of enterochromaffin cell-derived 5-HT to intestinal inflammation. Because peripheral TPH inhibitors evidently do not enter the murine ENS, they lack deleterious effects on constitutive intestinal motility in mice.

Ribosomal protein (RP) mutations in diseases such as 5q− syndrome both disrupt hematopoiesis and increase the risk of developing hematologic malignancy. However, the mechanism by which RP mutations increase cancer risk has remained an important unanswered question. We show here that monoallelic, germline inactivation of the ribosomal protein L22 (Rpl22) predisposes T-lineage progenitors to transformation. Indeed, RPL22 was found to be inactivated in ∼ 10% of human T-acute lymphoblastic leukemias. Moreover, monoallelic loss of Rpl22 accelerates development of thymic lymphoma in both a mouse model of T-cell malignancy and in acute transformation assays in vitro. We show that Rpl22 inactivation enhances transformation potential through induction of the stemness factor, Lin28B. Our finding that Rpl22 inactivation promotes transformation by inducing expression of Lin28B provides the first insight into the mechanistic basis by which mutations in Rpl22, and perhaps some other RP genes, increases cancer risk.

Sphingolipid metabolism involves the synthesis and degradation of a number of structurally similar molecules that function as building blocks of cell membranes as well as signalling molecules. Sphingosine, derived from the breakdown of ceramide, is phosphorylated by two sphingosine kinases (SK), SK1 and SK2, producing Sphingosine-1-Phosphate (S1P). S1P is an important signalling agent present in all mammalian cells as well as in the circulation. It can serve both as a chemotactic ligand of G-protein coupled receptors and as a second messenger in signal transduction pathways, which control cell differentiation, proliferation and apoptosis. S1P levels are regulated by two other catalytic processes in addition to the kinase activities. Sphingosine 1-phosphate lyase (S1PL) irreversibly degrades S1P through a retro-aldol reaction, and two classes of phosphatases, acting on lipids, dephosphorylate S1P to regenerate sphingosine. It has become evident that tipping the balance of S1P production can augment as well as inhibit inflammation in a context-dependent manner. This dual function places S1P in the company of other secreted factors that display a Yin-Yang role in the inflammatory process, such as INF-γ, TGF-β and members of the IL-17 family of cytokines. Therefore, enzymes of sphingolipid metabolism have become important new drug targets for the control of inflammation, autoimmune disorders and cancer. This chapter covers the immunology of S1PL, SK1 and SK2, and summarizes the drug-discovery efforts aimed at exploiting the potential of these enzymes as novel anti-inflammatory drug targets.

Characterization of biological and immunological properties of human immunodeficiency virus type 1 (HIV-1) is critical to developing effective therapies and vaccines for AIDS. With the use of a novel CD4+ T-cell line (PM-1) permissive to infection by both monocytotropic (MT) and T-cell-tropic virus types, we present a comparative analysis of the immunological properties of a prototypic primary MT isolate of HIV-1 strain JR-CSF (MT-CSF) with those of a T-cell-tropic variant (T-CSF) of the same virus, which emerged spontaneously in vitro. The parental MT-CSF infected only PM-1 cells and was markedly resistant to neutralization by sera from HIV-1-infected individuals, rabbit antiserum to recombinant MT-CSF gp120, and anti-V3 monoclonal antibodies. The T-CSF variant infected a variety of CD4+ T-cell lines, contained positively charged amino acid substitutions in the gp120 V3 region, and was highly sensitive to antibody neutralization. Neutralization and antibody staining of T-CSF-expressing cells were significantly inhibited by HIV-1 V3 peptides; in contrast, the MT strain showed only weak V3-specific binding of polyclonal and monoclonal antibodies. Exposure of PM-1 cells to a mixture of both viruses in the presence of human anti-HIV-1 neutralizing antiserum resulted in infection with only MT-CSF. These results demonstrate that although the V3 region of MT viruses is immunogenic, the target epitopes in the V3 principal neutralizing domain on the membrane form of the MT envelope appear to be cryptic or hidden from blocking antibodies.

The human proton coupled folate transporter (PCFT) is involved in low pH-dependent intestinal folate transport. In this report, we describe a new murine model of the hereditary folate malabsorption syndrome that we developed through targeted disruption of the first 3 coding exons of the murine homolog of the PCFT gene. By 4 weeks of age, PCFT-deficient (PCFT−/−) mice developed severe macrocytic normochromic anemia and pancytopenia. Immature erythroblasts accumulated in the bone marrow and spleen of PCFT−/− mice and failed to differentiate further, showing an increased rate of apoptosis in intermediate erythroblasts and reduced release of reticulocytes. In response to the inefficient hematologic development, the serum of the PCFT−/− animals contained elevated concentrations of erythropoietin, soluble transferrin receptor (sCD71), and thrombopoietin. In vivo folate uptake experiments demonstrated a systemic folate deficiency caused by disruption of PCFT-mediated intestinal folate uptake, thus confirming in vivo a critical and nonredundant role of the PCFT protein in intestinal folate transport and erythropoiesis. The PCFT-deficient mouse serves as a model for the hereditary folate malabsorption syndrome and is the most accurate animal model of folate deficiency anemia described to date that closely captures the spectrum of pathology typical of this disease.

Sphingosine 1-phosphate lyase (S1PL) has been characterized as a novel target for the treatment of autoimmune disorders using genetic and pharmacological methods. Medicinal chemistry efforts targeting S1PL by direct in vivo evaluation of synthetic analogues of 2-acetyl-4(5)-(1(R),2(S),3(R),4-tetrahydroxybutyl)-imidazole (THI, 1) led to the discovery of 2 (LX2931) and 4 (LX2932). The immunological phenotypes observed in S1PL deficient mice were recapitulated by oral administration of 2 or 4. Oral dosing of 2 or 4 yielded a dose-dependent decrease in circulating lymphocyte numbers in multiple species and showed a therapeutic effect in rodent models of rheumatoid arthritis (RA). Phase I clinical trials indicated that 2, the first clinically studied inhibitor of S1PL, produced a dose-dependent and reversible reduction of circulating lymphocytes and was well tolerated at dose levels of up to 180 mg daily. Phase II evaluation of 2 in patients with active rheumatoid arthritis is currently underway.

A series of potent piperidine-linked cytosine derivatives were prepared as inhibitors of deoxycytidine kinase (dCK). Compound 9h was discovered to be a potent inhibitor of dCK and shows a good combination of cellular potency and pharmacokinetic parameters. Compound 9h blocks the incorporation of radiolabeled cytosine into mouse T-cells in vitro, as well as in vivo in mice following a T-cell challenge.

A series of deoxycytidine kinase inhibitors was simultaneously optimized for potency and PK properties. A co-crystal structure then allowed merging this series with a high throughput screening hit to afford a highly potent, selective and orally bioavailable inhibitor, compound 10. This compound showed dose dependent inhibition of deoxycytidine kinase in vivo.

BACKGROUND: S1PL is an aldehyde-lyase that irreversibly cleaves sphingosine 1-phosphate (S1P) in the terminal step of sphingolipid catabolism. Because S1P modulates a wide range of physiological processes, its concentration must be tightly regulated within both intracellular and extracellular environments. METHODOLOGY: In order to better understand the function of S1PL in this regulatory pathway, we assessed the in vivo effects of different levels of S1PL activity using knockout (KO) and humanized mouse models. PRINCIPAL FINDINGS: Our analysis showed that all S1PL-deficient genetic models in this study displayed lymphopenia, with sequestration of mature T cells in the thymus and lymph nodes. In addition to the lymphoid phenotypes, S1PL KO mice (S1PL(-/-)) also developed myeloid cell hyperplasia and significant lesions in the lung, heart, urinary tract, and bone, and had a markedly reduced life span. The humanized knock-in mice harboring one allele (S1PL(H/-)) or two alleles (S1PL(H/H)) of human S1PL expressed less than 10 and 20% of normal S1PL activity, respectively. This partial restoration of S1PL activity was sufficient to fully protect both humanized mouse lines from the lethal non-lymphoid lesions that developed in S1PL(-/-) mice, but failed to restore normal T-cell development and trafficking. Detailed analysis of T-cell compartments indicated that complete absence of S1PL affected both maturation/development and egress of mature T cells from the thymus, whereas low level S1PL activity affected T-cell egress more than differentiation. SIGNIFICANCE: These findings demonstrate that lymphocyte trafficking is particularly sensitive to variations in S1PL activity and suggest that there is a window in which partial inhibition of S1PL could produce therapeutic levels of immunosuppression without causing clinically significant S1P-related lesions in non-lymphoid target organs.

Throughout evolution, mammals have developed an elaborate network of positive and negative regulatory mechanisms, which provide balance between defensive measures against bacterial and viral pathogens and protective measures against unwarranted destruction of the host by the activated immune system. Kinases and phosphatases encompassing the MAPK pathway are key players in the orderly action of pro- and anti-inflammatory processes, forming numerous promiscuous interactions. Several lines of evidence demonstrate that the phosphorylation and activation status of kinases in the MAPK system has crucial impact on the outcome of downstream events that regulate cytokine production. At least 13 members of the family of dual-specificity phosphatases (DUSP) display unique substrate specificities for MAPKs. Despite the considerable amount of information obtained about the contribution of the different DUSP to MAPK-mediated signaling and innate immunity, the interpretation of available data remains problematic. The in vitro and ex vivo findings are often complicated by functional redundancy of signaling molecules and do not always accurately predict the situation in vivo. Until recently, DUSP research has been hampered by the lack of relevant mammalian knockout (KO) models, which is a powerful tool for delineating in vivo function and redundancy in gene families. This situation changed dramatically over the last year, and this review integrates recent insights into the precise biological role of the DUSP family in innate immunity gained from a comprehensive analysis of mammalian KO models.

TLR-induced innate immunity and inflammation are mediated by signaling cascades leading to activation of the MAPK family of Ser/Thr protein kinases, including p38 MAPK, which controls cytokine release during innate and adoptive immune responses. Failure to terminate such inflammatory reactions may lead to detrimental systemic effects, including septic shock and autoimmunity. In this study, we provide genetic evidence of a critical and nonredundant role of MAPK phosphatase (MKP)-1 in the negative control of MAPK-regulated inflammatory reactions in vivo. MKP-1−/− mice are hyperresponsive to low-dose LPS-induced toxicity and exhibit significantly increased serum TNF-α, IL-6, IL-12, MCP-1, IFN-γ, and IL-10 levels after systemic administration of LPS. Furthermore, absence of MKP-1 increases systemic levels of proinflammatory cytokines and exacerbates disease development in a mouse model of rheumatoid arthritis. When activated through TLR2, TLR3, TLR4, TLR5, and TLR9, bone marrow-derived MKP-1−/− macrophages exhibit increased cytokine production and elevated expression of the differentiation markers B7.2 (CD86) and CD40. MKP-1-deficient macrophages also show enhanced constitutive and TLR-induced activation of p38 MAPK. Based on these findings, we propose that MKP-1 is an essential component of the intracellular homeostasis that controls the threshold and magnitude of p38 MAPK activation in macrophages, and inflammatory conditions accentuate the significance of this regulatory function.

Autotaxin (ATX), a lysophospholipase D that generates the growth factor-like mediator lysophosphatidic acid LPA, is up-regulated in metastatic and chemotherapy-resistant carcinomas. Many cancer cells secrete ATX, and it contributes to their invasive properties. The biological functions of ATX are mediated by LPA acting on G protein-coupled receptors (LPAR1-8). Many cancers overexpress multiple subtypes of LPAR and the overexpression of LPAR leads to malignant transformation, metastasis, and resistance to therapy. We have previously shown that knockdown of ATX expression in B16F10 melanoma cells decreased tumor invasion in vitro and lung metastasis in vivo. In our present study, we sought to understand the roles of LPAR both in the tumor cells and stromal cells of the tumor microenvironment. B16F10 melanoma cells predominantly express the LPA5 receptor. We found that these cells do not invade across a matrigel layer in response to LPA. shRNA knockdown of LPA5 receptors in B16F10 attenuates this inhibition, suggesting that the LPA5 receptor acts as an anti-invasive receptor in these tumor cells. Interestingly, we identified a different role for host LPA5 receptor. We found that the incidence of B16F10 lung metastasis was significantly reduced in the LPA5 knockout mice compared to wild type mice. In addition, LPA1 knockout mice also showed diminished B16F10 lung metastasis suggesting that host LPA1 and LPA5 receptors play critical roles in the seeding of metastasis. The decrease in tumor cell residence in the lungs of these knockout animals was apparent at 24 hours after injection. However, deletion of LPA1 or LPA5 did not affect the subcutaneous growth of B16F10 tumors. In summary, our results suggest that LPAR, in particular LPA5 and LPA1 receptors in tumor and stromal cells may play different roles in invasion and the seeding of metastasis. Citation Format: Sue-Chin Lee, Yuko Fujiwara, Jianxiong Liu, Junming Yue, Ryoko Tsukahara, Erzsebet Szabo, Renukadevi Patil, Duane D. Miller, Louisa Balazs, Tamas Oravecz, Gabor J. Tigyi. Autotaxin, LPA1 and LPA5 receptors in the tumor microenvironment determine melanoma invasion and metastasis. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B28. doi:10.1158/1538-7445.CHTME14-B28

Phorbol 12-myristate-13-acetate (PMA), a potent tumor promoter and activator of most protein kinase C (PKC) isotypes, was found to significantly inhibit the growth of low population density (1-5% confluency) NIH 3T3 cells. Higher cell population density (above 10% confluency) provided protection from this growth inhibitory effect of PMA. PMA-induced growth arrest is accompanied by an elevation in the level of p21(Cip1) protein, along with cell cycle arrest at the G1/S transition. Activation of PKC is required for this growth inhibitory response since the pan PKC inhibitor GF109203 blocked this effect of PMA. However, the classical PKC inhibitor Gö6976 had no effect, strongly suggesting the involvement of novel PKC isotypes (delta and/or epsilon). Overexpression of PKCepsilon, but not PKCdelta, was found to potentiate PMA-induced growth inhibition. Overexpression of a kinase-inactive dominant-negative mutant of PKCepsilon (K437R) decreased the growth inhibitory effect of PMA and also blocked the PMA-induced increase in the level of p21(Cip1) protein. Taken together, these results indicate that PMA has a cell population density-dependent effect on the growth of NIH 3T3 cells and that the PMA growth inhibitory effect at low cell population density is mediated through activation of PKCepsilon.