Constantinos M. Mikelis

CX3CR1-dependent renal macrophage survival promotes Candida control and host survival

Systemic Candida albicans infection causes high morbidity and mortality and is associated with neutropenia; however, the roles of other innate immune cells in pathogenesis are poorly defined. Here, using a mouse model of systemic candidiasis, we found that resident macrophages accumulated in the kidney, the main target organ of infection, and formed direct contacts with the fungus in vivo mainly within the first few hours after infection. Macrophage accumulation and contact with Candida were both markedly reduced in mice lacking chemokine receptor CX3CR1, which was found almost exclusively on resident macrophages in uninfected kidneys. Infected Cx3cr1-/- mice uniformly succumbed to Candida-induced renal failure, but exhibited clearance of the fungus in all other organs tested. Renal macrophage deficiency in infected Cx3cr1-/- mice was due to reduced macrophage survival, not impaired proliferation, trafficking, or differentiation. In humans, the dysfunctional CX3CR1 allele CX3CR1-M280 was associated with increased risk of systemic candidiasis. Together, these data indicate that CX3CR1-mediated renal resident macrophage survival is a critical innate mechanism of early fungal control that influences host survival in systemic candidiasis.

Decreased Lymphangiogenesis and Lymph Node Metastasis by mTOR Inhibition in Head and Neck Cancer

Despite our improved understanding of cancer, the 5-year survival rate for head and neck squamous cell carcinomas (HNSCC) patients remains relatively unchanged at 50% for the past three decades. HNSCCs often metastasize to locoregional lymph nodes, and lymph node involvement represents one of the most important prognostic factors of poor clinical outcome. Among the multiple dysregulated molecular mechanism in HNSCCs, emerging basic, preclinical, and clinical findings support the importance of the mTOR signaling route in HNSCC progression. Indeed, we observed here that the activation of mTOR is a widespread event in clinical specimens of HNSCCs invading locoregional lymph nodes. We developed an orthotopic model of HNSCC consisting of the implantation of HNSCC cells into the tongues of immunocompromised mice. These orthotopic tumors spontaneously metastasize to the cervical lymph nodes, where the presence of HNSCC cells can be revealed by histologic and immunohistochemical evaluation. Both primary and metastatic experimental HNSCC lesions exhibited elevated mTOR activity. The ability to monitor and quantitate lymph node invasion in this model system enabled us to explore whether the blockade of mTOR could impact HNSCC metastasis. We found that inhibition of mTOR with rapamycin and the rapalog RAD001 diminished lymphangiogenesis in the primary tumors and prevented the dissemination of HNSCC cancer cells to the cervical lymph nodes, thereby prolonging animal survival. These findings may provide a rationale for the future clinical evaluation of mTOR inhibitors, including rapamycin and its analogues, as part of a molecular-targeted metastasis preventive strategy for the treatment of patients with HNSCC.

Integrin ανβ3 is a pleiotrophin receptor required for pleiotrophin-induced endothelial cell migration through receptor protein tyrosine phosphatase β/ζ

We have previously shown that the angiogenic growth factor pleiotrophin (PTN) induces migration of endothelial cells through binding to its receptor protein tyrosine phosphatase β/£ (RPTPβ/ζ). In this study, we show that a monoclonal antibody against αvβ3 but not α5β1 integrin abolished PTN‐induced human endothelial cell migration in a concentration‐dependent manner. Integrin αvβ3 was found to directly interact with PTN in an RGD‐independent manner, whereas a synthetic peptide corresponding to the specificity loop of the α3 integrin extracellular domain (177CYD‐ MKTTC184) inhibited PTN‐αvβ3 interaction and totally abolished PTN‐induced endothelial cell migration. Interestingly, αvβ3 was also found to directly interact with RPTPβ/ζ, and PTN‐induced Y773 phosphorylation of β3 integrin was dependent on both RPTPβ/ζ and the downstream c‐src kinase activation. Midkine was found to interact with RPTPβ/ζ, but not with αvβ3, and caused a small but statistically significant decrease in cell migration. In the same line, PTN decreased migration of different glioma cell lines that express RPTPP/ζ but do not express αvβ3, while it stimulated migration of U87MG cells that express αvβ3 on their cell membrane. Overexpression or down‐regulation of β3 stimulated or abolished, respectively, the effect of PTN on cell migration. Collectively, these data suggest that αvβ3 is a key molecule that determines the stimulatory or inhibitory effect of PTN on cell migration.— Mikelis, C., Sfaelou, E., Koutsioumpa, M., Kieffer, N., Papadimitriou, E. Integrin ovP3 is a pleiotrophin receptor required for pleiotrophin‐induced endothelial cell migration through receptor protein tyrosine phosphatase P/£. FASEBJ. 23, 1459–1469 (2009)

RhoA and ROCK mediate histamine-induced vascular leakage and anaphylactic shock.

Histamine-induced vascular leakage is an integral component of many highly prevalent human diseases, including allergies, asthma, and anaphylaxis. Yet, how histamine induces the disruption of the endothelial barrier is not well defined. By using genetically modified animal models, pharmacologic inhibitors, and a synthetic biology approach, here we show that the small GTPase RhoA mediates histamine-induced vascular leakage. Histamine causes the rapid formation of focal adherens junctions, disrupting the endothelial barrier by acting on H1R Gαq-coupled receptors, which is blunted in endothelial Gαq/11 KO mice. Interfering with RhoA and ROCK function abolishes endothelial permeability, while phospholipase Cβ plays a limited role. Moreover, endothelial-specific RhoA gene deletion prevents vascular leakage and passive cutaneous anaphylaxis in vivo, and ROCK inhibitors protect from lethal systemic anaphylaxis. This study supports a key role for the RhoA signaling circuitry in vascular permeability, thereby identifying novel pharmacological targets for many human diseases characterized by aberrant vascular leakage.

CARD9-Dependent Neutrophil Recruitment Protects against Fungal Invasion of the Central Nervous System.

Candida is the most common human fungal pathogen and causes systemic infections that require neutrophils for effective host defense. Humans deficient in the C-type lectin pathway adaptor protein CARD9 develop spontaneous fungal disease that targets the central nervous system (CNS). However, how CARD9 promotes protective antifungal immunity in the CNS remains unclear. Here, we show that a patient with CARD9 deficiency had impaired neutrophil accumulation and induction of neutrophil-recruiting CXC chemokines in the cerebrospinal fluid despite uncontrolled CNS Candida infection. We phenocopied the human susceptibility in Card9 -/- mice, which develop uncontrolled brain candidiasis with diminished neutrophil accumulation. The induction of neutrophil-recruiting CXC chemokines is significantly impaired in infected Card9 -/- brains, from both myeloid and resident glial cellular sources, whereas cell-intrinsic neutrophil chemotaxis is Card9-independent. Taken together, our data highlight the critical role of CARD9-dependent neutrophil trafficking into the CNS and provide novel insight into the CNS fungal susceptibility of CARD9-deficient humans.

Roles of pleiotrophin in tumor growth and angiogenesis

Pleiotrophin (PTN) is a heparin-binding growth factor with diverse biological activities, the most studied of these being those related to the nervous system, tumor growth and angiogenesis. Although interest in the involvement of PTN in tumor growth is increasing, many questions remain unanswered, particularly concerning the receptors and the signaling pathways involved. In this review, we briefly introduce PTN, and summarize data on its involvement in tumor growth and angiogenesis, and on what is known to date concerning the receptors and pathways involved.

Extrapulmonary Aspergillus infection in patients with CARD9 deficiency

Invasive pulmonary aspergillosis is a life-threatening mycosis that only affects patients with immunosuppression, chemotherapy-induced neutropenia, transplantation, or congenital immunodeficiency. We studied the clinical, genetic, histological, and immunological features of 2 unrelated patients without known immunodeficiency who developed extrapulmonary invasive aspergillosis at the ages of 8 and 18. One patient died at age 12 with progressive intra-abdominal aspergillosis. The other patient had presented with intra-abdominal candidiasis at age 9, and developed central nervous system aspergillosis at age 18 and intra-abdominal aspergillosis at age 25. Neither patient developed Aspergillus infection of the lungs. One patient had homozygous M1I CARD9 (caspase recruitment domain family member 9) mutation, while the other had homozygous Q295X CARD9 mutation; both patients lacked CARD9 protein expression. The patients had normal monocyte and Th17 cell numbers in peripheral blood, but their mononuclear cells exhibited impaired production of proinflammatory cytokines upon fungus-specific stimulation. Neutrophil phagocytosis, killing, and oxidative burst against Aspergillus fumigatus were intact, but neither patient accumulated neutrophils in infected tissue despite normal neutrophil numbers in peripheral blood. The neutrophil tissue accumulation defect was not caused by defective neutrophil-intrinsic chemotaxis, indicating that production of neutrophil chemoattractants in extrapulmonary tissue is impaired in CARD9 deficiency. Taken together, our results show that CARD9 deficiency is the first known inherited or acquired condition that predisposes to extrapulmonary Aspergillus infection with sparing of the lungs, associated with impaired neutrophil recruitment to the site of infection.

Interplay between αvβ3 Integrin and Nucleolin Regulates Human Endothelial and Glioma Cell Migration

Background: Cell surface nucleolin (NCL) is a promising target for development of anticancer agents. Results: A novel pathway that includes ανβ3 integrin and leads to cell surface NCL localization and cell migration has been identified. Conclusion: ανβ3 can be used as a biomarker for the use of NCL antagonists. Significance: This pathway is active in endothelial and glioma cells, as well as in human glioblastomas. The multifunctional protein nucleolin (NCL) is overexpressed on the surface of activated endothelial and tumor cells and mediates the stimulatory actions of several angiogenic growth factors, such as pleiotrophin (PTN). Because αvβ3 integrin is also required for PTN-induced cell migration, the aim of the present work was to study the interplay between NCL and αvβ3 by using biochemical, immunofluorescence, and proximity ligation assays in cells with genetically altered expression of the studied molecules. Interestingly, cell surface NCL localization was detected only in cells expressing αvβ3 and depended on the phosphorylation of β3 at Tyr773 through receptor protein-tyrosine phosphatase β/ζ (RPTPβ/ζ) and c-Src activation. Downstream of αvβ3, PI3K activity mediated this phenomenon and cell surface NCL was found to interact with both αvβ3 and RPTPβ/ζ. Positive correlation of cell surface NCL and αvβ3 expression was also observed in human glioblastoma tissue arrays, and inhibition of cell migration by cell surface NCL antagonists was observed only in cells expressing αvβ3. Collectively, these data suggest that both expression and β3 integrin phosphorylation at Tyr773 determine the cell surface localization of NCL downstream of the RPTPβ/ζ/c-Src signaling cascade and can be used as a biomarker for the use of cell surface NCL antagonists as anticancer agents.

Nitric oxide stimulates migration of human endothelial and prostate cancer cells through up‐regulation of pleiotrophin expression and its receptor protein tyrosine phosphatase β/ζ

Pleiotrophin (PTN) is a secreted growth factor involved in angiogenesis and tumor growth. We have recently shown that low concentrations of hydrogen peroxide (HP) stimulate PTN expression, through activation of the transcription factor AP‐1. In the present work, we studied the possible involvement of endothelial nitric oxide synthase (eNOS) and the role of nitric oxide (NO) in the regulation of PTN expression, as well as involvement of the latter in the NO‐induced human endothelial and prostate cancer cell migration. Inhibition of eNOS or the downstream effector soluble guanylate cyclase (sGC) completely suppressed HP‐induced AP‐1 activities that lead to PTN expression and cell migration. The NO donor sodium nitroprusside (SNP) through activation of sGC significantly and concentration‐dependently increased expression of PTN, through transcriptional activation of the corresponding gene. Moreover, SNP had no effect on the migration of stably transfected prostate cancer cells that do not express PTN and knockdown of PTN receptor protein tyrosine phosphatase β/ζ (RPTPβ/ζ) completely abolished SNP‐induced cell migration. NO added exogenously or produced endogenously by low concentrations of HP through stimulation of sGC activates extracellular signal‐regulated kinase[1/2] (ERK[1/2]) and leads to PTN expression and cell migration. On the other hand, p38, which also intervenes in the up‐regulation of PTN expression by low concentrations of HP, seems to act upstream of eNOS and does not intervene in the SNP‐induced PTN expression and cell migration. The above data suggest that PTN through its receptor RPTPβ/ζ is a mediator of the stimulatory effects of eNOS/NO on human endothelial and prostate cancer cell migration.

PDZ-RhoGEF and LARG Are Essential for Embryonic Development and Provide a Link between Thrombin and LPA Receptors and Rho Activation

Background: Many Rho GEFs have been proposed to link GPCRs to Rho activation. Results: PDZ-RhoGEF and LARG double deficiency leads to embryonic lethality and, along with p115 RhoGEF, are required for thrombin-induced cell migration, proliferation, and RhoA activation. Conclusion: PDZ-RhoGEF, LARG, and p115 RhoGEF mediate Gα12/13 signaling to RhoA. Significance: These findings identify novel targets for pharmacological intervention in diseases involving aberrant GPCR signaling. G protein-coupled receptors (GPCRs) linked to both members of the Gα12 family of heterotrimeric G proteins α subunits, Gα12 and Gα13, regulate the activation of Rho GTPases, thereby contributing to many key biological processes. Multiple Rho GEFs have been proposed to link Gα12/13 GPCRs to Rho activation, including PDZ-RhoGEF (PRG), leukemia-associated Rho GEF (LARG), p115-RhoGEF (p115), lymphoid blast crisis (Lbc), and Dbl. PRG, LARG, and p115 share the presence of a regulator of G protein signaling homology (RGS) domain. There is limited information on the biological roles of this RGS-containing family of RhoGEFs in vivo. p115-deficient mice are viable with some defects in the immune system and gastrointestinal motor dysfunctions, whereas in an initial study we showed that mice deficient for Larg are viable and resistant to salt-induced hypertension. Here, we generated knock-out mice for Prg and observed that these mice do not display any overt phenotype. However, deficiency in Prg and Larg leads to complex developmental defects and early embryonic lethality. Signaling from Gα11/q-linked GPCRs to Rho was not impaired in mouse embryonic fibroblasts defective in all three RGS-containing RhoGEFs. However, a combined lack of Prg, Larg, and p115 expression abolished signaling through Gα12/13 to Rho and thrombin-induced cell proliferation, directional migration, and nuclear signaling through JNK and p38. These findings provide evidence of an essential role for the RGS-containing RhoGEF family in signaling to Rho by Gα12/13-coupled GPCRs, which may likely play a critical role during embryonic development.