Regulation of Dendritic Cell Migration to the Draining Lymph Node: Impact on T Lymphocyte Traffic and Priming

Antigen-pulsed dendritic cells (DCs) are used as natural adjuvants for vaccination, but the factors that influence the efficacy of this treatment are poorly understood. We investigated the parameters that affect the migration of subcutaneously injected mouse-mature DCs to the draining lymph node. We found that the efficiency of DC migration varied with the number of injected DCs and that CCR7+/+ DCs migrating to the draining lymph node, but not CCR7−/− DCs that failed to do so, efficiently induced a rapid increase in lymph node cellularity, which was observed before the onset of T cell proliferation. We also report that DC migration could be increased up to 10-fold by preinjection of inflammatory cytokines that increased the expression of the CCR7 ligand CCL21 in lymphatic endothelial cells. The magnitude and quality of CD4+ T cell response was proportional to the number of antigen-carrying DCs that reached the lymph node and could be boosted up to 40-fold by preinjection of tumor necrosis factor that conditioned the tissue for increased DC migration. These results indicate that DC number and tissue inflammation are critical parameters for DC-based vaccination

The role of PGC-1α and mitochondrial biogenesis in kidney diseases

Chronic kidney disease (CKD) is one of the fastest growing causes of death worldwide, emphasizing the need to develop novel therapeutic approaches. CKD predisposes to acute kidney injury (AKI) and AKI favors CKD progression. Mitochondrial derangements are common features of both AKI and CKD and mitochondria-targeting therapies are under study as nephroprotective agents. PGC-1α is a master regulator of mitochondrial biogenesis and an attractive therapeutic target. Low PGC-1α levels and decreased transcription of its gene targets have been observed in both preclinical AKI (nephrotoxic, endotoxemia, and ischemia-reperfusion) and in experimental and human CKD, most notably diabetic nephropathy. In mice, PGC-1α deficiency was associated with subclinical CKD and predisposition to AKI while PGC-1α overexpression in tubular cells protected from AKI of diverse causes. Several therapeutic strategies may increase kidney PGC-1α activity and have been successfully tested in animal models. These include AMP-activated protein kinase (AMPK) activators, phosphodiesterase (PDE) inhibitors, and anti-TWEAK antibodies. In conclusion, low PGC-1α activity appears to be a common feature of AKI and CKD and recent characterization of nephroprotective approaches that increase PGC-1α activity may pave the way for nephroprotective strategies potentially effective in both AKI and CKD.Supported by ISCIII-FIS, FEDER funds, CP14/00133, PI16/02057, PI16/01900, PI18/01366, PI19/00588, PI19/00815, DTS18/00032, ERA-PerMed-JTC2018 (KIDNEY ATTACK AC18/00064 and PERSTIGAN AC18/00071, ISCIII-RETIC REDinREN RD016/0009, Sociedad Española de Nefrología, Fundacion Renal Iñigo Álvarez de Toledo (FRIAT), ISCIII Miguel Servet (A.B.S., M.D.S.-N.), ISCIII Sara Borrell (J.M.M.-M.), Comunidad de Madrid CIFRA2 B2017/BMD-3686 (M.F.-B. and D.M.-S.

Urinary cyclophilin A as marker of tubular cell death and kidney injury

Background: Despite the term acute kidney injury (AKI), clinical biomarkers for AKI re-flect function rather than injury and independent markers of injury are needed. Tubular cell death, including necroptotic cell death, is a key feature of AKI. Cyclophilin A (CypA) is an intracellular protein that has been reported to be released during necroptosis. We have now explored CypA as a potential marker for kidney injury in cultured tubular cells and in clinical settings of ischemia-reperfusion injury (IRI), characterized by limitations of current diagnostic criteria for AKI. Meth-ods: CypA was analyzed in cultured human and murine proximal tubular epithelial cells exposed to chemical hypoxia, hypoxia/reoxygenation (H/R) or other cell death (apoptosis, necroptosis, fer-roptosis) inducers. Urinary levels of CypA (uCypA) were analyzed in patients after nephron sparing surgery (NSS) in which the contralateral kidney is not disturbed and kidney grafts with initial function. Results: Intracellular CypA remained unchanged while supernatant CypA increased in parallel to cell death induction. uCypA levels were higher in NSS patients with renal artery clamping (that is, with NSS-IRI) than in no clamping (NSS-no IRI), and in kidney transplantation (KT) recipients (KT-IRI) even in the presence of preserved or improving kidney function, while this was not the case for urinary Neutrophil gelatinase-associated lipocalin (NGAL). Furthermore, higher uCypA levels in NSS patients were associated with longer surgery duration and the incidence of AKI increased from 10% when using serum creatinine (sCr) or urinary output criteria to 36% when using high uCypA levels in NNS clamping patients. Conclusions: CypA is released by kidney tubular cells during different forms of cell death, and uCypA increased during IRI-induced clinical kidney injury independently from kidney function parameters. Thus, uCypA is a potential bi-omarker of kidney injury, which is independent from decreased kidney functionResearch by the authors was funded by FIS/ FEDER funds (PI17/00257, PI18/01386, PI19/00588, PI19/00815, DTS18/00032, ERA-PerMed-JTC2018 (KIDNEY ATTACK AC18/00064 and PERSTIGAN AC18/00071, ISCIII-RETIC REDinREN RD016/0009), Sociedad Española de Nefrología, FRIAT, Comunidad de Madrid en Biomedicina B2017/BMD-3686 CIFRA2-CM. Salary support: ISCIII Miguel Servet to A.B.S., MICIN Ramon y Cajal to M.D.S.-N., REDinREN RD016/0009 to M.F.-B.,SENEFRO to D.M.-S. and Consejería de Educación, Juventud y Deporte (Comunidad de Madrid/FSE) to A.M.L.-

Dysregulation of Chemokine/Chemokine Receptor Axes and NK Cell Tissue Localization during Diseases.

Chemokines are small chemotactic molecules that play key roles in physiological and pathological conditions. Upon signaling via their specific receptors, chemokines regulate tissue mobilization and trafficking of a wide array of immune cells, including natural killer (NK) cells. Current research is focused on analyzing changes in chemokine/chemokine receptor expression during various diseases to interfere with pathological trafficking of cells or to recruit selected cell types to specific tissues. NK cells are a heterogeneous lymphocyte population comprising several subsets endowed with distinct functional properties and mainly representing distinct stages of a linear development process. Because of their different functional potential, the type of subset that accumulates in a tissue drives the final outcome of NK cell-regulated immune response, leading to either protection or pathology. Correspondingly, chemokine receptors, including CXCR4, CXCR3, and CX3CR1, are differentially expressed by NK cell subsets, and their expression levels can be modulated during NK cell activation. At first, this review will summarize the current knowledge on the contribution of chemokines to the localization and generation of NK cell subsets in homeostasis. How an inappropriate chemotactic response can lead to pathology and how chemokine targeting can therapeutically affect tissue recruitment/localization of distinct NK cell subsets will also be discussed

Regulatory T cells inhibit Fas ligand-induced innate and adaptive tumour immunity

CD4+CD25+ regulatory T cells (Treg) are known to influence T cell responses to tumours. Here we have explored the role of Treg in inhibiting not only adaptive, but also innate immune responses to tumours. To this end we used a Fas ligand (FasL)-expressing melanoma cell line in a mouse model. In this system, innate immunity is sufficient to reject the tumour. All mice depleted of Treg and challenged with FasL-expressing melanoma remained tumour-free. Investigation of the underlying cellular effector mechanisms revealed that depletion of Treg enhanced an NK cell response capable of tumour lysis. Furthermore, this initial innate immune response primed mice to make an effective adaptive immune response leading to complete rejection of challenge with the parental melanoma. Both antigen-specific antibody and CD4+ T cells were implicated in protection via adaptive immunity. We conclude that removal of Treg and vaccination with whole tumour cells expressing FasL activates multiple arms of the immune system, leading to efficient tumour rejection. These findings highlight a novel role for FasL in inducing innate immune responses that are normally inhibited by Treg and uncover an adjuvant effect of FasL that can be used to stimulate tumour immunity after depletion of Treg

Epigenetic modifiers as potential therapeutic targets in diabetic kidney disease

Diabetic kidney disease is one of the fastest growing causes of death worldwide. Epigenetic regulators control gene expression and are potential therapeutic targets. There is functional interventional evidence for a role of DNA methylation and the histone post-translational modifications—histone methylation, acetylation and crotonylation—in the pathogenesis of kidney disease, including diabetic kidney disease. Readers of epigenetic marks, such as bromodomain and extra terminal (BET) proteins, are also therapeutic targets. Thus, the BD2 selective BET inhibitor apabetalone was the first epigenetic regulator to undergo phase-3 clinical trials in diabetic kidney disease with an endpoint of kidney function. The direct therapeutic modulation of epigenetic features is possible through pharmacological modulators of the specific enzymes involved and through the therapeutic use of the required substrates. Of further interest is the characterization of potential indirect effects of nephroprotective drugs on epigenetic regulation. Thus, SGLT2 inhibitors increase the circulating and tissue levels of β-hydroxybutyrate, a molecule that generates a specific histone modification, β-hydroxybutyrylation, which has been associated with the beneficial health effects of fasting. To what extent this impact on epigenetic regulation may underlie or contribute to the so-far unclear molecular mechanisms of cardio-and nephroprotection offered by SGLT2 inhibitors merits further in-depth studies.This research was funded by FIS/FEDER funds (PI15/00298, CP14/00133, PI16/01900, PI18/01386, PI18/0133, PI19/00588, PI19/00815, DTS18/00032, ERA-PerMed-JTC2018 (KIDNEY ATTACK AC18/00064 and PERSTIGAN AC18/00071), ISCIII-RETIC REDinREN RD016/0009), Sociedad Española de Nefrología, FRIAT, Comunidad de Madrid en Biomedicina B2017/BMD- 3686 CIFRA2-CM. Salary support: ISCIII Miguel Servet to ABS and MDS-N, ISCIII Sara Borrell to JM-MM, REDinREN RD016/0009 to MF-B, and MICIU to JG-M

Quantification and characterization of granulocyte macrophage colony-stimulating factor activated human peripheral blood mononuclear cells by fluorine-19 cellular MRI in an immunocompromised mouse model

PURPOSE: The purpose of this study was to test fluorine-19 (19F) cellular magnetic resonance (MRI) as a non-invasive imaging modality to track therapeutic cell migration as a surrogate marker of immunotherapeutic effectiveness. MATERIALS AND METHODS: Human peripheral blood mononuclear cell- (PBMC)-derived antigen presenting cell (APC) were labeled with a 19F-perfluorocarbon (PFC) and/or activated with granulocyte macrophage colony-stimulating factor (GM-CSF). Viability, phenotype and cell lineage characterization preceded 19F cellular MRI of PFC RESULTS: A high proportion of PBMC incorporated PFC without affecting viability, phenotype or cell lineage composition. PFC CONCLUSION: 19F cellular MRI is a non-invasive imaging technique capable of detecting and quantifying in vivo cell migration in conjunction with an established APC-based immunotherapy model. 19F cellular MRI can function as a surrogate marker for assessing and improving upon the therapeutic benefit that this immunotherapy provides

Locally Triggered Release of the Chemokine CCL21 Promotes Dendritic Cell Transmigration across Lymphatic Endothelia

Trafficking cells frequently transmigrate through epithelial and endothelial monolayers. How monolayers cooperate with the penetrating cells to support their transit is poorly understood. We studied dendritic cell (DC) entry into lymphatic capillaries as a model system for transendothelial migration. We find that the chemokine CCL21, which is the decisive guidance cue for intravasation, mainly localizes in the trans-Golgi network and intracellular vesicles of lymphatic endothelial cells. Upon DC transmigration, these Golgi deposits disperse and CCL21 becomes extracellularly enriched at the sites of endothelial cell-cell junctions. When we reconstitute the transmigration process in vitro, we find that secretion of CCL21-positive vesicles is triggered by a DC contact-induced calcium signal, and selective calcium chelation in lymphatic endothelium attenuates transmigration. Altogether, our data demonstrate a chemokine-mediated feedback between DCs and lymphatic endothelium, which facilitates transendothelial migration.Peer reviewe

Frequency and phenotype of natural killer cells and natural killer cell subsets in bovine lymphoid compartments and blood

Natural killer (NK) cells are widely distributed in lymphoid and non‐lymphoid tissues, but little is known about the recirculation of NK cells between blood and tissues. This is relevant to understanding recirculation in the steady‐state and also for determining the roles for NK cells in vaccine‐induced immunity and responses to infection. Therefore, the percentage of NK cells and their phenotype across peripheral blood, afferent lymph and lymph nodes in steady‐state conditions was investigated in cattle using the pseudo‐afferent lymphatic cannulation model. CD2(+) CD25(lo) NK cells were the predominant subset of NK cells within the blood. In contrast, CD2(−) CD25(hi) NK cells were the main subset present within the skin‐draining afferent lymphatic vessels and lymph nodes, indicating that CD2(−) NK cells are the principal NK cell subset trafficking to lymph nodes via the afferent lymphatic vessel. Furthermore, a low percentage of NK cells were present in efferent lymph, which were predominantly of the CD2(−) subset, indicating that NK cells can egress from lymph nodes and return to circulation in steady‐state conditions. These compartmentalization data indicate that NK cells represent a population of recirculating lymphocytes in steady‐state conditions and therefore may be important during immune responses to vaccination or infection