Upregulation Of Chemokine Receptors Research Articles

Cardiovascular risk in ANCA-associated vasculitis: Monocyte phenotyping reveals distinctive signatures between serological subsets

Background and aimsAnti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitides (AAV) is associated with an increased cardiovascular risk, particularly the myeloperoxidase AAV serotype (MPO-AAV). Distinct alterations in monocyte phenotypes may cause accelerated atherosclerotic disease in AAV. MethodsA cohort including 43 AAV patients and 19 healthy controls was included for downstream analyses. Extensive phenotyping of monocytes and monocyte-derived macrophages was performed using bulk RNA-sequencing and flow cytometry. An in vitro transendothelial migration assay reflecting intrinsic adhesive and migratory capacities of monocytes was employed. Subsequent sub-analyses were performed to investigate differences between serological subtypes. ResultsMonocyte subset analysis showed increased classical monocytes during active disease, whereas non-classical monocytes were decreased compared to healthy controls (HC). RNA-sequencing revealed upregulation of distinct inflammatory pathways and lipid metabolism-related markers in monocytes of active AAV patients. No differences were detected in the intrinsic monocyte adhesion and migration capacity. Compared to proteinase-3(PR3)-AAV, monocytes of MPO-AAV patients in remission expressed genes related to inflammation, coagulation, platelet-binding and interferon signalling, whereas the expression of chemokine receptors indicative of acute inflammation and monocyte extravasation (i.e., CCR2 and CCR5) was increased in monocytes of PR3-AAV patients. During active disease, PR3-AAV was linked with elevated serum CRP and increased platelet counts compared to MPO-AAV. ConclusionsThese findings highlight changes in monocyte subset composition and activation, but not in the intrinsic migration capacity of AAV monocytes. MPO-AAV monocytes are associated with sustained upregulation of inflammatory genes, whereas PR3-AAV monocytes exhibit chemokine receptor upregulation. These molecular changes may play a role in elevating cardiovascular risk as well as in the underlying pathophysiology of AAV.

MRNAs encoding self-DNA reactive cGAS enhance the immunogenicity of lipid nanoparticle vaccines.

Nucleic acid-based vaccines hold promise in preventing infections and treating cancer. The most common use of this technology is to encode antigenic proteins on mRNAs that are delivered to cells via lipid nanoparticle (LNP) formulations. In this study, we discovered that immunostimulatory proteins can also be encoded on mRNAs in LNPs. We found that an active mutant of the enzyme cGAS, referred to as cGAS∆N, acts as a catalytic adjuvant in LNP-encapsulated mRNA vaccines. The delivery of cGAS∆N mRNA via LNPs in combination with antigen mRNA-LNPs led to durable antigen-specific IFNγ-producing T cells that exceeded the efficiency of antigen-LNPs similar to those currently used in the clinic. This strategy did not compromise B cell responses; rather it induced Th1-biased antibody isotypes. This work unveils new vaccine design strategies using mRNA-encoded catalytic adjuvants that could be ideal for generating CD8+ T cell and B cell responses for immunotherapies.

Interaction between γδTCR signaling and the E protein-Id axis in γδ T cell development.

γδ T cells acquire their functional properties in the thymus, enabling them to exert rapid innate-like responses. To understand how distinct γδ T cell subsets are generated, we have developed a Two-Stage model for γδ T cell development. This model is predicated on the finding that γδTCR signal strength impacts E protein activity through graded upregulation of Id3. Our model proposes that cells enter Stage 1 in response to a γδTCR signaling event in the cortex that activates a γδ T cell-specific gene network. Part of this program includes the upregulation of chemokine receptors that guide them to the medulla. In the medulla, Stage 1 cells receive distinct combinations of γδTCR, cytokine, and/co-stimulatory signals that induce their transit into Stage 2, either toward the γδT1 or the γδT17 lineage. The intersection between γδTCR and cytokine signals can tune Id3 expression, leading to different outcomes even in the presence of strong γδTCR signals. The thymic signaling niches required for γδT17 development are segregated in time and space, providing transient windows of opportunity during ontogeny. Understanding the regulatory context in which E proteins operate at different stages will be key in defining how their activity levels impose functional outcomes.

Histone deacetylase 1 (HDAC1): A key player of T cell-mediated arthritis

Rheumatoid Arthritis (RA) represents a chronic T cell-mediated inflammatory autoimmune disease. Studies have shown that epigenetic mechanisms contribute to the pathogenesis of RA. Histone deacetylases (HDACs) represent one important group of epigenetic regulators. However, the role of individual HDAC members for the pathogenesis of arthritis is still unknown. In this study we demonstrate that mice with a T cell-specific deletion of HDAC1 (HDAC1-cKO) are resistant to the development of Collagen-induced arthritis (CIA), whereas the antibody response to collagen type II was undisturbed, indicating an unaltered T cell-mediated B cell activation. The inflammatory cytokines IL-17 and IL-6 were significantly decreased in sera of HDAC1-cKO mice. IL-6 treated HDAC1-deficient CD4+ T cells showed an impaired upregulation of CCR6. Selective inhibition of class I HDACs with the HDAC inhibitor MS-275 under Th17-skewing conditions inhibited the upregulation of chemokine receptor 6 (CCR6) in mouse and human CD4+ T cells. Accordingly, analysis of human RNA-sequencing (RNA-seq) data and histological analysis of synovial tissue samples from human RA patients revealed the existence of CD4+CCR6+ cells with enhanced HDAC1 expression. Our data indicate a key role for HDAC1 for the pathogenesis of CIA and suggest that HDAC1 and other class I HDACs might be promising targets of selective HDAC inhibitors (HDACi) for the treatment of RA.

Filming the airways

Innate Lymphoid Cells Group 2 innate lymphoid cells (ILC2s) are key drivers of immune responses in the lung. Puttur et al. used an interleukin-13 (IL-13) reporter mouse strain to visualize migration of ILC2s in allergic airway inflammation. ILC2s exhibited dynamic ameboid-like movement in response to IL-33. IL-3 induced up-regulation of chemokine receptor 8 on ILC2s, which promoted their homing to deposits of chemokine ligand 8 within the airways. Collagen-I, a component of the extracellular matrix (ECM), played a role in directly regulating ILC2 cell migration. Thus, structural cues from the ECM work in conjunction with immune mediators to influence immune cell functions in tissues. Sci. Immunol. 4 , eaav7638 (2019).

P66Shc deficiency in the Eμ-TCL1 mouse model of chronic lymphocytic leukemia enhances leukemogenesis by altering the chemokine receptor landscape.

The Shc family adaptor p66Shc acts as a negative regulator of proliferative and survival signals triggered by the B-cell receptor and, by enhancing the production of reactive oxygen species, promotes oxidative stress-dependent apoptosis. Additionally, p66Shc controls the expression and function of chemokine receptors that regulate lymphocyte traffic. Chronic lymphocytic leukemia cells have a p66Shc expression defect which contributes to their extended survival and correlates with poor prognosis. We analyzed the impact of p66Shc ablation on disease severity and progression in the Eμ-TCL1 mouse model of chronic lymphocytic leukemia. We showed that Eμ-TCL1/p66Shc−/− mice developed an aggressive disease that had an earlier onset, occurred at a higher incidence and led to earlier death compared to that in Eμ-TCL1 mice. Eμ-TCL1/p66Shc−/− mice displayed substantial leukemic cell accumulation in both nodal and extranodal sites. The target organ selectivity correlated with upregulation of chemokine receptors whose ligands are expressed therein. This also applied to chronic lymphocytic leukemia cells, where chemokine receptor expression and extent of organ infiltration were found to correlate inversely with these cells’ level of p66Shc expression. p66Shc expression declined with disease progression in Eμ-TCL1 mice and could be restored by treatment with the Bruton tyrosine kinase inhibitor ibrutinib. Our results highlight p66Shc deficiency as an important factor in the progression and severity of chronic lymphocytic leukemia and underscore p66Shc expression as a relevant therapeutic target.

MiR-505-3p controls chemokine receptor up-regulation in macrophages: role in familial hypercholesterolemia.

Familial hypercholesterolemia (FH) conveys a high risk of premature atherosclerosis as a result of lifelong exposure to high LDL cholesterol levels that are not fully reduced by standard-of-care lipid-lowering treatment. Inflammatory mediators have played a role in the progression of atherosclerotic lesions. Here, we investigated whether innate immunity cells in patients with FH have a specific proinflammatory phenotype that is distinct from that of cells in normal participants. To this end, miR-505-3p-a microRNA related to chronic inflammation-and its target genes were investigated in monocyte-derived macrophages (MACs) of patients with FH (FH-MACs) and non-FH controls (co-MACs). On the basis of the profiler PCR array analysis of agomiR-505-3p-transfected MACs, we identified the chemokine receptors, CCR3, CCR4, and CXCR1, as genes that are regulated by miR-505-3p via the transcription factor, RUNX1. miR-505-3p was significantly down-regulated, whereas CCR3, CCR4, CXCR, and RUNX1 were increased in FH-MAC compared with co-MAC, with the increase being more evident in the proinflammatory M1-like FH-MAC. Chemokine receptor levels were unrelated to LDL plasma levels at entry, but correlated with age in patients with FH, not in controls. In summary, we demonstrate for first time to our knowledge that MACs from FH-MACs have an inflammatory phenotype that is characterized by the up-regulation of CCR3, CCR4, and CXCR1 under the control of miR-505-3p. These results suggest a chronic inflammatory condition in FH innate immunity cells that is not reverted by standard lipid-lowering treatment.-Escate, R., Mata, P., Cepeda, J. M., Padró, T., Badimon, L. miR-505-3p controls chemokine receptor up-regulation in macrophages: role in familial hypercholesterolemia.

MiR-505-3p controls chemokine receptor up-regulation in macrophages: role in familial hypercholesterolemia.

Familial hypercholesterolemia (FH) conveys a high risk of premature atherosclerosis as a result of lifelong exposure to high LDL cholesterol levels that are not fully reduced by standard-of-care lipid-lowering treatment. Inflammatory mediators have played a role in the progression of atherosclerotic lesions. Here, we investigated whether innate immunity cells in patients with FH have a specific proinflammatory phenotype that is distinct from that of cells in normal participants. To this end, miR-505-3p-a microRNA related to chronic inflammation-and its target genes were investigated in monocyte-derived macrophages (MACs) of patients with FH (FH-MACs) and non-FH controls (co-MACs). On the basis of the profiler PCR array analysis of agomiR-505-3p-transfected MACs, we identified the chemokine receptors, CCR3, CCR4, and CXCR1, as genes that are regulated by miR-505-3p via the transcription factor, RUNX1. miR-505-3p was significantly down-regulated, whereas CCR3, CCR4, CXCR, and RUNX1 were increased in FH-MAC compared with co-MAC, with the increase being more evident in the proinflammatory M1-like FH-MAC. Chemokine receptor levels were unrelated to LDL plasma levels at entry, but correlated with age in patients with FH, not in controls. In summary, we demonstrate for first time to our knowledge that MACs from FH-MACs have an inflammatory phenotype that is characterized by the up-regulation of CCR3, CCR4, and CXCR1 under the control of miR-505-3p. These results suggest a chronic inflammatory condition in FH innate immunity cells that is not reverted by standard lipid-lowering treatment.-Escate, R., Mata, P., Cepeda, J.M., Padró, T., Badimon, L. miR-505-3p controls chemokine receptor up-regulation in macrophages: role in familial hypercholesterolemia. FASEB J. 32, 601-612 (2018). www.fasebj.org.

PET/CT Imaging of Chemokine Receptors in Inflammatory Atherosclerosis Using Targeted Nanoparticles.

Atherosclerosis is inherently an inflammatory process that is strongly affected by the chemokine-chemokine receptor axes regulating the trafficking of inflammatory cells at all stages of the disease. Of the chemokine receptor family, some specifically upregulated on macrophages play a critical role in plaque development and may have the potential to track plaque progression. However, the diagnostic potential of these chemokine receptors has not been fully realized. On the basis of our previous work using a broad-spectrum peptide antagonist imaging 8 chemokine receptors together, the purpose of this study was to develop a targeted nanoparticle for sensitive and specific detection of these chemokine receptors in both a mouse vascular injury model and a spontaneously developed mouse atherosclerosis model. The viral macrophage inflammatory protein-II (vMIP-II) was conjugated to a biocompatible poly(methyl methacrylate)-core/polyethylene glycol-shell amphiphilic comblike nanoparticle through controlled conjugation and polymerization before radiolabeling with (64)Cu for PET imaging in an apolipoprotein E-deficient (ApoE(-/-)) mouse vascular injury model and a spontaneous ApoE(-/-) mouse atherosclerosis model. Histology, immunohistochemistry, and real-time reverse transcription polymerase chain reaction were performed to assess the plaque progression and upregulation of chemokine receptors. The chemokine receptor-targeted (64)Cu-vMIP-II-comb showed extended blood retention and improved biodistribution. PET imaging showed specific tracer accumulation at plaques in ApoE(-/-) mice, confirmed by competitive receptor blocking studies and assessment in wild-type mice. Histopathologic characterization showed the progression of plaque including size and macrophage population, corresponding to the elevated concentration of chemokine receptors and more importantly increased PET signals. This work provides a useful nanoplatform for sensitive and specific detection of chemokine receptors to assess plaque progression in mouse atherosclerosis models.

Over-Expression of HSP47 Augments Mouse Embryonic Stem Cell Smooth Muscle Differentiation and Chemotaxis

In the recent decade, embryonic stem cells (ESC) have emerged as an attractive cell source of smooth muscle cells (SMC) for vascular tissue engineering owing to their unlimited self-renewal and differentiation capacities. Despite their promise in therapy, their efficacy is still hampered by the lack of definitive SMC differentiation mechanisms and difficulties in successful trafficking of the ESC towards a site of injury or target tissue. Heat shock protein 47 (HSP47) is a 47-kDa molecular chaperone that is required for the maturation of various types of collagen and has been shown to be a critical modulator of different pathological and physiological processes. To date, the role of HSP47 on ESC to SMC differentiation or ESC chemotaxis is not known and may represent a potential molecular approach by which ESC can be manipulated to increase their efficacy in clinic. We provide evidence that HSP47 is highly expressed during ESC differentiation into the SMC lineage and that HSP47 reduction results in an attenuation of the differentiation. Our experiments using a HSP47 plasmid transfection system show that gene over-expression is sufficient to induce ESC-SMC differentiation, even in the absence of exogenous stimuli. Furthermore, HSP47 over-expression in ESC also increases their chemotaxis and migratory responses towards a panel of chemokines, likely via the upregulation of chemokine receptors. Our findings provide direct evidence of induced ESC migration and differentiation into SMC via the over-expression of HSP47, thus identifying a novel approach of molecular manipulation that can potentially be exploited to improve stem cell therapy for vascular repair and regeneration.

CXCR4 drives the metastatic phenotype in breast cancer through induction of CXCR2 and activation of MEK and PI3K pathways

Aberrant expression of CXCR4 in human breast cancer correlates with metastasis to tissues secreting CXCL12. To understand the mechanism by which CXCR4 mediates breast cancer metastasis, MCF-7 breast carcinoma cells were transduced to express wild-type CXCR4 (CXCR4WT) or constitutively active CXCR4 (CXCR4ΔCTD) and analyzed in two-dimensional (2D) cultures, three-dimensional reconstituted basement membrane (3D rBM) cultures, and mice using intravital imaging. Two-dimensional cultures of MCF-7 CXCR4ΔCTD cells, but not CXCR4WT, exhibited an epithelial-to-mesenchymal transition (EMT) characterized by up-regulation of zinc finger E box-binding homeobox 1, loss of E-cadherin, up-regulation of cadherin 11, p120 isoform switching, activation of extracellular signal-regulated kinase 1/2, and matrix metalloproteinase-2. In contrast to the 2D environment, MCF-7 CXCR4WT cells cultured in 3D rBM exhibited an EMT phenotype, accompanied by expression of CXCR2, CXCR7, CXCL1, CXCL8, CCL2, interleukin-6, and granulocyte-macrophage colony stimulating factor. Dual inhibition of CXCR2 with CXCR4, or inhibition of either receptor with inhibitors of mitogen-activated protein kinase 1 or phosphatidylinositol 3-kinase, reversed the aggressive phenotype of MCF-7 CXCR4-expressing or MDA-MB-231 cells in 3D rBM. Intravital imaging of CXCR4-expressing MCF-7 cells revealed that tumor cells migrate toward blood vessels and metastasize to lymph nodes. Thus CXCR4 can drive EMT along with an up-regulation of chemokine receptors and cytokines important in cell migration, lymphatic invasion, and tumor metastasis.

PET imaging of chemokine receptors in vascular injury-accelerated atherosclerosis.

Atherosclerosis is the pathophysiologic process behind lethal cardiovascular diseases. It is a chronic inflammatory progression. Chemokines can strongly affect the initiation and progression of atherosclerosis by controlling the trafficking of inflammatory cells in vivo through interaction with their receptors. Some chemokine receptors have been reported to play an important role in plaque development and stability. However, the diagnostic potential of chemokine receptors has not yet been explored. The purpose of this study was to develop a positron emitter-radiolabeled probe to image the upregulation of chemokine receptor in a wire-injury-accelerated apolipoprotein E knockout (ApoE(-/-)) mouse model of atherosclerosis. A viral macrophage inflammatory protein II (vMIP-II) was used to image the upregulation of multiple chemokine receptors through conjugation with DOTA for (64)Cu radiolabeling and PET. Imaging studies were performed at 2 and 4 wk after injury in both wire-injured ApoE(-/-) and wild-type C57BL/6 mice. Competitive PET blocking studies with nonradiolabeled vMIP-II were performed to confirm the imaging specificity. Specific PET blocking with individual chemokine receptor antagonists was also performed to verify the upregulation of a particular chemokine receptor. In contrast, (18)F-FDG PET imaging was performed in both models to evaluate tracer uptake. Immunohistochemistry on the injury and sham tissues was performed to assess the upregulation of chemokine receptors. (15)O-CO PET showed decreased blood volume in the femoral artery after the injury. (64)Cu-DOTA-vMIP-II exhibited fast in vivo pharmacokinetics with major renal clearance. PET images showed specific accumulation around the injury site, with consistent expression during the study period. Quantitative analysis of tracer uptake at the injury lesion in the ApoE(-/-) model showed a 3-fold increase over the sham-operated site and the sites in the injured wild-type mouse. (18)F-FDG PET showed significantly less tracer accumulation than (64)Cu-DOTA-vMIP-II, with no difference observed between injury and sham sites. PET blocking studies identified chemokine receptor-mediated (64)Cu-DOTA-vMIP-II uptake and verified the presence of 8 chemokine receptors, and this finding was confirmed by immunohistochemistry. (64)Cu-DOTA-vMIP-II was proven a sensitive and useful PET imaging probe for the detection of 8 up-regulated chemokine receptors in a model of injury-accelerated atherosclerosis.

The use of hypoxic cultured mesenchymal stem cell for oncolytic virus therapy

The safety of oncolytic viruses, such as conditionally replicative adenoviruses (CRAds), has been validated in clinical trials for cancer therapy. Their antitumor efficacy is limited by the presence of preexisting neutralizing antibodies (NAbs). Mesenchymal stem cells (MSCs) are attractive as a cellular vehicle to carry antitumor agents, not only because they are easily obtained and expanded to great numbers in vitro, but also because of their ability to migrate and engraft to tumors. MSCs expanded under hypoxic conditions decrease in replicative senescence and increase in proliferation capacity and differentiation potentials. However it remains to be clarified whether these hypoxic MSCs also are good carriers for the delivery of CRAds to tumor cells in the presence of NAbs. This study firstly demonstrated hypoxic MSCs with an increased ability to migrate toward tumors through the upregulation of chemokine receptors, such as CXCR4 and CX3CR1. It is then demonstrated that hypoxic MSCs has the capacity to carry CRAds, without inducing apoptosis, for up to one week. Using an in vitro coculture with human colon cancer cells and with intraperitoneally (i.p.) and subcutaneously (s.c.) developed human colon cancer xenografts, it is demonstrated that hypoxic MSCs are able to protect CRAds from attack by NAbs, thereby successfully delivering them to the target tumor cells. These results show that hypoxic MSCs can serve as cell carriers for CRAds and may help to develop new strategies against cancer.

P40phoxExpression Regulates Neutrophil Recruitment and Function during the Resolution Phase of Intestinal Inflammation

NADPH oxidase is a multisubunit complex that assembles during phagocytosis to generate reactive oxygen species. Several components of this complex have been implicated in chronic granulomatous disease and Crohn's disease, highlighting the importance of reactive oxygen species in regulating host immune response. In this study, we use genetically deficient mice to elucidate how p40(phox), one subunit of the NADPH oxidase complex, functions during intestinal inflammation. We show that p40(phox) deficiency enhances inflammation in both dextran sulfate sodium-induced and innate immune-mediated murine colitis models. This inflammation is characterized by severe colonic tissue injury, increased proinflammatory cytokines, and increased neutrophil recruitment. We demonstrate that neutrophils are essential during the recovery phase of intestinal inflammation and that p40(phox) expression is necessary for this restitution. Lastly, using an integrative bioinformatic approach, we show that p40(phox) deficiency leads to upregulation of chemokine receptor 1 and downregulation of enzymes involved in glycan modifications, including fucosyltransferases and sialyltransferases, during inflammation. We propose that p40(phox) deficiency enhances intestinal inflammation through the dysregulation of these two pathways in neutrophils.

IL-15 differentially affects the migratory capacity of young and aged memory CD8 T cells to mucosal sites. (46.5)

Abstract Given that CD4 T cells are targets of depletion by HIV and required for the homing capacity of CD8 T cells, enhancing recruitment of both CD4 and CD8’s could potentially enhance protection against HIV infection. CD8 T cells at the site of infection are critical for clearance of HIV-infected cells. Memory CD8 T cells migrate into the periphery in the absence of “CD4 help”, homeostatically. However, migration to mucosal tissues such as the gut, require up-regulation of chemokine receptors, intergrins and other signals to gain entry. γC cytokines are key components of CD4 and CD8 T cell survival and maintenance. To date, the specific role of γC cytokines on migration of antigen-specific memory CD8 T cells to mucosal tissues is poorly understood. We show that IL-15 complex treatment drives migration of memory CD8 T cells to mucosa tissues such as the lung and lamina propria of the small intestine (SI LP). However, if the memory cells are aged then migration of CD8 T cells to the mucosal tissues is limited to the lung. These data reveal a novel role of for IL-15 for use as a potential vaccine adjuvant to enhance migration of young memory CD8 T cells to mucosal sites of infection and enhance protection against HIV infection.

Multiparameter Flow Cytometry Analysis of Peripheral Blood T, NK and Dendritic Cells From High-Risk Smoldering Multiple Myeloma Patients Treated with Lenalidomide and Dexamethasone

AbstractAbstract 1906Lenalidomide is an immunomodulatory agent that interacts with different components of the immune system by altering cytokine production, regulating T cells and increasing NK cell cytotoxicity. In multiple myeloma (MM), lenalidomide is approved for use in combination with dexamethasone in patients who have received at least one prior therapy. Recent observations have shown that dexamethasone enhances the anti-myeloma effect of lenalidomide; however, dexamethasone may also antagonize the immunomodulatory properties of lenalidomide.In the present study we evaluated by multiparameter flow cytometry (MFC) peripheral blood (PB) T, NK and dendritic (plasmacytoid, myeloid and monocytic) cells (DC) from high-risk smoldering MM (SMM) patients, defined by the presence of at least 2 of the 3 following criteria at diagnosis: bone marrow plasma cell (BMPC) infiltration ≥10%; and/or high M-component (IgG≥30g/L or IgA≥20g/L or B-J Protein>10g/L); and/or ≥95% myelomatous-PC/BMPC and immune paresis. SMM patients were treated according to the QuiReDex trial (NCT 00480363): an induction phase of nine four-week cycles of lenalidomide plus dexamethasone (LenDex) followed by maintenance with lenalidomide until disease progression. In this ongoing study, immunophenotypic data is available in 53 patients at diagnosis (baseline), 30 after 3 cycles of LenDex and 22 at the end of induction therapy (9th cycle). Here we will focus on the 22 cases with information at the 3 time points. For MFC analysis, PB samples were stained using a four-color direct immunofluorescence technique that allowed the quantification and characterization of T, NK and DC cells, including cell cycle analysis.The percentage of PB T cells in total PB cellularity was stable from baseline vs 3 vs 9 cycles of LenDex (22% vs 21% vs 21%; respectively, NS), with similar results also obtained for T CD4 (12% vs 11% vs 9%; respectively, NS) and T CD8 (8% vs 6% vs 8%; respectively, NS) cells. NK cells were slightly increased after 9 cycles of LenDex for both the CD56dim (4.1%, 3.4% and 6%; respectively; NS) and CD56bright (0.05%, 0.04% and 0.15%; respectively; NS) NK cell compartments. Similarly, the percentage of DC slightly increased along treatment, especially for plasmacytoid DC (0.2% at baseline vs 0.4% after 9 cycles; p=0.09). However, when a more detailed immunophenotypic characterization of T and NK cells was carried out significant differences emerged following LenDex treatment (Figure 1A). Accordingly, after 3 and 9 cycles of LenDex both T CD4 and CD8 cells showed increased expression of activation markers such as CD69 (p=.03), CD25 (p=.02 and NS, respectively), CD54 (p<.001), CD28 (p≤.03) and CD120b (p≤.01), together with increased production of IFNγ (p=.03) and IL-2 (p=.1 and p=.008, respectively). Interestingly, after induction therapy an up-regulation of chemokine receptors related to the Th1 (CCR5; p<.001) but also Th2 (CCR4; p≤.002) immune response was detectable in CD4 and CD8 T cells. T CD4 cells displayed a clear maturation into a central memory phenotype following LenDex treatment (38% at baseline vs 50% and 66% at 3 and 9 cycles, respectively; p<.001) while T CD8 cells displayed an increased effector memory phenotype (44% vs 59% vs 62%; p=.004). Further analysis showed increased expression of HLA-DR (p≤.008), the antibody-dependent cell-mediated cytotoxicity associated receptor CD16 (p≤.03), and the adhesion molecules CD11a (p'.006) and CD11b (p≤.004) both on NK (CD56dim and CD56bright) and T cells. No consistent changes were observed in other NK cell receptors, such as CD94 and the immunoglobulin like receptors CD158a, CD161, NKB1 (3DL1) and NKAT2 (2DL3). Concerning cell cycle analysis, the percentage of cells in S-phase was significantly increased from baseline vs 3 vs 9 cycles of LenDex in T CD4 (0.05% vs 0.15% vs 0.16%; p<.001), CD8 (0.05% vs 0.11% vs 0.23%; p<.001) and NK cells (0.09% vs 0.17% vs 0.20%; p=.001). Finally, an unsupervised cluster analysis of the overall immunophenotypic profile obtained after 9 cycles of LenDex (Figure 1B) was able to discriminate two groups of patients (A and B). Interestingly, within the group with higher activation profile (A) 50% of patients achieved ≥VGPR vs 23% in group B (p=.2).In summary, these preliminary results show that in high risk SMM patients the combination of lenalidomide and dexamethasone modulates PB T and NK cells, with increased activation status that may contribute to disease control. [Display omitted] Disclosures:Off Label Use: Lenalidomide is not approved for the treatment of smoldering multiple myeloma. De La Rubia:Janssen-Cilag: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Rosiñol:Celgene: Honoraria. Oriol:Celgene: Consultancy; Janssen-Cilag: Consultancy; Novartis: Consultancy. Hernández:Celgene: Honoraria. de Arriba:Janssen-Cilag: Honoraria; Celgene: Honoraria. Mateos:Celgene: Honoraria. San Miguel:Janssen-Cilag: Honoraria; Celgene: Honoraria, Speakers Bureau.

Differential activation and maturation of two porcine DC populations following TLR ligand stimulation

Dendritic cells (DCs) are at the interface of innate and adaptive immune responses. Once activated via triggering of their pattern recognition receptors (PRRs), they acquire a mature state and migrate to the lymph nodes where they activate T cells and direct the immune response. Compounds that trigger PRRs are potential vaccine adjuvants, hence in this study we stimulated two porcine DC populations, namely monocyte-derived DCs (MoDCs) and blood DCs (BDCs), with a broad range of toll-like receptors (TLRs) ligands and assessed the activation/maturation state of these porcine DCs. In order to determine if TLR ligands would have an effect on porcine DCs, we characterized the expression of TLRs and demonstrated that MoDCs and BDCs expressed the same set of TLRs but at different levels. Of the TLR ligands examined, lipopolysaccharide (LPS) and poly I:C were the most potent activators of MoDCs, inducing the up-regulation of co-stimulatory molecules CD80/86 and the chemokine receptor CCR7, and production of pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)α. The most effective in inducing BDCs activation were LPS and class A CpG oligodeoxynucleotide (ODN), resulting in up-regulation of chemokine receptor (CCR)7 and down-regulation of CCR2 and CCR5, production of IL-12p40, and expression of a broad range of chemokines that were able to attract porcine immune cells.

Moderate kidney disease inhibits atherosclerosis regression

Chronic kidney disease (CKD) accelerates cardiovascular disease. The mechanisms that explain this independent, excess risk associated with CKD have not been fully elucidated. ObjectivesWe propose that impaired regression of atherosclerosis in renal disease represents a novel risk factor for the heightened morbidity and mortality and resistance to treatment observed in patients with CKD. Methods and resultsUsing a transplant model to study atherosclerosis regression, we transplanted atheromatous aortic segments generated in Apolipoprotein E knock-out (ApoE−/−) mice, into either control or moderately uremic, normolipidemic, wild-type mice. In non-uremic mice, lesions regressed 55%, whereas lesions in uremic mice increased in size by 17% (p<0.01 for control vs. uremic). The lesions in uremic mice were also characterized by a greater presence of macrophages (36,300μm2 vs. 12,600μm2, p<0.01). This finding was despite upregulation of chemokine receptor 7 (CCR7), normally a migration factor, in uremic lesion macrophages. Gene expression analysis of lesion macrophages showed relative down-regulation of serum response factor (SRF) target genes in the uremic group, consistent with impaired CCR7 signaling. ConclusionModerate kidney disease inhibits regression of atherosclerosis in a mouse transplant model. This inhibition may be a result of impaired CCR7 signaling.

Understanding the molecular mechanism of blood–brain barrier damage in an experimental model of Japanese encephalitis: Correlation with minocycline administration as a therapeutic agent

The blood–brain barrier (BBB) serves to protect the central nervous system (CNS) from damage by exogenous molecules. Japanese encephalitis (JE), caused by a neurotropic flavivirus, leads to inflammation in the CNS, neuronal death and also compromises the structural and functional integrity of the BBB. Minocycline, a semisynthetic tetracycline, has been found to be broadly protective in neurological disease models featuring inflammation and cell death and at present, is being evaluated in clinical trials. In the present study, we propose that the neuroprotective role of minocycline in experimental models of JE extends also to the protection of the BBB. Damage to the BBB was assessed by Evan's blue dye exclusion test after minocycline treatment following Japanese encephalitis virus (JEV) infection. A breakdown of the BBB occurred in mice inoculated intravenously with JEV. This resulted in leakage of protein-bound Evan's blue dye into the brain tissue. Semi-quantitative RT-PCR revealed an up-regulation of chemokine receptors and adhesion molecules following JEV infection. Immunostaining showed leukocyte and neutrophil infiltration following JEV infection. Intraperitoneal injection of minocycline, beginning 24 h post-JEV infection, abrogated the effects by reducing BBB damage, decreasing expression of iNOS, Cox-2, VEGF and also by reducing the elevated level of transcript of chemokine receptors and adhesion molecules in the brain. Matrix metalloproteinases (MMPs) are known to disrupt the BBB and minocycline was found to significantly decrease the activity of MMP-9 in brain tissue homogenates. Thus, minocycline, administered at a clinically relevant time, appears to maintain blood–brain barrier integrity following JEV infection.

KLF2 Transcription-Factor Deficiency in T Cells Results in Unrestrained Cytokine Production and Upregulation of Bystander Chemokine Receptors

The transcription factor KLF2 regulates T cell trafficking by promoting expression of the lipid-binding receptor S1P(1) and the selectin CD62L. Recently, it was proposed that KLF2 also represses the expression of chemokine receptors. We confirmed the upregulation of the chemokine receptor CXCR3 on KLF2-deficient T cells. However, we showed that this was a cell-nonautonomous effect, as revealed by CXCR3 upregulation on wild-type bystander cells in mixed bone-marrow chimeras with KLF2-deficient cells. Furthermore, KLF2-deficient T cells overproduced IL-4, leading to the upregulation of CXCR3 through an IL-4-receptor- and eomesodermin-dependent pathway. Consistent with the increased IL-4 production, we found high concentrations of serum IgE in mice with T cell-specific KLF2 deficiency. Our findings support a model where KLF2 regulates T cell trafficking by direct regulation of S1P(1) and CD62L and restrains spontaneous cytokine production in naive T cells.