Complex evaluation of human monocyte‐derived dendritic cells for cancer immunotherapy

Dendritic cell (DC) immunotherapy is capable of generating tumour‐specific immune responses. Different maturation strategies were previously tested to obtain DC capable of anti‐cancer responses in vitro, usually with limited clinical benefit. Mutual comparison of currently used maturation strategies and subsequent complex evaluation of DC functions and their stimulatory capacity on T cells was performed in this study to optimize the DC vaccination strategy for further clinical application. DC were generated from monocytes using granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and interleukin (IL)‐4, pulsed with whole tumour cell lysate and then matured with one of five selected maturation strategies or cultured without additional maturation stimulus. DC were characterized with regard to their surface marker expression, cytokine profiles, migratory capacity, allogeneic and autologous T cell stimulatory capacity as well as their specific cytotoxicity against tumour antigens. We were able to demonstrate extensive variability among different maturation strategies currently used in DC immunotherapeutic protocols that may at least partially explain limited clinical benefit of some clinical trials with such DC. We identified DC matured with interferon‐γ and lipopolysaccharide as the most attractive candidate for future clinical trials in cancer immunotherapy.     

Modulation of the development of human monocyte‐derived dendritic cells by lithium chloride

Lithium has been used or explored to treat psychiatric and neurodegenerative diseases that are frequently associated with an abnormal immune status. It is likely that lithium may work through modulation of immune responses in these patients. Because dendritic cells (DC) play a central role in regulating immune responses, this study investigated the influence of lithium chloride (LiCl) on the development and function of DC. Exposure to LiCl during the differentiation of human monocyte‐derived immature DCs (iDC) enhances CD86 and CD83 expression and increases the production of IL‐1β, IL‐6, IL‐8, IL‐10, and TNF‐α. However, the presence of LiCl during LPS‐induced maturation of iDC has the opposite effect. During iDC differentiation, LiCl suppresses the activity of glycogen synthase kinase (GSK)‐3β, and activates PI3K and MEK. In addition, LiCl activates peroxisome proliferator‐activated receptor γ (PPARγ) during iDC differentiation, a pathway not described before. Each of these signaling pathways appears to have distinct impact on the differentiating iDC. The enhanced CD86 expression by LiCl involves the PI3K/AKT and GSK‐3β pathway. LiCl modulates the expression of CD83 in iDC mainly through MEK/ERK, PI3K/AKT, and PPARγ pathways, while the increased production of IL‐1β and TNF‐α mainly involves the MEK/ERK pathway. The effect of LiCl on IL‐6/IL‐8/IL‐10 secretion in iDC is mediated through inhibition of GSK‐3β. We have also demonstrated that PPARγ is downstream of GSK‐3β and is responsible for the LiCl‐mediated modulation of CD86/83 and CD1 expression, but not IL‐6/8/10 secretion. The combined influence of these molecular signaling pathways may account for certain clinical effect of lithium. J. Cell. Physiol. 226: 424–433, 2011. © 2010 Wiley‐Liss, Inc.     

Differential phagocytosis of Leishmania mexicana promastigotes and amastigotes by monocyte‐derived dendritic cells

Leishmania species are dimorphic protozoan parasites that live and replicate in the gut of sand flies as promastigotes or in mammalian hosts as amastigotes. Different immune cells, including DCs, and receptors differ in their involvement in phagocytosis of promastigotes and amastigotes and in recognition of different Leishmania species. In the case of L. mexicana, differences in phagocytosis of promastigotes and amastigotes by DCs and participation of C‐type lectin receptors (CLRs) have not been established. In the present study, flow cytometry and confocal microscopy were used to investigate the phagocytosis by monocyte‐derived dendritic cells (moDCs) of L. mexicana promastigotes and amastigotes in the presence or absence of immune serum during various periods of time. Blocking antibodies against mannose receptors and DC‐SIGN were used to explore the participation of these receptors in the phagocytosis of L. mexicana by moDC. The major differences in interactions of L. mexicana promastigotes and amastigotes with moDC were found to occur within the first 3 hr, during which phagocytosis of promastigotes predominated as compared with opsonization of promastigotes and amastigotes. However, after 6 hr of incubation, opsonized promastigotes were preferentially phagocytosed as compared with unopsonized promastigotes and amastigotes and after 24 hr of incubation there were no differences in the phagocytosis of promastigotes and amastigotes. Finally, after 3 hr incubation, DC‐SIGN was involved in the phagocytosis of promastigotes, but not of amastigotes.     

Distinct inflammatory phenotypes of microglia and monocyte‐derived macrophages in Alzheimer's disease models: effects of aging and amyloid pathology

Alzheimer's disease (AD) is a neurodegenerative disease characterized by formation of amyloid‐β (Aβ) plaques, activated microglia, and neuronal cell death leading to progressive dementia. Recent data indicate that microglia and monocyte‐derived macrophages (MDM) are key players in the initiation and progression of AD, yet their respective roles remain to be clarified. As AD occurs mostly in the elderly and aging impairs myeloid functions, we addressed the inflammatory profile of microglia and MDM during aging in TgAPP/PS1 and TgAPP/PS1dE9, two transgenic AD mouse models, compared to WT littermates. We only found MDM infiltration in very aged mice. We determined that MDM highly expressed activation markers at basal state. In contrast, microglia exhibited an activated phenotype only with normal aging and Aβ pathology. Our study showed that CD14 and CD36, two receptors involved in phagocytosis, were upregulated during Aβ pathogenesis. Moreover, we observed, at the protein levels in AD models, higher production of pro‐inflammatory mediators: IL‐1β, p40, iNOS, CCL‐3, CCL‐4, and CXCL‐1. Taken together, our data indicate that microglia and MDM display distinct phenotypes in AD models and highlight the specific effects of normal aging vs Aβ peptides on inflammatory processes that occur during the disease progression. These precise phenotypes of different subpopulations of myeloid cells in normal and pathologic conditions may allow the design of pertinent therapeutic strategy for AD.     

Piscirickettsia salmonis induces apoptosis in macrophages and monocyte‐like cells from rainbow trout

Piscirickettsia salmonis is the etiologic agent of the salmonid rickettsial septicemia (SRS) which causes significant losses in salmon production in Chile and other and in other regions in the southern hemisphere. As the killing of phagocytes is an important pathogenic mechanism for other bacteria to establish infections in vertebrates, we investigated whether P. salmonis kills trout macrophages by apoptosis. Apoptosis in infected macrophages was demonstrated by techniques based on morphological changes and host cell DNA fragmentation. Transmission electron microcopy showed classic apoptotic characteristics and terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling showed fragmented DNA. Programmed cell death type I was further confirmed by increased binding of annexin V to externalized phosphatidylserine in infected macrophages. Moreover, significant increases of caspase 3 activation were detected in infected cells and treatment with caspase inhibitor caused a decrease in levels of apoptosis. This is the first evidence that P. salmonis induces cell death in trout macrophages. This could lead to bacterial survival and evasion of the host immune response and play an important role in the establishment of infection in the host. J. Cell. Biochem. 110: 468–476, 2010. © 2010 Wiley‐Liss, Inc.     

Highly pathogenic avian influenza H5N1 virus induces cytokine dysregulation with suppressed maturation of chicken monocyte‐derived dendritic cells

One of the major causes of death in highly pathogenic avian influenza virus (HPAIV) infection in chickens is acute induction of pro‐inflammatory cytokines (cytokine storm), which leads to severe pathology and acute mortality. DCs and respiratory tract macrophages are the major antigen presenting cells that are exposed to mucosal pathogens. We hypothesized that chicken DCs are a major target for induction of cytokine dysregulation by H5N1 HPAIV. It was found that infection of chicken peripheral blood monocyte‐derived dendritic cells (chMoDCs) with H5N1 HPAIV produces high titers of progeny virus with more rounding and cytotoxicity than with H9N2 LPAIV. Expression of maturation markers (CD40, CD80 and CD83) was weaker in both H5N1 and H9N2 groups than in a LPS control group. INF‐α, ‐β and ‐γ were significantly upregulated in the H5N1 group. Pro‐inflammatory cytokines (IL‐1β, TNF‐α and IL‐18) were highly upregulated in early mid (IL‐1), and late (IL‐6) phases of H5N1 virus infection. IL‐8 (CXCLi2) mRNA expression was significantly stronger in the H5N1 group from 6 hr of infection. TLR3, 7, 15 and 21 were upregulated 24 hr after infection by H5N1 virus compared with H9N2 virus, with maximum expression of TLR 3 mRNA. Similarly, greater H5N1 virus‐induced apoptotic cell death and cytotoxicity, as measured by terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling and lactate dehydrogenase assays, respectively, were found. Thus, both H5N1 and H9N2 viruses evade the host immune system by inducing impairment of chMoDCs maturation and enhancing cytokine dysregulation in H5N1 HPAIV‐infected cells.     

Knock‐down of HEXA and HEXB genes correlate with the absence of the immunostimulatory function of HSC‐derived dendritic cells

In an attempt to investigate whether the genetic defect in the HEXA and HEXB genes (which causes the absence of the lysosomal β‐N‐acetyl‐hexosaminidase), are related to the wide inflammation in GM2 gangliosidoses (Tay‐Sachs and Sandhoff disease), we have chosen the dendritic cells (DCs) as a study model. Using the RNA interference approach, we generated an in vitro model of HEXs knock‐down immunogenic DCs (i‐DCs) from CD34⁺‐haemopoietic stem cells (CD34⁺‐HSCs), thus mimicking the Tay‐Sachs (HEXA?/?) and Sandhoff (HEXB?/?) cells. We showed that the absence of β‐N‐acetyl‐hexosaminidase activity does not alter the differentiation of i‐DCs from HSCs, but it is critical for the activation of CD4⁺T cells because knock‐down of HEXA or HEXB gene causes a loss of function of i‐DCs. Notably, the silencing of the HEXA gene had a stronger immune inhibitory effect, thereby indicating a major involvement of β‐N‐acetyl‐hexosaminidase A isoenzyme within this mechanism. Copyright © 2011 John Wiley & Sons, Ltd.     

Functionality of insect‐cell‐derived colorectal cancer vaccine candidate protein EpCAM‐Fc in human dendritic cells

The baculovirus–insect cell expression system is widely used to produce recombinant proteins for various biomedical applications. Our previous study demonstrated that EpCAM, a colorectal cancer vaccine candidate protein, can be expressed in the baculovirus–insect cell expression system. However, its functionality (the ability to elicit an immune response), which is important for its possible use as a colorectal cancer vaccine for immunotherapy, still needed to be confirmed. In this study, we examined the ability of recombinant EpCAM to induce maturation of immature dendritic cells (DCs) derived from CD34⁺ cells isolated from human umbilical cord blood. We demonstrated that EpCAM induces the expression of four DC maturation markers: CD80, CD83, CD86 and MHC II. These results suggest that EpCAM produced in the baculovirus–insect cell expression system is functional in terms of its ability to trigger maturation of human DCs.     

The relationship between energy status and AMP‐activated protein kinase in human H460 lung cancer cells

In cultured cells, glucose and serum provide constant sources of energy and growth factors, both of which are important for cell survival and proliferation. AMP‐activated protein kinase (AMPK) plays a key role in sensing intracellular ATP levels and acts as a critical regulator of energy homeostasis. To investigate the relationship between energy status and AMPK activity in lung cancer, H460 cells were starved in either glucose‐free or serum‐free medium and then re‐stimulated with glucose and serum, respectively. The levels of ATP and lactate and the activities of AMPK and lactate dehydrogenase (LDH) were analyzed at different time intervals. During glucose treatment, the activity of AMPK was induced by glucose and showed biphasic reaction kinetics. The ATP level was gradually increased up to 2‐fold compared with that in serum treatment after 24 h and lactate level was decreased to approximately 60%. The LDH activity slightly increased and reached a peak after 6 h. During serum treatment, the activity of AMPK was suppressed and the ATP level showed a dramatic 30% increase after 1 h. In contrast, the lactate level was gradually increased and then reverted to the background level after 24 h. The activity of LDH was slightly decreased after 12 h and eventually returned to the background level. This study showed the alteration of energy status in lung cancer cells in response to altered levels of glucose and serum. We suggest that the activation of AMPK and inhibition of glycolysis might be exploited as therapeutic tactics in cancer treatment. Copyright © 2010 John Wiley & Sons, Ltd.     

Induction of hepatocyte‐like cells from human umbilical cord‐derived mesenchymal stem cells by defined microRNAs

Generating functional hepatocyte‐like cells (HLCs) from mesenchymal stem cells (MSCs) is of great urgency for bio‐artificial liver support system (BALSS). Previously, we obtained HLCs from human umbilical cord‐derived MSCs by overexpressing seven microRNAs (HLC‐7) and characterized their liver functions in vitro and in vivo. Here, we aimed to screen out the optimal miRNA candidates for hepatic differentiation. We sequentially removed individual miRNAs from the pool and examined the effect of transfection with remainder using RT‐PCR, periodic acid—Schiff (PAS) staining and low‐density lipoprotein (LDL) uptake assays and by assessing their function in liver injury models. Surprisingly, miR‐30a and miR‐1290 were dispensable for hepatic differentiation. The remaining five miRNAs (miR‐122, miR‐148a, miR‐424, miR‐542‐5p and miR‐1246) are essential for this process, because omitting any one from the five‐miRNA combination prevented hepatic trans‐differentiation. We found that HLCs trans‐differentiated from five microRNAs (HLC‐5) expressed high level of hepatic markers and functioned similar to hepatocytes. Intravenous transplantation of HLC‐5 into nude mice with CCl₄‐induced fulminant liver failure and acute liver injury not only improved serum parameters and their liver histology, but also improved survival rate of mice in severe hepatic failure. These data indicated that HLC‐5 functioned similar to HLC‐7 in vitro and in vivo, which have been shown to resemble hepatocytes. Instead of using seven‐miRNA combination, a simplified five‐miRNA combination can be used to obtain functional HLCs in only 7 days. Our study demonstrated an optimized and efficient method for generating functional MSC‐derived HLCs that may serve as an attractive cell alternative for BALSS.     

Up‐regulation of galectin‐9 induces cell migration in human dendritic cells infected with dengue virus

Galectin‐9 (Gal‐9) exerts immunosuppressive effects by inducing apoptosis in T cells that produce interferon‐γ and interleukin (IL)‐17. However, Gal‐9 can be pro‐inflammatory in lipopolysaccharide‐stimulated monocytes. Using microarray analysis, we observed that Gal‐9 was up‐regulated in human dendritic cells (DCs) after dengue virus (DV) infection. The investigation into the immunomodulatory effects and mechanisms of Gal‐9 in DCs exposed to DV revealed that DV infection specifically increased mRNA and protein levels of Gal‐9 but not those of Gal‐1 or Gal‐3. Blocking p38, but not c‐Jun N‐terminal kinase or extracellular signal‐regulated kinase (ERK), inhibited DV‐induced expression of Gal‐9. Reduction in Gal‐9 by small interference RNA treatment suppressed DV‐stimulated migration of DCs towards the chemoattractants CCL19 and CCL21. In addition, DV‐induced IL‐12p40 production was reduced after knockdown of Gal‐9 in DCs. Furthermore, Gal‐9 deficiency suppressed DV‐induced activation of nuclear factor‐κB. Inhibition of DV‐induced DC migration under conditions of Gal‐9 deficiency was mediated through suppressing ERK activation but not by regulating the expression of CCR7, the receptor for CCL19 and CCL21. Both the reduction in IL‐12 production and the suppression of ERK activity might account for the inhibition of DV‐induced DC migration after knockdown of Gal‐9. In summary, this study reveals the roles of Gal‐9 in DV‐induced migration of DCs. The findings indicate that Gal‐9 might be a therapeutic target for preventing immunopathogenesis induced by DV infection.     

Malaria‐derived hemozoin exerts early modulatory effects on the phenotype and maturation of human dendritic cells

Plasmodium falciparum (P. falciparum)‐induced effects on the phenotype of human dendritic cells (DC) could contribute to poor induction of long‐lasting protective immunity against malaria. DC ability to present antigens to naïve T cells, thus initiating adaptive immune responses depends on complex switches in chemokine receptors, production of soluble mediators and expression of molecules enabling antigen‐presentation and maturation. To examine the cellular basis of these processes in the context of malaria, we performed detailed analysis of early events following exposure of human monocyte‐derived DC to natural hemozoin (nHZ) and the synthetic analog of its heme core, β‐hematin. DC exposed to either molecule produced high levels of the inflammatory chemokine MCP‐1, showed continuous high expression of the inflammatory chemokine receptor CCR5, no upregulation of the lymphoid homing receptor CCR7 and no cytoskeletal actin redistribution with loss of podosomes. DC partially matured as indicated by increased expression of major histocompatibility complex (MHC) class II and CD86 following nHZ and β‐hematin exposure, however there was a lack in expression of the maturation marker CD83 following nHZ but not β‐hematin exposure. Overall our data demonstrate that exposure to nHZ partially impairs the capacity of DC to mature, an effect in part differential to β‐hematin.     

Productive infection of Piscirickettsia salmonis in macrophages and monocyte‐like cells from rainbow trout,a possible survival strategy

Piscirickettsia salmonis is the etiologic agent of the salmonid rickettsial septicemia (SRS), an endemic disease which causes significant losses in salmon production. This intracellular bacterium is normally cultured in salmonid epithelial cell lines inducing characteristic cytopathic effects (CPEs). In this study we demonstrate that P. salmonis is able to infect, survive, replicate, and propagate in the macrophages/monocytes cell line RTS11 derived from rainbow trout spleen, without inducing the characteristic CPEs and the host cells showing the same expression levels as non‐infected control cell. On the other hand, bacteria were capable of expressing specific proteins within infected cells. Infected macrophages cease proliferation and a fraction of them detached from the plate, transform to non‐adhesive, monocyte‐like cells with proliferative activity. Productive infection of P. salmonis into salmonid macrophage/monocyte cells in culture provides an excellent model for the study of host–pathogen interactions, almost unknown in the case of P. salmonis. Our results suggest that the infection of cells from the salmonid innate immune system without inducing an important cell death response should lead to the persistence of the bacteria and consequently their dissemination to other tissues, favoring the evasion of the first line of defense against pathogens. J. Cell. Biochem. 108: 631–637, 2009. © 2009 Wiley‐Liss, Inc.     

Visualizing and quantifying the differential cleavages of the eukaryotic translation initiation factors eIF4GI and eIF4GII in the enterovirus‐infected cell

Enterovirus (EV) infection has been shown to cause a marked shutoff of host protein synthesis, an event mainly achieved through the cleavages of eukaryotic translation initiation factors eIF4GI and eIF4GII that are mediated by viral 2A protease (2A^pro). Using fluorescence resonance energy transfer (FRET), we developed genetically encoded and FRET‐based biosensors to visualize and quantify the specific proteolytic process in intact cells. This was accomplished by stable expression of a fusion substrate construct composed of the green fluorescent protein 2 (GFP²) and red fluorescent protein 2 (DsRed2), with a cleavage motif on eIF4GI or eIF4GII connected in between. The FRET biosensor showed a real‐time and quantifiable impairment of FRET upon EV infection. Levels of the reduced FRET closely correlated with the cleavage kinetics of the endogenous eIF4Gs isoforms. The FRET impairments were solely attributed to 2A^pro catalytic activity, irrespective of other viral‐encoded protease, the activated caspases or general inhibition of protein synthesis in the EV‐infected cells. The FRET biosensors appeared to be a universal platform for several related EVs. The spatiotemporal and quantitative imaging enabled by FRET can shed light on the protease–substrate behaviors in their normal milieu, permitting investigation into the molecular mechanism underlying virus‐induced host translation inhibition. Biotechnol. Bioeng. 2009; 104: 1142–1152. © 2009 Wiley Periodicals, Inc.