Role of LncRNA MIR99AHG in breast cancer: Bioinformatic analysis and preliminary verification.

Objective: This research was aimed to preliminarily explore the clinical roles and potential molecular mechanisms of MIR99AHG and its significant transcripts in breast cancer (BRCA). Methods: Public databases were utilized to analyze the expression and prognostic roles of MIR99AHG and its transcripts. Relationships between MIR99AHG expression and immune cells infiltration were analyzed in Xiantao platform. In addition, co-expressed genes and interacting proteins of MIR99AHG were predicted. CancerSEA analyzed its relationship with functional states. Next, CNV status, DNA methylation, interacting transcription factors (TFs) and ceRNA network were analyzed to explore its possible mechanisms. Then, RNA ISH and FISH assays were used to detect its expression and location in BRCA tissues and cell lines, respectively. Finally, qRT-PCR was utilized to investigate MIR99AHG expression in cell lines. Results: Compared with the corresponding normal tissues, MIR99AHG expression levels were lower in all BRCA subtypes, and luminal B's was the lowest one. And MIR99AHG expression was negatively related to the tumor stage. In addition, 4 transcripts (ENST00000619222.4, ENST00000418813.6, ENST00000602901.5 and ENST00000453910.5) of MIR99AHG showed significant differences in the expression. Databases also suggested that the high MIR99AHG expression levels indicated good prognosis, especially in patients without lymph node metastasis. Xiantao found that MIR99AHG was positively related to 17 immune cells and negatively linked with 2 immune cells. CancerSEA analysis showed no relationships between MIR99AHG and functional states. From GEPIA and BCIP databases, 19 co-expressed genes were highly related to these four significant transcripts of MIR99AHG. StarBase, RNAct and HDOCK showed that several tumor-associated proteins, including U2AF65, hnRNPC, AEBP2, CHIC1 and so on, might interact with MIR99AHG. Genetically, BRCA had a higher proportion of MIR99AHG CNV loss than CNV gain, and the high level of DNA methylation indicated a good prognosis. Furthermore, 19 TFs were predicted to combine with the promoter of MIR99AHG. Then, we screened out 10 miRNAs potentially interacting with the significant transcripts of MIR99AHG, and five were significantly increased in breast tumors compared to normal tissues, including miR-194-5p, miR-320 b and so on, which could combine 14 mRNAs. Through ISH and FISH assays, we verified that MIR99AHG was down-regulated in BRCA samples and cell lines in comparison to non-tumor tissues and mammary epithelial cell line (MCF10A), and MIR99AHG was located both in cytoplasm and nucleus. qRT-PCR assay also showed the lower expression of MIR99AHG in breast cancer cells than that in MCF10A. Conclusion: These results indicate that MIR99AHG can be a favorable prognostic indicator for BRCA. ENST00000619222.4, ENST00000418813.6, ENST00000602901.5 and ENST00000453910.5 are significant transcripts and their down-regulation may play crucial roles in the progression of BRCA.

Pan-cancer analysis of LncRNA XIST and its potential mechanisms in human cancers.

Background: X-inactive specific transcript (XIST), it has been found, is abnormal expression in various neoplasms. This work aims to explore its potential molecular mechanisms and prognostic roles in types of malignancies. Methods: This research comprehensively investigated XIST transcription across cancers from Oncomine, TIMER 2.0 and GEPIA2. Correlations of XIST expression with prognosis, miRNAs, interacting protens, immune infiltrates, checkpoint markers, mutations of tumor-associated genes and promoter methylation were also analyzed by public databases. In addition, 98 BRCA samples were collected to investigate XIST expression and evaluate its clinicopathological value. Results: In public databases, compared to normal tissues, XIST was lower in BRCA, CESC, COAD and so on, but increased in KIRC and PRAD. Databases also showed that XIST was a good indicator of prognosis in BRCA, COAD and so on, but a bad one in KIRC, KIRP and so on. From starBase, we found 29 proteins interacting with XIST, and identified 4 miRNAs which might be sponged by XIST in cancers. Furthermore, XIST was linked with immune infiltration, especially T cell CD4+, and was related to over 20 immune checkpoint markers. Moreover, several tumor-associated gene mutations and promoter methylation were negatively related to its expression. In addition, IHC showed that XIST in BRCA was obviously lower in comparison of normal tissues and was negatively related to lymph node invasion and TNM stage. Conclusion: In summary, abnormal expression of XIST influenced prognosis, miRNAs and immune infiltration across cancers, especially BRCA.

RFX5 promotes the progression of hepatocellular carcinoma through transcriptional activation of KDM4A.

Regulatory factor X-5 (RFX5) represents a key transcription regulator of MHCII gene expression in the immune system. This study aims to explore the molecular mechanisms and biological significance of RFX5. Firstly, by analyzing ENCODE chromatin immunoprecipitation (ChIP)-seq in HepG2 and TCGA RNA-seq data, we discovered lysine-specific demethylase 4A (KDM4A), also named JMJD2A, to be a major downstream target gene of RFX5. Moreover, RFX5 was verified to bind directly to the KDM4A's promoter region and sequentially promoted its transcription determined by the ChIP-PCR assay and luciferase assay. In addition, RFX5-dependent regulation of KDM4A was demonstrated in HCC. Compared with adjacent non-tumor tissues, the expression levels of KDM4A were significantly raised in HCC tumor tissues. Notably, elevated levels of KDM4A were strongly correlated with HCC patient prognosis. Functionally, KDM4A overexpression largely rescued the growth inhibitory effects of RFX5 deletion, highlighting KDM4A as a downstream effector of RFX5. Mechanistically, the RFX5-KDM4A pathway promoted the progression of the cell cycle from G0/G1 to S phase and was protective against cell apoptosis through regulation of p53 and its downstream genes in HCC. In conclusion, RFX5 could promote HCC progression via transcriptionally activating KDM4A expression.

Roles of eIF3m in the tumorigenesis of triple negative breast cancer.

BACKGROUND: Without targets, triple negative breast cancer (TNBC) has the worst prognosis in all subtypes of breast cancer (BC). Recently, eukaryotic translation initiation factor 3 m (eIF3m) has been declared to be involved in the malignant progression of various neoplasms. The aim of this study is to explore biological functions of eIF3m in TNBC. METHODS: Multiple databases, including Oncomine, KM-plotter and so on, were performed to analyze prognosis and function of eIF3m in TNBC. After transfection of eIF3m-shRNA lentivirus, CCK-8, colony formation assay, cell cycle analysis, wound healing assay, transwell assays, mitochondrial membrane potential assay and cell apoptosis analysis were performed to explore the roles of eIF3m in TNBC cell bio-behaviors. In addition, western blotting was conducted to analyze the potential molecular mechanisms of eIF3m. RESULTS: In multiple databases, up-regulated eIF3m had lower overall survival, relapse-free survival and post progression survival in BC. EIF3m expression in TNBC was obviously higher than in non-TNBC or normal breast tissues. Its expression in TNBC was positively related to differentiation, lymph node invasion and distant metastasis. After knockdown of eIF3m, cell proliferation, migration, invasion and levels of mitochondrial membrane potential of MDA-MB-231 and MDA-MB-436 were all significantly suppressed, while apoptosis rates of them were obviously increased. In addition, eIF3m could regulate cell-cycle, epithelial-mesenchymal transition and apoptosis-related proteins. Combined with public databases and RT-qPCR, 14 genes were identified to be modulated by eIF3m in the development of TNBC. CONCLUSIONS: eIF3m is an unfavorable indicator of TNBC, and plays a vital role in the process of TNBC tumorigenesis.

Regulatory factor X5 promotes hepatocellular carcinoma progression by transactivating tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein theta and suppressing apoptosis.

BACKGROUND: Our previous studies have shown that regulatory factor X5 (RFX5), a classical transcription regulator of MHCII genes, was obviously overexpressed in hepatocellular carcinoma (HCC) tumors. However, the role of RFX5 in the carcinogenesis and progress of HCC remains unknown. This study aimed to reveal its biological significance and the underlying mechanism in HCC. METHODS: RFX5 mRNA expression level and copy number variation in HCC tumors and cell lines were determined by analyzing deposited data sets in the Cancer Genome Atlas and Gene Expression Omnibus database. The biological significance of RFX5 in HCC was investigated by monitoring the colony formation and subcutaneous tumor growth capacity when RFX5 was silenced with lentiviral short hairpin RNA and CRISPR/Cas9 system in HCC cell lines. The downstream gene transcriptionally activated by RFX5 in HCC cells was determined by chromatin immunoprecipitation and luciferase reporter assay. The involvement of tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein theta (YWHAQ) in HCC development was further determined by performing colony formation rescue assay and subcutaneous tumor growth rescue experiment. The association of YWHAQ with recurrence-free survival of patients with HCC was assessed by Kaplan-Meier analysis. Moreover, apoptosis level and the protein level of p53 pathway were determined to reveal the mechanism of RFX5 in driving HCC development. RESULTS: RFX5 was amplified and highly overexpressed in HCC tumor tissues compared with the corresponding non-tumor tissues. The mRNA expression level of RFX5 was significantly correlated with its DNA copy number (r = 0.4, P < 0.001). Functional study demonstrated that RFX5 was required for both clonogenic forming in vitro and subcutaneous tumor growth in vivo of HCC cells. Further study identified YWHAQ, namely 14-3-3 tau, as a key downstream transcriptional target gene of RFX5, which was tightly regulated by RFX5 in HCC. Moreover, overexpression of YWHAQ largely rescued the clonogenic growth of HCC cells that was suppressed by RFX5 knockdown. In addition, overexpression of YWHAQ in primary tumor was linked to poor prognosis of patients with HCC. These results demonstrated that YWHAQ was a downstream effector of RFX5 in HCC. Notably, RFX5-YWHAQ pathway could protect cells from apoptosis by suppressing the p53 and Bax in HCC. CONCLUSION: RFX5 is a putative HCC driver gene that plays an important role in the development and progression of HCC by transactivating YWHAQ and suppressing apoptosis.

Overexpression of PLK3 Mediates the Degradation of Abnormal Prion Proteins Dependent on Chaperone-Mediated Autophagy.

Polo-like kinase 3 (PLK3) is the main cause of cell cycle reentry-related neuronal apoptosis which has been implicated in the pathogenesis of prion diseases. Previous work also showed the regulatory activity of exogenous PLK3 on the degradation of PrP (prion protein) mutants and pathogenic PrPSc; however, the precise mechanisms remain unknown. In this study, we identified that the overexpression of PLK3-mediated degradation of PrP mutant and PrPSc was repressed by lysosome rather than by proteasomal and macroautophagy inhibitors. Core components of chaperone-mediated autophagy (CMA) effectors, lysosome-associated membrane protein type 2A (LAMP2a), and heat shock cognate protein 70 (Hsc70) are markedly decreased in the HEK293T cells expressing PrP mutant and scrapie-infected cell line SMB-S15. Meanwhile, PrP mutant showed ability to interact with LAMP2a and Hsc70. Overexpression of PLK3 sufficiently increased the cellular levels of LAMP2a and Hsc70, accompanying with declining the accumulations of PrP mutant and PrPSc. The kinase domain (KD) of PLK3 was responsible for elevating LAMP2a and Hsc70. Knockdown of endogenous PLK3 enhanced the activity of macroautophagy in the cultured cells. Moreover, time-dependent reductions of LAMP2a and Hsc70 were also observed in the brain tissues of hamster-adapted scrapie agent 263K-infected hamsters, indicating an impairment of CMA during prion infection. Those data indicate that the overexpression of PLK3-mediated degradation of abnormal PrP is largely dependent on CMA pathway.

Activation of the AMPK-ULK1 pathway plays an important role in autophagy during prion infection.

AMPK is a serine/threonine protein kinase that acts as a positive regulator of autophagy, by phosphorylating ULK1 at specific sites. A previous study demonstrated activation of the macroautophagic system in scrapie-infected experimental rodents and in certain human prion diseases, in which the essential negative regulator mTOR is severely inhibited. In this study, AMPK and ULK1 in the brains of hamsters infected with scrapie strain 263 K and in the scrapie-infected cell line SMB-S15 were analysed. The results showed an up-regulated trend of AMPK and AMPK-Thr172, ULK1 and ULK1-Ser555. Increases in brain AMPK and ULK1 occurred at an early stage of agent 263 K infection. The level of phosphorylated ULK1-Ser757 decreased during mid-infection and was only negligibly present at the terminal stage, a pattern that suggested a close relationship of the phosphorylated protein with altered endogenous mTOR. In addition, the level of LKB1 associated with AMPK activation was selectively increased at the early and middle stages of infection. Knockdown of endogenous ULK1 in SMB-S15 cells inhibited LC3 lipidation. These results showed that, in addition to the abolishment of the mTOR regulatory pathway, activation of the AMPK-ULK1 pathway during prion infection contributes to autophagy activation in prion-infected brain tissues.

Fish Oil-Rich Diet Promotes Hematopoiesis and Alters Hematopoietic Niche.

The self-renewal and differentiation of hematopoietic stem cells (HSCs) in bone marrow are essential to replenish all blood cell types, but how this process is influenced by diet remains largely unclear. Here we show that a diet rich in fish oils promotes self-renewal of HSCs and extramedullary hematopoiesis. Chronic intake of a fish oil-rich diet increases the abundance of HSCs, alters the hematopoietic microenvironment, and, intriguingly, induces the expression of matrix metalloproteinase 12 (MMP12) in the bone marrow. Pointing to a direct effect of fish oil on MMP12 expression, omega-3 polyunsaturated fatty acids induce the expression of MMP12 in a dose-dependent manner in bone marrow cells. Importantly, down-regulation of MMP12 activity using an MMP12-specific inhibitor attenuates diet-induced myelopoiesis in both bone marrow and spleen. Thus, a fish oil-rich diet promotes hematopoiesis in the bone marrow and spleen, in part via the activity of MMP12. Taken together, these data provide new insights into diet-mediated regulation of hematopoiesis.

Polo-like kinase 3 (PLK3) mediates the clearance of the accumulated PrP mutants transiently expressed in cultured cells and pathogenic PrP(Sc) in prion infected cell line via protein interaction.

Polo-like kinases (PLKs) family has long been known to be critical for cell cycle and recent studies have pointed to new dimensions of PLKs function in the nervous system. Our previous study has verified that the levels of PLK3 in the brain are severely downregulated in prion-related diseases. However, the associations of PLKs with prion protein remain unclear. In the present study, we confirmed that PrP protein constitutively interacts with PLK3 as determined by both in vitro and in vivo assays. Both the kinase domain and polo-box domain of PLK3 were proved to bind PrP proteins expressed in mammalian cell lines. Overexpression of PLK3 did not affect the level of wild-type PrP, but significantly decreased the levels of the mutated PrPs in cultured cells. The kinase domain appeared to be responsible for the clearance of abnormally aggregated PrPs, but this function seemed to be independent of its kinase activity. RNA-mediated knockdown of PLK3 obviously aggravated the accumulation of cytosolic PrPs. Moreover, PLK3 overexpression in a scrapie infected cell line caused notable reduce of PrP(Sc) level in a dose-dependent manner, but had minimal effect on the expression of PrP(C) in its normal partner cell line. Our findings here confirmed the molecular interaction between PLK3 and PrP and outlined the regulatory activity of PLK3 on the degradation of abnormal PrPs, even its pathogenic isoform PrP(Sc). We, therefore, assume that the recovery of PLK3 in the early stage of prion infection may be helpful to prevent the toxic accumulation of PrP(Sc) in the brain tissues.

Abortive cell cycle events in the brains of scrapie-infected hamsters with remarkable decreases of PLK3/Cdc25C and increases of PLK1/cyclin B1.

Polo-like kinases (PLKs) consist of a family of kinases which play critical roles during multiple stages of cell cycle progression. Increase of PLK1 and decrease of PLK3 are associated with the developments and metastases of many types of human malignant tumors; however, the situations of PLKs in prion diseases are less understood. Using Western blots and immunohistochemical and immunofluorescent assays, marked increase of PLK1 and decrease of PLK3 were observed in the brains of scrapie strain 263K-infected hamsters, presenting obviously a time-dependent phenomenon along with disease progression. Similar alterations of PLKs were also detected in a scrapie infectious cell line SMB-S15. Both PLK1 and PLK3 were observed in neurons by confocal microscopy. Accompanying with the changes of PLKs in the brains of 263K-infected hamsters, Cdc25C and its phosphorylated forms (p-Cdc25C-Ser198 and p-Cdc25C-Ser216) were significantly down-regulated, whereas Cyclin B1 and PCNA were obviously up-regulated, while phospho-histone H3 remained almost unchanged. Moreover, exposure of the cytotoxic peptide PrP106-126 on the primary cultured cortical neuron cells induced similar changes of cellular PLKs and some cell cycle-related proteins, such as Cdc25C and its phosphorylated forms, phospho-histone H3. Those results illustrate obviously aberrant expressions of cell cycle regulatory proteins in the prion-infected neurons, which may lead to the cell cycle arrest at M phase. Possibly due to the ill-regulation of some key cell cycle events during prion infection, together with the fact that neurons are unable to complete mitosis, the cell cycle reentry in prion-infected neurons is definitely abortive, which may lead to neuron apoptosis and neuron degeneration.

Doxycycline up-regulates the expression of IL-6 and GM-CSF via MAPK/ERK and NF-κB pathways in mouse thymic epithelial cells.

Thymic epithelial cells (TECs) constitute a major component of the thymic stroma which provides a microenvironment critical for developing thymocytes. We have previously demonstrated that doxycycline (Dox), a tetracycline derivative, enhances the proliferation of the mouse thymic epithelial cell line 1 (MTEC1) via MAPK/ERK signal pathway. Herein we provide evidence that Dox also has profound impact on the cytokine production by MTEC1. Specifically, the expression of IL-6 and GM-CSF, both at mRNA and protein levels, was found to be increased in a time- and dose-dependent manner with the addition of Dox. Western blotting analysis revealed that treatment with Dox-induced phosphorylation of the p65 subunit of NF-κB and ERK. Notably, Dox-induced up-regulation of IL-6 and GM-CSF was largely abolished after pretreatment of MTEC1 with either NF-κB inhibitor BAY11-7082 or MEK1/2 inhibitor U0126, supporting the involvement of the two pathways in the process. These findings warrant further investigation into the potential application of Dox in T-cell reconstitution in such situations as chemotherapy, radiotherapy, bone marrow transplantation and HIV infection.

[The expression, identification of human HMGB1 B box and preparation of monoclonal antibodies against HMGB1 B box].

AIM: To express human HMGB1 B box protein and obtain monoclonal antibodies (mAbs) against HMGB1 B box for further study of the function of human HMGB1 protein. METHODS: pET28-HMGB1 B box plasmid transfected the DH5α, then expressed. And the extracted protein was purified by protein purification system. BALB/c mice were immunized with recombinant human HMGB1 B box protein. Hybridoma cell lines secreting mAb against human HMGB1 B box protein were screened by ELISA and subcloning approach. The characteristics of these mAbs were identified by ELISA and Western blot. RESULTS: Two hybridoma cell lines (1D2F4E3 and 2D4E3A2) stable secreting specific mAbs were successfully obtained.Western blot exhitited the two mAbs binded specifically to human HMGB1 B box protein. The immunoglobulin (Ig) class of two mAbs belonged to IgG, their titers were 1×10(6);, and the A(450); of mAb1D2F4E3, 2D4E3A2 were 0.324±0.093, 0.296±0.085, respectively. CONCLUSION: Two of high specificity mAbs against human HMGB1 B box protein have been successfully prepared, which laid the foundation for further study of biological function of human HMGB1 protein.

Immunosuppressive effects of mesenchymal stem cells in collagen-induced mouse arthritis.

OBJECTIVE: The objective of this study was to investigate the efficacy of mesenchymal stem cell (MSC) in the treatment of arthritis. METHODS: Mesenchymal stem cells were injected intravenously into mice with collagen-induced arthritis (CIA). Arthritic indexes were evaluated, and the levels of the pro- and anti-inflammatory cytokines interleukin-10 (IL-10), gamma interferon (IFN-gamma)-inducible protein 10 (IP-10), chemokine (C-X-C motif) receptor 3 (CXCR3), interleukin-17A (IL-17A), and tumor necrosis factor alpha (TNF-alpha) in serum or splenic cells were determined using real-time RT-PCR, Western blotting, and enzyme-linked immunosorbent assay (ELISA). The proliferation of dendritic cell line D2SC cells was determined using (3)H-TdR incorporation assay. RESULTS: Upon injection of MSCs, overall arthritis symptoms were significantly improved in the CIA mouse models as indicated by the paw edema. Consistent with this observation, serum levels of pro-inflammatory cytokine TNF-alpha and inflammatory cell infiltration decreased significantly 12 days after MSC injection, while the expression of anti-inflammatory cytokines IL-10, IP-10, and CXCR3 was increased in splenocytes. In addition, we provided evidence that MSCs may directly promote the proliferation of D2SC cells and the expression of IP-10 in D2SC cells in vitro. CONCLUSION: Mesenchymal stem cells significantly enhance the efficacy of collagen-induced arthritis treatment, likely through the modulation of the expression of various cytokines.

[Construction of MyD88-Pseudomonas aeruginosa epitope DNA vaccine and its expression in eukaryotic cells].

AIM: To construct the MyD88-Pseudomonas aeruginosa epitope vaccine and study its expression in eukaryotic cells. METHODS: To design and synthesize an epigene containing three B cell epitopes of OprF and one foreign "promiscuous" T cell epitope by overlapping extension PCR. tPA signal encoding sequence was amplified by PCR and then it was inserted into the 5' terminus of the epigene to construct tPA-OprF. tPA-OprF and MyD88 were cloned into the expression vector pIRES and the recombinant plasmid pIRES-tPAOprF-MyD88 was constructed. The recombinant plasmid was transfected into COS-7 cells by electroporation. The expression protein of tPA-OprF and MyD88 was detected by Western blot. RESULTS: The recombinant plasmid pIRES-tPA-OprF-MyD88 was successfully constructed. Western blot analysis indicated the tPA-OprF fusion protein was expressed in supertanant of COS-7 cells and MyD88 protein in COS-7 cells. CONCLUSION: The recombinant plasmid pIRES-tPA-OprF-MyD88 has been successfully constructed and tPA-OprF and MyD88 protein can be highly expressed in transfected cells. It may be used as a potential candidate of preventive vaccine of pseudomonas aeruginosa.

[Cloning and expression of human Runx3 gene obtained from human peripheral blood CD8(+) T lymphocyte].

AIM: Runx3, a type of Runt family member, plays an important role in immune regulation. In this study, we cloned and analyzed the cDNA encoding human Runx3 from T lymphocyte, expressed Runx3 protein in E.coli system, and studied the relation between Runx3 and some immune disorders or tumors. METHODS: The CD8(+) T were isolated from human peripheral blood with MACS, Runx3 cDNA was amplified by RT-PCR and cloned into pMD19-T vector, and recombinant was transformed into competent cells DH5alpha and recombinant sequencing were performed. The identical was subcloned into pQE30 vector and expressed in E.coli M15. The fusion protein was identified by Western blot. RESULTS: The 1,248 bp fragment amplified by RT-PCR was the same as the anticipated one in size and encodes 415 amino acids. Runx3 protein was gained. CONCLUSION: Human Runx3 gene was cloned and expressed in E.coli system successfully, which brought a foundation for further research on its biological function.

[Construction and identification of the recombination adeno-associated virus carrying murine FoxP3 gene].

AIM: To construct the recombination adeno-associated virus (rAAV) carrying murine forkhead box P3 (FoxP3) gene, and then detect the expression in NIH3T3 cells. METHODS: The recombination adeno-associated virus (rAAV) vector containing internal ribosome entry site which could coordinate the expression of FoxP3 gene and enhance green fluorescent protein gene was constructed. HEK293 cells were transfected by the transfection cocktail containing the constructed rAAV vector, trans plasmid and helper plasmid by the calcium phosphate method. The infectious rAAV carrying FoxP3 gene (rAAV/FoxP3) suspensions were harvested and purified by heparin affinity chromatography. The purity and titre of rAAV were detected by SDS-PAGE electrophoresis and real-time PCR, respectively. NIH3T3 cells were transfected by rAAV, the transfection efficiency was analyzed by flow cytometry and FoxP3 mRNA expression was detected by real-time PCR. RESULTS: The titre of the infectious rAAV was 5x 10(12) vg/mL by real-time PCR analysis. After NIH3T3 cells were transfected, the transfection efficiency of the infectious rAAV/FoxP3 was up to 92.88% and high level of FoxP3 mRNA was detected. CONCLUSION: rAAV carrying FoxP3 gene was successfully constructed and expressed in NIH3T3 cells, which will be of benefit to further researches on the function of FoxP3.

Cutting edge: generation of splenic CD8+ and CD8- dendritic cell equivalents in Fms-like tyrosine kinase 3 ligand bone marrow cultures.

We demonstrate that functional and phenotypic equivalents of mouse splenic CD8(+) and CD8(-) conventional dendritic cell (cDC) subsets can be generated in vitro when bone marrow is cultured with fms-like tyrosine kinase 3 (flt3) ligand. In addition to CD45RA(high) plasmacytoid DC, two distinct CD24(high) and CD11b(high) cDC subsets were present, and these subsets showed equivalent properties to splenic CD8(+) and CD8(-) cDC, respectively, in the following: 1) surface expression of CD11b, CD24, and signal regulatory protein-alpha; 2) developmental dependence on, and mRNA expression of, IFN regulatory factor-8; 3) mRNA expression of TLRs and chemokine receptors; 4) production of IL-12 p40/70, IFN-alpha, MIP-1alpha, and RANTES in response to TLR ligands; 5) expression of cystatin C; and 6) cross-presentation of exogenous Ag to CD8 T cells. Furthermore, despite lacking surface CD8 expression, the CD24(high) subset contained CD8 mRNA and up-regulated surface expression when transferred into mice. This culture system allows access to bona fide counterparts of the splenic DC subsets.

Development of the dendritic cell system during mouse ontogeny.

Based on the view that the efficacy of the immune system is associated with the maturation state of the immune cells, including dendritic cells (DC), we investigated the development and functional potential of conventional DC and plasmacytoid pre-DC (p-preDC) in spleen, thymus, and lymph nodes during mouse development. Both CD11c+ DC and CD45RA+ p-preDC were detected in small numbers in the thymus as early as embryonic day 17. The ratio of DC to thymocytes reached adult levels by 1 wk, although the normal CD8alpha+ phenotype was not acquired until later. Significant, but low, numbers of DC and p-preDC were present in the spleen of day 1 newborn mice. The full complement of DC and p-preDC was not acquired until 5 wk of age. The composition of DC populations in the spleen of young mice differed significantly from that found in adult mice, with a much higher percentage (50-60% compared with 20-25%) of the CD4-CD8alpha+ DC population and a much lower percentage (10-20% compared with 50-60%) of the CD4+CD8alpha- DC population. Although the p-preDC of young mice showed a capacity to produce IFN-alpha comparable with that of adult mice, the conventional DC of young mice were less efficient than those of their adult counterparts in IL-12p70 and IFN-gamma production and in Ag presentation. These results suggest that the neonatal DC system is not fully developed, and innate immunity is the dominant form of response. The complete DC system required for adaptive immunity in the mouse is not fully developed until 5 wk of age.