To-Ha Thai

MiR-150 controls B cell differentiation by targeting the transcription factor c-Myb.

MiR-150 is a microRNA (miRNA) specifically expressed in mature lymphocytes, but not their progenitors. A top predicted target of miR-150 is c-Myb, a transcription factor controlling multiple steps of lymphocyte development. Combining loss- and gain-of-function gene targeting approaches for miR-150 with conditional and partial ablation of c-Myb, we show that miR-150 indeed controls c-Myb expression in vivo in a dose-dependent manner over a narrow range of miRNA and c-Myb concentrations and that this dramatically affects lymphocyte development and response. Our results identify a key transcription factor as a critical target of a stage-specifically expressed miRNA in lymphocytes and suggest that this and perhaps other miRNAs have evolved to control the expression of just a few critical target proteins in particular cellular contexts.

Foxp3-Dependent MicroRNA155 Confers Competitive Fitness to Regulatory T Cells by Targeting SOCS1 Protein

Foxp3(+) regulatory T (Treg) cells limit pathogenic immune responses to self-antigens and foreign antigens. An essential role for microRNA (miRNA) in the maintenance and function of Treg cells, revealed by the Treg cell-specific Dicer ablation, raised a question as to a specific miRNA contribution. We found that Foxp3 controlled the elevated miR155 expression required for maintaining Treg cell proliferative activity and numbers under nonlymphopenic conditions. Moreover, miR155 deficiency in Treg cells resulted in increased suppressor of cytokine signaling 1 (SOCS1) expression accompanied by impaired activation of signal transducer and activator of transcription 5 (STAT5) transcription factor in response to limiting amounts of interleukin-2. Our studies suggest that Foxp3-dependent regulation of miR155 maintains competitive fitness of Treg cell subsets by targeting SOCS1, and they provide experimental support for a proposed role for miRNAs in ensuring the robustness of cellular phenotypes.

MicroRNA profiling of the murine hematopoietic system.

BackgroundMicroRNAs (miRNAs) are a class of recently discovered noncoding RNA genes that post-transcriptionally regulate gene expression. It is becoming clear that miRNAs play an important role in the regulation of gene expression during development. However, in mammals, expression data are principally based on whole tissue analysis and are still very incomplete.ResultsWe used oligonucleotide arrays to analyze miRNA expression in the murine hematopoietic system. Complementary oligonucleotides capable of hybridizing to 181 miRNAs were immobilized on a membrane and probed with radiolabeled RNA derived from low molecular weight fractions of total RNA from several different hematopoietic and neuronal cells. This method allowed us to analyze cell type-specific patterns of miRNA expression and to identify miRNAs that might be important for cell lineage specification and/or cell effector functions.ConclusionThis is the first report of systematic miRNA gene profiling in cells of the hematopoietic system. As expected, miRNA expression patterns were very different between hematopoietic and non-hematopoietic cells, with further subtle differences observed within the hematopoietic group. Interestingly, the most pronounced similarities were observed among fully differentiated effector cells (Th1 and Th2 lymphocytes and mast cells) and precursors at comparable stages of differentiation (double negative thymocytes and pro-B cells), suggesting that in addition to regulating the process of commitment to particular cellular lineages, miRNAs might have an important general role in the mechanism of cell differentiation and maintenance of cell identity.

Terminal deoxynucleotidyl transferase and repertoire development

Summary: In mice, the absence of terminal deoxynucleotidyl transferase (Tdt) expression during fetal and neonatal life provides a window in development where clones of lymphocytes are generated that provide protective immunity. Introducing premature Tdt activity interferes with the development of these clones and results in an impaired ability to make protective antibodies. Conversely, gene‐targeted disruption of Tdt prevents N additions at all stages of T and B‐lymphocyte development and promotes the development of fetal‐like T and B‐cell clones into adulthood, with accompanying alterations in repertoire. The alternative splice forms of Tdt may be necessary to provide regulatory mechanisms to restrict N addition to appropriate stages of the developmental pathways, the details of which are being revealed. The evidence continues to build that Tdt is a key player in influencing the outcome of V(D)J recombination during lymphocyte and repertoire development.

LNA-mediated anti–miR-155 silencing in low-grade B-cell lymphomas

miR-155 acts as an oncogenic miR in B-cell lymphoproliferative disorders, including Waldenstrom macroglobulinemia (WM) and chronic lymphocytic leukemia, and is therefore a potential target for therapeutic intervention. However, efficient targeting of miRs in tumor cells in vivo remains a significant challenge for the development of miR-155-based therapeutics for the treatment of B-cell malignancies. In the present study, we show that an 8-mer locked nucleic acid anti-miR-155 oligonucleotide targeting the seed region of miR-155 inhibits WM and chronic lymphocytic leukemia cell proliferation in vitro. Moreover, anti-miR-155 delivered systemically showed uptake in the BM CD19(+) cells of WM-engrafted mice, resulting in the up-regulation of several miR-155 target mRNAs in these cells, and decreased tumor growth significantly in vivo. We also found miR-155 levels to be elevated in stromal cells from WM patients compared with control samples. Interestingly, stromal cells from miR-155-knockout mice led to significant inhibition of WM tumor growth, indicating that miR-155 may also contribute to WM proliferation through BM microenvironmental cells. The results of the present study highlight the therapeutic potential of anti-miR-155-mediated inhibition of miR-155 in the treatment of WM.

Deletion of microRNA-155 reduces autoantibody responses and alleviates lupus-like disease in the Fas(lpr) mouse

Significance The host immune system provides diverse defense mechanisms to fight harmful bacteria and viruses. One of these mechanisms is the production of antibodies targeting infectious agents. However, the production of antibodies has to be tightly controlled. Insufficient control of the immune system may result in the development of autoimmune diseases, including lupus. Lupus is characterized by the production of antibodies attacking the kidneys, leading to life-threatening kidney failure if untreated. In this study, we show that microRNA-155, one member of a family of regulatory molecules, promotes the production of antibodies. By deleting microRNA-155, we can prevent the production of harmful antibodies and alleviate lupus-like disease in mice. Our results suggest the possibility of targeting microRNA-155 to treat autoimmune diseases. MicroRNA-155 (miR-155) regulates antibody responses and subsequent B-cell effector functions to exogenous antigens. However, the role of miR-155 in systemic autoimmunity is not known. Using the death receptor deficient (Faslpr) lupus-prone mouse, we show here that ablation of miR-155 reduced autoantibody responses accompanied by a decrease in serum IgG but not IgM anti-dsDNA antibodies and a reduction of kidney inflammation. MiR-155 deletion in Faslpr B cells restored the reduced SH2 domain-containing inositol 5′-phosphatase 1 to normal levels. In addition, coaggregation of the Fc γ receptor IIB with the B-cell receptor in miR-155−/−-Faslpr B cells resulted in decreased ERK activation, proliferation, and production of switched antibodies compared with miR-155 sufficient Faslpr B cells. Thus, by controlling the levels of SH2 domain-containing inositol 5′-phosphatase 1, miR-155 in part maintains an activation threshold that allows B cells to respond to antigens.

Distinct and opposite diversifying activities of terminal transferase splice variants

The short splice variant of mouse terminal deoxynucleotidyl transferase (TdTS) catalyzes the addition of nontemplated nucleotides (N addition) at the coding joins of B cell and T cell antigen receptor genes. However, the activity and function of the long isoform of TdT (TdTL) have not been determined. We show here, in vitro and in vivo, that TdTL is a 3′→5′ exonuclease that catalyzes the deletion of nucleotides at coding joins. These findings suggest that the two TdT isoforms may act in concert to preserve the integrity of the variable region of antigen receptors while generating diversity.

Cytosolic DNA-activated Human Dendritic Cells are Potent Activators of the Adaptive Immune Response

Recent studies in cell lines and genetically engineered mice have demonstrated that cytosolic dsDNA could activate dendritic cells (DCs) to become effector APCs. Recognition of DNA might be a major factor in antimicrobial immune responses against cytosolic pathogens and also in human autoimmune diseases such as systemic lupus erythematosus. However, the role of cytosolic dsDNA in human DC activation and its effects on effector T and B cells are still elusive. In this study, we demonstrate that intracellular dsDNA is a potent activator of human monocyte-derived DCs as well as primary DCs. Activation by dsDNA depends on NF-κB activation, partially on the adaptor molecule IFN-promoter stimulator-1 and the novel cytosolic dsDNA receptor IFI16, but not on the previously recognized dsDNA sentinels absent in melanoma 2, DNA-dependent activator of IFN regulatory factor 3, RNA polymerase III, or high-mobility group boxes. More importantly, we report for the first time, to our knowledge, that human dsDNA-activated DCs, rather than LPS- or inflammatory cytokine mixture-activated DCs, represent the most potent inducers of naive CD4+ T cells to promote Th1-type cytokine production and generate CD4+ and CD8+ cytotoxic T cells. dsDNA-DCs, but not LPS- or mixture-activated DCs, induce B cells to produce complement-fixing IgG1 and IgG3 Abs. We propose that cytosolic dsDNA represents a novel, more effective approach to generate DCs to enhance vaccine effectiveness in reprogramming the adaptive immune system to eradicate infectious agents, autoimmunity, allergy, and cancer.

Depletion of gut commensal bacteria attenuates intestinal ischemia/reperfusion injury

Gut commensal bacteria play important roles in the development and homeostasis of intestinal immunity. However, the role of gut commensals in intestinal ischemia/reperfusion (I/R) injury is unclear. To determine the roles of gut commensal bacteria in intestinal IR injury, we depleted gut microbiota with a broad-spectrum antibiotic cocktail and performed mesenteric I/R (M I/R). First, we confirmed that antibiotic treatment completely depleted gut commensal bacteria and diminished the size of secondary lymphoid tissues such as the Peyers patches. We next found that antibiotic treatment attenuated intestinal injury following M I/R. Depletion of gut commensal bacteria reduced the expression of Toll-like receptor (TLR)2 and TLR4 in the intestine. Both are well-known receptors for gram-positive and -negative bacteria. Decreased expression of TLR2 and TLR4 led to the reduction of inflammatory mediators, such as TNF, IL-6, and cyclooxygenase-2. Intestinal I/R injury is initiated when natural antibodies recognize neo-antigens that are revealed on ischemic cells and activate the complement pathway. Thus we evaluated complement and immunoglobulin (Ig) deposition in the damaged intestine and found that antibiotic treatment decreased the deposition of both C3 and IgM. Interestingly, we also found that the deposition of IgA also increased in the intestine following M I/R compared with control mice and that antibiotic treatment decreased the deposition of IgA in the damaged intestine. These results suggest that depletion of gut commensal bacteria decreases B cells, Igs, and TLR expression in the intestine, inhibits complement activation, and attenuates intestinal inflammation and injury following M I/R.

Distinct and Opposite Activities of Human Terminal Deoxynucleotidyltransferase Splice Variants

Evidence for potential human TdT (hTdT) isoforms derived from hTdT genomic sequences led us to identify the short isoform (hTdTS), as well as mature long transcripts containing exon XII (hTdTL1) and another including exon VII (hTdTL2) in lymphoid cells. Normal B and T lymphocytes express exclusively hTdTS and hTdTL2, whereas hTdTL1 expression appears to be restricted to transformed lymphoid cell lines. In in vitro recombination and primer assays, both long isoforms were shown to have 3′→5′ exonuclease activity. Overexpression of hTdTS or hTdTL2 greatly reduced the efficiency of recombination, which was reverted to normal levels by the simultaneous expression of both enzymes. Therefore, alternative splicing may prevent the adverse effects of unchecked elongation or diminution of coding ends during V(D)J recombination, thus affecting the survival of a B or T cell precursor during receptor gene rearrangements. Finally, the newly discovered hTdT isoforms should be considered in future screening of human leukemias.