Jiaqiang Huang

Bax Deficiency Partially Corrects Interleukin-7 Receptor α Deficiency

The requirement for cytokines in hematopoiesis is partly attributable to the protection of cells from apoptosis. Since IL-7 is required for normal T cell development, we evaluated the role of Bax in vivo by generating mice deficient in both Bax and the IL-7 receptor α chain (IL-7R). Starting at birth, we observed complete recovery of all stages of αβ thymocyte development up to 4 weeks of age. However, by 12 weeks of age, thymic cellularity had reverted to that of mice deficient in IL-7R alone. The BH3 only proteins, Bad and Bim, were also part of the death pathway repressed by IL-7. Thus, in young mice, Bax emerges as an essential protein in the death pathway induced by IL-7 deficiency.

Transcriptional Inactivation of STAT3 by PPARγ Suppresses IL-6-Responsive Multiple Myeloma Cells

Multiple myeloma (MM) remains largely incurable despite conventional and high-dose therapies. Therefore, novel biologically based treatment approaches are urgently required. Here we demonstrate that expression of peroxisome proliferator-activated receptor gamma (PPARgamma) in MM cells and its agonists 15-d-PGJ2 and troglitazone completely abolished IL-6-inducible MM cell proliferation and induced apoptosis through affecting expression of multiple cell cycle or apoptosis genes, whereas PPARgamma antagonist GW9662 and PPARalpha agonist WY14643 did not display this inhibitory effect. These PPARgamma agonists significantly inhibited DNA binding and transactivation of STAT3 bound to the promoter of target genes in chromatin, but did not affect the expression of IL-6 receptor and phosphorylation of JAK/STAT3, MAPK, and PI3K/Akt. Interestingly, although inactivation of STAT3 by PPARgamma agonists is in a PPARgamma-dependent manner, the molecular mechanism by which two structurally distinct PPARgamma agonists suppress IL-6-activated STAT3 shows the divergent interactions between PPARgamma and STAT3 including direct or SMRT-mediated association.

Lsh, a modulator of CpG methylation, is crucial for normal histone methylation

Methylation of histone tails and CpG methylation are involved in determining heterochromatin structure, but their cause and effect relationship has not been resolved as yet in mammals. Here we report that Lsh, a member of the SNF2 chromatin remodeling family, controls both types of epigenetic modifications. Lsh has been shown to be associated with pericentromeric heterochromatin and to be required for normal CpG methylation at pericentromeric sequences. Loss of Lsh, in Lsh‐deficient mice, results in accumulation of di‐ and tri‐methylated histone 3 at lysine 4 (H3‐K4me) at pericentromeric DNA and other repetitive sequences. In contrast, di‐ or tri‐methylation of H3‐K9 and distribution of HP1 appear unchanged after Lsh deletion, suggesting independent regulatory mechanisms for H3‐K4 or K9 methylation. Experimental DNA demethylation with 5′‐azacytidine results in a similar increase of H3‐K4me. These results support the model that loss of CpG methylation caused by Lsh deficiency antecedes elevation of H3‐K4me. Thus, Lsh is crucial for the formation of normal heterochromatin, implying a functional role for Lsh in the regulation of transcription and mitosis.

B7-H4 deficient mice display augmented neutrophil-mediated innate immunity

B7-H4 is an immunoglobulin superfamily molecule and shown to be inhibitory for T-cell responses. To explore physiologic roles of B7-H4, we created B7-H4-deficient (KO) mice by genetic targeting. B7-H4KO mice are healthy and their T- and B-cell responses to polyclonal antigens are in normal range. However, B7-H4KO mice are more resistant to infection by Listeria monocytogenes than their littermates. Within 3 days after infection, bacterial colonies in livers and spleens are significantly lower than the controls, suggesting a role of B7-H4 in enhancing innate immunity. Further studies demonstrate that neutrophils increase in peripheral organs of B7-H4KO mice more so than their littermates but their bactericidal functions remain unchanged. Augmented innate resistance is completely dependent on neutrophils, even in the absence of adaptive immunity. In vitro B7-H4 inhibits the growth of bone marrow-derived neutrophil progenitors, suggesting an inhibitory function of B7-H4 in neutrophil expansion. Our results identify B7-H4 as a negative regulator of the neutrophil response to infection and provide a new target for manipulation of innate immunity.

Cutting Edge: Histone Acetylation and Recombination at the TCRγ Locus Follows IL-7 Induction

IL-7 signaling is required for V(D)J recombination at the TCRγ locus. We have recently reported that IL-7 controls chromatin accessibility for RAG-mediated cleavage. Inhibition of histone deacetylase substituted for the IL-7 signal, indicating a role for histone acetylation in altering chromatin accessibility. We found a greatly reduced histone 3 and histone 4 acetylation level in IL-7Rα−/− thymocytes in comparison with RAG−/− thymocytes or fetal thymocytes. Sterile transcripts, indicating an open chromatin configuration, were suppressed in IL-7Rα−/− and IL-7−/−RAG−/− thymocytes. Moreover, exogenously added IL-7 induced sterile transcripts from the TCRγ constant region in cultured thymocytes from IL-7−/−RAG−/− mice. This induction correlated with increased histone acetylation at the J-promoter and C-enhancer regulatory elements at the TCRγ locus. These results suggest that IL-7 regulates chromatin accessibility for V(D)J recombination by specifically altering histone acetylation within the TCRγ locus.

TNF optimally activatives regulatory T cells by inducing TNF receptor superfamily members TNFR2, 4-1BB and OX40

TNF is a pleiotropic cytokine with intriguing biphasic pro‐inflammatory and anti‐inflammatory effects. Our previous studies demonstrated that TNF up‐regulated FoxP3 expression and activated and expanded CD4+FoxP3+ regulatory T cells (Tregs) via TNFR2. Furthermore, TNFR2‐expressing Tregs exhibited maximal suppressive activity. In this study, we show that TNF, in concert with IL‐2, preferentially up‐regulated mRNA and surface expression of TNFR2, 4‐1BB and OX40 on Tregs. Agonistic antibodies against 4‐1BB and OX40 also induced the proliferation of suppressive Tregs. Thus, TNF amplifies its stimulatory effect on Tregs by inducing TNF receptor superfamily (TNFRSF) members. In addition, administration of neutralizing anti‐TNF Ab blocked LPS‐induced expansion of splenic Tregs and up‐regulation of TNFR2, OX40 and 4‐1BB receptors on Tregs in vivo, indicating that the expansion of Tregs expressing these co‐stimulatory TNFRSF members in response to LPS is mediated by TNF. Altogether, our novel data indicate that TNF preferentially up‐regulates TNFR2 on Tregs, and this is amplified by the stimulation of 4‐1BB and OX40, resulting in the optimal activation of Tregs and augmented attenuation of excessive inflammatory responses.

Lsh, chromatin remodeling family member, modulates genome-wide cytosine methylation patterns at nonrepeat sequences.

DNA methylation is critical for normal development and plays important roles in genome organization and transcriptional regulation. Although DNA methyltransferases have been identified, the factors that establish and contribute to genome-wide methylation patterns remain elusive. Here, we report a high-resolution cytosine methylation map of the murine genome modulated by Lsh, a chromatin remodeling family member that has previously been shown to regulate CpG methylation at repetitive sequences. We provide evidence that Lsh also controls genome-wide cytosine methylation at nonrepeat sequences and relate those changes to alterations in H4K4me3 modification and gene expression. Deletion of Lsh alters the allocation of cytosine methylation in chromosomal regions of 50 kb to 2 Mb and, in addition, leads to changes in the methylation profile at the 5′ end of genes. Furthermore, we demonstrate that loss of Lsh promotes—as well as prevents—cytosine methylation. Our data indicate that Lsh is an epigenetic modulator that is critical for normal distribution of cytosine methylation throughout the murine genome.

Control of chromatin accessibility for V(D)J recombination by interleukin‐7

IL‐7 is a key factor for lymphoid development, and it contributes to V(D)J recombination at multiple loci in immune‐receptor genes. IL‐7 signal transduction, involving γc and Jak3, is required for successful recombination at the TCR‐γ locus. IL‐7 signaling controls the initiation phase of V(D)J recombination by controlling access of the V(D)J recombinase to the locus. In the absence of IL‐7, the TCR‐γ locus is methylated and packaged in a repressed form of chromatin consisting of hypoacetylated histones. IL‐7 signaling likely increases the acetylation state of the nucleosomal core histones resulting in an “open” form of chromatin. This opening leads to a higher accessibility for the transcription machinery and increased accessibility of the Rag heterodimer that performs the cleavage of DNA.

The Formylpeptide Receptor 2 (Fpr2) and Its Endogenous Ligand Cathelin-related Antimicrobial Peptide (CRAMP) Promote Dendritic Cell Maturation

Background: Chemoattractant receptor Fpr2 interacts with host-derived agonist CRAMP and promotes dentritic cell maturation in immune responses. Results: Deficiency in Fpr2 or CRAMP results in impaired maturation of dendritic cells in vitro and in vivo. Conclusion: Fpr2 and its agonist CRAMP play a nonredundant role in DC maturation. Significance: Fpr2 and its agonist CRAMP are potential targets for disease intervention. Mouse formylpeptide receptor 2 (Fpr2) is a homologue of the human G-protein coupled chemoattractant receptor FPR2, which interacts with pathogen and host-derived chemotactic agonists. Our previous studies revealed reduced allergic airway inflammation and immune responses in Fpr2-deficient (Fpr2−/−) mice in association with diminished dendritic cell (DC) recruitment into the airway and draining lymph nodes. These defects prompted us to investigate the potential changes in the differentiation and maturation of DCs caused by Fpr2 deficiency. Bone marrow monocytes from Fpr2−/− mouse mice incubated with GM-CSF and IL-4 in vitro showed normal expression of markers of immature DCs. However, upon stimulation with the TLR4 agonist LPS, Fpr2−/− mouse DCs failed to express normal levels of maturation markers with reduced production of IL-12 and diminished chemotaxis in response to the DC homing chemokine CCL21. Fpr2−/− DCs also failed to induce allogeneic T-cell proliferation in vitro, and their recruitment into the T-cell zones of the spleen was reduced after antigen immunization. The capacity of Fpr2 to sustain normal DC maturation was dependent on its interaction with an endogenous ligand CRAMP expressed by DCs, because neutralization of either Fpr2 or CRAMP inhibited DC maturation in response to LPS. We additionally observed that the presence of exogenous CRAMP in culture increased the sensitivity of WT mouse DCs to LPS stimulation. The importance of CRAMP for DC maturation was further demonstrated by the observations that DCs from CRAMP−/− mice expressed lower levels of costimulatory molecules and MHC II and exhibited poor chemotaxis in response to CCL21 after LPS stimulation. Our observations indicate a nonredundant role for Fpr2 and its agonist CRAMP in DC maturation in immune responses.

IL-7Rα deficiency in p53 null mice exacerbates thymocyte telomere erosion and lymphomagenesis

Interleukin-7 (IL-7) is an essential T-cell survival cytokine. IL-7 receptor (IL-7Rα) deficiency severely impairs T-cell development due to substantial apoptosis. We hypothesized that IL-7Rαnull-induced apoptosis is partially contributed by an elevated p53 activity. To investigate the genetic association of IL-7/IL-7Rα signaling with the p53 pathway, we generated IL-7Rαnullp53null (DKO) mice. DKO mice exhibited a marked reduction of apoptosis in developing T cells and an augmented thymic lymphomagenesis with telomere erosions and exacerbated chromosomal anomalies, including chromosome duplications, breaks, and translocations. In particular, Robertsonian translocations, in which telocentric chromosomes fuse at the centromeric region, and a complete loss of telomeres at the fusion site occurred frequently in DKO thymic lymphomas. Cellular and molecular investigations revealed that IL-7/IL-7Rα signaling withdrawal diminished the protein synthesis of protection of telomere 1 (POT1), a subunit of telomere protective complex shelterin, leading to telomere erosion and the activation of the p53 pathway. Blockade of IL-7/IL-7Rα signaling in IL-7-dependent p53null cells reduced POT1 expression and caused telomere and chromosome abnormalities similar to those observed in DKO lymphomas. This study underscores a novel function of IL-7/IL-7Rα during T-cell development in regulating telomere integrity via POT1 expression and provides new insights into cytokine-mediated survival signals and T-cell lymphomagenesis.