Adedayo Hanidu

Epstein-Barr virus-induced gene 3 negatively regulates IL-17, IL-22 and RORγt

Epstein‐Barr virus‐induced gene 3 (EBI3) associates with p28 to form IL‐27 and with IL‐12p35 to form IL‐35. IL‐27Rα–/– mice studies indicate that IL‐27 negatively regulates Th17 cell differentiation. However, no EBI3, p28 or p35‐deficiency studies that directly address the role of EBI3, p28 or p35 on Th17 cells have been done. Here, we demonstrate that spleen cells derived from EBI3–/– mice produce significantly higher levels of IL‐17 as well as IL‐22 upon stimulation with OVA. In vitro derived EBI3–/– Th17 cells also produced significantly higher levels of IL‐17 and IL‐22 than WT cells. The frequency of IL‐17‐producing cells was also elevated when EBI3–/– cells were cultured under Th17 conditions. In addition, spleen cells from EBI3–/– mice immunized with Listeria monocytogenes produced significantly elevated levels of IL‐17 and IL‐22. Furthermore, the Th17 transcription factor RORγt was significantly enhanced in EBI3–/– cells. Finally, EBI3–/– mice exhibited a reduced bacterial load following an acute challenge with L. monocytogenes or a re‐challenge of previously immunized mice, suggesting that EBI3 negatively regulates both innate and adaptive immunity. Taken together, these data provide direct evidence that EBI3 negatively regulates the expression of IL‐17, IL‐22 and RORγt as well as protective immunity against L. monocytogenes.

MEK5 is Activated by Shear Stress, Activates ERK5 and Induces KLF4 to Modulate TNF Responses in Human Dermal Microvascular Endothelial Cells

Please cite this paper as: Clark, Jensen, Kluger, Morelock, Hanidu, Qi, Tatake, Pober (2011). MEK5 is Activated by Shear Stress, Activates ERK5 and Induces KLF4 to Modulate TNF Responses in Human Dermal Microvascular Endothelial Cells. Microcirculation18(2), 102–117.

Sustained NF-κB activation produces a short-term cell proliferation block in conjunction with repressing effectors of cell cycle progression controlled by E2F or FoxM1

NF‐κB transcription factors induce a host of genes involved in pro‐inflammatory/stress‐like responses; but the collateral effects and consequences of sustained NF‐κB activation on other cellular gene expression programming remain less well understood. Here enforced expression of a constitutively active IKKβ T‐loop mutant (IKKβca) drove murine fibroblasts into transient growth arrest that subsided within 2–3 weeks of continuous culture. Proliferation arrest was associated with a G1/S phase block in immortalized and primary early passage MEFs. Molecular analysis in immortalized MEFs revealed that inhibition of cell proliferation in the initial 1–2 weeks after their IKKβca retroviral infection was linked to the transient, concerted repression of essential cell cycle effectors that are known targets of either E2F or FoxM1. Co‐expression of a phosphorylation resistant IκBα super repressor and IKKβca abrogated growth arrest and cell cycle effector repression, thereby linking IKKβcas effects to canonical NF‐κB activation. Transient growth arrest of IKKβca cells was associated with enhanced p21 (cyclin‐dependent kinase inhibitor 1A) protein expression, due in part to transcriptional activation by NF‐κB and also likely due to strong repression of Skp2 and Csk1, both of which are FoxM1 direct targets mediating proteasomal dependent p21 turnover. Ablation of p21 in immortalized MEFs reduced their IKKβca mediated growth suppression. Moreover, trichostatin A inhibition of HDACs alleviated the repression of E2F and FoxM1 targets induced by IKKβca, suggesting chromatin mediated gene silencing in IKKβcas short term repressive effects on E2F and FoxM1 target gene expression. J. Cell. Physiol. 218: 215–227, 2009.

Gene Expression Profiling in Conjunction with Physiological Rescues of IKKα-null Cells with Wild Type or Mutant IKKα Reveals Distinct Classes of IKKα/NF-κB-dependent Genes

Cellular responses to stress-like stimuli require the IκB kinase (IKK) signalsome (IKKα, IKKβ, and NEMO/IKKγ) to activate NF-κB-dependent genes. IKKβ and NEMO/IKKγ are required to release NF-κB p65/p50 heterodimers from IκBα, resulting in their nuclear migration and sequence-specific DNA binding; but IKKα was found to be dispensable for this initial phase of canonical NF-κB activation. Nevertheless, IKKα(-/-) mouse embryonic fibroblasts (MEFs) fail to express NF-κB targets in response to proinflammatory stimuli, uncovering a nuclear role for IKKα in NF-κB activation. However, it remains unknown whether the global defect in NF-κB-dependent gene expression of IKKα(-/-) cells is caused by the absence of IKKα kinase activity. We show by gene expression profiling that rescue of near physiological levels of wild type IKKα in IKKα(-/-) MEFs globally restores expression of their canonical NF-κB target genes. To prove that the kinase activity of IKKα was required on a genomic scale, the same physiological rescue was performed with a kinase-dead, ATP binding domain IKKα mutant (IKKα(K44M)). Remarkably, the IKKα(K44M) protein rescued ∼28% of these genes, albeit in a largely stimulus-independent manner with the notable exception of several genes that also acquired tumor necrosis factor-α responsiveness. Thus the IKKα-containing signalsome unexpectedly functions in the presence and absence of extracellular signals in both kinase-dependent and -independent modes to differentially modulate the expression of five distinct classes of IKKα/NF-κB-dependent genes.

Sphingosine kinase 1 is a negative regulator of CD4+ Th1 cells

CD4+ Th1 cells produce IFN-γ, TNF-α, and IL-2. These Th1 cytokines play critical roles in both protective immunity and inflammatory responses. In this study we report that sphingosine kinase 1 (SPHK1), but not SPHK2, is highly expressed in DO11.10 Th1 cells. The expression of SPHK1 in Th1 cells requires TCR signaling and new protein synthesis. SPHK1 phosphorylates sphingosine to form sphingosine-1-phosphate. Sphingosine-1-phosphate plays important roles in inhibition of apoptosis, promotion of cell proliferation, cell migration, calcium mobilization, and activation of ERK1/2. When SPHK1 expression was knocked down by SPHK1 short interfering RNA, the production of IL-2, TNF-α, and IFN-γ by Th1 cells in response to TCR stimulation was enhanced. Consistently, overexpression of dominant-negative SPHK1 increased the production of IL-2, TNF-α, and IFN-γ in Th1 cells. Furthermore, overexpression of SPHK1 in Th1 and Th0 cells decreased the expression of IL-2, TNF-α, and IFN-γ. Several chemokines, including Th2 chemokines CCL17 and CCL22, were up-regulated by SPHK1 short interfering RNA and down-regulated by overexpression of SPHK1. We also showed that Th2 cells themselves express CCL17 and CCL22. Finally, we conclude that SPHK1 negatively regulates the inflammatory responses of Th1 cells by inhibiting the production of proinflammatory cytokines and chemokines.

Kcnn4 is a regulator of macrophage multinucleation in bone homeostasis and inflammatory disease.

Summary Macrophages can fuse to form osteoclasts in bone or multinucleate giant cells (MGCs) as part of the immune response. We use a systems genetics approach in rat macrophages to unravel their genetic determinants of multinucleation and investigate their role in both bone homeostasis and inflammatory disease. We identify a trans-regulated gene network associated with macrophage multinucleation and Kcnn4 as being the most significantly trans-regulated gene in the network and induced at the onset of fusion. Kcnn4 is required for osteoclast and MGC formation in rodents and humans. Genetic deletion of Kcnn4 reduces macrophage multinucleation through modulation of Ca2+ signaling, increases bone mass, and improves clinical outcome in arthritis. Pharmacological blockade of Kcnn4 reduces experimental glomerulonephritis. Our data implicate Kcnn4 in macrophage multinucleation, identifying it as a potential therapeutic target for inhibition of bone resorption and chronic inflammation.

Involvement of GATA3 in Protein Kinase C θ-induced Th2 cytokine expression

Protein kinase C θ (PKCθ) is essential for T cell activation, as it is required for the activation of NF‐κB and expression of IL‐2. PKCθ has also been shown to affect NFAT activation and Th2 differentiation. To better understand the role of PKCθ in the regulation of T helper cells, we used PKCθ‐deficient DO11.10 transgenic T cells to study its role in vitro. DO11.10 Th1 cells deficient in PKCθ produced significantly less TNF‐α and IL‐2. The expression of Th2 cytokines, including IL‐4, IL‐5, IL‐10, IL‐13 and IL‐24 was significantly reduced in PKCθ‐deficient T cells. Moreover, the expression of the Th2 transcription factor, GATA3, was significantly reduced in PKCθ‐deficient T cells. Overexpression of GATA3 by retroviral infection in PKCθ‐deficient T cells resulted in increased expansion of IL‐4‐producing T cells and higher IL‐4 production than that of wild type Th2 cells. IL‐5, IL‐10, IL‐13 and IL‐24 expressions were also rescued by GATA3 overexpression. Our observations suggest that PKCθ regulates Th2 cytokine expression via GATA3.

Genome-Wide Expression Profiling Identifies an Impairment of Negative Feedback Signals in the Crohn’s Disease-Associated NOD2 Variant L1007fsinsC

NOD2 is an intracellular receptor for the bacterial cell wall component muramyl dipeptide (MDP), and variants of NOD2 are associated with chronic inflammatory diseases of barrier organs (e.g., Crohn’s disease, asthma, and atopic eczema). It is known that activation of NOD2 induces a variety of inflammatory and antibacterial factors. The exact transcriptomal signatures that define the cellular programs downstream of NOD2 activation and the influence of the Crohn-associated variant L1007fsinsC are yet to be defined. To describe the MDP-induced activation program, we analyzed the transcriptomal reactions of isogenic HEK293 cells expressing NOD2wt or NOD2L1007fsinsC to stimulation with MDP. Importantly, a clear loss of function could be observed in the cells carrying the Crohn-associated variant L1007fsinsC, whereas the NOD2wt cells showed differential regulation of growth factors, chemokines, and several antagonists of NF-κB (e.g., TNFAIP3 [A20] and IER3). This genotype-dependent regulation pattern was confirmed in primary human myelomonocytic cells. The influence of TNFAIP3 and IER3 in the context of NOD2 signaling was characterized, and we could validate the predicted role as inhibitors of NOD2-induced NF-κB activation. We show that IER3 impairs the protective effect of NOD2wt against bacterial cytoinvasion. These results further our understanding of NOD2 as a first-line defense molecule and emphasize the importance of simultaneous upregulation of counterregulatory anti-inflammatory factors as an integral part of the NOD2-induced cellular program. Lack of these regulatory events due to the L1007fsinsC variant may pivotally contribute to the induction and perpetuation of chronic inflammation.

Interferon-γ Represses M2 Gene Expression in Human Macrophages by Disassembling Enhancers Bound by the Transcription Factor MAF

&NA; Mechanisms by which interferon (IFN)‐&ggr; activates genes to promote macrophage activation are well studied, but little is known about mechanisms and functions of IFN‐&ggr;‐mediated gene repression. We used an integrated transcriptomic and epigenomic approach to analyze chromatin accessibility, histone modifications, transcription‐factor binding, and gene expression in IFN‐&ggr;‐primed human macrophages. IFN‐&ggr; suppressed basal expression of genes corresponding to an “M2”‐like homeostatic and reparative phenotype. IFN‐&ggr; repressed genes by suppressing the function of enhancers enriched for binding by transcription factor MAF. Mechanistically, IFN‐&ggr; disassembled a subset of enhancers by inducing coordinate suppression of binding by MAF, lineage‐determining transcription factors, and chromatin accessibility. Genes associated with MAF‐binding enhancers were suppressed in macrophages isolated from rheumatoid‐arthritis patients, revealing a disease‐associated signature of IFN‐&ggr;‐mediated repression. These results identify enhancer inactivation and disassembly as a mechanism of IFN‐&ggr;‐mediated gene repression and reveal that MAF regulates the macrophage enhancer landscape and is suppressed by IFN‐&ggr; to augment macrophage activation. Graphical Abstract Figure. No caption available. HighlightsIFN‐&ggr; suppresses basal expression of M2‐like genes in human macrophagesIFN‐&ggr; downregulates MAF and targets MAF‐binding enhancers for suppressionA subset of IFN‐&ggr;‐inactivated enhancers loses TF binding and chromatin accessibilityLow MAF expression correlates with a “negative IFN‐&ggr; signature” in RA macrophages &NA; Kang et al. demonstrate that IFN‐&ggr; represses basal expression of M2‐like genes by targeting enhancers. IFN‐&ggr; induces loss of enhancer binding by MAF and lineage‐determining transcription factors, with concomitant “disassembly” and loss of chromatin accessibility. These results provide new insights into how IFN‐&ggr; regulates gene expression and activates macrophages.

Proviral integration site 2 is required for interleukin-6 expression induced by interleukin-1, tumour necrosis factor-α and lipopolysaccharide.

PIM (proviral integration site) kinases are a distinct class of serine/threonine‐specific kinases consisting of PIM1, PIM2 and PIM3. PIM2 is known to function in apoptosis pathways. Expression of PIM2 is highly induced by pro‐inflammatory stimuli but the role of PIM2 in the expression of pro‐inflammatory cytokines is unclear. In this study, we showed that over‐expression of PIM2 in HeLa cells as well as in human umbilical vein endothelial cells enhanced interleukin‐1β (IL‐1β) ‐induced and tumour necrosis factor‐α‐induced IL‐6 expression, whereas over‐expression of a kinase‐dead PIM2 mutant had the opposite effect. Studies with small interfering RNA specific to PIM2 further confirmed that IL‐6 expression in HeLa cells requires PIM2. To investigate the function of PIM2 further, we generated PIM2‐deficient mice. It was found that IL‐6 production was significantly decreased from PIM2‐deficient spleen cells after stimulation with lipopolysaccharide. Taken together, we demonstrated an important function of PIM2 in controlling the expression of the pro‐inflammatory cytokine IL‐6. PIM2 inhibitors may be beneficial for IL‐6‐mediated diseases such as rheumatoid arthritis.