192 research outputs found
Contributions of the inhibitor of kappa B kinases (IKKs) in macrophages and neutrophils after Francisella tularensis live vaccine strain (LVS) infection.
No full textThe immune system is a complex network of cells regulated by a number of signaling pathways to drive specific host defenses against invading pathogens. The NF-&kappaB (nuclear factor of the kappa light chain enhancer in B cells) family of inducible transcription factors is a critical regulator of many innate and adaptive immune responses. NF-&kappaB activation by several receptors converges upon two upstream kinases, IKK&alpha and IKK&beta, that regulate gene induction. Many studies report that IKK&beta is the critical kinase involved in NF--&kappaB activation, although at a transcriptional level, both kinases are needed to obtain the full spectrum of gene induction. More recently, in an in vivo bacterial infection model both IKK&alpha and IKK&beta were reported to have anti-inflammatory properties through different mechanisms.The intracellular bacterium Francisella tularensis is able to down modulate inflammatory reactions within macrophages and neutrophils to provide a niche for bacterial growth. To this end, I asked whether disruptions of NF-&kappaB signaling, specifically through IKK&alpha and IKK&beta, could promote inflammation and allow the host more resistance to infection.I used conditional gene targeting of IKK&alpha and IKK&beta to generate mice with specific deletions for either kinase in these cells. These mice were used in a tularemia infection using the live vaccine strain of Francisella tularensis (Ft. LVS) as a model to elucidate the specific contributions of each kinase.Contrary to what has been previously reported for Group B Streptococcus infection models, IKK&beta but not IKK&alpha, was required for host resistance and survival in tularemia infection as 100% of Ft. LVS infected mice succumbed to lethal infection within 10 days.In a sublethal model of infection, both IKK&alpha and IKK&beta contributed in different ways to the maintenance of hepatic granulomas after infection. Depletion of IKK&alpha led to fewer, but sometimes, large necrotic granuloma formation indicating a potential role for IKK&alpha in the clearance of apoptotic cells. IKK&beta depletion resulted disordered granuloma structures and elevated bacterial colonization and growth throughout the infection. This led to increased inflammation as early as 2 days post infection as evidenced by a polarization towards M1 macrophages and IL-12 production. Compensation mechanisms to reduce inflammation such as an increase in myeloid derived suppressor cells or a subsequent M2a macrophage polarization occurred, but were not able to control inflammation or bacterial growth in these mice. In addition, IKK&beta loss resulted in protracted IFN-&gamma production by cytotoxic T lymphocytes.Overall, in an Ft. LVS infection model, IKK&alpha may be more important in neutrophils for the clearance of apoptotic cells, while IKK&beta is required more globally to prevent inflammation and control bacterial colonization that cannot be compensated by anti-inflammatory mechanisms. These functions appear to be correlated with early activation of both macrophages, neutrophils and extrinsic activation of cytotoxic T lymphocytes.Advisor(s): Kenneth B. Marcu. Michael Hadjiargyrou. Committee Member(s): Martha B. Furie; Richard R. Kew; William P. Tansey.Stony Brook University Libraries. SBU Graduate School in Department of Genetics. Lawrence Martin (Dean of Graduate School)
Cell migration to CXCL12 requires simultaneous IKKα and IKKβ-dependent NF-κB signaling
No full textAbstractCXCL12 and its unique receptor CXCR4, is critical for the homing of a variety of cell lineages during both development and tissue repair. CXCL12 is particularly important for the recruitment of hemato/lymphopoietic cells to their target organs. In conjunction with the damage-associated alarmin molecule HMGB1, CXCL12 mediates immune effector and stem/progenitor cell migration towards damaged tissues for subsequent repair. Previously, we showed that cell migration to HMGB1 simultaneously requires both IKKβ and IKKα-dependent NF-κB activation. IKKβ-mediated activation maintains sufficient expression of HMGB1's receptor RAGE, while IKKα-dependent NF-κB activation ensures continuous production of CXCL12, which complexes with HMGB1 to engage CXCR4. Here using fibroblasts and primary mature macrophages, we show that IKKβ and IKKα are simultaneously essential for cell migration in response to CXCL12 alone. Non-canonical NF-κB pathway subunits RelB and p52 are also both essential for cell migration towards CXCL12, suggesting that IKKα is required to drive non-canonical NF-κB signaling. Flow cytometric analyses of CXCR4 expression show that IKKβ, but not IKKα, is required to maintain a critical threshold level of this CXCL12 receptor. Time-lapse video microscopy experiments in primary MEFs reveal that IKKα is required both for polarization of cells towards a CXCL12 gradient and to establish a basal level of velocity towards CXCL12. In addition, CXCL12 modestly up-regulates IKKα-dependent p52 nuclear translocation and IKKα-dependent expression of the CXCL12 gene. On the basis of our collective results we posit that IKKα is needed to maintain the basal expression of a critical protein co-factor required for cell migration to CXCL12
The cytidine deaminases AID and APOBEC-1 exhibit distinct functional properties in a novel yeast selectable system
No full textActivation-induced cytidine deaminase (AID) is indispensable for immunoglobulin maturation by somatic hypermutations and class switch recombination and is supposed to deaminate cytidines in DNA, while its homolog APOBEC-1 edits apolipoprotein (apo) B mRNA by cytidine deamination. We studied the editing activity of APOBEC-1 and AID in yeast using the selectable marker Gal4 linked to its specific inhibitor protein Gal80 via an apo B cassette (Gal4-C) or via the variable region of a mouse immunoglobulin heavy chain gene (Gal4-VH). Expression of APOBEC-1 induced C to U editing in up to 15% of the Gal4-C transcripts, while AID was inactive in this reaction even in the presence of the APOBEC-1 complementation factor. After expression of APOBEC-1 as well as AID approximately 10(-3) of yeast cells survived low stringency selection and expressed beta-galactosidase. Neither AID nor APOBEC-1 mutated the VH sequence of Gal4-VH, and consequently the yeast colonies did not escape high stringent selection. AID, however, induced frequent plasmid recombinations that were only rarely observed with APOBEC-1. In conclusion, AID cannot substitute APOBEC-1 to edit the apo B mRNA, and the expression of AID in yeast is not sufficient for the generation of point mutations in a highly transcribed Gal4-VH sequence. Cofactors for AID induced somatic hypermutations of immunoglobulin variable regions, that are present in B cells and a variety of non-B cells, appear to be missing in yeast. In contrast to APOBEC-1, AID alone does not exhibit an intrinsic specificity for its target sequences
IKKα/CHUK regulates extracellular matrix remodeling independent of its kinase activity to facilitate articular chondrocyte differentiation.
No full textThe non-canonical NF-κB activating kinase IKKα, encoded by CHUK (conserved-helix-loop-helix-ubiquitous-kinase), has been reported to modulate pro- or anti- inflammatory responses, cellular survival and cellular differentiation. Here, we have investigated the mechanism of action of IKKα as a novel effector of human and murine chondrocyte extracellular matrix (ECM) homeostasis and differentiation towards hypertrophy.IKKα expression was ablated in primary human osteoarthritic (OA) chondrocytes and in immature murine articular chondrocytes (iMACs) derived from IKKα(f/f):CreERT2 mice by retroviral-mediated stable shRNA transduction and Cre recombinase-dependent Lox P site recombination, respectively. MMP-10 was identified as a major target of IKKα in chondrocytes by mRNA profiling, quantitative RT-PCR analysis, immunohistochemistry and immunoblotting. ECM integrity, as assessed by type II collagen (COL2) deposition and the lack of MMP-dependent COL2 degradation products, was enhanced by IKKα ablation in mice. MMP-13 and total collagenase activities were significantly reduced, while TIMP-3 (tissue inhibitor of metalloproteinase-3) protein levels were enhanced in IKKα-deficient chondrocytes. IKKα deficiency suppressed chondrocyte differentiation, as shown by the quantitative inhibition of.Alizarin red staining and the reduced expression of multiple chondrocyte differentiation effectors, including Runx2, Col10a1 and Vegfa,. Importantly, the differentiation of IKKα-deficient chondrocytes was rescued by a kinase-dead IKKα protein mutant.IKKα acts independent of its kinase activity to help drive chondrocyte differentiation towards a hypertrophic-like state. IKKα positively modulates ECM remodeling via multiple downstream targets (including MMP-10 and TIMP-3 at the mRNA and post-transcriptional levels, respectively) to maintain maximal MMP-13 activity, which is required for ECM remodeling leading to chondrocyte differentiation. Chondrocytes are the unique cell component in articular cartilage, which are quiescent and maintain ECM integrity during tissue homeostasis. In OA, chondrocytes reacquire the capacity to proliferate and differentiate and their activation results in pronounced cartilage degeneration. Τηυσ, our findings are also of potential relevance for defining the onset and/or progression of OA disease
Regulated transcription of human matrix metalloproteinase 13 (MMP13) and interleukin -1B (IL 1B) genes in chondrocytes depends on methylation of specific proximal promoter CpG sites
No full textThe role of DNA methylation in the regulation of catabolic genes such as MMP13 and IL1B, which have sparse CpG islands, is poorly understood in the context of musculoskeletal diseases. We report that demethylation of specific CpG sites at ?110 bp and ?299 bp of the proximal MMP13 and IL1B promoters, respectively, detected by in situ methylation analysis of chondrocytes obtained directly from human cartilage, strongly correlated with higher levels of gene expression. The methylation status of these sites had a significant impact on promoter activities in chondrocytes, as revealed in transfection experiments with site-directed CpG mutants in a CpG-free luciferase reporter. Methylation of the ?110 and ?299 CpG sites, which reside within a hypoxia-inducible factor (HIF) consensus motif in the respective MMP13 and IL1B promoters, produced the most marked suppression of their transcriptional activities. Methylation of the ?110 bp CpG site in the MMP13 promoter inhibited its HIF-2?-driven transactivation and decreased HIF-2? binding to the MMP13 proximal promoter in chromatin immunoprecipitation assays. In contrast to HIF-2?, MMP13 transcriptional regulation by other positive (RUNX2, AP-1, ELF3) and negative (Sp1, GATA1, and USF1) factors was not affected by methylation status. However, unlike the MMP13 promoter, IL1B was not susceptible to HIF-2? transactivation, indicating that the ?299 CpG site in the IL1B promoter must interact with other transcription factors to modulate IL1B transcriptional activity. Taken together, our data reveal that the methylation of different CpG sites in the proximal promoters of the human MMP13 and IL1B genes modulates their transcription by distinct mechanisms
IKKβ in myeloid cells controls the host response to lethal and sublethal Francisella tularensis LVS infection.
No full textThe NF-κB activating kinases, IKKα and IKKβ, are key regulators of inflammation and immunity in response to infection by a variety of pathogens. Both IKKα and IKKβ have been reported to modulate either pro- or anti- inflammatory programs, which may be specific to the infectious organism or the target tissue. Here, we analyzed the requirements for the IKKs in myeloid cells in vivo in response to Francisella tularensis Live Vaccine Strain (Ft. LVS) infection.In contrast to prior reports in which conditional deletion of IKKβ in the myeloid lineage promoted survival and conferred resistance to an in vivo group B streptococcus infection, we show that mice with a comparable conditional deletion (IKKβ cKO) succumb more rapidly to lethal Ft. LVS infection and are unable to control bacterial growth at sublethal doses. Flow cytometry analysis of hepatic non-parenchymal cells from infected mice reveals that IKKβ inhibits M1 classical macrophage activation two days post infection, which has the collateral effect of suppressing IFN-γ(+) CD8(+) T cells. Despite this early enhanced inflammation, IKKβ cKO mice are unable to control infection; and this coincides with a shift toward M2a polarized macrophages. In comparison, we find that myeloid IKKα is dispensable for survival and bacterial control. However, both IKKα and IKKβ have effects on hepatic granuloma development. IKKα cKO mice develop fewer, but well-contained granulomas that accumulate excess necrotic cells after 9 days of infection; while IKKβ cKO mice develop numerous micro-granulomas that are less well contained.Taken together our findings reveal that unlike IKKα, IKKβ has multiple, contrasting roles in this bacterial infection model by acting in an anti-inflammatory capacity at early times towards sublethal Ft. LVS infection; but in spite of this, macrophage IKKβ is also a critical effector for host survival and efficient pathogen clearance
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
No full textNF-kappaB transcription factors induce a host of genes involved in pro-inflammatory/stress-like responses; but the collateral effects and consequences of sustained NF-kappaB activation on other cellular gene expression programming remain less well understood. Here enforced expression of a constitutively active IKKbeta T-loop mutant (IKKbetaca) 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 IKKbetaca 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 IkappaBalpha super repressor and IKKbetaca abrogated growth arrest and cell cycle effector repression, thereby linking IKKbetaca's effects to canonical NF-kappaB activation. Transient growth arrest of IKKbetaca cells was associated with enhanced p21 (cyclin-dependent kinase inhibitor 1A) protein expression, due in part to transcriptional activation by NF-kappaB 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 IKKbetaca mediated growth suppression. Moreover, trichostatin A inhibition of HDACs alleviated the repression of E2F and FoxM1 targets induced by IKKbetaca, suggesting chromatin mediated gene silencing in IKKbetaca's short term repressive effects on E2F and FoxM1 target gene expression
Reactivity of <i>Ft</i>. LVS induced granulomas.
No full text<p>Mice were challenged i.d. with 10<sup>6</sup> CFU of <i>Ft</i>. LVS and livers were analyzed by immunohistochemistry for anti-<i>Ft</i>. LVS antigen, anti-iNOS and anti-cleaved caspase-3 (CC3) at days 2 and 9 post infection. <i>Ft</i>. LVS antigens were detected within (<b>A–B</b>) IKK<i><sup>f/f</sup></i>, (<b>C–D</b>) IKKα cKO and (<b>E–F</b>) IKKβ cKO granulomas at days 2 and 9, respectively. iNOS positivity was determined for (<b>G–H</b>) IKK<i><sup>f/f</sup></i>, (<b>I–J</b>) IKKα cKO and (<b>K-L</b>) IKKβ cKO mice. Activated caspase-3, an early indicator of apoptosis, was detected in (<b>M-N</b>) IKK<i><sup>f/f</sup></i>, (<b>O–P</b>) IKKα cKO and (<b>Q–R</b>) in IKKβ cKO granulomas at days 2 and 9 post infection, respectively. 200× magnification, scale bar = 50 µm; d.p.i.: days, post-infection, (n = 4).</p
Dissemination of <i>Ft</i>. LVS leads to lymphocytic aggregate development in IKKβ cKO mouse lung.
No full text<p>H & E stained lung tissue was examined for histological changes at days 2 and 9 (<b>A–F</b>) after i.d. infection with 10<sup>6</sup> CFU of <i>Ft</i>. LVS. (<b>A</b>) IKK<i><sup>f/f</sup></i> and (<b>B</b>) IKKα cKO mice show evolution and (<b>D, E</b>) resolution of inflammation, while (<b>C and F</b>) IKKβ cKO mice exhibit a delay in resolution concordant with the appearance of (<b>F, inset</b>) lymphocytic aggregates. Figure insets are taken from regions marked by rectangles. Yellow arrows indicate neutrophils, blue arrows indicate lymphocytic infiltrate, yellow arrowheads indicate lymphocytic aggregates. Scale bar = 500 µm, 20× magnification, inset scale bar = 50 µm, 400× magnification). Representative sections are shown from at least three independent experiments.</p
Loss of either IKK kinase results in defects in hepatic granuloma development.
No full text<p>Comparison of early and late granuloma development after sublethal i.d. infection with 10<sup>6</sup> CFU of <i>Ft.</i> LVS. Representative H & E stained liver sections are shown for (<b>A</b>) IKK<i><sup>f/f</sup></i>, (<b>B</b>) IKKα cKO and (<b>C</b>) IKKβ cKO infected mice at two days post-infection. Granulomas, indicated by yellow arrows (40× magnification, scale bars = 500 µm), are magnified in the inset of each panel (400× magnification, scale bars = 30 µm). Panels (<b>D–F</b>) are representative granulomas at 9 days post-infection (panels D and F are 200× magnification, scale bar = 50 µm; Panel E is 100×magnification, scale bar = 100 µm). (<b>G</b>) Granulomas were quantified as counts per 200× field and were analyzed by one-way ANOVA and Tukey’s ad hoc post-test. Data are pooled from two independent experiments (each with n = 4 mice per group) for a total of n = 8 mice per group; ***P<0.0001.</p
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