scholarly journals Brd4 regulates NLRC4 inflammasome activation by facilitating IRF8-mediated transcription of Naips

2021 ◽  
Vol 220 (3) ◽  
Author(s):  
Xingchen Dong ◽  
Xiangming Hu ◽  
Yan Bao ◽  
Guo Li ◽  
Xiao-dong Yang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cytokine Production ◽  
Conditional Knockout ◽  
Inflammasome Activation ◽  
Cytokine Release ◽  
Rt Pcr ◽  
Binding Motifs ◽  
Epigenetic Regulator ◽  
Important Regulator ◽  
Protection Against Infection

NLRC4 inflammasome activation and the subsequent maturation of IL-1β and IL-18 are critical for protection against infection by bacterial pathogens. The epigenetic regulator Brd4 has emerged as a key player in inflammation by regulating the expression of inflammatory cytokines. However, whether Brd4 has any role in inflammasome activation remains undetermined. Here, we demonstrated that Brd4 is an important regulator of NLRC4 inflammasome activation in response to Salmonella typhimurium infection. Brd4-deficient bone marrow–derived macrophages (BMDMs) displayed impaired caspase-1 activation, ASC oligomerization, IL-1β maturation, gasdermin-D cleavage, and pyroptosis in response to S. typhimurium infection. RNA sequencing and RT-PCR results revealed that the transcription of Naips was decreased in Brd4-deficient BMDMs. Brd4 formed a complex with IRF8/PU.1 and bound to the IRF8 and PU.1 binding motifs on the promoters of Naips to maintain the expression of Naips. Furthermore, myeloid lineage–specific Brd4 conditional knockout mice were more susceptible to S. typhimurium infection with increased mortality, bacterial loads, and tissue damage; impaired inflammasome-dependent cytokine production; and pyroptosis. Our studies identify a novel function of Brd4 in innate immunity by controlling inflammasome-mediated cytokine release and pyroptosis to effectively battle S. typhimurium infection.

Macrophage Migration Inhibitory Factor Drives Multiple Myeloma IL-6/8 Pro-Survival Signals in the Tumor Microenvironment

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2988-2988 ◽  
Author(s):  
Rachel E Piddock ◽  
Amina M Abdul-Aziz ◽  
Martin J Auger ◽  
Kristian M Bowles ◽  
Stuart A Rushworth
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Stromal Cells ◽  
Chemotherapeutic Agents ◽  
Inhibitory Factor ◽  
Rna Expression ◽  
Cytokine Release ◽  
Rt Pcr ◽  
Myeloma Cells ◽  
Cytokine Array

Abstract Background Multiple myeloma (MM) accounts for 10-15% of all newly diagnosed hematological malignancies in the western world and despite recent significant progress in the treatment of MM, it presently remains an incurable disease. Average survival following diagnosis is currently approximately 5 years. Current treatments are effective at reducing malignant cell number (and therefore alleviating symptoms), however a small sub-population of MM cells will survive within the bone marrow milieu and relapse is inevitable. Interactions between the MM and bone marrow mesenchymal stromal cells (BM-MSCs) have been shown to be highly beneficial to the MM cells, protecting them from chemotherapeutic agents and upregulating the expression of genes associated with increased survival and proliferation. We hypothesize that the myeloma cells initiate the re-programming of the BM-MSCs for this very purpose, providing themselves with a more favorable environment. The cytokine MIF (Macrophage Migratory Inhibitory Factor) has been shown to be present in abnormally high levels in many cancer types. Here we show that MIF is aberrantly produced by primary myeloma cells and induces transcriptional changes and cytokine release from the BM-MSC. We also show these signals contribute to MM cell survival and drug resistance. Methods Primary MM and BM-MSC were obtained from patient's bone marrow as previously described. Primary MM cells at 1x106 were co-cultured on confluent autologous BM-MSC. Conditioned media from MM alone, MM/BM-MSC and BM-MSC cultures were collected and analyzed using Proteome Profiler Human XL Cytokine Array and cytokine specific ELISAs. Real-time PCR was used to analyze the changes in BM-MSC transcriptional levels in response to specific cytokine stimulation or inhibition. Recombinant MIF and the MIF inhibitor ISO-1 were used to verify the MM-BMSC findings. Results Here we report that bone marrow, aspirated from MM patients, had a higher level of MIF expression when compared to samples obtained from healthy donors. We examined the cytokine profile in human primary MM monoculture compared to MM cultured with autologous BM-MSC or BM-MSC alone and found that MIF was highly expressed by primary MM and that IL-6 and IL-8 were also increased in MM/BM-MSC co-cultures. RT-PCR confirmed that the majority of MIF transcription within the bone marrow microenvironment occurs within the myeloma cells themselves, with relatively minimal levels found within the bone marrow stromal cells. Next BM-MSCs from MM patients (n=5) were stimulated with recombinant MIF and cytokine release was measured using the XL cytokine array. The results showed that that recombinant human MIF stimulated the release of IL-6 and IL-8 to the media from BM-MSC monocultures. These observed changes were confirmed with the use of cytokine specific ELISA assays. RT-PCR also showed that IL-6 and IL-8 RNA expression were induced in BM-MSCs in response to recombinant MIF. These results confirm that MM derived MIF is instrumental in the transcriptional regulation of IL-6 and IL-8 in BM-MSCs. Next we found that the MIF inhibitor ISO-1 inhibited MM and MIF induced BM-MSC IL-6 and IL-8 RNA expression and cytokine release. Finally we found that MIF inhibition in MM/BM-MSC co-cultures reduced MM survival as well as enhancing the therapeutic potential of proteasome inhibition. Summary Together, these data suggest that MM signals via MIF to initiate the re-programming of the BM-MSCs, which creates a pro-tumoral environment in which MM can thrive and be protected from anti-MM therapeutics. Disclosures No relevant conflicts of interest to declare.

Disseminated tumor cells in bone marrow of breast cancer patients–Comparison of immunocytochemistry and RT-PCR

2006 ◽  
Vol 66 (S 01) ◽  
Author(s):  
T Fehm ◽  
S Becker ◽  
MJ Banys ◽  
G Becker-Pergola ◽  
S Duerr-Stoerzer ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Bone Marrow ◽  
Cancer Patients ◽  
Tumor Cells ◽  
Disseminated Tumor Cells ◽  
Breast Cancer Patients ◽  
Rt Pcr

Abstract 4296: Allogeneic Bone Marrow Mesenchymal Cells Improved Heart Function but Were Rejected after Myogenic Differentiation

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Xi-Ping Huang ◽  
Zhuo Sun ◽  
Yasuo Miyagi ◽  
Shafie Fazel ◽  
Richard D Weisel ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cardiac Function ◽  
Cell Survival ◽  
Myogenic Differentiation ◽  
Heart Function ◽  
Allogeneic Bone Marrow ◽  
Male Rats ◽  
Functional Improvement ◽  
Allogeneic Bone ◽  
Rt Pcr

Rationale: Allogeneic bone marrow mesenchymal stem cells (MSC) are currently undergoing clinical trials to test their potential to repair the heart after a myocardial infarction (MI). MSCs can improve function, but neither the host immune responses nor the fate of these cells has been extensively investigated. This study compared the outcomes of allogeneic and syngeneic MSC transplantation after an MI. Methods: Female Lewis rat underwent coronary ligation. Three weeks later, allogeneic or syngeneic MSCs (3×10 6 /rat) from male rats (Wister or Lewis, respectively) were implanted into the infracted region. We evaluated implanted cell survival (real-time PCR to quantify Y-chromosomes), cytokines (RT-PCR) and infiltrating cells (immunostaining) in the heart, and allogeneic antibodies in the blood. Cardiac function was assessed by echocardiography and pressure-volume catheters. Immune antigen expression (RT-PCR, immunostaining and flow cytometry) was characterized in the MSCs before and after myogenic differentiation. Results: At 1 week post implantation, cell survival was similar in the allogeneic and syngeneic groups. Gene expression of 11 cytokines and T-cell infiltration into the cell-implanted region were also similar, but more B-cells were observed in the allogeneic group (p<0.05 vs. syngeneic group). Allo-antibodies (IgG1) against Wistar MSCs were detected in the peripheral blood of animals that received allogeneic cells. At 5 weeks after delivery, implanted MSCs were detected only in the syngeneic group. Cardiac function was equally restored in both cell groups (ejection fraction: allogeneic=30.4±1.8%; syngeneic=29.5±2.8%; p<0.05 vs. media controls; control=24.8±3.2%); these data were supported by the pressure-volume analysis. Myogenic differentiation (expression of Nkx2.5, MyoD, MHCβ), CD86 expression and MHC class II molecules were observed in the MSCs after prolonged culture, which could explain why the cells were rejected. Conclusions: Allogeneic BMSC transplantation produced a robust cardiac functional improvement, but following myogenic differentiation the cells were no longer immunoprivileged. Rejection of the allogeneic cells could limit their long term benefit on cardiac remodeling after an MI.

scholarly journals Phagocytes from Mice Lacking the Sts Phosphatases Have an Enhanced Antifungal Response to Candida albicans

mBio ◽  
2018 ◽  
Vol 9 (4) ◽  
Author(s):  
David Frank ◽  
Shamoon Naseem ◽  
Gian Luigi Russo ◽  
Cindy Li ◽  
Kaustubh Parashar ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Immune Response ◽  
Candida Albicans ◽  
Tyrosine Kinase ◽  
Critical Role ◽  
Inflammasome Activation ◽  
Wild Type ◽  
Content Type ◽  
Enhanced Production ◽  
Immunological Mechanisms

ABSTRACT Mice lacking expression of the homologous phosphatases Sts-1 and Sts-2 (Sts−/− mice) are resistant to disseminated candidiasis caused by the fungal pathogen Candida albicans. To better understand the immunological mechanisms underlying the enhanced resistance of Sts−/− mice, we examined the kinetics of fungal clearance at early time points. In contrast to the rapid C. albicans growth seen in normal kidneys during the first 24 h postinfection, we observed a reduction in kidney fungal CFU within Sts−/− mice beginning at 12 to 18 h postinfection. This corresponds to the time period when large numbers of innate leukocytes enter the renal environment to counter the infection. Because phagocytes of the innate immune system are important for host protection against pathogenic fungi, we evaluated responses of bone marrow leukocytes. Relative to wild-type cells, Sts−/− marrow monocytes and bone marrow-derived dendritic cells (BMDCs) displayed a heightened ability to inhibit C. albicans growth ex vivo. This correlated with significantly enhanced production of reactive oxygen species (ROS) by Sts−/− BMDCs downstream of Dectin-1, a C-type lectin receptor that plays a critical role in stimulating host responses to fungi. We observed no visible differences in the responses of other antifungal effector pathways, including cytokine production and inflammasome activation, despite enhanced activation of the Syk tyrosine kinase downstream of Dectin-1 in Sts−/− cells. Our results highlight a novel mechanism regulating the immune response to fungal infections. Further understanding of this regulatory pathway could aid the development of therapeutic approaches to enhance protection against invasive candidiasis. IMPORTANCE Systemic candidiasis caused by fungal Candida species is becoming an increasingly serious medical problem for which current treatment is inadequate. Recently, the Sts phosphatases were established as key regulators of the host antifungal immune response. In particular, genetic inactivation of Sts significantly enhanced survival of mice infected intravenously with Candida albicans. The Sts−/− in vivo resistance phenotype is associated with reduced fungal burden and an absence of inflammatory lesions. To understand the underlying mechanisms, we studied phagocyte responses. Here, we demonstrate that Sts−/− phagocytes have heightened responsiveness to C. albicans challenge relative to wild-type cells. Our data indicate the Sts proteins negatively regulate phagocyte activation via regulating selective elements of the Dectin-1–Syk tyrosine kinase signaling axis. These results suggest that phagocytes lacking Sts respond to fungal challenge more effectively and that this enhanced responsiveness partially underlies the profound resistance of Sts−/− mice to systemic fungal challenge.

Temporary increased cytokine mRNA levels in bone marrow after ovariectomy; using competitive RT-PCR

Bone ◽  
1995 ◽  
Vol 17 (6) ◽  
pp. 587
Author(s):  
R.L. van Bezooiien ◽  
S.E. Papapoulos ◽  
C.W.G.M. Löwik
Keyword(s):  
Bone Marrow ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Cytokine Mrna

Molecular cloning and expression of visinin-like protein 1 from Gekko japonicus spinal cord

Biologia ◽  
2010 ◽  
Vol 65 (1) ◽  
Author(s):  
Lijie Ren ◽  
Xiaosong Gu ◽  
Yan Liu ◽  
Fei Ding ◽  
Xingxing Gu ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Rna Binding ◽  
Cloning And Expression ◽  
Rt Pcr ◽  
Binding Motifs ◽  
Reading Frame ◽  
Dsrna Binding ◽  
Gekko Japonicus ◽  
Ef Hand ◽  
The Central Nervous System

AbstractVisinin-like protein 1 (VILIP-1), a myristoylated calcium sensor protein of the EF-hand Ca2+-binding protein superfamily, plays multiple physiological roles in the central nervous system and peripheral organs. In present study, the cDNA encoding VILIP-1 was identified from the brain and spinal cord cDNA library of Gekko japonicus. It contains a 573 bp open reading frame corresponding to a deduced protein of 191 amino acids. Gecko VILIP-1 shares more than 95.3% identity with vertebrate VILIP-1 proteins, and structurally consists of conserved four EF-hand Ca2+-binding motifs and one dsRNA-binding domain, suggesting that selective pressure must have been extremely high for the conservation of VILIP-1 during vertebrate evolution. Northern blot and RT-PCR showed that gecko VILIP-1 was ubiquitously expressed in all tissues examined. In situ hybridization revealed that the VILIP-1 transcript mainly appeared in the gray matter of the spinal cord, with less distribution in the white matter. Semiquantitative RT-PCR also showed that VILIP-1 expression in spinal cord after tail amputation remained stable at 1 day and 1 week, but decreased at 2 weeks, a time coinciding with regeneration bud formation. This suggests that VILIP-1 may function as a regeneration-associated factor in the form of a monomer or/and RNA-binding complex.

Lidocaine depresses splenocyte immune functions following trauma-hemorrhage in mice

2006 ◽  
Vol 291 (5) ◽  
pp. C1049-C1055 ◽  
Author(s):  
Takashi Kawasaki ◽  
Mashkoor A. Choudhry ◽  
Martin G. Schwacha ◽  
Kirby I. Bland ◽  
Irshad H. Chaudry
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
Immune Responses ◽  
Cytokine Production ◽  
Bone Marrow Cells ◽  
Marrow Cell ◽  
Sham Operation ◽  
Mapk Activation ◽  
Midline Laparotomy ◽  
Marrow Cells

Traumatic and/or surgical injury as well as hemorrhage induces profound suppression of cellular immunity. Although local anesthetics have been shown to impair immune responses, it remains unclear whether lidocaine affects lymphocyte functions following trauma-hemorrhage (T-H). We hypothesized that lidocaine will potentiate the suppression of lymphocyte functions after T-H. To test this, we randomly assigned male C3H/HeN (6–8 wk) mice to sham operation or T-H. T-H was induced by midline laparotomy and ∼90 min of hemorrhagic shock (blood pressure 35 mmHg), followed by fluid resuscitation (4× shed blood volume in the form of Ringer lactate). Two hours later, the mice were killed and splenocytes and bone marrow cells were isolated. The effects of lidocaine on concanavalin A-stimulated splenocyte proliferation and cytokine production in both sham-operated and T-H mice were assessed. The effects of lidocaine on LPS-stimulated bone marrow cell proliferation and cytokine production were also assessed. The results indicate that T-H suppresses cell proliferation, Th1 cytokine production, and MAPK activation in splenocytes. In contrast, cell proliferation, cytokine production, and MAPK activation in bone marrow cells were significantly higher 2 h after T-H compared with shams. Lidocaine depressed immune responses in splenocytes; however, it had no effect in bone marrow cells in either sham or T-H mice. The enhanced immunosuppressive effects of lidocaine could contribute to the host's enhanced susceptibility to infection following T-H.

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