Immunoproteasome Activity and Content Determine Hematopoietic Cell Sensitivity to ONX-0914 and to the Infection of Cells with Lentiviruses

Proteasomes are intracellular structures responsible for protein degradation. The 20S proteasome is a core catalytic element of the proteasome assembly. Variations of catalytic subunits generate different forms of 20S proteasomes including immunoproteasomes (iPs), which are present mostly in the immune cells. Certain cells of the immune system are primary targets of retroviruses. It has been shown that several viral proteins directly affect proteasome functionality, while inhibition of proteasome activity with broad specificity proteasome inhibitors stimulates viral transduction. Here we specifically addressed the role of the immunoproteasomes during early stages of viral transduction and investigated the effects of specific immunoproteasome inhibition and activation prior to infection using a panel of cell lines. Inhibition of iPs in hematopoietic cells with immunoproteasome-specific inhibitor ONX-0914 resulted in increased infection by VSV-G pseudotyped lentiviruses. Moreover, a tendency for increased infection of cloned cells with endogenously decreased proteasome activity was revealed. Conversely, activation of iPs by IFN-γ markedly reduced the viral infectivity, which was rescued upon simultaneous immunoproteasome inhibition. Our results indicate that immunoproteasome activity might be determinative for the cellular antiretroviral resistance at least for the cells with high iP content. Finally, therapeutic application of immunoproteasome inhibitors might promote retroviral infection of cells in vivo.

Download Full-text

A Yes-Associated Protein (YAP) and Insulin-Like Growth Factor 1 Receptor (IGF-1R) Signaling Loop Is Involved in Sorafenib Resistance in Hepatocellular Carcinoma

Cancers ◽

10.3390/cancers13153812 ◽

2021 ◽

Vol 13 (15) ◽

pp. 3812

Author(s):

Mai-Huong T. Ngo ◽

Sue-Wei Peng ◽

Yung-Che Kuo ◽

Chun-Yen Lin ◽

Ming-Heng Wu ◽

...

Keyword(s):

Nuclear Translocation ◽

Combination Index ◽

Specific Inhibitor ◽

In Vivo Model ◽

Mrna Levels ◽

Margin Distribution ◽

Related Proteins ◽

Emt Markers

The role of a YAP-IGF-1R signaling loop in HCC resistance to sorafenib remains unknown. Method: Sorafenib-resistant cells were generated by treating naïve cells (HepG2215 and Hep3B) with sorafenib. Different cancer cell lines from databases were analyzed through the ONCOMINE web server. BIOSTORM–LIHC patient tissues (46 nonresponders and 21 responders to sorafenib) were used to compare YAP mRNA levels. The HepG2215_R-derived xenograft in SCID mice was used as an in vivo model. HCC tissues from a patient with sorafenib failure were used to examine differences in YAP and IGF-R signaling. Results: Positive associations exist among the levels of YAP, IGF-1R, and EMT markers in HCC tissues and the levels of these proteins increased with sorafenib failure, with a trend of tumor-margin distribution in vivo. Blocking YAP downregulated IGF-1R signaling-related proteins, while IGF-1/2 treatment enhanced the nuclear translocation of YAP in HCC cells through PI3K-mTOR regulation. The combination of YAP-specific inhibitor verteporfin (VP) and sorafenib effectively decreased cell viability in a synergistic manner, evidenced by the combination index (CI). Conclusion: A YAP-IGF-1R signaling loop may play a role in HCC sorafenib resistance and could provide novel potential targets for combination therapy with sorafenib to overcome drug resistance in HCC.

Download Full-text

Resistance to Acute Babesiosis Is Associated with Interleukin-12- and Gamma Interferon-Mediated Responses and Requires Macrophages and Natural Killer Cells

Infection and Immunity ◽

10.1128/iai.71.4.2002-2008.2003 ◽

2003 ◽

Vol 71 (4) ◽

pp. 2002-2008 ◽

Cited By ~ 43

Author(s):

Irma Aguilar-Delfin ◽

Peter J. Wettstein ◽

David H. Persing

Keyword(s):

Nk Cells ◽

Gamma Interferon ◽

Interleukin 12 ◽

No Production ◽

Cytokine Responses ◽

Early Production ◽

Ifn Γ ◽

Crucial Part

ABSTRACT We examined the role of the cytokines gamma interferon (IFN-γ) and interleukin-12 (IL-12) in the model of acute babesiosis with the WA1 Babesia. Mice genetically deficient in IFN-γ-mediated responses (IFNGR2KO mice) and IL-12-mediated responses (Stat4KO mice) were infected with the WA1 Babesia, and observations were made on the course of infection and cytokine responses. Levels of IFN-γ and IL-12 in serum increased 24 h after parasite inoculation. The augmented susceptibility observed in IFNGR2KO and Stat-4KO mice suggests that the early IL-12- and IFN-γ-mediated responses are involved in protection against acute babesiosis. Resistance appears to correlate with an increase in nitric oxide (NO) production. In order to assess the contribution of different cell subsets to resistance against the parasite, we also studied mice lacking B cells, CD4+ T cells, NK cells, and macrophages. Mice genetically deficient in B lymphocytes or CD4+ T lymphocytes were able to mount protective responses comparable to those of immunosufficient mice. In contrast, in vivo depletion of macrophages or NK cells resulted in elevated susceptibility to the infection. Our observations suggest that a crucial part of the response that protects from the pathogenic Babesia WA1 is mediated by macrophages and NK cells, probably through early production of IL-12 and IFN-γ, and induction of macrophage-derived effector molecules like NO.

Download Full-text

Exploiting GRK2 Inhibition as a Therapeutic Option in Experimental Cancer Treatment: Role of p53-Induced Mitochondrial Apoptosis

Cancers ◽

10.3390/cancers12123530 ◽

2020 ◽

Vol 12 (12) ◽

pp. 3530

Author(s):

Jessica Gambardella ◽

Antonella Fiordelisi ◽

Gaetano Santulli ◽

Michele Ciccarelli ◽

Federica Andrea Cerasuolo ◽

...

Keyword(s):

Cell Proliferation ◽

Cancer Cell ◽

Nude Mice ◽

Specific Inhibitor ◽

Cancer Cell Proliferation ◽

In Vivo Models ◽

P53 Expression

The involvement of GRK2 in cancer cell proliferation and its counter-regulation of p53 have been suggested in breast cancer even if the underlying mechanism has not yet been elucidated. Furthermore, the possibility to pharmacologically inhibit GRK2 to delay cancer cell proliferation has never been explored. We investigated this possibility by setting up a study that combined in vitro and in vivo models to underpin the crosstalk between GRK2 and p53. To reach this aim, we took advantage of the different expression of p53 in cell lines of thyroid cancer (BHT 101 expressing p53 and FRO cells, which are p53-null) in which we overexpressed or silenced GRK2. The pharmacological inhibition of GRK2 was achieved using the specific inhibitor KRX-C7. The in vivo study was performed in Balb/c nude mice, where we treated BHT-101 or FRO-derived tumors with KRX-C7. In our in vitro model, FRO cells were unaffected by GRK2 expression levels, whereas BHT-101 cells were sensitive, thus suggesting a role for p53. The regulation of p53 by GRK2 is due to phosphorylative events in Thr-55, which induce the degradation of p53. In BHT-101 cells, the pharmacologic inhibition of GRK2 by KRX-C7 increased p53 levels and activated apoptosis through the mitochondrial release of cytochrome c. These KRX-C7-mediated events were also confirmed in cancer allograft models in nude mice. In conclusion, our data showed that GRK2 counter-regulates p53 expression in cancer cells through a kinase-dependent activity. Our results further corroborate the anti-proliferative role of GRK2 inhibitors in p53-sensitive tumors and propose GRK2 as a therapeutic target in selected cancers.

Download Full-text

In vivo role of IFN-γ produced by antigen-presenting cells in early host defense against intracellular pathogens

European Journal of Immunology ◽

10.1002/eji.200323292 ◽

2003 ◽

Vol 33 (10) ◽

pp. 2666-2675 ◽

Cited By ~ 36

Author(s):

Kazutomo Suzue ◽

Takashi Asai ◽

Tsutomu Takeuchi ◽

Shigeo Koyasu

Keyword(s):

Host Defense ◽

Antigen Presenting Cells ◽

Intracellular Pathogens ◽

Ifn Γ ◽

Antigen Presenting

Download Full-text

Investigation of the Role of CD8+ T Cells in Bovine Tuberculosis In Vivo

Infection and Immunity ◽

10.1128/iai.71.8.4297-4303.2003 ◽

2003 ◽

Vol 71 (8) ◽

pp. 4297-4303 ◽

Cited By ~ 43

Author(s):

B. Villarreal-Ramos ◽

M. McAulay ◽

V. Chance ◽

M. Martin ◽

J. Morgan ◽

...

Keyword(s):

Lymph Node ◽

Bovine Tuberculosis ◽

The Other ◽

Cd8 Cells ◽

Cd8 Cell ◽

Ifn Γ ◽

Mycobacterial Antigens

ABSTRACT Mycobacterium bovis is the causative agent of bovine tuberculosis (TB), and it has the potential to induce disease in humans. CD8+ T cells (CD8 cells) have been shown to respond to mycobacterial antigens in humans, cattle, and mice. In mice, CD8 cells have been shown to play a role in protection against mycobacterial infection. To determine the role of CD8 cells in bovine TB in vivo, two groups of calves were infected with the virulent M. bovis strain AF2122/97. After infection, one group was injected with a CD8 cell-depleting monoclonal antibody (MAb), and the other group was injected with an isotype control MAb. Immune responses to mycobacterial antigens were measured weekly in vitro. After 8 weeks, the animals were killed, and postmortem examinations were carried out. In vitro proliferation responses were similar in both calf groups, but in vitro gamma interferon (IFN-γ) production in 24-h whole-blood cultures was significantly higher in control cattle than in CD8 cell-depleted calves. Postmortem examination showed that calves in both groups had developed comparable TB lesions in the lower respiratory tract and associated lymph nodes. Head lymph node lesion scores, on the other hand, were higher in control calves than in CD8 cell-depleted calves. Furthermore, there was significant correlation between the level of IFN-γ and the head lymph node lesion score. These experiments indicate that CD8 cells play a role in the immune response to M. bovis in cattle by contributing to the IFN-γ response. However, CD8 cells may also play a deleterious role by contributing to the immunopathology of bovine TB.

Download Full-text

The role of endothelial PI3Kγ activity in neutrophil trafficking

Blood ◽

10.1182/blood-2005-01-0023 ◽

2005 ◽

Vol 106 (1) ◽

pp. 150-157 ◽

Cited By ~ 118

Author(s):

Kamal D. Puri ◽

Teresa A. Doggett ◽

Ching-Yu Huang ◽

Jason Douangpanya ◽

Joel S. Hayflick ◽

...

Keyword(s):

Critical Role ◽

Fold Increase ◽

Necrosis Factor Alpha ◽

Catalytic Subunits ◽

Injury Model ◽

Lung Injury Model ◽

Factor Alpha ◽

Phosphoinositide 3 Kinase

Phosphoinositide 3-kinase gamma (PI3Kγ) in neutrophils plays a critical role in the directed migration of these cells into inflamed tissues. In this study, we demonstrate the importance of the endothelial component of PI3Kγ activity relative to its leukocyte counterpart in supporting neutrophil interactions with the inflamed vessel wall. Despite the reconstitution of class-Ib PI3K function in neutrophils of p110γ–/– mice, we observed a 45% reduction in accumulation of these cells in an acute lung injury model. Mechanistically, this appears to result from a perturbation in selectin-mediated adhesion as manifested by a 70% reduction in wild-type (WT) neutrophil attachment to and 17-fold increase in rolling velocities on p110γ–/– microvessels in vivo in response to tumor necrosis factor alpha (TNFα). This alteration in adhesion was further augmented by a deficiency in p110δ, suggesting that the activity of both catalytic subunits is required for efficient capture of neutrophils by cytokine-stimulated endothelium. Interestingly, E-selectin–mediated adhesion in p110γ–/– mice was impaired by more than 95%, but no defect in nuclear factor kappa B (NF-κB)–induced gene expression was observed. These findings suggest a previously unrecognized partnership between class-I PI3Ks expressed in leukocytes and endothelium, the combination of which is required for the efficient trafficking of immunocompetent cells to sites of inflammation.

Download Full-text

Role of SLC12A10.2, a Na-Cl cotransporter-like protein, in a Cl uptake mechanism in zebrafish (Danio rerio)

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00119.2009 ◽

2009 ◽

Vol 296 (5) ◽

pp. R1650-R1660 ◽

Cited By ~ 81

Author(s):

Yi-Fang Wang ◽

Yung-Che Tseng ◽

Jia-Jiun Yan ◽

Junya Hiroi ◽

Pung-Pung Hwang

Keyword(s):

Specific Inhibitor ◽

In Vivo Model ◽

Zebrafish Embryos ◽

Uptake Mechanism ◽

Ion Regulation ◽

Specific Expression ◽

Net Uptake ◽

First Time

The thiazide-sensitive Na+-Cl− cotransporter (NCC), a member of the SLC12 family, is mainly expressed in the apical membrane of the mammalian distal convoluted tubule (DCT) cells, is responsible for cotransporting Na+ and Cl− from the lumen into DCT cells and plays a major role in the mammalian renal NaCl reabsorption. The NCC has also been reported in fish, but the functional role in fish ion regulation is yet unclear. The present study used zebrafish as an in vivo model to test the hypothesis of whether the NCC plays a role in Na+ and/or Cl− uptake mechanisms. Four NCCs were cloned, and only one of them, zebrafish (z) slc12a10.2 was found to predominately and specifically be expressed in gills. Double in situ hybridization/immunocytochemistry in zebrafish skin/gills demonstrated that the specific expression of zslc12a10.2 mRNA in a novel group of ionocytes differed from those of the previously-reported H+-ATPase-rich (HR) cells and Na+-K+-ATPase-rich (NaR) cells. Gill mRNA expression of zslc12a10.2 was induced by a low-Cl environment that stimulated fish Cl− influx, while a low-Na environment suppressed this expression. Incubation with metolazone, a specific inhibitor of the NCC, impaired both Na+ and Cl− influx in 5-day postfertilization (dpf) zebrafish embryos. Translational knockdown of zslc12a10.2 with a specific morpholino caused significant decreases in both Cl− influx and Cl− content of 5-dpf zebrafish embryos, suggesting that the operation of zNCC-like 2 results in a net uptake of Cl− in zebrafish. On the contrary, zslc12a10.2 morphants showed increased Na+ influx and content that resulted from upregulation of mRNA expressions of Na+-H+ exchanger 3b and carbonic anhydrase 15a in HR cells. These results for the first time provide in vivo molecular physiological evidence for the possible role of the NCC in the Cl− uptake mechanism in zebrafish skin/gills.

Download Full-text

In Vivo Regulation of Replicative Legionella pneumophila Lung Infection by Endogenous Interleukin-12

Infection and Immunity ◽

10.1128/iai.66.1.65-69.1998 ◽

1998 ◽

Vol 66 (1) ◽

pp. 65-69 ◽

Cited By ~ 52

Author(s):

J. K. Brieland ◽

D. G. Remick ◽

M. L. LeGendre ◽

N. C. Engleberg ◽

J. C. Fantone

Keyword(s):

Legionella Pneumophila ◽

Lung Infection ◽

Interleukin 12 ◽

Necrosis Factor Alpha ◽

Protein Levels ◽

Tnf Α ◽

Factor Alpha ◽

Ifn Γ

ABSTRACT The in vivo role of endogenous interleukin 12 (IL-12) in modulating intrapulmonary growth of Legionella pneumophila was assessed by using a murine model of replicative L. pneumophila lung infection. Intratracheal inoculation of A/J mice with virulent bacteria (106 L. pneumophilacells per mouse) resulted in induction of IL-12, which preceded clearance of the bacteria from the lung. Inhibition of endogenous IL-12 activity, via administration of IL-12 neutralizing antiserum, resulted in enhanced intrapulmonary growth of the bacteria within 5 days postinfection (compared to untreated L. pneumophila-infected mice). Because IL-12 has previously been shown to modulate the expression of cytokines, including gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and IL-10, which regulate L. pneumophila growth, immunomodulatory effects of endogenous IL-12 on intrapulmonary levels of these cytokines during replicative L. pneumophila lung infection were subsequently assessed. Results of these experiments demonstrated that TNF-α activity was significantly lower, while protein levels of IFN-γ and IL-10 in the lung were similar, in L. pneumophila-infected mice administered IL-12 antiserum, compared to similarly infected untreated mice. Together, these results demonstrate that IL-12 is critical for resolution of replicativeL. pneumophila lung infection and suggest that regulation of intrapulmonary growth of L. pneumophila by endogenous IL-12 is mediated, at least in part, by TNF-α.

Download Full-text

β-Chemokines Enhance Parasite Uptake and Promote Nitric Oxide-Dependent Microbiostatic Activity in Murine Inflammatory Macrophages Infected with Trypanosoma cruzi

Infection and Immunity ◽

10.1128/iai.67.9.4819-4826.1999 ◽

1999 ◽

Vol 67 (9) ◽

pp. 4819-4826 ◽

Cited By ~ 101

Author(s):

Júlio C. S. Aliberti ◽

Fabiana S. Machado ◽

Janeusa T. Souto ◽

Ana P. Campanelli ◽

Mauro M. Teixeira ◽

...

Keyword(s):

Trypanosoma Cruzi ◽

Neutralizing Antibodies ◽

Specific Inhibitor ◽

No Production ◽

Dependent Manner ◽

Macrophage Infection ◽

Parasite Replication ◽

Ifn Γ

ABSTRACT In the present study, we describe the ability of Trypanosoma cruzi trypomastigotes to stimulate the synthesis of β-chemokines by macrophages. In vivo infection with T. cruzi led to MIP-1α, RANTES, and JE/MCP1 mRNA expression by cells from peritoneal inflammatory exudate. In addition, in vitro infection with T. cruzi resulted in expression of β-chemokine MIP-1α, MIP-1β, RANTES, and JE mRNA by macrophages. The expression of the β-chemokine MIP-1α, MIP-1β, RANTES, and JE proteins by murine macrophages cultured with trypomastigote forms ofT. cruzi was confirmed by immunocytochemistry. Interestingly, macrophage infection with T. cruzi also resulted in NO production, which we found to be mediated mainly by β-chemokines. Hence, treatment with anti-β-chemokine-specific neutralizing antibodies partially inhibited NO release by macrophages incubated with T. cruzi parasites. Further, the addition of the exogenous β-chemokines MIP-1α, MIP-1β, RANTES, and JE/MCP-1 induced an increased T. cruzi uptake, leading to enhanced NO production and control of parasite replication in a dose-dependent manner. l-NMMA, a specific inhibitor of thel-arginine–NO pathway, caused a decrease in NO production and parasite killing when added to cultures of macrophages stimulated with β-chemokines. Among the β-chemokines tested, JE was more potent in inhibiting parasite growth, although it was much less efficient than gamma interferon (IFN-γ). Nevertheless, JE potentiates parasite killing by macrophages incubated with low doses of IFN-γ. Together, these results suggest that in addition to their chemotactic activity, murine β-chemokines may also contribute to enhancing parasite uptake and promoting control of parasite replication in macrophages and may play a role in resistance to T. cruziinfection.

Download Full-text

S100A8/A9 modulates inflammatory collateral tissue damage during intraperitoneal origin systemic candidiasis

10.1101/2020.03.30.017491 ◽

2020 ◽

Author(s):

Madhu Shankar ◽

Nathalie Uwamahoro ◽

Sandra Holmberg ◽

Maria Joanna Niemiec ◽

Johannes Roth ◽

...

Keyword(s):

Tissue Damage ◽

Critical Role ◽

Specific Inhibitor ◽

Surgical Intensive Care ◽

Inflammatory Protein ◽

Systemic Level ◽

Combating Infection ◽

Deficient Mice

AbstractPeritonitis is a leading cause of severe sepsis in surgical intensive care units, as over 70% of patients diagnosed with peritonitis develop septic shock. A critical role of the immune system is to return to homeostasis after combating infection. S100A8/A9 (calprotectin) is an antimicrobial, pro-inflammatory protein complex often used as a biomarker for diagnosis of disease activities in many inflammatory disorders. Here we describe the role of S100A8/A9 on inflammatory collateral tissue damage (ICTD).We performed an in vivo Candida albicans disseminated peritonitis mouse model using WT and S100A9-deficient mice and stimulated primary macrophages with recombinant S100A8/A9 in the presence or absence of the compound paquinimod, a specific inhibitor of S100A9. In addition, the effects on ICTD and fungal clearance were investigated. S100A9-deficient mice developed less ICTD than wildtype mice. Restoration of S100A8/A9 in S100A9 knockout mice resulted in increased ICTD and fungal clearance comparable to wildtype levels. Treatment with paquinimod abolished ICTD.The data indicated that S100A8/A9 controls ICTD levels and host antimicrobial modulation at a systemic level during intra-abdominal candidiasis (IAC).

Download Full-text