Immunotherapy for breast cancer using EpCAM aptamer tumor-targeted gene knockdown.

New strategies for cancer immunotherapy are needed since most solid tumors do not respond to current approaches. Here we used epithelial cell adhesion molecule EpCAM (a tumor-associated antigen highly expressed on common epithelial cancers and their tumor-initiating cells) aptamer-linked small-interfering RNA chimeras (AsiCs) to knock down genes selectively in EpCAM+ tumors with the goal of making cancers more visible to the immune system. Knockdown of genes that function in multiple steps of cancer immunity was evaluated in aggressive triple-negative and HER2+ orthotopic, metastatic, and genetically engineered mouse breast cancer models. Gene targets were chosen whose knockdown was predicted to promote tumor neoantigen expression (Upf2, Parp1, Apex1), phagocytosis, and antigen presentation (Cd47), reduce checkpoint inhibition (Cd274), or cause tumor cell death (Mcl1). Four of the six AsiC (Upf2, Parp1, Cd47, and Mcl1) potently inhibited tumor growth and boosted tumor-infiltrating immune cell functions. AsiC mixtures were more effective than individual AsiC and could synergize with anti-PD-1 checkpoint inhibition.

Regulatory role and mechanism of the inhibition of the Mcl-1 pathway during apoptosis and polarization of H37Rv-infected macrophages.

BACKGROUND: Myeloid cell leukemia-1 (Mcl-1) plays an important role in the clearance of Mycobacterium tuberculosis (MTB) infection. It has the effect of anti-apoptosis, protecting macrophages that have engulfed pathogens and preventing pathogen clearance. Meanwhile, the MAPK signaling pathway plays a significant role in regulating Mcl-1 expression during tuberculosis infection. In the case of latent infection and active infection, the apoptosis and polarization of macrophages have a great influence during MTB infection, so we discussed the effect of Mcl-1 on apoptosis and polarization. Then, further discussed its mechanism. METHODS: An infected RAW264.7 macrophage model was established to investigate the regulatory role and mechanism of the Mcl-1 pathway inhibition during apoptosis and polarization of H37Rv infection. First, Mcl-1 protein and mRNA was identified by western blotting and Real-Time Polymerase Chain Reaction (RT-PCR). RAW264.7 macrophage apoptosis was detected by flow cytometry. RT-PCR was utilized to detect Bax, Caspase-3, Cyt-c and Bcl-2 mRNA expression. Next, Then the expression levels of inflammation factors CD86, CD206, iNOS, Fizz1, IL-6, IL-10, TNF-α, and TGF-ß was detected by ELISA. SEM was used to observe macrophages phenotype. Finally, Bax, Bcl-2 and Bcl-xl the expression was detected by western blotting. Confocal microscopy was used to analyze mitochondrial membrane potential using the JC-10 kit. RESULTS: In this study, we found that inhibiting the Mcl-1 expression signaling pathway led to infection by different virulence Mycobacterium tuberculosis, as well as changes in Mcl-1 protein and mRNA expression. Concomitantly macrophage apoptosis rate also changed, While, two phenotypic states of M1 and M2 appeared in the infected cells. We also found that the mitochondrial pathway was activated, the expression of its related genes Bax, casepase3, and Cyt-c, increased, whereas that of Bcl-2 decreased, and the mitochondrial membrane depolarization function was changed. CONCLUSIONS: We found that Mcl-1 affected the apoptosis and polarization of macrophages infected by Mycobacterium tuberculosis, mainly M1 in the early stage and M2 in the later stage. In addition, mitochondria played a crucial role in this process.

Chymase-Cre; Mcl-1fl/fl Mice Exhibit Reduced Numbers of Mucosal Mast Cells.

Mast cells (MCs) are considered as key effector cells in the elicitation of allergic symptoms, and they are essential players in innate and adaptive immune responses. In mice, two main types of MCs have been described: connective tissue MCs (CTMCs) and mucosal MCs (MMCs). However, little is known about the biological functions of MMCs, which is due to the lack of suitable models to investigate MMCs in vivo. Here, we aimed to generate a mouse model selectively deficient in MMCs. It has been previously described that Cre expressed under the control of the baboon α-chymase promotor is predominantly localized in MMCs. Therefore, we mated α-chymase-Cre transgenic mice with mice bearing a floxed allele of the myeloid cell leukemia sequence 1 (Mcl-1). Mcl-1 encodes for an intracellular antiapoptotic factor in MCs; hence, a selective reduction in MMCs was expected. Our results show that this new mouse model contains markedly reduced numbers of MMCs in mucosal tissues, whereas numbers of CTMCs are normal. Thus, Chm-Cre; Mcl-1fl/fl mice are a useful tool for the investigation of the pathophysiological functions of MMCs in vivo.

Low-molecular-mass hyaluronan induces pulmonary inflammation by up-regulation of Mcl-1 to inhibit neutrophil apoptosis via PI3K/Akt1 pathway.

Although low-molecular-mass hyaluronan (LMMHA) has been implicated in pulmonary inflammatory diseases, the signalling pathway of LMMHA (200 000 molecular weight) that initiates the inflammatory response in lung is still unknown. In this study, we evaluate the role of phosphoinositide 3-kinase (PI3K) and its downstream signalling pathway in LMMHA-induced lung inflammatory responses. Our results indicate that pharmacological inhibition of PI3K or genetic deletion of Akt1 enhances neutrophil apoptosis, attenuates neutrophil influx into the lungs of mice and diminishes the expression of pro-inflammatory factors such as interleukin-6, keratinocyte cell-derived chemokine and pro-matrix metalloproteinase-9 in bronchoalveolar lavage fluid after intratracheal administration of LMMHA. More importantly, we found that PI3K/Akt1 participates in LMMHA-induced inflammatory responses, which are mainly mediated by the myeloid leukaemia cell differentiation protein (Mcl-1). Our study suggests that LMMHA induced significantly increased levels of inflammatory factors in bronchoalveolar lavage fluid and activation of the PI3K/Akt1 pathway, which up-regulates the expression of the anti-apoptotic protein Mcl-1 and inhibits the activation of caspase-3, thereby suppressing neutrophil apoptosis to trigger lung inflammation. These findings reveal a novel molecular mechanism underlying sterile inflammation and provides a new potential target for the treatment of pulmonary disease.

Recombinant MDA-7/IL24 Suppresses Prostate Cancer Bone Metastasis through Downregulation of the Akt/Mcl-1 Pathway.

Prostate cancer is a principal cause of cancer-associated morbidity in men. Although 5-year survival of patients with localized prostate cancer approaches 100%, survival decreases precipitously after metastasis. Bone is the preferred site for disseminated prostate cancer cell colonization, altering the equilibrium of bone homeostasis resulting in weak and fragile bones. Currently, no curative options are available for prostate cancer bone metastasis. Melanoma differentiation associated gene-7 (MDA-7)/IL24 is a well-studied cytokine established as a therapeutic in a wide array of cancers upon delivery as a gene therapy. In this study, we explored the potential anticancer properties of MDA-7/IL24 delivered as a recombinant protein. Using bone metastasis experimental models, animals treated with recombinant MDA-7/IL24 had significantly less metastatic lesions in their femurs as compared with controls. The inhibitory effects of MDA-7/IL24 on bone metastasis resulted from prostate cancer-selective killing and inhibition of osteoclast differentiation, which is necessary for bone resorption. Gain- and loss-of-function genetic approaches document that prosurvival Akt and Mcl-1 pathways are critically important in the antibone metastatic activity of MDA-7/IL24. Our previous findings showed that MDA-7/IL24 gene therapy plus Mcl-1 inhibitors cooperate synergistically. Similarly, an Mcl-1 small-molecule inhibitor synergized with MDA-7/IL24 and induced robust antibone metastatic activity. These results expand the potential applications of MDA-7/IL24 as an anticancer molecule and demonstrate that purified recombinant protein is nontoxic in preclinical animal models and has profound inhibitory effects on bone metastasis, which can be enhanced further when combined with an Mcl-1 inhibitory small molecule. Mol Cancer Ther; 17(9); 1951-60. ©2018 AACR.

SYK Inhibition Induces Apoptosis in Germinal Center-Like B Cells by Modulating the Antiapoptotic Protein Myeloid Cell Leukemia-1, Affecting B-Cell Activation and Antibody Production.

B cells play a major role in the antibody-mediated rejection (AMR) of solid organ transplants, a major public health concern. The germinal center (GC) is involved in the generation of donor-specific antibody-producing plasma cells and memory B cells, which are often poorly controlled by current treatments. Myeloid cell leukemia-1 (Mcl-1), an antiapoptotic member of the B-cell lymphoma-2 family, is essential for maintenance of the GC reaction and B-cell differentiation. During chronic AMR (cAMR), tertiary lymphoid structures resembling GCs appear in the rejected organ, suggesting local lymphoid neogenesis. We report the infiltration of the kidneys with B cells expressing Mcl-1 in patients with cAMR. We modulated GC viability by impairing B-cell receptor signaling, by spleen tyrosine kinase (SYK) inhibition. SYK inhibition lowers viability and Mcl-1 protein levels in Burkitt's lymphoma cell lines. This downregulation of Mcl-1 is coordinated at the transcriptional level, possibly by signal transducer and activator of transcription 3 (STAT3), as shown by (1) the impaired translocation of STAT3 to the nucleus following SYK inhibition, and (2) the lower levels of Mcl-1 transcription upon STAT3 inhibition. Mcl-1 overproduction prevented cells from entering apoptosis following SYK inhibition. In vitro studies with primary tonsillar B cells confirmed that SYK inhibition impaired cell survival and decreased Mcl-1 protein levels. It also impaired B-cell activation and immunoglobulin G secretion by tonsillar B cells. These findings suggest that the SYK-Mcl-1 pathway could be targeted, to improve graft survival by manipulating the humoral immune response.

Apoptosis Modulation in the Immune System Reveals a Role of Neutrophils in Tissue Damage in a Murine Model of Chlamydial Genital Infection.

Background: Chlamydial infection frequently causes damage to the female genital tract. The precise mechanisms of chlamydial clearance and tissue damage are unknown, but studies suggest immunopathology with a particular role of neutrophils. The goal of this study was to understand the contribution of the immune system, in particular neutrophils. Methods: Using Chlamydia muridarum, we infected mice with a prolonged immune response due to expression of B-cell lymphoma 2 (Bcl-2) in hematopoietic cells (Bcl-2 mice), and mice where mature neutrophils are lacking due to the deletion of Myeloid cell leukemia 1 (Mcl-1) in myeloid cells (LysM-cre-mcl-1-flox mice; Mcl-1 mice). We monitored bacterial clearance, cellular infiltrate, and long-term tissue damage. Results: Both mutant strains showed slightly delayed clearance of the acute infection. Bcl-2 mice had a strongly increased inflammatory infiltrate concerning almost all cell lineages. The infection of Bcl-2 mice caused increased tissue damage. The loss of neutrophils in Mcl-1 mice was associated with substantial quantitative and qualitative alterations of the inflammatory infiltrate. Mcl-1 mice had higher chlamydial burden and reduced tissue damage, including lower incidence of hydrosalpinx and less uterine dilation. Conclusions: Inhibition of apoptosis in the hematopoietic system increases inflammation and tissue damage. Neutrophils have broad functions, including a role in chlamydial clearance and in tissue destruction.

Myeloid cell leukemia-1 protein expression and myeloid cell leukemia-1 gene amplification in non small cell lung cancer.

BACKGROUND: Myeloid cell leukemia-1 (Mcl-1) is a member of the B-cell lymphoma 2 family known to play a significant role in the regulation of apoptosis. Mcl-1 expression has been studied in nonsmall cell lung cancer (NSCLC) cell lines but has not been previously evaluated as a prognostic factor in clinical samples. MATERIALS AND METHODS: Formalin-fixed, paraffin-embedded sections from 119 NSCLC, including 33 squamous cell carcinomas (SCC), 55 adenocarcinomas (AC), and 31 either pure adenocarcinoma in situ (AIS) or AC with lepidic features were immunostained by an automated method with rabbit polyclonal Mcl-1. Cytoplasmic Mcl-1 (cMcl-1) immunoreactivity was scored based on intensity and percentage of positive tumor cells in both tumor and adjacent benign epithelium in each case. MCL1 amplification was determined by hybrid capture-based comprehensive genomic profiling (CGP) on a separate cohort of 9393 NSCLC samples. RESULTS: Intense diffuse cMcl-1 overexpression was noted in 35/119 (29%) tumors overall and correlated with tumor type (52% AIS vs. 31% AC vs. 6% SCC, P < 0.0001), tumor grade (48% grade 1 vs. 14% grade 2 vs. 31% grade 3, P = 0.007), small tumor size (36% ≤3.0 cm vs. 16% >3.0 cm, P = 0.016), and lengthened survival within the AIS subgroup (100% alive vs. 42% expired, P = 0.018) while showing a trend toward correlation with nonrecurrent disease overall (32% nonrecurrent vs. 11% recurrent, P = 0.072) and within the AC subgroup (33% nonrecurrent vs. 0% recurrent, P = 0.092). MCL1 amplification was identified in 569 (6%) of 9393 NSCLC by CGP. CONCLUSIONS: cMcl-1 overexpression appears to occur independently from MCL1 gene amplification in NSCLC and correlates with AIS histologic type, lower tumor grade, smaller tumor size, nonrecurrent disease, and increased survival.

Anti-apoptotic proteins BCL-2, MCL-1 and A1 summate collectively to maintain survival of immune cell populations both in vitro and in vivo.

Survival of various immune cell populations has been proposed to preferentially rely on a particular anti-apoptotic BCL-2 family member, for example, naive T cells require BCL-2, while regulatory T cells require MCL-1. Here we examined the survival requirements of multiple immune cell subsets in vitro and in vivo, using both genetic and pharmacological approaches. Our findings support a model in which survival is determined by quantitative participation of multiple anti-apoptotic proteins rather than by a single anti-apoptotic protein. This model provides both an insight into how the sum of relative levels of anti-apoptotic proteins BCL-2, MCL-1 and A1 influence survival of T cells, B cells and dendritic cells, and a framework for ascertaining how these different immune cells can be optimally targeted in treatment of immunopathology, transplantation rejection or hematological cancers.

Mcl-1 regulates effector and memory CD8 T-cell differentiation during acute viral infection.

Mcl-1, an anti-apoptotic member of Bcl-2 family maintains cell viability during clonal expansion of CD8 T cells, but the cell intrinsic role of Mcl-1 in contraction of effectors or the number of memory CD8 T cells is unknown. Mcl-1 levels decline during the contraction phase but rebound to high levels in memory CD8 T cells. Therefore, by overexpressing Mcl-1 in CD8 T cells we asked whether limiting levels of Mcl-1 promote contraction of effectors and constrain CD8 T-cell memory. Mcl-1 overexpression failed to affect CD8 T-cell expansion, contraction or the magnitude of CD8 T-cell memory. Strikingly, high Mcl-1 levels enhanced mTOR phosphorylation and augmented the differentiation of terminal effector cells and effector memory CD8 T cells to the detriment of poly-cytokine-producing central memory CD8 T cells. Taken together, these findings provided unexpected insights into the role of Mcl-1 in the differentiation of effector and memory CD8 T cells.

Mouse cytotoxic T cell-derived granzyme B activates the mitochondrial cell death pathway in a Bim-dependent fashion.

Cytotoxic T cells (Tc) use perforin and granzyme B (gzmB) to kill virus-infected cells and cancer cells. Recent evidence suggests that human gzmB primarily induces apoptosis via the intrinsic mitochondrial pathway by either cleaving Bid or activating Bim leading to the activation of Bak/Bax and subsequent generation of active caspase-3. In contrast, mouse gzmB is thought to predominantly induce apoptosis by directly processing pro-caspase-3. However, in certain mouse cell types gzmB-mediated apoptosis mainly occurs via the mitochondrial pathway. To investigate whether Bim is involved under the latter conditions, we have now employed ex vivo virus-immune mouse Tc that selectively kill by using perforin and gzmB (gzmB(+)Tc) as effector cells and wild type as well as Bim- or Bak/Bax-deficient spontaneously (3T9) or virus-(SV40) transformed mouse embryonic fibroblast cells as targets. We show that gzmB(+)Tc-mediated apoptosis (phosphatidylserine translocation, mitochondrial depolarization, cytochrome c release, and caspase-3 activation) was severely reduced in 3T9 cells lacking either Bim or both Bak and Bax. This outcome was related to the ability of Tc cells to induce the degradation of Mcl-1 and Bcl-XL, the anti-apoptotic counterparts of Bim. In contrast, gzmB(+)Tc-mediated apoptosis was not affected in SV40-transformed mouse embryonic fibroblast cells lacking Bak/Bax. The data provide evidence that Bim participates in mouse gzmB(+)Tc-mediated apoptosis of certain targets by activating the mitochondrial pathway and suggest that the mode of cell death depends on the target cell. Our results suggest that the various molecular events leading to transformation and/or immortalization of cells have an impact on their relative resistance to the multiple gzmB(+)Tc-induced death pathways.

Unmethylated CpG motifs in the L. donovani DNA regulate TLR9-dependent delay of programmed cell death in macrophages.

Regulation of macrophage PCD plays an important role in pathogenesis of leishmaniasis. However, the precise involvement of any parasite molecule in this process remains uncertain. In the current study, in silico wide analysis demonstrated that genes in the Leishmania donovani genome are highly enriched for CpG motifs, with sequence frequency of 8.7%. Here, we show that unmethylated species-specific CpG motifs in LdDNA significantly (P = 0.01) delay macrophage PCD by endosomal interaction with TLR9 via the adaptor protein MyD88. Importantly, LdDNA triggered high levels of luciferase activity (P = 0.001) under NF-κB-dependent transcription in HEK-TLR9 cells. Furthermore, the activation of caspases in macrophages was inhibited (P = 0.001) in the presence of LdDNA. Notably, the delay of PCD was mediated by modulation of the antiapoptotic proteins, Mcl-1 and Bfl-1, and impairment of loss of Δψm in macrophages through the neutralization of oxidative and nitrosative stress. The inhibition of caspase activation and up-regulation of Mcl-1 by LdDNA were TLR9 dependent. Analysis of the targets of LdDNA identified an early activation of the TLR9-dependent PI3K/Akt and SFK pathways, which were required for the observation of the antiapoptotic effects in macrophages. Moreover, we demonstrate that LdDNA modulates the TLR9-IκB-α pathway by promoting the tyrosine phosphorylation of TLR9 and the TLR9-mediated recruitment of Syk kinase. The results have identified a novel, TLR9-dependent antiapoptotic function of LdDNA, which will provide new opportunities for discovering and evaluating molecular targets for drug and vaccine designing against VL.

Neutrophils are required for both the sensitization and elicitation phase of contact hypersensitivity.

Allergic contact dermatitis and its animal model, contact hypersensitivity (CHS), are T cell-mediated inflammatory skin diseases induced by contact allergens. Though numerous cellular and molecular players are known, the mechanism of chemical-induced sensitization remains poorly understood. Here, we identify neutrophils as crucial players in the sensitization phase of CHS. Genetic deficiency of neutrophils caused by myeloid-specific deletion of Mcl-1 or antibody-mediated depletion of neutrophils before sensitization abrogated the CHS response. Neutrophil deficiency reduced contact allergen-induced cytokine production, gelatinase release, and reactive oxygen species production in naive mice. Mast cell deficiency inhibited neutrophil accumulation at the site of sensitization. In turn, neutrophils were required for contact allergen-induced release of further neutrophil-attracting chemokines, migration of DCs to the draining lymph nodes, and priming of allergen-specific T cells. Lymph node cells from mice sensitized in the absence of neutrophils failed to transfer sensitization to naive recipients. Furthermore, no CHS response could be induced when neutrophils were depleted before elicitation or when normally sensitized lymph node cells were transferred to neutrophil-deficient recipients, indicating an additional role for neutrophils in the elicitation phase. Collectively, our data identify neutrophils to be critically involved in both the sensitization and elicitation phase of CHS.

IL-10/Janus kinase/signal transducer and activator of transcription 3 signaling dysregulates Bim expression in autoimmune lymphoproliferative syndrome.

BACKGROUND: Autoimmune lymphoproliferative syndrome (ALPS) is a human disorder of T cell homeostasis caused by mutations that impair FAS-mediated apoptosis. A defining characteristic of ALPS is the expansion of double negative T cells (DNTC). Relatively little is known about how defective FAS-driven cell death and the Bcl-2 apoptotic pathway intersect in ALPS patients. OBJECTIVE: We studied changes in Bcl-2 family member expression in ALPS to determine whether the Bcl-2 pathway might provide a therapeutic target. METHODS: We used flow cytometry to analyze the expression of pro- and anti-apoptotic Bcl-2 family members in T cells from 12 ALPS patients and determined the in vitro sensitivity of ALPS DNTC to the pro-apoptotic BH3 mimetic, ABT-737. RESULTS: The pro-apoptotic molecule, Bim, was significantly elevated in DNTC. Although no general pattern of individual anti-apoptotic Bcl-2 family members emerged, increased expression of Bim was always accompanied by increased expression of at least 1 anti-apoptotic Bcl-2 family member. Strikingly, Bim levels in DNTC correlated significantly with serum IL-10 in ALPS patients, and IL-10 was sufficient to mildly induce Bim in normal and ALPS T cells via a Janus kinase/signal transducer and activator of transcription 3-dependent mechanism. Finally, ABT-737 preferentially killed ALPS DNTC in vitro. CONCLUSION: Combined, these data show that an IL-10/Janus kinase/signal transducer and activator of transcription 3 pathway drives Bim expression in ALPS DNTC, which renders them sensitive to BH3 mimetics, uncovering a potentially novel therapeutic approach to ALPS.

IL-32γ delays spontaneous apoptosis of human neutrophils through MCL-1, regulated primarily by the p38 MAPK pathway.

IL-32γ is a multifunctional cytokine involved in various inflammatory and auto-immune diseases in which neutrophils can affect the evolution of these diseases. To persist at inflammatory sites, neutrophils require inhibition of their rapid and constitutive apoptosis, an inhibitory effect that phlogogenic cytokines support. To date, the effects of IL-32γ on neutrophils remain unknown. We demonstrate that IL-32γ delays, in a dose-dependent manner, the spontaneous apoptosis of human blood neutrophils by activating mainly p38 MAPK through rapid p38 phosphorylation. PI3-K and ERK1/2 MAPK are also involved, but to a lesser extent. Most of cytokines that induce retardation of neutrophil apoptosis activate the expression of MCL-1 at both mRNA and protein levels. IL-32γ added to human blood neutrophils in vitro is associated with sustained levels of MCL-1 protein. This effect in neutrophils corresponds to a decrease of MCL-1 protein degradation without any effect on MCL-1 mRNA levels. The sustained levels of MCL-1 induced by IL-32γ are only abrogated by the p38ß MAPK inhibitor SB202190. Additionally, IL-32γ induces a reduction in caspase 3 activity in neutrophils. In conclusion, IL-32γ affects human blood neutrophils in vitro by increasing their survival, suggesting that this cytokine could have profound effects on the deleterious functions of neutrophils in several diseases.

The survival and differentiation of pro-B and pre-B cells in the bone marrow is dependent on IL-7Rα Tyr449.

IL-7 is critical for murine T and B cell development and survival and plays a significant role in lymphoblastic leukemia in both humans and mice. We evaluated the role of the IL-7Rα Tyr(449) cytoplasmic SH2-binding motif in IL-7-mediated B cell development using a knock-in mouse with a Tyr to Phe mutation (IL-7Rα(449F/449F) mouse). IL-7Rα(449F/449F) and IL-7Rα(-/-) mice showed no defect in the number of pre-pro-B cells, although IL-7Rα(449F/449F) mice had decreased Ebf1 in pre-pro-B cells and impairment in B cell-committed CLPs. We identified that IL-7Rα Tyr(449) was critical for both pro-B and pre-B stages of development in the bone marrow. IL-7Rα(449F/449F) and IL-7Rα(-/-) mice had comparable precursor B cell defects, indicating that signaling from the IL-7Rα required this motif. Although the defect in IL-7Rα(449F/449F) pro-B cells was associated with loss of STAT5 activation and diminished expression of Mcl1, this was not rescued by overexpression of Bcl-2. IL-7Rα(449F/449F) and IL-7Rα(-/-) pre-B cells also showed defective cyto-Igµ and CD25 expression, associated with reduced levels of Rag1, Rag2, and Irf4. Pre-B cells from IL-7Rα(449F/449F) mice also failed to proliferate, perhaps as a result of the failure to rearrange Igµ. Our data suggest that IL-7Rα Tyr(449) was essential for IL-7Rα signaling in bone marrow B cell development and survival.

Notch signaling regulates expression of Mcl-1 and apoptosis in PPD-treated macrophages.

Macrophages are cellular targets for infection by bacteria and viruses. The fate of infected macrophages plays a key role in determining the outcome of the host immune response. Apoptotic cell death of macrophages is considered to be a protective host defense that eliminates pathogens and infected cells. In this study, we investigated the involvement of Notch signaling in regulating apoptosis in macrophages treated with tuberculin purified protein derivative (PPD). Murine bone marrow-derived macrophages (BMMs) treated with PPD or infected with Mycobacterium bovis Bacillus Calmette-Guérin (BCG) induced upregulation of Notch1. This upregulation correlated well with the upregulation of the anti-apoptotic gene mcl-1 both at the transcriptional and translational levels. Decreased levels of Notch1 and Mcl-1 were observed in BMM treated with PPD when a gamma secretase inhibitor (GSI), which inhibits the processing of Notch receptors, was used. Moreover, silencing Notch1 in the macrophage-like cell line RAW264.7 decreased Mcl-1 protein expression, suggesting that Notch1 is critical for Mcl-1 expression in macrophages. A significant increase in apoptotic cells was observed upon treatment of BMM with PPD in the presence of GSI compared to the vehicle-control treated cells. Finally, analysis of the mcl-1 promoter in humans and mice revealed a conserved potential CSL/RBP-Jκ binding site. The association of Notch1 with the mcl-1 promoter was confirmed by chromatin immunoprecipitation. Taken together, these results indicate that Notch1 inhibits apoptosis of macrophages stimulated with PPD by directly controlling the mcl-1 promoter.

Targeting BCL2 family in human myeloid dendritic cells: a challenge to cure diseases with chronic inflammations associated with bone loss.

Rheumatoid arthritis (RA) and Langerhans cell histiocytosis (LCH) are common and rare diseases, respectively. They associate myeloid cell recruitment and survival in inflammatory conditions with tissue destruction and bone resorption. Manipulating dendritic cell (DC), and, especially, regulating their half-life and fusion, is a challenge. Indeed, these myeloid cells display pathogenic roles in both diseases and may be an important source of precursors for differentiation of osteoclasts, the bone-resorbing multinucleated giant cells. We have recently documented that the proinflammatory cytokine IL-17A regulates long-term survival of DC by inducing BCL2A1 expression, in addition to the constitutive MCL1 expression. We summarize bibliography of the BCL2 family members and their therapeutic targeting, with a special emphasis on MCL1 and BCL2A1, discussing their potential impact on RA and LCH. Our recent knowledge in the survival pathway, which is activated to perform DC fusion in the presence of IL-17A, suggests that targeting MCL1 and BCL2A1 in infiltrating DC may affect the clinical outcomes in RA and LCH. The development of new therapies, interfering with MCL1 and BCL2A1 expression, to target long-term surviving inflammatory DC should be translated into preclinical studies with the aim to increase the well-being of patients with RA and LCH.

Expression of G protein αq Subunit is Decreased in Lymphocytes from Patients with Rheumatoid Arthritis and is Correlated with Disease Activity.

Gαq, the alpha subunit of Gq, a member of the Gq/11 sub-family, was reported to inhibit phosphatidylinositol-3-Kinase (PI3K) activation and prevent the activation of Akt. Previous studies demonstrated that mice losing Gαq in their immune system could spontaneously develop inflammatory arthritis. In this study, we showed that the Gαq expressions at mRNA and protein levels in the peripheral blood lymphocytes (PBLs) from patients with rheumatoid arthritis (RA) were significantly decreased in comparison of which in healthy individuals. The expression levels of Gαq mRNA in PBLs from patients with RA were correlated with RA disease activity (DAS28), anti-cyclic citrullinated protein antibodies, C-reactive protein and rheumatoid factor. We also demonstrated that Gαq controlled the apoptosis of RA PBLs through regulating the activity of Mcl-1 and caspase-3. These data suggested that Gαq might be involved in the pathogenesis of RA by regulating PBLs apoptosis.