DNA hypomethylating agents increase activation and cytolytic activity of CD8+ T cells.

We demonstrate that DNA hypomethylating agent (HMA) treatment can directly modulate the anti-tumor response and effector function of CD8+ T cells. In vivo HMA treatment promotes CD8+ T cell tumor infiltration and suppresses tumor growth via CD8+ T cell-dependent activity. Ex vivo, HMAs enhance primary human CD8+ T cell activation markers, effector cytokine production, and anti-tumor cytolytic activity. Epigenomic and transcriptomic profiling shows that HMAs vastly regulate T cell activation-related transcriptional networks, culminating with over-activation of NFATc1 short isoforms. Mechanistically, demethylation of an intragenic CpG island immediately downstream to the 3' UTR of the short isoform was associated with antisense transcription and alternative polyadenylation of NFATc1 short isoforms. High-dimensional single-cell mass cytometry analyses reveal a selective effect of HMAs on a subset of human CD8+ T cell subpopulations, increasing both the number and abundance of a granzyme Bhigh, perforinhigh effector subpopulation. Overall, our findings support the use of HMAs as a therapeutic strategy to boost anti-tumor immune response.

BH3 mimetics and TKI combined therapy for Chronic Myeloid Leukemia.

Chronic myeloid leukemia (CML) was considered for a long time one of the most hostile leukemia that was incurable for most of the patients, predominantly due to the extreme resistance to chemotherapy. Part of the resistance to cell death (apoptosis) is the result of increased levels of anti-apoptotic and decreased levels of pro-apoptotic member of the BCL-2 family induced by the BCR-ABL1 oncoprotein. BCR-ABL1 is a constitutively active tyrosine kinase responsible for initiating multiple and oncogenic signaling pathways. With the development of specific BCR-ABL1 tyrosine kinase inhibitors (TKIs) CML became a much more tractable disease. Nevertheless, TKIs do not cure CML patients and a substantial number of them develop intolerance or become resistant to the treatment. Therefore, novel anti-cancer strategies must be developed to treat CML patients independently or in combination with TKIs. Here, we will discuss the mechanisms of BCR-ABL1-dependent and -independent resistance to TKIs and the use of BH3-mimetics as a potential tool to fight CML.

Long non-coding RNA INXS is a critical mediator of BCL-XS induced apoptosis.

BCL-X mRNA alternative splicing generates pro-apoptotic BCL-XS or anti-apoptotic BCL-XL gene products and the mechanism that regulates splice shifting is incompletely understood. We identified and characterized a long non-coding RNA (lncRNA) named INXS, transcribed from the opposite genomic strand of BCL-X, that was 5- to 9-fold less abundant in tumor cell lines from kidney, liver, breast and prostate and in kidney tumor tissues compared with non-tumors. INXS is an unspliced 1903 nt-long RNA, is transcribed by RNA polymerase II, 5'-capped, nuclear enriched and binds Sam68 splicing-modulator. Three apoptosis-inducing agents increased INXS lncRNA endogenous expression in the 786-O kidney tumor cell line, increased BCL-XS/BCL-XL mRNA ratio and activated caspases 3, 7 and 9. These effects were abrogated in the presence of INXS knockdown. Similarly, ectopic INXS overexpression caused a shift in splicing toward BCL-XS and activation of caspases, thus leading to apoptosis. BCL-XS protein accumulation was detected upon INXS overexpression. In a mouse xenograft model, intra-tumor injections of an INXS-expressing plasmid caused a marked reduction in tumor weight, and an increase in BCL-XS isoform, as determined in the excised tumors. We revealed an endogenous lncRNA that induces apoptosis, suggesting that INXS is a possible target to be explored in cancer therapies.

Cytosolic flagellin-induced lysosomal pathway regulates inflammasome-dependent and -independent macrophage responses.

NAIP5/NLRC4 (neuronal apoptosis inhibitory protein 5/nucleotide oligomerization domain-like receptor family, caspase activation recruitment domain domain-containing 4) inflammasome activation by cytosolic flagellin results in caspase-1-mediated processing and secretion of IL-1ß/IL-18 and pyroptosis, an inflammatory cell death pathway. Here, we found that although NLRC4, ASC, and caspase-1 are required for IL-1ß secretion in response to cytosolic flagellin, cell death, nevertheless, occurs in the absence of these molecules. Cytosolic flagellin-induced inflammasome-independent cell death is accompanied by IL-1α secretion and is temporally correlated with the restriction of Salmonella Typhimurium infection. Despite displaying some apoptotic features, this peculiar form of cell death do not require caspase activation but is regulated by a lysosomal pathway, in which cathepsin B and cathepsin D play redundant roles. Moreover, cathepsin B contributes to NAIP5/NLRC4 inflammasome-induced pyroptosis and IL-1α and IL-1ß production in response to cytosolic flagellin. Together, our data describe a pathway induced by cytosolic flagellin that induces a peculiar form of cell death and regulates inflammasome-mediated effector mechanisms of macrophages.

Pathogen-induced proapoptotic phenotype and high CD95 (Fas) expression accompany a suboptimal CD8+ T-cell response: reversal by adenoviral vaccine.

MHC class Ia-restricted CD8(+) T cells are important mediators of the adaptive immune response against infections caused by intracellular microorganisms. Whereas antigen-specific effector CD8(+) T cells can clear infection caused by intracellular pathogens, in some circumstances, the immune response is suboptimal and the microorganisms survive, causing host death or chronic infection. Here, we explored the cellular and molecular mechanisms that could explain why CD8(+) T cell-mediated immunity during infection with the human protozoan parasite Trypanosoma cruzi is not optimal. For that purpose, we compared the CD8(+) T-cell mediated immune responses in mice infected with T. cruzi or vaccinated with a recombinant adenovirus expressing an immunodominant parasite antigen. Several functional and phenotypic characteristics of specific CD8(+) T cells overlapped. Among few exceptions was an accelerated expansion of the immune response in adenoviral vaccinated mice when compared to infected ones. Also, there was an upregulated expression of the apoptotic-signaling receptor CD95 on the surface of specific T cells from infected mice, which was not observed in the case of adenoviral-vaccinated mice. Most importantly, adenoviral vaccine provided at the time of infection significantly reduced the upregulation of CD95 expression and the proapoptotic phenotype of pathogen-specific CD8(+) cells expanded during infection. In parallel, infected adenovirus-vaccinated mice had a stronger CD8 T-cell mediated immune response and survived an otherwise lethal infection. We concluded that a suboptimal CD8(+) T-cell response is associated with an upregulation of CD95 expression and a proapoptotic phenotype. Both can be blocked by adenoviral vaccination.

Evaluation of pyroptosis in macrophages using cytosolic delivery of purified flagellin.

Pyroptosis is a molecularly controlled form of cell death that exhibits some features of apoptosis as well of necrosis. Pyroptosis is induced by inflammasome-activated caspase-1 or caspase-11 (caspase-4 in humans), as a result of distinct pathogenic or damage stimuli. Although pyroptosis displays some morphological and biochemical features of apoptosis, it has an inflammatory outcome due to the loss of plasma membrane integrity and the consequent release of intracellular contents, reminiscent to necrosis. Here, we use cytosolic delivery of purified flagellin as an experimental tool to trigger pyroptosis and describe potential methods to study this form of cell death. Finally, we discuss the advantages and limitations of these methods.

Hypoxia inducible factor-dependent regulation of angiogenesis by nitro-fatty acids.

OBJECTIVE: Nitro-fatty acids (NO(2)-FAs) are emerging as a new class of cell signaling mediators. Because NO(2)-FAs are found in the vascular compartment and their impact on vascularization remains unknown, we aimed to investigate the role of NO(2)-FAs in angiogenesis. METHODS AND RESULTS: The effects of nitrolinoleic acid and nitrooleic acid were evaluated on migration of endothelial cell (EC) in vitro, EC sprouting ex vivo, and angiogenesis in the chorioallantoic membrane assay in vivo. At 10 µmol/L, both NO(2)-FAs induced EC migration and the formation of sprouts and promoted angiogenesis in vivo in an NO-dependent manner. In addition, NO(2)-FAs increased intracellular NO concentration, upregulated protein expression of the hypoxia inducible factor-1α (HIF-1α) transcription factor by an NO-mediated mechanism, and induced expression of HIF-1α target genes, such as vascular endothelial growth factor, glucose transporter-1, and adrenomedullin. Compared with typical NO donors such as spermine-NONOate and deta-NONOate, NO(2)-FAs were slightly less potent inducers of EC migration and HIF-1α expression. Short hairpin RNA-mediated knockdown of HIF-1α attenuated the induction of vascular endothelial growth factor mRNA expression and EC migration stimulated by NO(2)-FAs. CONCLUSION: Our data disclose a novel physiological role for NO(2)-FAs, indicating that these compounds induce angiogenesis in an NO-dependent mechanism via activation of HIF-1α.

BCR-ABL1 Tyrosine Kinase Complex Signaling Transduction: Challenges to Overcome Resistance in Chronic Myeloid Leukemia.

The constitutively active BCR-ABL1 tyrosine kinase, found in t(9;22)(q34;q11) chromosomal translocation-derived leukemia, initiates an extremely complex signaling transduction cascade that induces a strong state of resistance to chemotherapy. Targeted therapies based on tyrosine kinase inhibitors (TKIs), such as imatinib, dasatinib, nilotinib, bosutinib, and ponatinib, have revolutionized the treatment of BCR-ABL1-driven leukemia, particularly chronic myeloid leukemia (CML). However, TKIs do not cure CML patients, as some develop TKI resistance and the majority relapse upon withdrawal from treatment. Importantly, although BCR-ABL1 tyrosine kinase is necessary to initiate and establish the malignant phenotype of Ph-related leukemia, in the later advanced phase of the disease, BCR-ABL1-independent mechanisms are also in place. Here, we present an overview of the signaling pathways initiated by BCR-ABL1 and discuss the major challenges regarding immunologic/pharmacologic combined therapies.

Blockade of caspase cascade overcomes malaria-associated acute respiratory distress syndrome in mice.

Malaria is an enormous burden on global health that caused 409,000 deaths in 2019. Severe malaria can manifest in the lungs, an illness known as acute respiratory distress syndrome (ARDS). Not much is known about the development of malaria-associated ARDS (MA-ARDS), especially regarding cell death in the lungs. We had previously established a murine model that mimics various human ARDS aspects, such as pulmonary edema, hemorrhages, pleural effusion, and hypoxemia, using DBA/2 mice infected with Plasmodium berghei ANKA. Here, we explored the mechanisms and the involvement of apoptosis in this syndrome. We found that apoptosis contributes to the pathogenesis of MA-ARDS, primarily as facilitators of the alveolar-capillary barrier breakdown. The protection of pulmonary endothelium by inhibiting caspase activation could be a promising therapeutic strategy to prevent the pathogenicity of MA-ARDS. Therefore, intervention in the programmed death cell mechanism could help patients not to develop severe malaria.

A novel pathway for inducible nitric-oxide synthase activation through inflammasomes.

Innate immune recognition of flagellin is shared by transmembrane TLR5 and cytosolic Nlrc4 (NOD-like receptor family CARD (caspase activation recruitment domain) domain containing 4)/Naip5 (neuronal apoptosis inhibitory protein 5). TLR5 activates inflammatory genes through MYD88 pathway, whereas Nlrc4 and Naip5 assemble multiprotein complexes called inflammasomes, culminating in caspase-1 activation, IL-1ß/IL-18 secretion, and pyroptosis. Although both TLR5 and Naip5/Nlrc4 pathways cooperate to clear infections, little is known about the relative anti-pathogen effector mechanisms operating through each of them. Here we show that the cytosolic flagellin (FLA-BSDot) was able to activate iNOS, an enzyme previously associated with TLR5 pathway. Using Nlrc4- or Naip5-deficient macrophages, we found that both receptors are involved in iNOS activation by FLA-BSDot. Moreover, distinct from extracellular flagellin (FLA-BS), iNOS activation by intracellular flagellin is completely abrogated in the absence of caspase-1. Interestingly, IL-1ß and IL-18 do not seem to be important for FLA-BSDot-mediated iNOS production. Together, our data defined an additional anti-pathogen effector mechanism operated through Naip5 and Nlrc4 inflammasomes and illustrated a novel signaling transduction pathway that activates iNOS.

Comparative effect of FGF2, synthetic peptides 1-28 N-POMC and ACTH on proliferation in rat adrenal cell primary cultures.

There is evidence that pro-opiomelanocortin (POMC)-derived peptides other than adrenocorticotropic hormone (ACTH) have a role in adrenal cell proliferation. We compared the activity of synthetic rat N-terminal POMC fragment 1-28 with disulfide bridges (N-POMC(w)) and without disulfide bridges (N-POMC(w/o)), with the activity of fibroblast growth factor (FGF2), a widely studied adrenal growth factor, and ACTH, in well-characterized pure cultures of both isolated adrenal Glomerulosa (G) and Fasciculata/Reticularis (F/R) cells. Three days of FGF2-treatment had a proliferative effect similar to serum, and synthetic peptide N-POMC(w) induced proliferation more efficiently than N-POMC(w/o). Moreover, both induced proliferation via the ERK1/2 pathway. In contrast, sustained ACTH treatment decreased proliferation and viability through apoptosis induction, but not necrosis, and independently of PKA and PKC pathways. Further elucidation of 1-28 POMC signal transduction is of interest, and primary cultures of adrenal cells were found to be useful for examining the trophic activity of this peptide.

Absence of Bim sensitizes mice to experimental Trypanosoma cruzi infection.

Chagas disease is a life-threatening disorder caused by the protozoan parasite Trypanosoma cruzi. Parasite-specific antibodies, CD8+ T cells, as well as IFN-γ and nitric oxide (NO) are key elements of the adaptive and innate immunity against the extracellular and intracellular forms of the parasite. Bim is a potent pro-apoptotic member of the Bcl-2 family implicated in different aspects of the immune regulation, such as negative selection of self-reactive thymocytes and elimination of antigen-specific T cells at the end of an immune response. Interestingly, the role of Bim during infections remains largely unidentified. To explore the role of Bim in Chagas disease, we infected WT, Bim+/-, Bim-/- mice with trypomastigotes forms of the Y strain of T. cruzi. Strikingly, our data revealed that Bim-/- mice exhibit a delay in the development of parasitemia followed by a deficiency in the control of parasite load in the bloodstream and a decreased survival compared to WT and Bim+/- mice. At the peak of parasitemia, peritoneal macrophages of Bim-/- mice exhibit decreased NO production, which correlated with a decrease in the pro-inflammatory Small Peritoneal Macrophage (SPM) subset. A similar reduction in NO secretion, as well as in the pro-inflammatory cytokines IFN-γ and IL-6, was also observed in Bim-/- splenocytes. Moreover, an impaired anti-T. cruzi CD8+ T-cell response was found in Bim-/- mice at this time point. Taken together, our results suggest that these alterations may contribute to the establishment of a delayed yet enlarged parasitic load observed at day 9 after infection of Bim-/- mice and place Bim as an important protein in the control of T. cruzi infections.

RIPK3 and Caspase-1/11 Are Necessary for Optimal Antigen-Specific CD8 T Cell Response Elicited by Genetically Modified Listeria monocytogenes.

Efficient induction of effector and long-term protective antigen-specific CD8+ T memory response by vaccination is essential to eliminate malignant and pathogen-infected cells. Intracellular infectious bacteria, including Listeria monocytogenes, have been considered potent vectors to carry multiple therapeutic proteins and generate antigen-specific CD8+ T cell responses. Although the role of molecules involved in inflammatory cell death pathways, such as necroptosis (RIPK3-mediated) and pyroptosis (Caspase-1/11-mediated), as effectors of immune response against intracellular bacteria are relatively well understood, their contribution to the adjuvant effect of recombinant bacterial vectors in the context of antigen-specific CD8+ T cell response remained obscure. Therefore, we evaluated the impact of RIPK3 and Caspase-1/11 (Casp-1/11) individual and combined deficiencies on the modulation of antigen-specific CD8+ T cell response during vaccination of mice with ovalbumin-expressing L. monocytogenes (LM-OVA). We observed that Casp-1/11 but not RIPK3 deficiency negatively impacts the capacity of mice to clear LM-OVA. Importantly, both RIPK3 and Casp-1/11 are necessary for optimal LM-OVA-mediated antigen-specific CD8+ T cell response, as measured by in vivo antigen-specific CD8+ T cell proliferation, target cell elimination, and cytokine production. Furthermore, Casp-1/11 and Casp-1/11/RIPK3 combined deficiencies restrict the early initiation of antigen-specific CD8+ T cell memory response. Taken together, our findings demonstrate that RIPK3 and Casp-1/11 influence the quality of CD8+ T cell responses induced by recombinant L. monocytogenes vectors.

Apoptosis of macrophages during pulmonary Mycobacterium bovis infection: correlation with intracellular bacillary load and cytokine levels.

Apoptosis of macrophages infected with pathogenic mycobacteria is an alternative host defence capable of removing the environment supporting bacterial growth. In this work the influence of virulence and bacterial load on apoptosis of alveolar macrophages during the initial phase of infection by Mycobacterium bovis was investigated. BALB/c mice were infected intratracheally with high or low doses of the virulent (ATCC19274) or attenuated (bacillus Calmette-Guérin Moreau) strains of M. bovis. The frequency of macrophage apoptosis, the growth of mycobacteria in macrophages, and the in situ levels of the cytokines tumour necrosis factor-alpha (TNF-alpha), interleukin-10 (IL-10) and IL-12 and of the anti-apoptotic protein Bcl-2 were measured at day 3 and day 7 post-infection. An increase of macrophage apoptosis was observed after infection with both strains but the virulent strain induced less apoptosis than the attenuated strain. On the 3rd day after infection with the virulent strain macrophage apoptosis was reduced in the high-dose group, while on the 7th day post-infection macrophage apoptosis was reduced in the low-dose group. Inhibition of apoptosis was correlated with increased production of IL-10, reduced production of TNF-alpha and increased production of Bcl-2. In addition, the production of IL-12 was reduced at points where the lowest levels of macrophage apoptosis were observed. Our results indicate that virulent mycobacteria are able to modulate macrophage apoptosis to an extent dependent on the intracellular bacterial burden, which benefits its intracellular growth and dissemination to adjacent cells.

Sustained activation of p53 in confluent nucleotide excision repair-deficient cells resistant to ultraviolet-induced apoptosis.

P53 activation is one of the main signals after DNA damage, controlling cell cycle arrest, DNA repair and apoptosis. We have previously shown that confluent nucleotide excision repair (NER)-deficient cells are more resistant to apoptosis induced by ultraviolet irradiation (UV). Here, we further investigated the effect of cell confluence on UV-induced apoptosis in normal and NER-deficient (XP-A and XP-C) cells, as well as the effects of treatments with the ATM/ATR inhibitor caffeine, and the patterns of p53 activation. Strong p53 activation was observed in either proliferating or confluent cells. Caffeine increased apoptosis levels and inhibited p53 activation in proliferating cells, suggesting a protective role for p53. However, in confluent NER-deficient cells no effect of caffeine was observed. Transcription recovery measurements showed decreased recovery in proliferating XPA-deficient cells, but no recovery was observed in confluent cells. The levels of the cyclin/Cdk inhibitor, p21(Waf1/Cip1), correlated well with p53 activation in proliferating cells. Surprisingly, confluent cells also showed similar activation of p21(Waf1/Cip1). These results indicate that reduced apoptosis in confluent cells is associated with the deficiency in DNA damage removal, since this effect is not clearly observed in NER-proficient cells. Moreover, the strong activation of p53 in confluent cells, which barely respond to apoptosis, suggests that this protein, under these conditions, is not linked to UV-induced cell death signaling.

CPDs and 6-4PPs play different roles in UV-induced cell death in normal and NER-deficient human cells.

Ultraviolet (UV) light generates two major DNA lesions: cyclobutane pyrimidine dimers (CPDs) and pyrimidine-(6-4)-pyrimidone photoproducts (6-4PPs), but the specific participation of these two lesions in the deleterious effects of UV is a longstanding question. In order to discriminate the precise role of unrepaired CPDs and 6-4PPs in UV-induced responses triggering cell death, human fibroblasts were transduced by recombinant adenoviruses carrying the CPD-photolyase or 6-4PP-photolyase cDNAs. Both photolyases were able to prevent UV-induced apoptosis in cells deficient for nucleotide excision repair (NER) to a similar extent, while in NER-proficient cells UV-induced apoptosis was prevented only by CPD-photolyase, with no effects observed when 6-4PPs were removed by the specific photolyase. These results strongly suggest that both CPDs and 6-4PPs contribute to UV-induced apoptosis in NER-deficient cells, while in NER-proficient cells, CPDs are the only lesions responsible for UV-killing, probably due to the rapid repair of 6-4PPs by NER. As a consequence, the difference in skin photosensitivity, including carcinogenesis, of most of the xeroderma pigmentosum patients and of normal people is probably not only a quantitative aspect, but depends on the type of DNA damage induced by sunlight and its rate of repair.

Resistance to ultraviolet-induced apoptosis in DNA repair deficient growth arrested human fibroblasts is not related to recovery from RNA transcription blockage.

The impact of ultraviolet (UV-C) photoproducts on apoptosis induction was investigated in growth arrested (confluent) and proliferating human primary fibroblasts. Confluent fibroblasts were more resistant to UV-C-induced apoptosis than proliferating cells, and this was observed for normal human cells and for cells from patients with Cockayne and trichothiodystrophy syndromes, deficient in transcription coupled repair. This resistance was sustained for at least seven days and was not due to DNA repair efficiency, as the removal of CPDs in the genome was similar under both growth conditions. There was no correlation between reduced apoptosis and RNA synthesis recovery. Following UV-C treatment, proliferating and confluent fibroblasts showed a similar level of RNA synthesis inhibition and recovery from transcription blockage. These results support the hypothesis that the decrease of DNA replication, in growth arrested cells, protects cell from UV-C-induced apoptosis, even in the presence of DNA lesions.

Pattern Recognition Receptors and the Host Cell Death Molecular Machinery.

Pattern Recognition Receptors (PRRs) are proteins capable of recognizing molecules frequently found in pathogens (the so-called Pathogen-Associated Molecular Patterns-PAMPs), or molecules released by damaged cells (the Damage-Associated Molecular Patterns-DAMPs). They emerged phylogenetically prior to the appearance of the adaptive immunity and, therefore, are considered part of the innate immune system. Signals derived from the engagement of PRRs on the immune cells activate microbicidal and pro-inflammatory responses required to eliminate or, at least, to contain infectious agents. Molecularly controlled forms of cell death are also part of a very ancestral mechanism involved in key aspects of the physiology of multicellular organism, including the elimination of unwanted, damaged or infected cells. Interestingly, each form of cell death has its particular effect on inflammation and on the development of innate and adaptive immune responses. In this review article, we discuss some aspects of the molecular interplay between the cell death machinery and signals initiated by the activation of PRRs by PAMPs and DAMPs.

TLR3 Is a Negative Regulator of Immune Responses Against Paracoccidioides brasiliensis.

Toll-like receptors (TLRs) comprise the best-characterized pattern-recognition receptor (PRR) family able to activate distinct immune responses depending on the receptor/adaptor set assembled. TLRs, such as TLR2, TLR4 and TLR9, and their signaling were shown to be important in Paracoccidioides brasiliensis infections. However, the role of the endosomal TLR3 in experimental paracoccidioidomycosys remains obscure. In vitro assays, macrophages of the bone marrow of WT or TLR3-/- mice were differentiated for evaluation of their microbicidal activity. In vivo assays, WT or TLR3-/- mice were infected intratracheally with Paracoccidioides brasiliensis yeasts for investigation of the lung response type induced. The cytotoxic activity of CD8+ T cells was assessed by cytotoxicity assay. To confirm the importance of CD8+ T cells in the control of infection in the absence of tlr3, a depletion assay of these cells was performed. Here, we show for the first time that TLR3 modulate the infection against Paracoccidioides brasiliensis by dampening pro-inflammatory response, NO production, IFN+CD8+T, and IL-17+CD8+T cell activation and cytotoxic function, associated with granzyme B and perforin down regulation. As conclusion, we suggest that TLR3 could be used as an escape mechanism of the fungus in an experimental paracoccidioidomycosis.