AKT inhibition interferes with the expression of immune checkpoint proteins and increases NK-induced killing of HL60-AML cells.

OBJECTIVE: To determine the role of the AKT pathway in the regulating of natural Killer-induced apoptosis of acute myeloid leukemia cells and to characterize the associated molecular mechanisms. METHODS: BALB/c nude mice were injected with HL60 cells to induce a xenogenic model of subcutaneous leukemic tumors. Mice were treated with perifosine, and their spleens were analyzed using biometry, histopathology, and immunohistochemistry. Gene expression analysis in leukemia cells was performed by real-time PCR. Protein analysis of leukemia and natural Killer cells was performed by flow cytometry. AKT inhibition in HL60 cells, followed by co-culture with natural Killer cells was performed to assess cytotoxicity. Apoptosis rate was quantified using flow cytometry. RESULTS: Perifosine treatment caused a reduction in leukemic infiltration in the spleens of BALB/c nude mice. In vitro , AKT inhibition reduced HL60 resistance to natural Killer-induced apoptosis. AKT inhibition suppressed the immune checkpoint proteins PD-L1, galectin-9, and CD122 in HL60 cells, but did not change the expression of their co-receptors PD1, Tim3, and CD96 on the natural Killer cell surface. In addition, the death receptors DR4, TNFR1, and FAS were overexpressed by AKT inhibition, thus increasing the susceptibility of HL60 cells to the extrinsic pathway of apoptosis. CONCLUSION: The AKT pathway is involved in resistance to natural Killer-induced apoptosis in HL60 cells by regulating the expression of immune suppressor receptors. These findings highlight the importance of AKT in contributing to immune evasion mechanisms in acute myeloid leukemia and suggests the potential of AKT inhibition as an adjunct to immunotherapy.

AKT inhibition interferes with the expression of immune checkpoint proteins and increases NK-induced killing of HL60-AML cells

ABSTRACT Objective To determine the role of the AKT pathway in the regulating of natural Killer-induced apoptosis of acute myeloid leukemia cells and to characterize the associated molecular mechanisms. Methods BALB/c nude mice were injected with HL60 cells to induce a xenogenic model of subcutaneous leukemic tumors. Mice were treated with perifosine, and their spleens were analyzed using biometry, histopathology, and immunohistochemistry. Gene expression analysis in leukemia cells was performed by real-time PCR. Protein analysis of leukemia and natural Killer cells was performed by flow cytometry. AKT inhibition in HL60 cells, followed by co-culture with natural Killer cells was performed to assess cytotoxicity. Apoptosis rate was quantified using flow cytometry. Results Perifosine treatment caused a reduction in leukemic infiltration in the spleens of BALB/c nude mice. In vitro , AKT inhibition reduced HL60 resistance to natural Killer-induced apoptosis. AKT inhibition suppressed the immune checkpoint proteins PD-L1, galectin-9, and CD122 in HL60 cells, but did not change the expression of their co-receptors PD1, Tim3, and CD96 on the natural Killer cell surface. In addition, the death receptors DR4, TNFR1, and FAS were overexpressed by AKT inhibition, thus increasing the susceptibility of HL60 cells to the extrinsic pathway of apoptosis. Conclusion The AKT pathway is involved in resistance to natural Killer-induced apoptosis in HL60 cells by regulating the expression of immune suppressor receptors. These findings highlight the importance of AKT in contributing to immune evasion mechanisms in acute myeloid leukemia and suggests the potential of AKT inhibition as an adjunct to immunotherapy.

A probiotic has differential effects on allergic airway inflammation in A/J and C57BL/6 mice and is correlated with the gut microbiome.

The phenotypes of allergic airway diseases are influenced by the interplay between host genetics and the gut microbiota, which may be modulated by probiotics. We investigated the probiotic effects on allergic inflammation in A/J and C57BL/6 mice. C57BL/6 mice had increased gut microbiota diversity compared to A/J mice at baseline. Acetate producer probiotics differentially modulated and altered the genus abundance of specific bacteria, such as Akkermansia and Allistipes, in mouse strains. We induced airway inflammation followed by probiotic treatment and found that only A/J mice exhibited decreased inflammation, and the beneficial effects of probiotics in A/J mice were partially due to acetate production. To understand the relevance of microbial composition colonization in the development of allergic diseases, we implanted female C57BL/6 mice with A/J embryos to naturally modulate the microbial composition of A/J mice, which increased gut microbiota diversity and reduced eosinophilic inflammation in A/J. These data demonstrate the central importance of microbiota to allergic phenotype severity. Video Abstract.

Association Between Heart Rate Variability and Decompression-Induced Physiological Stress.

The purpose of this study was to analyze the correlation between decompression-related physiological stress markers, given by inflammatory processes and immune system activation and changes in Heart Rate Variability, evaluating whether Heart Rate Variability can be used to estimate the physiological stress caused by the exposure to hyperbaric environments and subsequent decompression. A total of 28 volunteers participated in the experimental protocol. Electrocardiograms were performed; blood samples were obtained for the quantification of red cells, hemoglobin, hematocrit, neutrophils, lymphocytes, platelets, aspartate transaminase (AST), alanine aminotransferase (ALT), and for immunophenotyping and microparticles (MP) research through Flow Cytometry, before and after each experimental protocol from each volunteer. Also, myeloperoxidase (MPO) expression and microparticles (MPs) deriving from platelets, neutrophils and endothelial cells were quantified. Negative associations between the standard deviation of normal-to-normal intervals (SDNN) in the time domain, the High Frequency in the frequency domain and the total number of circulating microparticles was observed (p-value = 0.03 and p-value = 0.02, respectively). The pre and post exposure ratio of variation in the number of circulating microparticles was negatively correlated with SDNN (p-value = 0.01). Additionally, a model based on the utilization of Radial Basis Function Neural Networks (RBF-NN) was created and was able to predict the SDNN ratio of variation based on the variation of specific inflammatory markers (RMSE = 0.06).

Rhipicephalus microplus serpins interfere with host immune responses by specifically modulating mast cells and lymphocytes.

Rhipicephalus microplus ticks feed on a bovine host for three weeks. At the attachment site, inflammatory and immune responses are triggered resulting in the recruitment of cells and production of a set of immunological mediators. To oppose the host's immune responses, ticks inoculate bioactive salivary molecules capable of interfering with these defense mechanisms. Serpins are among the most frequent molecules present in tick saliva and have been shown to negatively affect the host's anti-tick immunity. R. microplus has at least eighteen full-length serpins (RmS) and eleven are transcribed during blood feeding. Among them, RmS-3, RmS-6, and RmS-17 are present in the saliva of engorged females. Here, the effect of these serpins on the immune responses was evaluated in cells involved in innate/inflammatory (mast cells and macrophages) and adaptive (T cells) immunity. RmS-3 modulated mast cells due to its inhibitory activity on peritoneal rat chymase and on vascular permeability in acute inflammation. In addition, both RmS-6 and RmS-17 inhibited vascular permeability. Of the three serpins studied, neither affected activation nor inflammatory cytokine production by murine macrophages. On the other hand, RmS-3 and RmS-17 presented an inhibitory effect on the metabolic activity of lymphocytes, with the latter being the most potent, while RmS-6 had no effect on it. This activity was associated with a decrease in lymphocyte proliferation, but not with induction of cell death. The present study highlights the powerful modulatory role of tick salivary serpins in the host's immune system and inspire the discovery of targets for the treatment of inflammatory/immune disorders.

Amblyomma sculptum Salivary PGE2 Modulates the Dendritic Cell-Rickettsia rickettsii Interactions in vitro and in vivo.

Amblyomma sculptum is an important vector of Rickettsia rickettsii, causative agent of Rocky Mountain spotted fever and the most lethal tick-borne pathogen affecting humans. To feed on the vertebrate host's blood, A. sculptum secretes a salivary mixture, which may interact with skin resident dendritic cells (DCs) and modulate their function. The present work was aimed at depicting the A. sculptum saliva-host DC network and the biochemical nature of the immunomodulatory component(s) involved in this interface. A. sculptum saliva inhibits the production of inflammatory cytokines by murine DCs stimulated with LPS. The fractionation of the low molecular weight salivary content by reversed-phase chromatography revealed active fractions eluting from 49 to 55% of the acetonitrile gradient. Previous studies suggested that this pattern of elution matches with that observed for prostaglandin E2 (PGE2) and the molecular identity of this lipid mediator was unambiguously confirmed by a new high-resolution mass spectrometry methodology. A productive infection of murine DCs by R. rickettsii was demonstrated for the first time leading to proinflammatory cytokine production that was inhibited by both A. sculptum saliva and PGE2, a result also achieved with human DCs. The adoptive transfer of murine DCs incubated with R. rickettsii followed by treatment with A. sculptum saliva or PGE2 did not change the cytokine profile associated to cellular recall responses while IgG2a-specific antibodies were decreased in the serum of these mice. Together, these findings emphasize the role of PGE2 as a universal immunomodulator of tick saliva. In addition, it contributes to new approaches to explore R. rickettsii-DC interactions both in vitro and in vivo.

Amblyomma sculptum salivary PGE2 modulates the dendritic cell-Rickettsia rickettsii interactions in vitro and in vivo

Amblyomma sculptum is an important vector of Rickettsia rickettsii, causative agent of Rocky Mountain spotted fever and the most lethal tick-borne pathogen affecting humans. To feed on the vertebrate host’s blood, A. sculptum secretes a salivary mixture, which may interact with skin resident dendritic cells (DCs) and modulate their function. The present work was aimed at depicting the A. sculptum saliva-host DC network and the biochemical nature of the immunomodulatory component(s) involved in this interface. A. sculptum saliva inhibits the production of inflammatory cytokines by murine DCs stimulated with LPS. The fractionation of the low molecular weight salivary content by reversed-phase chromatography revealed active fractions eluting from 49 to 55% of the acetonitrile gradient. Previous studies suggested that this pattern of elution matches with that observed for prostaglandin E2 (PGE2) and the molecular identity of this lipid mediator was unambiguously confirmed by a new high-resolution mass spectrometry methodology. A productive infection of murine DCs by R. rickettsii was demonstrated for the first time leading to proinflammatory cytokine production that was inhibited by both A. sculptum saliva and PGE2, a result also achieved with human DCs. The adoptive transfer of murine DCs incubated with R. rickettsii followed by treatment with A. sculptum saliva or PGE2 did not change the cytokine profile associated to cellular recall responses while IgG2a-specific antibodies were decreased in the serum of these mice. Together, these findings emphasize the role of PGE2 as a universal immunomodulator of tick saliva. In addition, it contributes to new approaches to explore R. rickettsii-DC interactions both in vitro and in vivo.

Amblyomma sculptum salivary PGE2 modulates the dendritic cell-Rickettsia rickettsii interactions in vitro and in vivo

Amblyomma sculptum is an important vector of Rickettsia rickettsii, causative agent of Rocky Mountain spotted fever and the most lethal tick-borne pathogen affecting humans. To feed on the vertebrate host’s blood, A. sculptum secretes a salivary mixture, which may interact with skin resident dendritic cells (DCs) and modulate their function. The present work was aimed at depicting the A. sculptum saliva-host DC network and the biochemical nature of the immunomodulatory component(s) involved in this interface. A. sculptum saliva inhibits the production of inflammatory cytokines by murine DCs stimulated with LPS. The fractionation of the low molecular weight salivary content by reversed-phase chromatography revealed active fractions eluting from 49 to 55% of the acetonitrile gradient. Previous studies suggested that this pattern of elution matches with that observed for prostaglandin E2 (PGE2) and the molecular identity of this lipid mediator was unambiguously confirmed by a new high-resolution mass spectrometry methodology. A productive infection of murine DCs by R. rickettsii was demonstrated for the first time leading to proinflammatory cytokine production that was inhibited by both A. sculptum saliva and PGE2, a result also achieved with human DCs. The adoptive transfer of murine DCs incubated with R. rickettsii followed by treatment with A. sculptum saliva or PGE2 did not change the cytokine profile associated to cellular recall responses while IgG2a-specific antibodies were decreased in the serum of these mice. Together, these findings emphasize the role of PGE2 as a universal immunomodulator of tick saliva. In addition, it contributes to new approaches to explore R. rickettsii-DC interactions both in vitro and in vivo.

Hematobin is a novel immunomodulatory protein from the saliva of the horn fly Haematobia irritans that inhibits the inflammatory response in murine macrophages.

BACKGROUND: The horn fly Haematobia irritans is a blood-sucking ectoparasite responsible for substantial economic loss of livestock. Like other hematophagous arthropods species, the successful blood-feeding of H. irritans is highly dependent on the modulation of the host's hemostasis and immune system. Here, we evaluated the biological activity of hematobin (HTB), a protein recently identified in the H. irritans saliva, on macrophage biology. The goal was to understand the putative interactions between the components of H. irritans saliva and the early host immune responses. RESULTS: Thioglycolate-elicited peritoneal macrophages from BALB/c mice were stimulated by lipopolysaccharide (LPS) plus interferon-γ (IFN-γ) in the presence or absence of recombinant HTB. The presence of the salivary protein in the cultures inhibited nitric oxide production and decreased the inducible nitric oxide synthase (iNOS) expression induced by LPS plus IFN-γ. The tumor necrosis factor-α (TNF-α) and interleukin-12p40 (IL-12p40) levels were also reduced in the macrophages pre-incubated with HTB; these findings correlated to the decreased NF-κB expression. The biological activities described here were not associated with changes in annexin V binding to macrophages suggesting that HTB does not induce cell death. In addition, the activity of HTB seems to be specific to macrophages because no changes were observed in lymphocyte proliferation or cytokine production. CONCLUSIONS: We describe here the first bioactive salivary protein of H. irritans. We characterized its ability to modulate macrophage inflammatory response, and the results can help explain how horn flies modulate the host immune system to feed on blood.

Boosting Adaptive Immunity: A New Role for PAFR Antagonists.

We have previously shown that the Platelet-Activating Factor Receptor (PAFR) engagement in murine macrophages and dendritic cells (DCs) promotes a tolerogenic phenotype reversed by PAFR-antagonists treatment in vitro. Here, we investigated whether a PAFR antagonist would modulate the immune response in vivo. Mice were subcutaneously injected with OVA or OVA with PAFR-antagonist WEB2170 on days 0 and 7. On day 14, OVA-specific IgG2a and IgG1 were measured in the serum. The presence of WEB2170 during immunization significantly increased IgG2a without affecting IgG1 levels. When WEB2170 was added to OVA in complete Freund's adjuvant, enhanced IgG2a but not IgG1 production was also observed, and CD4+ FoxP3+ T cell frequency in the spleen was reduced compared to mice immunized without the antagonist. Similar results were observed in PAFR-deficient mice, along with increased Tbet mRNA expression in the spleen. Additionally, bone marrow-derived DCs loaded with OVA were transferred into naïve mice and their splenocytes were co-cultured with fresh OVA-loaded DCs. CD4+ T cell proliferation was higher in the group transferred with DCs treated with the PAFR-antagonist. We propose that the activation of PAFR by ligands present in the site of immunization is able to fine-tune the adaptive immune response.

Exposure to Aedes aegypti Bites Induces a Mixed-Type Allergic Response following Salivary Antigens Challenge in Mice.

Classical studies have shown that Aedes aegypti salivary secretion is responsible for the sensitization to mosquito bites and many of the components present in saliva are immunogenic and capable of inducing an intense immune response. Therefore, we have characterized a murine model of adjuvant-free systemic allergy induced by natural exposure to mosquito bites. BALB/c mice were sensitized by exposure to A. aegypti mosquito bites and intranasally challenged with phosphate-buffered saline only or the mosquito's salivary gland extract (SGE). Blood, bronchoalveolar lavage (BAL) and lung were collected and evaluated for cellularity, histopathological analyses, cytokines and antibody determination. Respiratory pattern was analyzed by Penh measurements and tracheal segments were obtained to study in vitro reactivity to methacholine. BAL recovered from sensitized mice following challenge with SGE showed an increased number of eosinophils and Th2 cytokines such as IL-4, IL-5 and IL-13. Peribronchoalveolar eosinophil infiltration, mucus and collagen were also observed in lung parenchyma of sensitized mice, suggesting the development of a typical Th2 response. However, the antibody profile in serum of these mice evidenced a mixed-type response with presence of both, IgG1/IgE (Th2-related) and IgG2a (Th1-related) isotypes. In addition, changes in breathing pattern and tracheal reactivity to methacholine were not found. Taken together, our results show that A. aegypti bites trigger an atypical allergic reaction, with some classical cellular and soluble Th2 components in the lung, but also systemic Th1 and Th2 antibody isotypes and no change in either the respiratory pattern or the trachea responsiveness to agonist.

The dual effect of paradoxical sleep deprivation on murine immune functions.

We aimed to evaluate the effect of paradoxical sleep deprivation on the cellular migration during inflammation, the peritoneal macrophage phenotype and the infectious stimulus outcomes. A/J mice were inoculated with thioglycollate and exposed to paradoxical sleep deprivation. Sleep-deprived animals presented decreased cell migration compared to controls. Nitric oxide production was reduced in macrophages from sleep-deprived mice compared to controls. Cell surface analysis showed that sleep deprivation reduced F4/80(+)/CD80(low) peritoneal cell population induced by thioglycollate injection. Sleep-deprived mice were not more susceptible to infection than control mice. Our findings challenge the general perception that sleep loss always increases infection susceptibility.

Protein malnutrition alters spleen cell proliferation and IL-2 and IL-10 production by affecting the STAT-1 and STAT-3 balance.

Protein malnutrition (PM) is an important public health problem that affects resistance to infection by impairing a number of physiological processes. PM induces structural changes in the lymphoid organs that affect the roles of the immune and inflammatory responses in a crucial way. The activation of different transcription factors, including signal transducer and activator of transcription (STAT) family members, leads to the production of different cytokines, which are mediators essential to mounting adequate immune and inflammatory responses. In this study, malnourished animals presented anemia, leukopenia, and a severe reduction in spleen cellularity, with reduced numbers of most cell populations, as well as increased percentages of CD3(+) and CD4(+) cells. The proliferation rates were reduced, and cells were increasingly observed in the G0/G1 cell cycle phase; further, IL-2 production was reduced, while IL-10 production was increased. In spleen cells from malnourished animals, STAT-3 protein expression was increased, with a concomitant reduction in STAT-1 expression. Knowing that STAT-1 and STAT-3 are key transcription factors in both immunity and inflammatory pathways, these results infer, at least in part, a mechanistic pathway that affects the manner or intensity of the immune response in malnourished individuals, increasing susceptibility to infection.

Tempol, an intracellular antioxidant, inhibits tissue factor expression, attenuates dendritic cell function, and is partially protective in a murine model of cerebral malaria.

BACKGROUND: The role of intracellular radical oxygen species (ROS) in pathogenesis of cerebral malaria (CM) remains incompletely understood. METHODS AND FINDINGS: We undertook testing Tempol--a superoxide dismutase (SOD) mimetic and pleiotropic intracellular antioxidant--in cells relevant to malaria pathogenesis in the context of coagulation and inflammation. Tempol was also tested in a murine model of CM induced by Plasmodium berghei Anka infection. Tempol was found to prevent transcription and functional expression of procoagulant tissue factor in endothelial cells (ECs) stimulated by lipopolysaccharide (LPS). This effect was accompanied by inhibition of IL-6, IL-8, and monocyte chemoattractant protein (MCP-1) production. Tempol also attenuated platelet aggregation and human promyelocytic leukemia HL60 cells oxidative burst. In dendritic cells, Tempol inhibited LPS-induced production of TNF-α, IL-6, and IL-12p70, downregulated expression of co-stimulatory molecules, and prevented antigen-dependent lymphocyte proliferation. Notably, Tempol (20 mg/kg) partially increased the survival of mice with CM. Mechanistically, treated mice had lowered plasma levels of MCP-1, suggesting that Tempol downmodulates EC function and vascular inflammation. Tempol also diminished blood brain barrier permeability associated with CM when started at day 4 post infection but not at day 1, suggesting that ROS production is tightly regulated. Other antioxidants-such as α-phenyl N-tertiary-butyl nitrone (PBN; a spin trap), MnTe-2-PyP and MnTBAP (Mn-phorphyrin), Mitoquinone (MitoQ) and Mitotempo (mitochondrial antioxidants), M30 (an iron chelator), and epigallocatechin gallate (EGCG; polyphenol from green tea) did not improve survival. By contrast, these compounds (except PBN) inhibited Plasmodium falciparum growth in culture with different IC50s. Knockout mice for SOD1 or phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (gp91(phox-/-)) or mice treated with inhibitors of SOD (diethyldithiocarbamate) or NADPH oxidase (diphenyleneiodonium) did not show protection or exacerbation for CM. CONCLUSION: Results with Tempol suggest that intracellular ROS contribute, in part, to CM pathogenesis. Therapeutic targeting of intracellular ROS in CM is discussed.

Effects of Aedes aegypti salivary components on dendritic cell and lymphocyte biology.

BACKGROUND: Saliva is a key element of interaction between hematophagous mosquitoes and their vertebrate hosts. In addition to allowing a successful blood meal by neutralizing or delaying hemostatic responses, the salivary cocktail is also able to modulate the effector mechanisms of host immune responses facilitating, in turn, the transmission of several types of microorganisms. Understanding how the mosquito uses its salivary components to circumvent host immunity might help to clarify the mechanisms of transmission of such pathogens and disease establishment. METHODS: Flow cytometry was used to evaluate if increasing concentrations of A. aegypti salivary gland extract (SGE) affects bone marrow-derived DC differentiation and maturation. Lymphocyte proliferation in the presence of SGE was estimated by a colorimetric assay. Western blot and Annexin V staining assays were used to assess apoptosis in these cells. Naïve and memory cells from mosquito-bite exposed mice or OVA-immunized mice and their respective controls were analyzed by flow cytometry. RESULTS: Concentration-response curves were employed to evaluate A. aegypti SGE effects on DC and lymphocyte biology. DCs differentiation from bone marrow precursors, their maturation and function were not directly affected by A. aegypti SGE (concentrations ranging from 2.5 to 40 µg/mL). On the other hand, lymphocytes were very sensitive to the salivary components and died in the presence of A. aegypti SGE, even at concentrations as low as 0.1 µg/mL. In addition, A. aegypti SGE was shown to induce apoptosis in all lymphocyte populations evaluated (CD4+ and CD8+ T cells, and B cells) through a mechanism involving caspase-3 and caspase-8, but not Bim. By using different approaches to generate memory cells, we were able to verify that these cells are resistant to SGE effects. CONCLUSION: Our results show that lymphocytes, and not DCs, are the primary target of A. aegypti salivary components. In the presence of A. aegypti SGE, naïve lymphocyte populations die by apoptosis in a caspase-3- and caspase-8-dependent pathway, while memory cells are selectively more resistant to its effects. The present work contributes to elucidate the activities of A. aegypti salivary molecules on the antigen presenting cell-lymphocyte axis and in the biology of these cells.

Activation of PAF-receptor induces regulatory dendritic cells through PGE2 and IL-10.

Activation of the platelet-activating factor receptor (PAFR) in macrophages is associated with suppressor phenotype. Here, we investigated the PAFR in murine dendritic cells (DC). Bone marrow-derived dendritic cells (BALB/c) were cultured with GM-CSF and maturation was induced by LPS. The PAFR antagonists (WEB2086, WEB2170, PCA4248) and the prostaglandin (PG) synthesis inhibitors (indomethacin, nimesulide and NS-398) were added before LPS. Mature and immature DCs expressed PAFR. LPS increased MHCII, CD40, CD80, CD86, CCR7 and induced IL-10, IL-12, COX-2 and PGE2 expression. IL-10, COX-2 and PGE2 levels were reduced by PAFR antagonists and increased by cPAF. The IL-10 production was independent of PGs. Mature DCs induced antigen-specific lymphocyte proliferation. PAFR antagonists or PG-synthesis inhibitors significantly increased lymphocyte proliferation. It is proposed that PAF has a central role in regulatory DC differentiation through potentiation of IL-10 and PGE2 production.