Relevance of Thymic Stromal Lymphopoietin on the Pathogenesis of Glioblastoma: Role of the Neutrophil.

Glioblastoma multiforme (GBM) is the most predominant and malignant primary brain tumor in adults. Thymic stromal lymphopoietin (TSLP), a cytokine primarily generated by activated epithelial cells, has recently garnered attention in cancer research. This study was aimed to elucidate the significance of TSLP in GBM cells and its interplay with the immune system, particularly focused on granulocyte neutrophils. Our results demonstrate that the tumor produces TSLP when stimulated with epidermal growth factor (EGF) in both the U251 cell line and the GBM biopsy (GBM-b). The relevance of the TSLP function was evaluated using a 3D spheroid model. Spheroids exhibited increased diameter, volume, and proliferation. In addition, TSLP promoted the generation of satellites surrounding the main spheroids and inhibited apoptosis in U251 treated with temozolomide (TMZ). Additionally, the co-culture of polymorphonuclear (PMN) cells from healthy donors with the U251 cell line in the presence of TSLP showed a reduction in apoptosis and an increase in IL-8 production. TSLP directly inhibited apoptosis in PMN from GBM patients (PMN-p). Interestingly, the vascular endothelial growth factor (VEGF) production was elevated in PMN-p compared with PMN from healthy donors. Under these conditions, TSLP also increased VEGF production, in PMN from healthy donors. Moreover, TSLP upregulated programed death-ligand 1 (PDL-1) expression in PMN cultured with U251. On the other hand, according to our results, the analysis of RNA-seq datasets from Illumina HiSeq 2000 sequencing platform performed with TIMER2.0 webserver demonstrated that the combination of TSLP with neutrophils decreases the survival of the patient. In conclusion, our results position TSLP as a possible new growth factor in GBM and indicate its modulation of the tumor microenvironment, particularly through its interaction with PMN.

Inhibition of hyperprogressive cancer disease induced by immune-checkpoint blockade upon co-treatment with meta-tyrosine and p38 pathway inhibitor.

BACKGROUND: Although immune-checkpoint inhibitors (ICI) are overall promissory for cancer treatment, they entail, in some cases, an undesired side-effect called hyperprogressive-cancer disease (HPD) associated with acceleration of tumor growth and shortened survival. METHODS: To understand the mechanisms of HPD we assayed the ICI therapy on two murine tumors widely different regarding immunogenicity and, subsequently, on models of local recurrences and metastases of these tumors. To potentiate the immune response (IR), we combined ICI with meta-tyrosine-that counteracts immune-suppressive signals-and a selective inhibitor of p38 pathway that proved to counteract the phenomenon of tumor-immunostimulation. RESULTS: ICI were therapeutically effective against both tumor models (proportionally to their immunogenicity) but only when they faced incipient tumors. In contrast, ICI produced acceleration of large and residual tumors. The combined treatment strongly inhibited the growth of large tumors and it managed to cure 80% of mice with local recurrences and 60% of mice bearing residual metastases. CONCLUSIONS: Tumor enhancement was paradoxically correlated to a weak increase of the antitumor IR suggesting that a weak IR - different from a strong tumor-inhibitory one-may produce stimulation of tumor growth, mimicking the HPD observed in some clinical settings.

Differential Outcome between BALB/c and C57BL/6 Mice after Escherichia coli O157:H7 Infection Is Associated with a Dissimilar Tolerance Mechanism.

Enterohemorrhagic Escherichia coli (EHEC) infections can result in a wide range of clinical presentations despite that EHEC strains belong to the O157:H7 serotype, one of the most pathogenic forms. Although pathogen virulence influences disease outcome, we emphasize the concept of host-pathogen interactions, which involve resistance or tolerance mechanisms in the host that determine total host fitness and bacterial virulence. Taking advantage of the genetic differences between mouse strains, we analyzed the clinical progression in C57BL/6 and BALB/c weaned mice infected with an E. coli O157:H7 strain. We carefully analyzed colonization with several bacterial doses, clinical parameters, intestinal histology, and the integrity of the intestinal barrier, as well as local and systemic levels of antibodies to pathogenic factors. We demonstrated that although both strains had comparable susceptibility to Shiga toxin (Stx) and the intestinal bacterial burden was similar, C57BL/6 showed increased intestinal damage, alteration of the integrity of the intestinal barrier, and impaired renal function that resulted in increased mortality. The increased survival rate in the BALB/c strain was associated with an early specific antibody response as part of a tolerance mechanism.

Tolerogenic signals delivered by dendritic cells to T cells through a galectin-1-driven immunoregulatory circuit involving interleukin 27 and interleukin 10.

Despite their central function in orchestrating immunity, dendritic cells (DCs) can respond to inhibitory signals by becoming tolerogenic. Here we show that galectin-1, an endogenous glycan-binding protein, can endow DCs with tolerogenic potential. After exposure to galectin-1, DCs acquired an interleukin 27 (IL-27)-dependent regulatory function, promoted IL-10-mediated T cell tolerance and suppressed autoimmune neuroinflammation. Consistent with its regulatory function, galectin-1 had its highest expression on DCs exposed to tolerogenic stimuli and was most abundant from the peak through the resolution of autoimmune pathology. DCs lacking galectin-1 had greater immunogenic potential and an impaired ability to halt inflammatory disease. Our findings identify a tolerogenic circuit linking galectin-1 signaling, IL-27-producing DCs and IL-10-secreting T cells, which has broad therapeutic implications in immunopathology.

Characterization of a Nanovaccine Platform Based on an α1,2-Mannobiose Derivative Shows Species-non-specific Targeting to Human, Bovine, Mouse, and Teleost Fish Dendritic Cells.

Dendritic cells serve as the main immune cells that trigger the immune response. We developed a simple and cost-effective nanovaccine platform based on the α1',2-mannobiose derivative for dendritic cell targeting. In previous work, we have formulated the α1,2-mannobiose-based nanovaccine platform with plasmid DNA and tested it in cattle against BoHV-1 infection. There, we have shown that the dendritic cell targeting using this nanovaccine platform in vivo can boost the immunogenicity, resulting in a long-lasting immunity. In this work, we aim to characterize the α1',2-mannobiose derivative, which is key in the nanovaccine platform. This DC-targeting strategy takes advantage of the specific receptor known as DC-SIGN and exploits its capacity to bind α1,2-mannobiose that is present at terminal ends of oligosaccharides in certain viruses, bacteria, and other pathogens. The oxidative conjugation of α1',2-mannobiose to NH2-PEG2kDa-DSPE allowed us to preserve the chemical structure of the non-reducing mannose of the disaccharide and the OH groups and the stereochemistry of all carbons of the reducing mannose involved in the binding to DC-SIGN. Here, we show specific targeting to DC-SIGN of decorated micelles incubated with the Raji/DC-SIGN cell line and uptake of targeted liposomes that took place in human, bovine, mouse, and teleost fish DCs in vitro, by flow cytometry. Specific targeting was found in all cultures, demonstrating a species-non-specific avidity for this ligand, which opens up the possibility of using this nanoplatform to develop new vaccines for various species, including humans.

B. abortus RNA is the component involved in the down-modulation of MHC-I expression on human monocytes via TLR8 and the EGFR pathway.

Despite eliciting a potent CD8+ T cell response, Brucella abortus is able to persist and establish a chronic infection inside its host. We have previously reported that the infection of human monocytes/macrophages with B. abortus inhibits the IFN-γ-induced MHC-I cell surface expression down-modulating cytotoxic CD8+ T cell responses. MHC-I down-modulation depends on bacterial viability and results from the capacity of B. abortus to retain the MHC-I molecules within the Golgi apparatus. Furthermore, we recently demonstrated that epidermal growth factor receptor (EGFR) pathway is involved in this phenomenon and that this is an early event during infection. However, the components and mechanisms whereby B. abortus is able to down-modulate MHC-I remained to be elucidated. In this study we demonstrated that the down-modulation of MHC-I expression is not mediated by well-known Brucella virulence factors but instead by B. abortus RNA, a PAMP associated to viability (vita-PAMP). Surprisingly, completely degraded RNA was also able to inhibit MHC-I expression to the same extent as intact RNA. Accordingly, B. abortus RNA and its degradation products were able to mimic the MHC-I intracellular retention within the Golgi apparatus observed upon infection. We further demonstrated that TLR8, a single-stranded RNA and RNA degradation products sensor, was involved in MHC-I inhibition. On the other hand, neutralization of the EGFR reversed the MHC-I inhibition, suggesting a connection between the TLR8 and EGFR pathways. Finally, B. abortus RNA-treated macrophages display diminished capacity of antigen presentation to CD8+ T cells. Overall, our results indicate that the vita-PAMP RNA as well as its degradation products constitute novel virulence factors whereby B. abortus, by a TLR8-dependent mechanism and through the EGFR pathway, inhibits the IFN-γ-induced MHC-I surface expression on human monocytes/macrophages. Thus, bacteria can hide within infected cells and avoid the immunological surveillance of cytotoxic CD8+ T cells.

Modulation of Dendritic Cell Apoptosis and CD8+ Cytotoxicity by Histamine: Role of Protein Kinase C.

Dendritic cells (DC) are able to present extracellular antigens associated with the molecules of the major histocompatibility complex class I. In a previous work, we demonstrated that the histamine (HIS), acting through H1/H4 receptors, increases the cross-presentation of soluble ovalbumin by murine DC and can enhance the recruitment of specific CD8+ T lymphocytes during the development of chronic inflammatory responses. Here, we studied in more depth the mechanisms underlying this enhancement. We showed that the cytotoxicity of specific CD8+ lymphocytes is increased in HIS-treated DC and it is lost by inhibition of vacuolar-ATPase that prevents endosome acidification. It is known that HIS acts through G protein-coupled receptors. The H1/H4 receptors are associated with a Gq subunit, which involves PKC signaling, a pathway related to the apoptotic process. Interestingly, we demonstrated for the first time that HIS prevents DC apoptosis induced by heat shock through the inhibition of caspase-3, a mechanism dependent on PKC activation, since it is reversed by its inhibition. By contrast, cytolytic activity of T lymphocytes induced by HIS-stimulated DC was independent of PKC pathway.

Leukotriene C4 increases the susceptibility of adult mice to Shiga toxin-producing Escherichia coli infection.

Shiga toxin-producing Escherichia coli (STEC) is a food-borne pathogen that causes hemorrhagic colitis. Under some circumstances, Shiga toxin (Stx) produced within the intestinal tract enters the bloodstream, leading to systemic complications that may cause the potentially fatal hemolytic-uremic syndrome (HUS). Despite STEC human infection is characterized by acute inflammation of the colonic mucosa, little is known regarding the role of proinflammatory mediators like cysteine leukotrienes (cysLTs) in this pathology. Thus, the aim of this work was to analyze whether leukotriene C4 (LTC4) influences STEC pathogenesis in mice. We report that exogenous LTC4 pretreatment severely affected the outcome of STEC gastrointestinal infection. LTC4-pretreated (LTC4+) and STEC-infected (STEC+) mice showed an increased intestinal damage by histological studies, and a decreased survival compared to LTC4-non-pretreated (LTC4-) and STEC+ mice. LTC4+/STEC+ mice that died after the infection displayed neutrophilia and high urea levels, indicating that the cause of death was related to Stx2-toxicity. Despite the differences observed in the survival between LTC4+ and LTC4- mice after STEC infection, both groups showed the same survival after Stx2-intravenous inoculation. In addition, LTC4 pretreatment increased the permeability of mucosal intestinal barrier, as assessed by FITC-dextran absorption experiments. Altogether these results suggest that LTC4 detrimental effect on STEC infection is related to the increased passage of pathogenic factors to the bloodstream. Finally, we showed that STEC infection per se increases the endogenous LTC4 levels in the gut, suggesting that this inflammatory mediator plays a role in the pathogenicity of STEC infection in mice, mainly by disrupting the mucosal epithelial barrier.

Elevated glycolytic metabolism of monocytes limits the generation of HIF1A-driven migratory dendritic cells in tuberculosis.

During tuberculosis (TB), migration of dendritic cells (DCs) from the site of infection to the draining lymph nodes is known to be impaired, hindering the rapid development of protective T-cell-mediated immunity. However, the mechanisms involved in the delayed migration of DCs during TB are still poorly defined. Here, we found that infection of DCs with Mycobacterium tuberculosis (Mtb) triggers HIF1A-mediated aerobic glycolysis in a TLR2-dependent manner, and that this metabolic profile is essential for DC migration. In particular, the lactate dehydrogenase inhibitor oxamate and the HIF1A inhibitor PX-478 abrogated Mtb-induced DC migration in vitro to the lymphoid tissue-specific chemokine CCL21, and in vivo to lymph nodes in mice. Strikingly, we found that although monocytes from TB patients are inherently biased toward glycolysis metabolism, they differentiate into poorly glycolytic and poorly migratory DCs compared with healthy subjects. Taken together, these data suggest that because of their preexisting glycolytic state, circulating monocytes from TB patients are refractory to differentiation into migratory DCs, which may explain the delayed migration of these cells during the disease and opens avenues for host-directed therapies for TB.

Nasal immunization with H7 flagellin protects mice against hemolytic uremic syndrome secondary to Escherichia coli O157:H7 gastrointestinal infection.

Introduction: Shiga-toxin (Stx) producing Escherichia coli (STEC) O157:H7 is the most frequent serotype associated with hemolytic uremic syndrome (HUS) after gastrointestinal infections. Protection against HUS secondary to STEC infections has been experimentally assayed through the generation of different vaccine formulations. With focus on patients, the strategies have been mainly oriented to inhibit production of Stx or its neutralization. However, few approaches have been intended to block gastrointestinal phase of this disease, which is considered the first step in the pathogenic cascade of HUS. The aim of this work was to assay H7 flagellin as a mucosal vaccine candidate to prevent the systemic complications secondary to E. coli O157:H7 infections. Materials and methods: The cellular and humoral immune response after H7 nasal immunization in mice were studied by the analysis of systemic and intestinal specific antibody production, as well as cytokine production and lymphocyte proliferation against H7 flagellin ex vivo. Results: Immunized mice developed a strong and specific anti-H7 IgG and IgA response, at systemic and mucosal level, as well as a cellular Th1/Th2/Th17 response. H7 induced activation of bone marrow derived dendritic cells in vitro and a significant delayed-type hypersensitivity (DTH) response in immunized mice. Most relevant, immunized mice were completely protected against the challenge with an E. coli O157:H7 virulent strain in vivo, and surviving mice presented high titres of anti-H7 and Stx antibodies. Discussion: These results suggest that immunization avoids HUS outcome and allows to elicit a specific immune response against other virulence factors.

Venetoclax resistance induced by activated T cells can be counteracted by sphingosine kinase inhibitors in chronic lymphocytic leukemia.

The treatment of chronic lymphocytic leukemia (CLL) patients with venetoclax-based regimens has demonstrated efficacy and a safety profile, but the emergence of resistant cells and disease progression is a current complication. Therapeutic target of sphingosine kinases (SPHK) 1 and 2 has opened new opportunities in the treatment combinations of cancer patients. We previously reported that the dual SPHK1/2 inhibitor, SKI-II enhanced the in vitro cell death triggered by fludarabine, bendamustine or ibrutinib and reduced the activation and proliferation of chronic lymphocytic leukemia (CLL) cells. Since we previously showed that autologous activated T cells from CLL patients favor the activation of CLL cells and the generation of venetoclax resistance due to the upregulation of BCL-XL and MCL-1, we here aim to determine whether SPHK inhibitors affect this process. To this aim we employed the dual SPHK1/2 inhibitor SKI-II and opaganib, a SPHK2 inhibitor that is being studied in clinical trials. We found that SPHK inhibitors reduce the activation of CLL cells and the generation of venetoclax resistance induced by activated T cells mainly due to a reduced upregulation of BCL-XL. We also found that SPHK2 expression was enhanced in CLL cells by activated T cells of the same patient and the presence of venetoclax selects resistant cells with high levels of SPHK2. Of note, SPHK inhibitors were able to re-sensitize already resistant CLL cells to a second venetoclax treatment. Our results highlight the therapeutic potential of SPHK inhibitors in combination with venetoclax as a promising treatment option for the patients.

Structure-anti-leukemic activity relationship study of ortho-dihydroxycoumarins in U-937 cells: key role of the δ-lactone ring in determining differentiation-inducing potency and selective pro-apoptotic action.

Previous studies indicated the need of at least one phenolic hydroxyl group in the coumarin core for induction of cytotoxicity in different cell lines. Herein, we present an exhaustive structure-activity relationship study including ortho-dihydroxycoumarins (o-DHC) derivatives, cinnamic acid derivatives (as open-chain coumarin analogues) and 1,2-pyrones (representative of the δ-lactone ring of the coumarin core), carried out to further identify the structural features of o-DHC required to induce leukemic cell differentiation and apoptosis in U-937 cells. Our results show for the first time that the δ-lactone ring positively influences the aforementioned biological effects, by conferring greater potency to compounds with an intact coumarin nucleus. Most tellingly, we reveal herein the crucial role of this molecular portion in determining the selective toxicity that o-DHC show for leukemic cells over normal blood cells. From a pharmacological perspective, our findings point out that o-DHC may be useful prototypes for the development of novel chemotherapeutic agents.

Mechanisms of unconventional CD8 Tc2 lymphocyte induction in allergic contact dermatitis: Role of H3/H4 histamine receptors.

Histamine (HA) is a potent mediator that plays a central role in inflammation and allergy, acting through four G-protein-coupled receptors (i.e. H1-H4). HA is an accepted promoter of type 2 immunity in CD4+ T cells during hypersensitivity. Previously, we demonstrated that HA can promote antigen cross-presentation, inducing the activation of antigen-specific CD8+ T cells in an asthmatic murine model. Non-classical CD8+ T-cell profiles, such as Tc2 or Tc17, are associated with allergic disease persistence and chronicity. In this paper, we focus on the role of the H3 receptor (H3R) and the H4 receptor (H4R) in the development of allergic contact dermatitis. We were able to show that induction of the type 2 profiles associated with interleukin 13 production, both by CD4 and CD8 lymphocytes, depend on the interaction of HA with H3R and H4R. Blocking both receptors using the selective H3/H4 receptor antagonist thioperamide or the selective H4R ligand JNJ777120 reduces the inflammatory response, inducing an immunosuppressive profile associated with the increased proportion of FOXp3+ regulatory T lymphocytes and CD11b+Gr-1+ myeloid suppressor cells. Interestingly, in dendritic cells, only H4R blockade, and not H3R blockade, is capable of modulating most of the inflammatory effects observed in our model.

Leukotriene C(4) prevents the complete maturation of murine dendritic cells and modifies interleukin-12/interleukin-23 balance.

Leukotriene C(4) is an important mediator in the development of inflammatory reactions and ischaemia. Previous studies have shown that leukotriene C(4) is able to modulate the function of dendritic cells (DCs) and induce their chemotaxis from skin to lymph node. In this study, we decided to evaluate the modulation exerted by leukotriene C(4) on DCs, depending on their status of activation. We showed for the first time that leukotriene C(4) stimulates endocytosis both in immature and lipopolysaccharide (LPS) -activated DCs. Moreover, it suppressed the interleukin-12p70 (IL-12p70) release, but induces the secretion of IL-23 by DCs activated with LPS and promotes the expansion of T helper type 17 (Th17) lymphocytes. Furthermore, blocking the release of IL-23 reduced the percentages of CD4(+) T cells producing IL-17 in a mixed lymphocyte reaction. Ours results suggest that leukotriene C(4) interferes with the complete maturation of inflammatory DCs in terms of phenotype and antigen uptake, while favouring the release of IL-23, the main cytokine involved in the maintenance of the Th17 profile.

An Improved DNA Vaccine Against Bovine Herpesvirus-1 Using CD40L and a Chemical Adjuvant Induces Specific Cytotoxicity in Mice.

Bovine herpesvirus-1 (BoHV-1) uses many mechanisms to elude the immune system; one of them is spreading intracellularly, even in the presence of specific antiviral antibodies. Cytotoxic T lymphocytes (CTLs) are necessary to eliminate the virus. The main preventive strategy is vaccination based on inactivated virus. These vaccines are poor inducers of cellular immune responses, and complicate serological diagnosis and determination of the real prevalence of infection. DNA vaccines are a good option because of the capacity of Differentiating Infected from Vaccinated Animals-(DIVA vaccine)-and may be the best way to induce cytotoxic responses. Although this type of vaccines leads to only weak "in vivo" expression and poor immune responses, incorporation of molecular and/or chemical adjuvants can improve the latter, both in magnitude and in direction. In this study, we have investigated the specific immune responses elicited in mice by DNA vaccines based on the BoHV-1 glycoprotein D (pCIgD) with and without two different adjuvants: a plasmid encoding for murine CD40L (pCD40L) or Montanide™ 1113101PR (101). Mice vaccinated with pCIgD+CD40L, pCIgD+101, and pCIgD+CD40L+101 developed significantly higher specific antibody titers against BoHV-1 than the pCIgD group (p < 0.01). The animals vaccinated with pCgD+pCD40L+101 raised significantly higher levels of IgG2a and IgG2b (p < 0.01 and p < 0.001, respectively) than mice vaccinated with pCIgD alone. On the contrary, when the activity of CTL against cells infected with BoHV-1 was measured, the vaccine pCgD+pCD40L+101 induced significantly higher levels of cytotoxicity activity (p < 0.001) than pCIgD alone. A significant increase in the CD4+ populations in the group receiving pCIgD+CD40L+101 in comparison with the pCIgD group was observed and, also, interferon gamma, interleukin (IL)-6, and IL-17A levels were higher. Considering the results obtained from this study for humoral and cellular responses in mice, the inclusion of pCD40L and 101 as adjuvants in a BoHV-1 DNA vaccine for cattle is highly recommendable.

Targeting Myeloid-Derived Suppressor Cells to Enhance a Trans-Sialidase-Based Vaccine Against Trypanosoma cruzi.

Trypanosoma cruzi (T. cruzi) is a hemoflagellate protozoan parasite that causes Chagas disease, a neglected tropical disease that affects more than 6 million people around the world, mostly in Latin America. Despite intensive research, there is no vaccine available; therefore, new approaches are needed to further improve vaccine efficacy. It is well established that experimental T. cruzi infection induces a marked immunosuppressed state, which includes notably increases of CD11b+ GR-1+ myeloid-derived suppressor cells (MDSCs) in the spleen, liver and heart of infected mice. We previously showed that a trans-sialidase based vaccine (TSf-ISPA) is able to confer protection against a virulent T. cruzi strain, stimulating the effector immune response and decreasing CD11b+ GR-1+ splenocytes significantly. Here, we show that even in the immunological context elicited by the TSf-ISPA vaccine, the remaining MDSCs are still able to influence several immune populations. Depletion of MDSCs with 5 fluorouracil (5FU) at day 15 post-infection notably reshaped the immune response, as evidenced by flow cytometry of spleen cells obtained from mice after 21 days post-infection. After infection, TSf-ISPA-vaccinated and 5FU-treated mice showed a marked increase of the CD8 response, which included an increased expression of CD107a and CD44 markers in CD8+ cultured splenocytes. In addition, vaccinated and MDSC depleted mice showed an increase in the percentage and number of CD4+ Foxp3+ regulatory T cells (Tregs) as well as in the expression of Foxp3+ in CD4+ splenocytes. Furthermore, depletion of MDSCs also caused changes in the percentage and number of CD11chigh CD8α+ dendritic cells as well as in activation/maturation markers such as CD80, CD40 and MHCII. Thus, the obtained results suggest that MDSCs not only play a role suppressing the effector response during T. cruzi infection, but also strongly modulate the immune response in vaccinated mice, even when the vaccine formulation has a significant protective capacity. Although MDSC depletion at day 15 post-infection did not ameliorated survival or parasitemia levels, depletion of MDSCs during the first week of infection caused a beneficial trend in parasitemia and mice survival of vaccinated mice, supporting the possibility to target MDSCs from different approaches to enhance vaccine efficacy. Finally, since we previously showed that TSf-ISPA immunization causes a slight but significant increase of CD11b+ GR-1+ splenocytes, here we also targeted those cells at the stage of immunization, prior to T. cruzi challenge. Notably, 5FU administration before each dose of TSf-ISPA vaccine was able to significantly ameliorate survival and decrease parasitemia levels of TSf-ISPA-vaccinated and infected mice. Overall, this work supports that targeting MDSCs may be a valuable tool during vaccine design against T. cruzi, and likely for other pathologies that are characterized by the subversion of the immune system.

Histamine-treated dendritic cells improve recruitment of type 2 CD8 T cells in the lungs of allergic mice.

Histamine controls the function of dendritic cells (DCs). It appears to be required for the normal development of DCs. It also induces the chemotaxis of immature DCs and promotes the differentiation of CD4(+) T cells into cells with a T helper type 2 (Th2) profile. Moreover, we have recently shown that histamine stimulates both the uptake and the cross-presentation of antigens by DCs, supporting the theory that histamine promotes activation of CD8(+) T cells during the development of allergic pathologies. Here, we investigated whether the course of an allergic response, in a well-defined model of ovalbumin (OVA)-induced allergic airway inflammation, could be modulated by intratracheal injection of OVA-pulsed DCs previously treated with histamine (DCHISs). Compared with control DCs, DCHISs induced: (i) greater recruitment of CD8(+) T cells in the lung, (ii) greater stimulation of the production of interleukin (IL)-5 by lung CD8(+) T cells, and (iii) increased recruitment of CD11c/CD8 double-positive DCs in the lungs of allergic mice. Moreover, mice treated with DCHISs showed increased levels of serum-specific immunoglobulin E (IgE) antibodies directed to OVA, and a higher proportion of eosinophils in bronchoalveolar lavage (BAL) compared with mice treated with OVA-pulsed control DCs. Our results support the notion that histamine, by acting on DCs, increases the severity of allergic processes.

Flagellin delays spontaneous human neutrophil apoptosis.

Neutrophils are short-lived cells that rapidly undergo apoptosis. However, their survival can be regulated by signals from the environment. Flagellin, the primary component of the bacterial flagella, is known to induce neutrophil activation. In this study we examined the ability of flagellin to modulate neutrophil apoptosis. Neutrophils cultured for 12 and 24 h in the presence of flagellin from Salmonella typhimurium at concentrations found in pathological situations underwent a marked prevention of apoptosis. In contrast, Helicobacter pylori flagellin did not affect neutrophil survival, suggesting that Salmonella flagellin exerts the antiapoptotic effect by interacting with TLR5. The delaying in apoptosis mediated by Salmonella flagellin was coupled to higher expression levels of the antiapoptotic protein Mcl-1 and lower levels of activated caspase-3. Analysis of the signaling pathways indicated that Salmonella flagellin induced the activation of the p38 and ERK1/2 MAPK pathways as well as the PI3K/Akt pathway. Furthermore, it also stimulated IkappaBalpha degradation and the phosphorylation of the p65 subunit, suggesting that Salmonella flagellin also triggers NF-kappaB activation. Moreover, the pharmacological inhibition of ERK1/2 pathway and NF-kappaB activation partially prevented the antiapoptotic effects exerted by flagellin. Finally, the apoptotic delaying effect exerted by flagellin was also evidenced when neutrophils were cultured with whole heat-killed S. typhimurium. Both a wild-type and an aflagellate mutant S. typhimurium strain promoted neutrophil survival; however, when cultured in low bacteria/neutrophil ratios, the flagellate bacteria showed a higher capacity to inhibit neutrophil apoptosis, although both strains showed a similar ability to induce neutrophil activation. Taken together, our results indicate that flagellin delays neutrophil apoptosis by a mechanism partially dependent on the activation of ERK1/2 MAPK and NF-kappaB. The ability of flagellin to delay neutrophil apoptosis could contribute to perpetuate the inflammation during infections with flagellated bacteria.

Impaired function of dendritic cells deficient in angiotensin II type 1 receptors.

Dendritic cells (DC) are highly specialized antigen-presenting cells with a unique ability to activate resting T lymphocytes and initiate primary immune responses. Angiotensin II (AII) is involved in key events of the inflammatory response. Because our previous work implicated an effect of AII on differentiation and function of murine and human DC, we investigated the impact of AII type 1 receptor (AT(1)) deficiency on the phenotypical and functional properties of mouse DC in vitro and in vivo. Bone marrow (BM) cells isolated from mice lacking AII subtype 1a receptor (AT(1a)), AII subtype 1b receptor (AT(1b)), or both receptor isoforms and control littermates [wild type (WT)] were cultured for 7 days in the presence of recombinant mouse granulocyte/macrophage colony-stimulating factor to generate myeloid DC in vitro. Generation of CD11c(+) cells was less efficient in both AT(1a)- and AT(1b)-deficient BM cells than in WT BM cell cultures. Moreover, DC generated from AT(1)-deficient progenitors showed lower levels of expression of major histocompatibility complex II (MHC-II) and CD11c (p < 0.01) and a marked reduction in their allostimulatory activity (p < 0.01 or 0.001). Although AT(1)-deficient DC released comparable levels of interleukin (IL)-10 and IL-12p70 to WT DC, they produced significantly lower levels of tumor necrosis factor alpha (TNF-alpha) (p < 0.05). Remarkably, CD11c(+) cells isolated from the spleen of AT(1) knockout mice challenged with lipopolysaccharide in vivo up-regulated MHC-II, CD40, and CD80 as did WT, but released significantly lower levels of TNF-alpha (p < 0.01). These data provide clear evidence that AT(1) controls differentiation and functionality of DC and thus may have a crucial impact on inflammatory processes where local angiotensinergic systems are known to be activated.