The influence of sex and gender on immunity, infection and vaccination.

Sex/gender significantly contribute to shape the immune responses, contributing to differences in the pathogenesis of infectious diseases in males and females, the response to viral vaccines and the prevalence of autoimmune diseases. Females typically develop higher innate, humoral and cellular immune responses to viral infections and in response to vaccine. At the same time, women are more prone to autoimmune diseases and experience more adverse reactions to vaccination. Hormonal, genetic and environmental factors between males and females may affect the immune responses and the sex-related outcome of vaccination. Knowledge of the mechanisms involved in sex disparity in immune responses will contribute to identify the ways to reduce adverse reactions in females and to improve the immune responses in males. This is necessary to adequately protect both sexes against the immune-mediated and infectious diseases with the long-term goal of personalizing the therapies for males and females.

ß2-Agonist clenbuterol hinders human monocyte differentiation into dendritic cells.

Clenbuterol (CLB) is a beta2-adrenergic agonist commonly used in asthma therapy, but is also a non-steroidal anabolic drug often abused in sport doping practices. Here we evaluated the in vitro impact of CLB on the physiology and function of human monocytes and dendritic cells (DCs), instrumental in the development of immune responses. We demonstrate that CLB inhibits the differentiation of monocytes into DCs and this effect is specific and dependent on ß2-adrenergic receptor (AR) activation. We found that CLB treatment reduced the percentage of CD1a(+) immature DCs, while increasing the frequency of monocytes retaining CD14 surface expression. Moreover, CLB inhibited tumor necrosis factor-alpha (TNF-alpha) enhanced IL-(interleukin)-10 and IL-6 production. In contrast, CLB did not modulate the phenotypic and functional properties of monocytes and DCs, such as the surface expression of HLA-DR, CD83, CD80 and CD86 molecules, cytokine production, immunostimulatory activity and phagocytic activity. Moreover, we found that CLB did not modulate the activation of NF-kB in DCs. Moreover, we found that the differentiation of monocytes into DCs was associated with a significant decrease of ß2-ARs mRNA expression. These results provide new insights on the effect of CLB on monocyte differentiation into DCs. Considering the frequent illegal use of CLB in doping, our work suggests that this drug is potentially harmful to immune responses decreasing the supply of DCs, thus subverting immune surveillance.

Two wheat decapeptides prevent gliadin-dependent maturation of human dendritic cells.

Celiac disease (CD) is a small intestinal enteropathy, triggered in susceptible individuals by the ingestion of dietary gluten. Dendritic cells (DC) are instrumental in the generation and regulation of immune responses and oversee intestinal immune homeostasis promoting and maintaining oral tolerance to food antigens. The aim of this study was to monitor the effect of peptic-tryptic digest of gliadin (PT-gliadin) on the maturation of human monocyte-derived DC and the impact of pDAV and pRPQ decapeptides in the modulation of PT-gliadin-induced phenotypic and functional DC maturation. Immature DC (iDC) were challenged in vitro with PT-gliadin. In some experiments iDC were pre-treated with pDAV or pRPQ and after 2h PT-gliadin was added to the cultures. We found that PT-gliadin up-regulates the expression of the maturation markers HLA-DR, CD83, CD80 and CD86. The functional consequence of PT-gliadin treatment of iDC is a significant increase in IL-12, TNF-alpha production as well as in their T cell stimulatory capacity. On the contrary, the digest of zein had no effect on DC maturation. Interestingly, we found that pre-treatment of iDC with pDAV or pRPQ decapeptides significantly prevents the functional maturation of DC induced by PT-gliadin. On the other hand, pDAV and pRPQ did not revert the PT-gliadin-induced phenotypic maturation of DC. Here we report, for the first time, that naturally occurring peptides are able to prevent the gliadin-dependent DC maturation. This finding could have implication for CD, raising the perspective of a potential therapeutic strategy alternative to a gluten free diet.

Does oxidative stress play a critical role in cardiovascular complications of Kawasaki disease?

The aim of the present work was to evaluate the contribution of the different reactive oxidizing species to systemic oxidative stress in the whole blood of patients with Kawasaki disease (KD). This is a rare generalized systemic vasculitis typical of the early childhood characterized by inflammation and endothelial dysfunction with a high risk for cardiovascular fatal events. We found that, compared to age-matched healthy donors, blood from KD patients showed increased production of oxygen- and nitrogen-derived species as detected by electron paramagnetic resonance (EPR) spin probing with the cyclic hydroxylamine 1-hydroxy-3-carboxy-pyrrolidine. The (•)NO pathway involvement was also confirmed by the decreased concentrations of the endogenous (•)NO synthase inhibitor asymmetric dimethyl-arginine and the increased amounts of 3-nitrotyrosine in plasma. Further, increased plasma yields of the proinflammatory enzyme myeloperoxidase were also observed. The appearance of circulating red blood cell alterations typically associated with oxidative imbalance and premature aging (e.g., decrease of total thiol content, glycophorin A, and CD47 expression, as well as increase of phosphatidylserine externalization) has also been detected. Collectively, our observations lead to hypothesize that the simultaneous oxidative and nitrative stress occurrence in the blood of KD patients may play a pathogenetic role in the cardiovascular complications often associated with this rare disease.

Exogenous HIV-1 Nef upsets the IFN-γ-induced impairment of human intestinal epithelial integrity.

BACKGROUND: The mucosal tissues play a central role in the transmission of HIV-1 infection as well as in the pathogenesis of AIDS. Despite several clinical studies reported intestinal dysfunction during HIV infection, the mechanisms underlying HIV-induced impairments of mucosal epithelial barrier are still unclear. It has been postulated that HIV-1 alters enterocytic function and HIV-1 proteins have been detected in several cell types of the intestinal mucosa. In the present study, we analyzed the effect of the accessory HIV-1 Nef protein on human epithelial cell line. METHODOLOGY/PRINCIPAL FINDINGS: We used unstimulated or IFN-γ-stimulated Caco-2 cells, as a model for homeostatic and inflamed gastrointestinal tracts, respectively. We investigated the effect of exogenous recombinant Nef on monolayer integrity analyzing its uptake, transepithelial electrical resistance, permeability to FITC-dextran and the expression of tight junction proteins. Moreover, we measured the induction of proinflammatory mediators. Exogenous Nef was taken up by Caco-2 cells, increased intestinal epithelial permeability and upset the IFN-γ-induced reduction of transepithelial resistance, interfering with tight junction protein expression. Moreover, Nef inhibited IFN-γ-induced apoptosis and up-regulated TNF-α, IL-6 and MIP-3α production by Caco-2 cells while down-regulated IL-10 production. The simultaneous exposure of Caco-2 cells to Nef and IFN-γ did not affect cytokine secretion respect to untreated cells. Finally, we found that Nef counteracted the IFN-γ induced arachidonic acid cascade. CONCLUSION/SIGNIFICANCE: Our findings suggest that exogenous Nef, perturbing the IFN-γ-induced impairment of intestinal epithelial cells, could prolong cell survival, thus allowing for accumulation of viral particles. Our results may improve the understanding of AIDS pathogenesis, supporting the discovery of new therapeutic interventions.

Effects of HIV-1 Nef on virus co-receptor expression and cytokine release in human bladder, laryngeal, and intestinal epithelial cell lines.

HIV infections are mainly acquired by mucosal transmission, through oral, rectal, or genital mucosa. Epithelial cells (EC) are the first cells encountered by HIV during infection through sexual transmission and breastfeeding. EC express several receptors critical for both primary HIV infection and secondary transmission. The regulation of co-receptor expression correlates with changes in susceptibility to infection by HIV-1 strains with different tropism. Moreover, inflammatory responses at mucosal surfaces after HIV-1 transmission may influence disease outcome. In the present study, we analyzed the effect of the accessory HIV-1 Nef protein on mucosal EC, using unstimulated or IFN-γ-stimulated HEp-2, T24, and Caco2 cell lines as models for homeostatic or inflamed mucosal tracts. We found that Nef significantly upregulated the expression of CXCR4 on the Caco-2 cell surface and the expression of galactosylceramide on the T24 cell surface. In addition, Nef significantly upregulated IL-6 production by T24 and Caco-2 cells, and TNF-α release by all three cell lines analyzed. Notably, Nef abrogated the IFN-γ-induced modulation of co-receptor expression and cytokine secretion. Our findings suggest that Nef differently regulates co-receptor expression and cytokine secretion at the epithelial level, depending on the anatomical derivation of the cells and the inflammatory status.

Docosahexaenoic acid modulates in vitro the inflammation of celiac disease in intestinal epithelial cells via the inhibition of cPLA2.

BACKGROUND & AIMS: The cytosolic phospolypase A(2) (cPLA(2)) - dependent release of arachidonic acid (AA) from the intra-epithelial lymphocytes plays a pivotal role in arming lymphocytes to cytolysis in the immune response of celiac disease. However, little is known about the role of enterocytes in releasing AA. Docosahexaenoic acid (DHA) is a long chain polyunsaturated fatty acid that counteracts many of the proinflammatory effect of AA. The aims of the present work were to evaluate if: 1) intestinal epithelial cells have a role in the celiac inflammation, releasing AA, and 2) if DHA is able to modulate the celiac inflammation, down-regulating the release of AA. METHODS: A human intestinal epithelial cell line (Caco-2) was exposed to gliadin peptides (PT-gl) (500 µg/ml) and DHA (2 µg/ml), both alone and simultaneously up to 24 h. RESULTS: The exposure of those cells to PT-gl alone resulted in an increased AA release, cycloxygenase-2 expression, cPLA(2) activity and prostaglandin E(2) and interleukin-8 release in culture medium, whereas the simultaneous exposure of the cells to DHA and PT-gl prevented the above-mentioned increases. CONCLUSIONS: These results suggest that intestinal epithelial cells sustain the celiac inflammation, releasing AA when stimulated with gliadin and that DHA inhibits the AA release by these cells.

Oxidative stress and defective platelet apoptosis in naïve patients with Kawasaki disease.

Kawasaki disease (KD) is a rare and often undiagnosed disease, at least in the western countries. It is characterized by an inflammatory acute febrile vasculitis of medium sized arteries with a propensity to damage the coronary arteries. It normally occurs in the early childhood and the diagnosis is based on clinical symptoms. During the progression of the disease thrombocytosis is usually detected. This can exert a pathogenetic role in the cardiovascular complications occurring in KD. In the present work peripheral blood plasma and platelets from twelve naïve patients with KD were analyzed in order to detect possible pathogenetic determinants or progression markers. Morphological, biochemical and flow cytometrical methods have been used. With respect to age-matched healthy donors, we found an increase of platelet activation markers, i.e. degranulation, phosphatidylserine (PS) externalization and leukocyte-red cell-platelet aggregates. Some significant alterations that could represent suitable diagnostic determinants have also been detected in patient plasma: (i) decreased antioxidant power, (ii) decreased levels of asymmetric dymethylarginine (ADMA), a naturally occurring chemical interfering with the production of nitric oxide, and (iii) increased levels of soluble P-Selectin and soluble annexin V. Since PS externalizing platelets are known to exert a pro-coagulant activity, our data suggest the hypothesis that increased risk of vascular complications in KD could depend on platelet stimulation and defective apoptosis probably related to nitrosative stress.

Immunoregulatory effects of HIV-1 Nef protein.

During HIV infection, the perturbation of the adaptive and innate immune responses contributes to the progressive immunosuppression leading to an increased susceptibility to opportunistic infections and neoplastic diseases. Several impairments observed in HIV-infected patients include a gradual loss of CD4(+) T cells, CD8(+) T cell dysfunction, and a decreased number and function of natural killer (NK) cells. Moreover, a functional impairment and variation in the number of DC and B cells were observed during HIV infection. HIV-1 codes for proteins, including the accessory Nef proteins, that interacting with immune cells may contribute to AIDS pathogenesis. Here, we review the recent progress on the immunomodulatory effect of the accessory Nef protein and its role in the pathogenesis of HIV-1 infection. (c) 2009 International Union of Biochemistry and Molecular Biology, Inc.

Leptin exerts an anti-apoptotic effect on human dendritic cells via the PI3K-Akt signaling pathway.

Leptin is an adipocyte-derived hormone/cytokine that modulates immune responses. It induces functional and morphological changes in human dendritic cells (DCs), licensing them towards Th1 priming and promoting DC survival. Here we found that leptin protects DCs from spontaneous, UVB and H(2)O(2)-induced apoptosis, by triggering the activation of nuclear factor-kappa B (NF-kappaB) and a parallel up-regulation of bcl-2 and bcl-XL gene expression and Akt activation. We found that leptin activates the PI3K-Akt signaling pathway as demonstrated by the suppression of the effect of leptin on DC survival by wortmannin and API-2, which suppress the leptin-induced activation of Akt, NF-kappaB, bcl-2, bcl-XL and protection from apoptosis. These results provide insights on the immunoregulatory function of leptin, supporting a potential application in immunotherapeutic approaches.

Leptin as an immunological adjuvant: enhanced migratory and CD8+ T cell stimulatory capacity of human dendritic cells exposed to leptin.

Leptin is an adipocyte-derived hormone/cytokine that links nutrition, metabolism, and immune homeostasis and is endowed to modulate several immune responses. We previously demonstrated that both immature and mature human dendritic cells (DCs) express a functional leptin receptor, and we found that leptin activates DCs, licenses them for Th1 priming, and promotes DC survival. Moreover, we found that leptin induces rearrangement of actin microfilaments, leading to uropod and ruffle formation. Here we monitor the effects of leptin on DC migratory capacities, focusing on the intracellular signaling driving cytoskeleton rearrangement. We found that leptin increases immature DC migratory performance both by favoring cytoskeleton dynamics and by up-regulating CCR7 surface expression, thus favoring chemotactic responsiveness. We found that in immature DCs, leptin activates cofilin, favoring the turnover of actin microfilaments, and, by triggering Vav phosphorylation, promotes Rac1 activation. Finally, we found that in immature DCs, leptin up-regulates interleukin-12p70 production on CD40 stimulation and, more importantly, increases their capacity to stimulate activation of autologous CD8(+) T cells. Taken altogether, the findings herein highlight the potential use of leptin as an adjuvant tool in vaccination protocols employing ex vivo-generated autologous DCs.

Cytotoxic necrotizing factor 1 prevents apoptosis via the Akt/IkappaB kinase pathway: role of nuclear factor-kappaB and Bcl-2.

Cytotoxic necrotizing factor 1 (CNF1) is a protein toxin produced by some pathogenic strains of Escherichia coli that specifically activates Rho, Rac, and Cdc42 GTPases. We previously reported that this toxin prevents the ultraviolet-B-induced apoptosis in epithelial cells, with a mechanism that remained to be defined. In this work, we show that the proteasomal degradation of the Rho GTPase is necessary to achieve cell death protection, because inhibition of Rho degradation abolishes the prosurvival activity of CNF1. We hypothesize that Rho inactivation allows the activity of Rac to become dominant. This in turn leads to stimulation of the phosphoinositide 3-kinase/Akt/IkappaB kinase/nuclear factor-kappaB prosurvival pathway and to a remarkable modification in the architecture of the mitochondrial network, mainly consisting in the appearance of elongated and interconnected mitochondria. Importantly, we found that Bcl-2 silencing reduces the ability of CNF1 to protect cells against apoptosis and that it also prevents the CNF1-induced mitochondrial changes. It is worth noting that the ability of a bacterial toxin to induce such a remodeling of the mitochondrial network is herein reported for the first time. The possible pathophysiological relevance of this finding is discussed.

HIV-1 Nef impairs the dynamic of DC/NK crosstalk: different outcome of CD56(dim) and CD56(bright) NK cell subsets.

Dendritic cells (DCs) and natural killer (NK) cells are essential components of the innate immunity and play a critical role in the first phase of host defense against infection. Interactions between DCs and NK cells have been demonstrated in a variety of settings, with evidence emerging of complex bidirectional crosstalk between the two cell types. The accessory HIV-1 Nef protein is a crucial determinant for viral replication and pathogenesis. We previously demonstrated that Nef, hijacking DC functional activity, subverts the DC arm of immune response to escape the adaptive immune attack. Here, we monitor the effect of Nef on the outcome of the innate immune response, focusing on the impact of Nef on DC/NK crosstalk. We demonstrate that Nef up-regulates the ability of DCs to stimulate the immunoregulatory NK cells (CD56(bright)) as assessed by the activated phenotype, up-regulation of their proliferative response and INF-gamma release. On the other hand, Nef-pulsed DCs inhibit cytotoxic NK cells (CD56(dim)), as assessed by the reduced HLA-DR surface expression, reduced proliferation and cytotoxic activity. Moreover, in the presence of Nef-pulsed DCs, we found a significant up-regulation of TNF-alpha secretion and a significant reduction of IL-10, GM-CSF, MIP-1alpha and RANTES secretion. Our findings suggest that the Nef-induced dysregulation in the DC/NK cell crosstalk may represent a potential mechanism through which HIV escapes innate immune surveillance.

The immunoregulatory effects of HIV-1 Nef on dendritic cells and the pathogenesis of AIDS.

Dendritic cells (DC) play a crucial role in the generation and regulation of immunity, and their interaction with HIV is relevant in the pathogenesis of AIDS favoring both the initial establishment and spread of the infection and the development of antiviral immunity. HIV-1 Nef is an essential factor for efficient viral replication and pathogenesis, and several studies have been addressed to assess the possible influence of endogenous or exogenous Nef on DC biology. Our findings and other reported data described in this review demonstrate that Nef subverts DC biology interfering with phenotypical, morphological, and functional DC developmental programs, thus representing a viral tool underlying AIDS pathogenesis. This review provides an overview on the mechanism by which Nef, hijacking DC functional activity, may favor both the replication of HIV-1 and the escape from immune surveillance. Overall, the findings described here may contribute to the understanding of Nef function, mechanism of action, and cellular partners. Further elucidation of genes induced through Nef signaling in DC could reveal pathways used by DC to drive HIV spread and will be critical to identify therapeutic strategies to bias the DC system toward activation of antiviral immunity instead of facilitating virus dissemination.

Differentiation of monocyte-derived dendritic cells is associated with upregulation and activation of Rac-1 small GTPase.

Critical changes occurring in Rac-1 molecule, a cytoskeleton organizing small GTPase associated with cell ruffling, have been analyzed in dendritic cells (DCs) derived from monocytes cultured with granulocyte-macrophage colony-stimulating factor and IFN-alpha or IL-4. Although with different kinetics, both agents induced activation of Rac-1 molecule and, more importantly, an upregulation of both protein expression and mRNA transcription. These findings strengthen the role of Rac-1 molecule in the induction of DC differentiation and suggest that, besides its activation, the upregulation of Rac-1 molecule might also play a role in the acquisition of DC mature phenotype.

Impaired NK-cell-mediated cytotoxic activity and cytokine production in patients with endometriosis: a possible role for PCBs and DDE.

Endometriosis is a gynaecological disorder characterized by the presence and growth of endometrial tissue in ectopic sites. In this study we examined the immunological functions of patients with endometriosis and serum level of PCBs and p,p'-DDE to verify the impact of these environmental contaminants on the dysregulation of immune functions. We found that proliferative responses and immunoglobulin production were not dysregulated in patients with endometriosis while NK cell activity was significantly down-regulated in these patients. Moreover, a significant down-regulation of IL-1beta and IL-12 production was found in patients with respect to controls. Serum levels of PCBs and p,p'-DDE were found to be significantly higher in women with endometriosis than in the control group, with respect to the sum of the congeners most prominent in human tissues. In particular, total PCBs concentration in patients with endometriosis and controls was respectively 330 and 160 ng/g fat with respect to the most abundant congeners, while p,p'-DDE concentration was of 770 and 310 ng/g fat. Moreover, we found that normal human PBMC pulsed with PCBs, p,p'-DDE and their combination showed a significant down-regulation of NK cell cytotoxic activity and IL-1beta and IL-12 production. These findings suggest that changes in specific immune parameters correlate with elevated serum PCBs and DDE levels and endometriosis.

Mildly oxidized low-density lipoprotein inhibits the in vitro induction of the specific antibody response to Candida albicans.

Oxidized low-density lipoprotein plays a critical role in the pathogenesis of atherosclerosis and exerts pleiotropic effects on various cellular functions. The present study was designed to evaluate the effects of mildly oxidized LDL (mLDL) on the induction and regulation of an in vitro specific antibody response. We found that mLDL significantly inhibited the induction of the anti-Candida albicans antibody response by human peripheral blood mononuclear cells (PBMC). mLDL-induced down-regulation of antibody production was abrogated by blocking the major receptors that bind and internalize modified LDL. In the mLDL-treated C. albicans-stimulated PBMC cultures an early increase in IL-1beta production was observed and the addition of anti-IL-1beta antibody abrogated the mLDL-induced inhibitory effect. Moreover, the addition of IL-1beta to the cultures inhibited the induction of the specific antibody response, similar to mLDL. On the other hand, mLDL up-regulated PWM-induced polyclonal immunoglobulin (Ig) production. In the same cultures IgM anti-mLDL was found. These results indicate that the up-regulation of IL-1beta production induced by mLDL may be involved in the hindering of B cell function, i.e., specific antibody production. This could be relevant in the pathogenesis of inflammatory diseases such as atherosclerosis.

Leptin promotes differentiation and survival of human dendritic cells and licenses them for Th1 priming.

Leptin is an adipocyte-derived hormone/cytokine that links nutrition, metabolism, and immune homeostasis. Leptin is capable of modulating several immune responses. However, the effect of leptin on dendritic cells (DCs) has not yet been recognized. Because DCs are instrumental in the development of immune responses, in this study, we evaluated the impact of leptin on DC activation. We demonstrated the presence of leptin receptor in human immature and mature DCs both at mRNA and protein level and its capacity to transduce leptin signaling leading to STAT-3 phosphorylation. We found no consistent modulation of DC surface molecules known to be critical for their APC function in response to leptin. In contrast, we found that leptin induces rearrangement of actin microfilaments, leading to uropod and ruffle formation. At a functional level, leptin up-regulates the IL-1beta, IL-6, IL-12, TNF-alpha, and MIP-1alpha production. Coincident with this, leptin-treated DCs stimulate stronger heterologous T cell responses. Furthermore, we found that leptin down-regulates IL-10 production by DCs and drives naive T cell polarization toward Th1 phenotype. Finally, we found that leptin partly protects DCs from spontaneous and UVB-induced apoptosis. Consistent with the antiapoptotic effect of leptin, we observed the activation of NF-kappaB and a parallel up-regulation of bcl-2 and bcl-x(L) gene expression. These results provide new insights on the immunoregulatory function of leptin demonstrating its ability to improve DC functions and to promote DC survival. This is of relevance considering a potential application of leptin in immunotherapeutic approaches and its possible use as adjuvant in vaccination protocols.

HIV-1 Nef equips dendritic cells to reduce survival and function of CD8+ T cells: a mechanism of immune evasion.

The accessory HIV-1 Nef protein is a crucial determinant for viral replication and pathogenesis. During HIV infection, loss of immune control in the setting of a strong and broad HIV-specific T-lymphocyte response, leads to a lethal outcome through AIDS. Moreover, dysfunction of dendritic cells (DCs) may contribute to the immune suppression associated with AIDS progression. We recently demonstrated that exogenous Nef selectively activates immature DCs manipulating their phenotypical, morphological, and functional developmental program. Here, we tracked whether Nef, targeting DCs, could be involved in the dysregulation of CD8+ T cell responses. We found that Nef inhibits the capacity of DCs to prime alloreactive CD8+ T cell responses down-regulating their proliferation and functional competence. This coincides with the induction of CD8+ T cell apoptosis. Nef oversees apoptotic killing of CD8+ T cells up-regulating TNF-alpha and FasL production by DCs and interfering with the death receptor pathway in CD8+ T cells and thus activating caspase 8. Our findings suggest that Nef may contribute to the immune evasion associated with HIV-1 infection, subverting DC biology. This may help explain the pleiotropic function that Nef plays during infection and makes this protein an attractive target for preventive and therapeutic intervention.