Journal of Cellular Immunology and Serum Biology | Volume 2 | Issue 2 | 2016-08-17

The Decrease of Dendritic Cells Activation by “PAMPS” and “DAMPS” Danger Signals in Presence of VIP Neuropeptide

Lubangi M.M. Ghislain

Abstract

The dendritic cells (DCs) play a fundamental role (messengers between innate and adaptive immunities) in the development of immune responses. They can be activated by the danger signals (DAMPs: Damage-associated molecular pattern molecules; or PAMPs: Pathogen-associated molecular pattern molecules) that modulate the expression of specific receptors on their surface, cytokines producing (pro-inflammatory agents) and release exosomes in an extracellular space. The majority of VIP (Vasoactive Intestinal Peptide) that is usually found in the digestive tract plays an anti- inflammatory role by reducing DCs activation by PAMPs. This article presents the results of a research whose objective was to study the effect of VIP on the modulation of DC responses after an-activation by lipopolysaccharide called LPS (PAMPs) and by Monosodium Urate Crystals MSU. (DAMPs) in the presence or absence of VIP various cytokines dosage done using HEK- BlueTM line indicator and ELISA method, show that LPS and MSU crystals induce the release of pro-inflammatory cytokines by DCs in presence of VIP. These observations showed that PAMPs and DAMPs could participate in DC activation during an infection as in the case of infection by HIV-1, and the presence of VIP could play an inhibitor effect on this activation and indirectly on the infection.

Keywords: HIV, Immune response, Dendritic cells, VIP, Exosomes, Danger signals

Full text

HIV (Human Immunodeficiency Virus) weakens the immune system and causes a sensitization to opportunistic infections. Approximately 35.5 million people worldwide are infected by this virus [1] . Its eradication remains a challenge [2] because there is no vaccine or curative treatment. During its intrusion into the body, HIV-1 crosses the epithelial mucous to reach lymphoid organs, the dendritic cells (primary targets of HIV-1, primary role in the pathogenesis) where they are processed and become antigen presenting cells (APC). The transmission of the virus to lymphocytes occurs in these secondary lymphoid organs [3,4] . HIV-1 is a retrovirus that causes progressive depletion of LT CD4+ through the DCs, leading to a weakening of the immune system. Immature dendritic cells (DC) which integrated HIV-1 migrate mainly to the lymph nodes in the gastro-intestinal tract, a major site of HIV-1 replication during acute infection. The rapid loss of LT CD4+ cells (within 15 days) compromises the development of both the humoral and cellular responses [5] . To improve their role of antigen presenting cells, the DCs release large amounts of exosomes into the extracellular medium [6] . These exosomes are endosomal bioactive vesicles (30 to 100 nm) capable of regulating the immune system in the presence of self or non-self-antigens, or by expressing various inhibitory or activator molecules on their surface which affect the immune system cells [7,8] . It was also observed that the virus from dendritic cell was expelled in association with exosomes, increasing infectivity of virus particles [9] . To maintain their integrity, tissues are made of immune cells which use a variety of receptors called Pattern Recognition Receptor (PRRs) that enable pathogens recognition. Activation of these receptors accelerates the innate immune response recognizing conserved structural patterns present on micro-organism envelope, PAMPs (Pathogen-Associated Molecular Patterns). These can be capsids, bacterial components such as LPS (Lipopolysaccharide: component of the wall of gram-negative bacteria), or even of the CpG (unmethylated Cytosine-Guanine nucleotide repeats) which are repetitions of non-methylated nucleotides abundantly found in DNA (deoxyribonucleic acid) of bacteria and viruses. However, innate immunity can also be triggered by endogenous molecules called DAMPs (Danger-Associated Molecular Patterns) or danger signals. These DAMPs are molecules released by cells that have undergone stress or damage [10] and can activate the immune response or PAMPs [11] . However, a disruption of these endogenous signals could interfere with the HIV-1 causing a disorder of the immune response. The DAMPs that catch our attention are MSU crystals (Monosodium Urate) because they were identified as the main endogenous danger signal released by damaged cells [12] . These crystals will enable APCs and release pro-inflammatory cytokines such as interleukin 1 [13,14] . The cytokine plays a main role in activating the immune system because it participates in the recruitment of inflammatory cells [13,15] , and is involved in cellular proliferation [16] , the differentiation [17] and in the apoptosis of inflammatory cells [6] . However, to maintain their integrity, tissues will consist of VIP neuropeptides that are widely distributed molecules in the body and have a wide biological activity spectrum. Among their outstanding features, there is their role as modulators of cell proliferation and differentiation, especially in the immune system [18] . They are produced by the innervation of lymphoid tissues but also by some cells particularly Th2 lymphocytes. The receptors for neuropeptides are expressed by different cell types of the immune system (DC, LB and LT) which regulate proliferation, differentiation and migration [19] . It was demonstrated in the literature that the DCs express VIP receptors and that VIP inhibits the expression of co-activator molecules, CD80/CD86 in mature DCs and reduce their ability to activate T lymphocytes [20] . Also, it has been observed that neuropeptides can directly affect the maturation and migration of DCs toward secondary lymphoid organs to prevent an immune response because they inhibit the migration of mature DCs in a dose-dependent manner on response to the chemokine CCL5 (Chemokine (c-c motif) Ligand 5) and CCL19 (Chemokine (c-c motif) Ligand 19) [21] . In the case of HIV-1 infection, the VIP would block the HIV-1 fusion with target cells [22,23] and reduce viral replication in the infected macrophages [24] . Our study shows that PAMPs trigger DCs maturation, the DAMPs increase the release of exosomes and danger signals stimulate the releasing of pro-inflammatory cytokines. We also observed that all these phenomena are reduced in the presence of VIP. Materials and Methods Reagents and antibodies Different reagents were used in particular, the lymphocyte separation medium (LSM), Hank s Buffered Salt Solution (HBSS), Phosphate Buffered Saline (PBS), RPMI 1640 medium (Roswell Park Memorial Institute) and Dulbecco s Modified Eagle s Medium (DMEM), Fetal Bovine Serum (FBS), penicillin, streptomycin and primocine (preventing the contamination of culture medium by inhibiting growth of Gram-positive and Gram-negative bacteria), glutamine (nutrient for cellular growth), LPS, GM-CSF (Granulocyte Macrophage Colony-Stimulating Factor), IL-4 interleukin, accutase, MSU and VIP. The FBS is decomplemented and ultra-centrifuged to remove exosomes that may be present. Anti-HLA-DR [anti-Human Leukocyte Antigen-DR (DA6.147)], antibodies and the secondary antibodies (anti-IGG1 from mice) were used for the isotype control. The LSM, HBSS, PBS, culture media RPMI 1640 and DMEM, the FBS, penicillin, streptomycin and glutamine were purchased from Wisent inc. (St-Bruno, QC, CAN). The primocine and LPS were purchased from InvivoGen (San Diego, CA, USA). GM-CSF was purchased from GenScript (Piscataway, NJ, .U) while IL-4 comes from R & D Systems (Minneapolis, MN, .U). Accutase is available at Innovative Cell Technology inc. (San Diego, CA, .U). MSU crystals were graciously provided by Dr. Paul Naccache of Laval University (Qu bec, QC, CAN). The VIP comes from the company Neuropeptide (Strasbourg, Alsace, FRA). The anti-HLA-DR (DA6.147) was provided us by the team of Michel J. Tremblay (Quebec, QC, CAN). The secondary antibodies were from Jackson Immuno Research Laboratories inc. (Baltimore, PA, U.S). Peripheral blood mononuclear cells (PBMCs) and dendritic cells PBMCs: Peripheral blood mononuclear cells (PBMCs) were isolated from healthy human blood by density gradient using LSM. The ring of mononuclear cells obtained was collected and washed three times with HBSS IX (Hanks Balanced Salt Solution). Then PBMCs were then cultured at a concentration of 5 x 10 6 cells/ml in RPMI 1640 supplemented with 10% FBS and in the presence of antibiotics like penicillin (100 U/ml), streptomycin (100 U/ml), primocin (100 pg/ml) and amino acids like glutamine (2 mM). Among these PBMCs that were cultured, a portion of PBMC were treated or not with the VIP (10 -6 M) for 2 days at 37 C. Another portion of PBMC were stimulated with MSU crystals (0.15 mg/ml), in presence or absence of VIP (10 -6 M) for 2 days at 37 C. Finally, a portion of PBMC was kept for DCs. DCs: To get dendritic cells, a positive selection of CD14+ monocytes has been performed from PBMCs obtained by the method described previously. The selection was made using magnetic microbeads to which anti-CD14 antibody is attached (Stem cells, Vancouver, Canada). Once isolated, CD14+ monocytes were cultured in 6-well plates, at a rate of 3 x 10 6 cells/well in 3 ml of RPMI 1640. IL4 (200 U/ml) and GM-CSF (1000 U/ml) on day 0,2 and 4 to ensure their differentiation into immature DCs. After 5 days of differentiation some immature DCs were grown at the rate of 500,000 cells/wells in a volume of 500 l in duplicate in a 24-well plate with or without MSU crystals (0.15 mg/ml) for 48hrs at 37 C in the presence or absence of VIP (10 -6 M). Then the other portion of DC s were cultured at the rate of 500,000 cells/wells in a volume of 500 l in duplicate in a 24-well plate with or without LPS/IFN (1 g/ml/1000 U/ml final) during 48 hrs at 37 C in the presence or absence of VIP (10 -6 M). Treatment of iDCs (i.e., monocytes treated with GM-CSF and IL-4) with LPS and IFN-g is a well-known method used to induce full maturation of DCs [25,26] . Methods Isolation of exosomes The supernatants recovered from DCs after 48hrs of activation and treatment were centrifuged for 10 min at 10.000 g then ultra-centrifuged 45 min at 100.000 g to isolate the exosomes. After ultra-centrifugation, the supernatants were recovered and stored at -80 C for subsequent dosage of cytokines, whereas the exosomes pellets were washed in the presence of PBS IX and ultra-centrifuged again for 45 min at 100.000 g. Finally, the resulting pellets were re-suspended in a final volume of 190 l with PBS IX. Immunoblotting Exosomes were lysed in SB2X and SB5X (sample buffer) respectively, and allowed to migrate on a 12% SDS-PAGE minigel 12% during 90 minutes at 120 V. A transfer was then performed on PolyVinylideneFluoride (PVDF) membrane and transfer verification was carried out by staining the membranes with red ponceau. Thereafter, the non-specific sites of the membranes were saturated with 5% blotto for 30 minutes. The membranes were then incubated with the primary antibody anti-HLA-DR (1/1000). Antibody solutions are diluted in the Tris Buffered Saline (TBS) buffer 0.1% Tween-20 overnight at 4 C. The next day, the membranes were washed three times for 5 min with TBS Tween-20 0.1% buffer then incubated with an anti-mouse secondary antibody (1/10000) diluted in a TBS Tween-20 0.1% for 30 minutes at room temperature. Finally, three washes of 10 minutes were made and the membranes were then incubated with a developing solution. Dosage of pro-inflammatory cytokines To dose TNF or IL1 cytokines present in the supernatants recovered after the first ultra-centrifugation when the isolating exosomes, an Enzyme-Linked Immunosorbent Assay (ELISA) (eBioscience, San Diego, CA) and an indicator line HEK-Blue TNF /IL1 (eBioscience, San Diego, CA) have been used: 1. The indicator line HEK-Blue TNF /IL1 : HEK-Blue TNF /IL1 cells are indicator lines for the detection of the presence of TNF or IL1 pro-inflammatory cytokines contained in the supernatant. These cells were transfected by a SEAP (Secreted Embryonic Alkaline Phosphatase) reporter gene which is under the control an NF- B promoter. SEAP secretion, specific for the activation of NF- B path by TNF or IL1 was measured by spectrophotometer through the use of Reagent Quanti-Blue (reagent which becomes blue/purple in the presence of SEAP) [27] . 2. ELISA TNF : The ELISA test used ( Human TNF alpha ELISA Ready-SET-GO! ) by eBioscience is an enzyme-linked immunosorbent assay for the quantitative detection of human TNF . It has been conducted according to the instructions of manufacturer. Statistical analysis Statistical analysis were performed by ANOVA (one-way ANOVA) followed tests of Tukey multiple comparisons. These analyzes were performed using the software Prism 5 (GraphPad Software, San Diego, California). P values less than 0.05 were considered significant. 1* = p < 0.05; 2* = p < 0.01; 3* = p < 0,001. Results VIP effect on the activation of DCs with the measurement of the production of TNF or IL1 cytokines by DCs in the response of LPS and IFN It is known that the production of TNF /IL1 cytokines is increased in DCs stimulated with LPS and IFN [28-30] but decreased by LPS in the presence of VIP [31,32] . However, it is not known if VIP may regulate the release of these cytokines by DCs stimulated with LPS and IFN . We are therefore in studying the effect of VIP on the production of pro-inflammatory cytokines TNF and IL1 . To do so, DCs were stimulated for 48 hours with LPS and IFN then the supernatants were recovered and cultured with a line of HEK-Blue TNF /IL1 cells for detection of the presence of TNF or IL1 active cytokines. The results presented in (figure 1) show that the VIP modulates the secretion of TNF or IL1 by activated DCs with LPS or IFN . In the absence of stimulation, no effect of VIP has been observed on the cytokines secretion. These results were confirmed by using ELISA for TNF . The results presented on (figure 2) indicate a decrease of secretion of this cytokine by DCs stimulated by LPS or IFN in the presence of VIP. Figure 1: VIP effect on the production of TNF or IL1 cytokines by DCs after a stimulation with LPS and IFN . The immature DCs (500.000 cells/500 l) have been incubated in the presence or absence of VIP (10 -6 M final), and/or LPS/ IFN (1 g/ml / 1000 U/ml final) during 48 h at 37 C. Then, the supernatants have been collected and cultured with HEK-Blue TNF /IL1 cells (Invivogen) in order to evaluate the active TNF or IL1 quantities. 2*= p < 0,01 ; 3* = p < 0,001.(3 donors). Figure 2: VIP effect on the production of TNF by DCs after a stimulation with LPS and IFN . The immature DCs (500.000 cells/500 l) have been incubated in the presence or absence of VIP (10 -6 M final) and/or LPS (1 g/ml final) and IFN (1000 U/ml final) during 48 h at 37 C. Then the supernatants have been collected and an ELISA test has been performed in order to evaluate the quantity of TNF . 3* = p < 0,001. (2 donors). VIP effect on the production of TNF or IL1 cytokines by PBMC and DCs in the presence of MSU crystals In 2006, Martinon and co-workers have shown that the stimulation of macrophages by MSU crystals causes an increase in the production of IL1 pro-inflammatory interleukins [15] . Our results show that activated PBMC with MSU crystals, synthesize a larger amount of TNF or IL1 pro-inflammatory cytokines activated versus non-activated PBMCs. It is noted that this increase on the cytokines synthesis is decreased when PBMC stimulated by MSU crystals are pretreated by VIP. However, VIP alone has no effect when PBMCs are not activated (figure 3). Figure 3: VIP effect on the production of TNF or IL1 cytokines by PBMC after a stimulation with MSU crystals. PBMCs (5 x 10 6 cell/ml) have been incubated in the presence or absence of VIP (10 -6 M final) and/or MSU crystals (0.15 mg/ml final) during 48 h at 37 C. Then the supernatants have been collected and cultured with HEK-Blue TNF /IL1 cells (Invivogen) in order to evaluate the quantity of activate TNF or IL1 . 3* = p < 0,001. (5 donors) It has been observed that the MSU crystals identified as one of the major endogens danger signals can cause an increase in the maturation of primary DCs derived from the bone marrow of mouse [12] . So we looked at whether the MSU crystals could cause activation of primary human immature DCs and if the peptide VIP could regulate their activation. Our results show an increase of the production of TNF pro-inflammatory cytokines after stimulation of DCs by the MSU crystals using the method of ELISA and that the VIP causes a decrease in the release of this cytokine. However, we can observe that the TNF secretion is less important than during stimulation of DCs by LPS and IFN . These results should be confirmed by the other healthy donor cells (figure 4) and/or by those infected by HIV. Figure 4: VIP effect on the production of TNF cytokines by DCs after a stimulation with MSU crystals. The immature DCs (500.000 cell/500 l) have been incubated in the presence or absence of VIP (10 -6 M final) and/or MSU crystals (0.15 mg/ml final) during 48 h at 37 C. Then the supernatants have been collected and an ELISA method has been performed in order to evaluate TNF quantity. 3* = p < 0,001. (1 donor). Quantification of exosomes produced by DCs stimulated with MSU crystals and treated by VIP To help them in their role as antigen presenting cells, DCs constitutively release large amounts of exosomes into the extracellular medium [6] . The exosomes participate in the regulation of various function of immune system [7,8] . The results show that DCs stimulated with MSU crystals are accompanied by an increase in the amount of extracellular exosomes produced by these DCs and the VIP could regulate the production of these exosomes. The results presented in (figure 5) show by means of immunoblotting with anti-HLA-DR antibody that the MSU crystals cause the release of exosomes by DCs stimulation. Indeed, we observed the presence of HLA-DR proteins in exosomes samples obtained from DCs stimulated with MSU crystals but not in control samples. We also observed that the presence of HLA-DR is decreased when DCs are stimulated with MSU crystals in the presence of VIP. This peptide helps reduce the production of exosomes by DCs stimulated with MSU crystals. In the future, it will be necessary to confirm these results with immunoblotting with other molecules such as actin or Lysosomal-associated membrane protein 2 (LAMP2) which are also found in large quantities in the exosomes. Figure 5: The effect of VIP on the production of exosomes by DCs after a stimulation with MSU crystals (2 donors). The immature DCs (500.000 cell/500 l) are incubated in the presence or absence of VIP (10 -6 M final) and/or MSU crystals (0.15 mg/ml final) during 48 h at 37 C. Then the supernatants are collected and centrifuged at 10.000 g then at 100.000 g. The obtained pellets are washed with PBSIX and re-ultra-centrifuged at 100.000 g. Finally, an immunoblottin g is performed on obtained exosomes samples with an antibody directed against the HLA-DR protein. Discussion Under normal physiological conditions, immune system activation is initiated by the recognition of endogenous danger signals called DAMPs and PAMPs respectively [33,34] . The DAMPs represent products released by damaged tissues while PAMPs are molecular patterns conserved from pathogens. They allow initiating an innate immune response through recognition and activation of the receptors of the PRRs family expressed in many immune cells like neutrophils and phagocytes especially including DCs. These phagocytes are important in removing pathogens and cellular debris, but also in the development of an adaptive immune response [35,36] . It is observed that in the presence of PAMPs or DAMPs, DCs undergo maturation and migrate thereafter to secondary lymphoid organs where they activate the T lymphocytes [37,38] . Among the PAMPs, of LPS could be found in the gastro-intestinal tract damaged during infection by HIV-1. Indeed, hundreds of billions of bacteria of the intestinal flora normally line the intestine under physiological conditions [39] . It is observed that LPS is present in large quantities in the blood circulation of patients infected by HIV-1. The presence of LPS in blood could be due to microbial translocation following damage of the gastro-intestinal tract and the intestinal epithelium [5,40-42] . Thus, bacteria may be found in the gastro-intestinal tract and participate in the depletion of LT-CD4+. This depletion caused as a result of a chronic immune activation by LPS or other bacterial patterns. In facts, it has been shown a correlation between increased LPS and chronic activation of innate and adaptive immune system [42] . This depletion could be also promoted by an alteration of VIP quantity and function which is usually found in large quantity in the gastro-intestinal tracts in a physiological condition and which contributes to tolerance [43] . However, several studies have shown that VIP can act as immunoregulatory on activation of DCs when they are stimulated with PAMPs. VIP significantly reduced the capacity of these cells stimulated with LPS to activate T lymphocytes [20] . We therefore sought to confirm these results but in the presence of IFN , because it is a cytokine released by activated T lymphocytes that helps strengthen the activation and survival of DCs [44] . Our results show that LPS and IFN activate an increase in the production of TNF or IL1 pro-inflammatory active cytokines through the use of indicator HEK-Blue . However, this technique does not differentiate TNF and IL1 cytokines but the advantage of this technique is that it can measure the active cytokines. Therefore, we used ELISA to quantify TNF and we have confirmed an increase of this cytokine s production. However, the results confirmed observations of other studies indicating that the production of TNF cytokines is increased in DCs stimulated with LPS and IFN [28-30] . It should be necessary to supplement these experiences with ELISA method which allows determining the IL1 dosage. Finally, we note that this activation may be diminished in the presence of VIP. Indeed, the impact of observed VIP on the activation of the DCs is that this peptide can block the production of TNF cytokines. Also, when a tissue is damaged, the necrosis cells can release endogenous danger signals called DAMPS and activate the immune response [11] . We therefore wanted to verify if VIP could also have an effect on the activation of DCs when they are exposed to endogenous danger signals. Our results show that the MSU crystals can trigger an increase in the production of TNF or IL1 pro-inflammatory cytokines by human PBMC and TNF by human DCs. However, the amount of TNF produced by DCs is less than when stimulated with LPS and IFN . It would therefore be necessary in subsequent experiments to see if MSU crystals can trigger an increase in the production by human of IL1 DCs. Also, it would be appropriate to understand the difference between the amounts TNF produced in response to MSU crystals versus those produced in response to LPS/IFN . Finally, we saw that exosomes could also participate in the inflammatory response. Exosomes are micro-vesicles which may have roles in immunity such as in the induction of tolerance [45] and in antigenic presentation [46] . This can be explained by the fact that exosomes produced by antigens presenter cells such as DCs, carry on their surface CMH I and CMH II molecules [47] or co-stimulation molecule such as CD86 [46] . The results obtained show that DCs stimulated with MSU crystals produce a large amount of exosomes integrated a large quantity of HLA-DR, and suggest that these exosomes have a role in the antigenic presentation and thus in enhancing the immune response. Therefore, exosomes produced as a result of stimulation of DCs with MSU crystals, could stimulate LT-CD4+. However, these effects could be decreased in the presence of VIP. Our results show that VIP may regulate the production of these exosomes following stimulation by MSU crystals. The VIP therefore presents an inhibitory effect on antigenic presentation by exosomes and so on activating the LT-CD4+ because it reduces the amount of exosomes produced in the extracellular medium. It would be interesting to verify these effects by collecting exosomes released from DCs stimulated with MSU crystals and by performing a co-culture of these exosomes with LT-CD4+. So VIP could have a protective effect vis- -vis the rapid depletion of LT-CD4+ during a HIV-1 infection through: a reduction of the effects of PAMPs and DAMPs, a decrease of DCs maturation, a decrease of the migration of DCs to the secondary lymphoid organs where the virus transmission to T CD4+ lymphocytes takes place, and a decrease of DCs role in the antigen presentation. Conclusion The DCs are important in removing pathogens and cellular debris. Our results show that LPS, in the presence of IFN , and MSU crystals result in an activation of DCs. Indeed, these two types of signals increase the production of TNF or IL1 pro-inflammatory active cytokines. Our results also show that MSU crystals cause an increase of exosomes production. Also, we have shown that VIP neuropeptide allows regulating the activation of DCs stimulated by LPS and IFN , or by MSU crystals. Indeed, this peptide reduces the production of TNF or IL1 cytokines by DCs stimulated by LPS and IFN or by MSU crystals. Also, we observe that the VIP peptide regulates the production of exosomes by DCs stimulated with MSU crystals. It is shown that these signals may have a role in the development of several autoimmune diseases and that they are also involved in viral infections such as HIV-1 infection. This study will increase our knowledge of the role of VIP molecules in the release of exosomes as well as the impact of danger signals. 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