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Alum adjuvants : discovering their hidden secrets

Alum adjuvants : discovering their hidden secrets. Ana Carolina Pagliarone Everton dos Santos Giuliano Bonfá. Non-living antigens vaccines ( purified or recombinant subunits ). Advantage : safety (no possibility of disease development ).

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Alum adjuvants : discovering their hidden secrets

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  1. Alumadjuvants : discoveringtheirhiddensecrets Ana Carolina Pagliarone Everton dos Santos Giuliano Bonfá

  2. Non-living antigensvaccines (purifiedorrecombinantsubunits) Advantage :safety (no possibilityofdiseasedevelopment) Adjuvantsenhancethestrengthanddurationofimmune responses andmodulatesthetypeofimmune response tothevaccineantigen Disadvantage:poorimmunogenicity ADJUVANTS !!!!

  3. Adjuvant :additionalvaccinescomponentthatenhancestheimmune response toantigensin vivo . Vaccineantigen Adjuvant amplificationofimmunecellssignals Immune response

  4. A B Signal 0 : antigenrecognitionby DC leads to cellactivationandmaturation Signal 1 : DC presentsantigenpeptidesthrough MHC class II molecule tothe TCR ofnaive CD4+T cell Signal 2: DC expresses high levelsof MHC, co-stimulatory (CD40, CD80 and CD86) andadhesionmolecules (CD54 and CD58).

  5. C CD4+ T cell differentiation Signal 3 : DC secretes high amountsofcytokinesthat are crucial for inflammationanddifferentiationof CD4+T cells. (modified)

  6. ADJUVANTS DELIVERY SYSTEMS IMMUNOMODULATORS -“Antigenvehicles” -Carrierstowhichantigens are associated bywayofadsorption,co-precipitationor encapsulation. -Immunogenicity : - antigenretentionat site ofinjection (antigendepoteffect); - increaseofuptakeby DC cellsand macrophages(slow release ofantigen) • -Tipicallysmallmoleculeswithadjuvantfuntionsthroughothermechanisms • thanantigenretention; • -Immununogenecity: • DirectstimulationofinnateimmunecellsthroughinteractionwithPRRsTLRs,NLRs, RLRsand C-typelectins.

  7. * * More used in humanvaccines

  8. Adjuvantactionofaluminiumsalts Adjuvantmechanismofaluminiumsalts :antigenprecipitadedontoinsolubleparticlesofthesesalts are releasedslowly in thebody (“depoteffect”-Alexander Glenny, 1926). HOWEVER ..... Studieshaveshownthattheadjuvantactivityofaluminiumis more complexthanthis.....

  9. Humanmacrophagespre-immunizedwithtoxoidtetanus + aluminiumhydroxideandfurtherco-culturedwithautologous T cells Increased IL-1 productionand T cellproliferation Aluminiumcanactivateantigen-specificimmune response byactivatingAPCs!

  10. HowaluminiumsaltscanstimulateAPCsandadaptiveimmune responses ? - In cultureoflymph node cellsfrommiceimmunizedwithalumit wasobserved increased IL-1 and IL-4 productionandproliferationof T cells. Treatmentwith anti-IL-4 decreasedtheproliferation (GRUN & MAURER, 1989) -Immunizationwithalum in miceenhacedonly Th2 antibodies(IgG1 and IgE). However, IL-4-/-micepresentedinduced IgG2a and Th1 cytokinesproduction (BREWER et al.,1996) Aluminiumhydroxideinducedchemokinessecretion ( CCL2/3 and 4 and CXCL-8) andinhibited CD14 expression in humanmonocytesandenhances MHC II and CD86 expression in DCs(monocyteactivation, recruitment tobloodanddifferentiationtowardDCs.) (SEUBERT et al., 2008).

  11. DCsactivationbyaluminiumsaltsoccursthrough specificreceptors??????? MyD88-deficient miceimmunizedwithantigenandaluminumsaltsproduced IgG1 andspecially more IgE, comparingtocontrolmice. Deficientmiceforboth MyD88 andTRIF immmunizedwithalum + TNP-Hyshowed Th2-induced IgG1 and IgE responses comparabletothoseobserved in controlmice.

  12. Accordingtotheseobservations, aluminiumsalts are both delivery systems andimmunomodulatorsadjuvants, althoughtheiractivities do notoccurthroughTLRs....

  13. IMMUNOMODULATORS • Are tipicallysmallmoleculesthatexerttheiradjuvantfuntionsthrough • mechanismsotherthanantigenretention Directstimulationofinnateimmunecells(monocytes, macrophages, NK and NKT cellsandDCs) speciallythroughinteractionwithPRRsofthesecells (TLRs, NLRs, RLRsand C-typelectins PAMPs

  14. However, its knownthatthere are ENDOGENOUScompoundswhich are also abletoberecognizedby host cells, inducingtheimmune response even in absenceofpathogens. Damage Associated Molecular Patterns DAMPs DAMPs =substances/compoundsreleasedbynecroticcellswhich are recognizedby innateimmunecells(tissueinjuryindicators). Theyactivateinnateimmunecells accordingtotheDangerHypothesis.

  15. Are DAMPsendogeousadjuvants ?

  16. Deadcellsco-administeredwithantigeninducedincreased antigen-specific CD4+ T cellsin vivo. Moreover, deadcells could lead toDCsmaturationin vitro. Uricacidstimulates DC maturationin vitroand stimulates CD8+ T cell response in antigen- immunizedmice HowDAMPs are recognizedbyDCs ?

  17. RecognitionofendogenousDAMPsbyTLRs There are studiesthatdefendtheideaof TLR ligandscontamination(such as LPS) in studied DAMPs, othersshowedthat TLR knockoutmicepresentedreduction in inflammatory response tonecroticcelldeathin vivo (CHEN & NUÑEZ, 2010).

  18. MSU: monosodiumurate Humanmonocytes Humanmonocytes MRU-induced peritonitis in mice

  19. Mechanisms for DAMPs –inducedinflammation cellrecruitment

  20. DAMPsandPAMPs are recognizedbyinnateimmunecellsleadingtofurther adaptivecellsactivation (adjuvantactivity!!).

  21. AluminiumadjuvantscanindirectlyactivateAPCsbycausing tissuedamageduetonecrosisofskeletalmusclefibers (SHY et al., 2003) Uricacidisabletoinduce NLRP3 (MARTINON et al., 2006) Aluminiumadjuvantscanactivatetheinflammasome NALP 3?

  22. Micesubmitedto OVA orOVA+aluminjectionsi.p.

  23. Nalp 3or ASC 3 orCasp 3 deficientmiceinjectedi.pwith OVA

  24. Possiblemechanismsofalumaction Indirectactivation ex: TLRs Directactivation

  25. Everton dos Santos Giuliano Bonfá

  26. Show that silica crystal and aluminum induces activation of MAPK beyond of NALP3 Everton dos Santos

  27. Couldsilicaandaluminduceinflamossomeactivationofmacrophages? Primedwith LPS (1ng/mL)/3h Stimulation for 2h ELISA (IL-1β) (culture Supernatants) - Silica ( 50 -100µg/mL) ; - Aluminium (200-400µg/mL); - ATP (1mM). Macrophages (5x105/mL) Macrophages (5x105/mL) Silica, alum and ATP induced inflamossome activity on LPS-primed macrophages

  28. Besidesof IL-1B production, couldmacrophagesproduceothersinflammatoryfactors? Primedwith LPS (1ng/mL)/3h Stimulation for 2h ELISA (PGE2, TNF-α, IL-1β, 6, 12, 18) (culture Supernatants) - Silica ( 50 -100µg/mL) ; - Aluminium (200-400µg/mL); - ATP (1mM). Macrophages (5x105/mL) Macrophages (5x105/mL) These results showed that in addition to IL-1βand IL-18, macrophagesalsoproduce PGE2 in response tosilica, alumand ATP.

  29. Can DCs produce IL-1βand PGE2 in response stimulation for silica or alum? GM-CSF (10ng/mL) DCs (5x105/mL) DC cells (5x105/mL) Primedwith LPS (1ng/mL)/3h Stimulation for 6h ELISA (PGE2,IL-1β) (culture Supernatants) - Silica (100µg/mL) ; - Aluminium (400µg/mL); BM cells M-CSF (10ng/mL) Macrophages (5x105/mL) Macrophages (5x105/mL) DCs can produce IL-1βand PGE2, but macrophages produce higher amounts of PGE2 than DCs *B6/Balb; PBMC

  30. Aluminium, Silica In short... Macrophages MB; Peritoneal DC MB; • Activityinflammasome; • Productionof IL-β • Prostaglandin E2 Does isnecessarythephagocytosis for productionof IL-1βandprostaglandin?

  31. Could the engulfment of particulates, lysosomal rupture, and release of lysosomal enzymes induce PGE2 and IL-1βproduction in macrophages? Primedwith LPS (1ng/mL)/3h Stimulation for 2h ELISA (PGE2, IL-1β) (culture Supernatants) • - Silica (100µg/mL) ; • Alum (400µg/mL); • Cyt D (2μM orCat B (10μM) Macrophages (5x105/mL) Macrophages (5x105/mL) Phagocytosis, lysosomaldamage and release of enzymes triggers PGE2 production in macrophages.

  32. In short... Phagocytosis Macrophages Aluminium, Silica lysosomalrupture release of lysosomal enzymes prostaglandin E2 and IL-1βproduction Does prostaglandinproductionisdependentofinflammasome?

  33. The productionof PGE2 isdependentofactivityofinflammasome? Primedwith LPS (1ng/mL)/3h Stimulation for 2h ELISA (PGE2, IL-1β) (culture Supernatants) - Silica (100µg/mL) ; - TiO2 (100µg/mL); Macrophages (5x105/mL) Macrophages (5x105/mL)

  34. The productionof PGE2 isdependentofactivityofinflammasome? Aluminium, Silica Macrophages MB; Peritoneal Primedwith LPS (1ng/mL)/3h Stimulation for 6h ELISA (PGE2) (culture Supernatants) • Silica (50μg/mL) • -Alum (200μg/mL) Macrophages (5x105/mL) M-CSF (10ng/mL) WT; Nalp3, Asc, Casp1 (-/-) BM cells • Activityinflammasome; • Productionof IL-β • Prostaglandin E2 PGE2 production in macrophages is independent of inflammasome

  35. In short... Phagocytosis Macrophages Aluminium, Silica lysosomalrupture release of lysosomal enzymes prostaglandin E2 and IL-1βproduction The PGE2 productionwasindependentofinflammasomeactivity. Prostaglandin E2 (PGE2) is generated by the sequential metabolism of arachidonic acid by cyclo-oxygenase and prostaglandin E synthase (Needleman et al., 1986; Smith. 1992). Couldalumandsilicainducetheactivityof cyclooxygenase(COX) and synthase PGE?

  36. Couldsilicaandaluminduceactivityof COX orPGE synthaseenzymes? Primedwith LPS (1ng/mL)/3h Stimulation for 2h ELISA (PGE2, IL-1β) (culture Supernatants) - Silica (100µg/mL) ; - COX-2 inhibitor (1µM); Macrophages (5x105/mL) Macrophages (5x105/mL) WT PTGES (+/+;-/-) Silica- and alum-induce PGE2 production in macrophages mediated by the COX-2 and PTGES.

  37. In short... Phagocytosis Macrophages Aluminium, Silica lysosomalrupture release of lysosomal enzymes COX-2 and PTGES Activationoftheinflammasome Prostaglandin E2 production IL-1βproduction Which signaling pathway is involved in this process?

  38. Which signaling pathway is involved in the production of PGE2? Primedwith LPS (1ng/mL)/3h Stimulation for 2h ELISA (PGE2, IL-1β) (culture Supernatants) Absence of inhibitors were used as the 100% controls • - Silica (100µg/mL) ; • Inhibitors Macrophages (5x105/mL) Macrophages (5x105/mL) These results showed that P38 MAP kinase is involved with in the PGE2 production and the lysosomal rupture is necessary for your activation.

  39. In short... Phagocytosis Macrophages Aluminium, Silica lysosomalrupture release of lysosomal enzymes P38 MAPK phosphorilation Activationoftheinflammasome Phospholipase A2??? IL-1βproduction COX-2 and PTGES Prostaglandin E2 production

  40. Phosphorylation of p38 MAPK could activate phospholipase A2 after phagocytosis of silica or aluminium by macrophages? Primedwith LPS (1ng/mL)/3h Stimulation for 2h ELISA (PGE2, IL-1β) (culture Supernatants) Absence of inhibitors were used as the 100% controls • - Silica (100µg/mL) ; • Inhibitors Macrophages (5x105/mL) Macrophages (5x105/mL) Phosphorylation of p38 MAPK active phospholipase A2 The Syk can be activated by phagocytosis de silica or aluminum??

  41. The Syk can be activated by phagocytosis de silica or aluminum? Primedwith LPS (1ng/mL)/3h Stimulation for 2h ELISA (PGE2, IL-1β) orblot • - Silica (100µg/mL) ; • Inhibitors Macrophages (5x105/mL) Macrophages (5x105/mL) These results suggest that lysosomal damage triggers Syk activation, and then activated Sykupregulates cPLA2 activity via the phosphorylation of p38 MAP kinase.

  42. Phagocytosis In short... lysosomalrupture Macrophages Aluminium, Silica release of lysosomal enzymes Syk Activationoftheinflammasome P38 MAPK phosphorilation IL-1βproduction Phospholipase A2 COX-2 and PTGES Prostaglandin E2 production

  43. - PGE2 inhibits production of cytokines such as TNF-α, and IL-12 (Scales et al., 1989; Van der PouwKraan et al., 1995). - PGE2 may polarize cellular response toward a Th2 phenotype enhancing IL-4 and IL-5 production (Betz and Fox, 1991; Katamura et al., 1995) and facilitating immunoglobulin class switching to IgE(Roper et al., 1995). - PGE2modulates the functions of cell populations, such as T cells and macrophages (Nataraj et al., 2001). Prostaglandinsynthases Silica- and Alum-Induced Production of PGE2 by Macrophages Regulates Immune Responses In Vivo?

  44. Silica- and Alum-Induced Production of PGE2 by Macrophages Regulates Immune Responses In Vivo? OVA+AlumorOVA+Silica 0 7 17 ELISA IgG1, IgG2c, IgE Seracollected Days Ptges+/+orPtges-/- PGE2 production but not inflammasome activation in macrophages, positively regulates the generation of IgE antibodies in vivo.

  45. Phagocytosis Conclusion lysosomalrupture Macrophages Aluminium, Silica release of lysosomal enzymes Syk Activationoftheinflammasome P38 MAPK phosphorilation IL-1βproduction Phospholipase A2 A.A COX-2  PTGES Prostaglandin E2 production IgE

  46. Aim: To identifythecitotoxicandadjuvanteffectofalumonimmuneresponses

  47. Is alumcytotoxicon local ofinjection? i.m.andi.p. OVA + Alum Peritoneal lavagefluid Musclelavagefluid days 0, 14 and 21 Celldeath rate i.p. Aluminducecelldeathand release of host DNA at sites ofinjection!

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