Presentation is loading. Please wait.

Presentation is loading. Please wait.

Alum adjuvants : Alum adjuvants : discovering their hidden secrets Ana Carolina Pagliarone Everton dos Santos Giuliano Bonfá.

Similar presentations


Presentation on theme: "Alum adjuvants : Alum adjuvants : discovering their hidden secrets Ana Carolina Pagliarone Everton dos Santos Giuliano Bonfá."— Presentation transcript:

1 Alum adjuvants : Alum adjuvants : discovering their hidden secrets Ana Carolina Pagliarone Everton dos Santos Giuliano Bonfá

2 Non-living antigens vaccines (purified or recombinant subunits) Advantage : safety (no possibility of disease development) ADJUVANTS !!!! Disadvantage: poor immunogenicity Adjuvants enhance the strength and duration of immune responses and modulates the type of immune response to the vaccine antigen

3 Vaccine antigen Adjuvant amplification of immune cells signals Immune response Adjuvant : additional vaccines component that enhances the immune response to antigens in vivo.

4 Signal 0 : antigen recognition by DC leads to cell activation and maturation AB Signal 1 : DC presents antigen peptides through MHC class II molecule to the TCR of naive CD4 + T cell Signal 2: DC expresses high levels of MHC, co-stimulatory (CD40, CD80 and CD86) and adhesion molecules (CD54 and CD58).

5 Signal 3 : DC secretes high amounts of cytokines that are crucial for inflammation and differentiation of CD4 + T cells. C CD4 + T cell differentiation (modified)

6 ADJUVANTS DELIVERY SYSTEMSIMMUNOMODULATORS -“Antigen vehicles” -Carriers to which antigens are associated by way of adsorption,co-precipitation or encapsulation. -Immunogenicity : - antigen retention at site of injection (antigen depot effect); - increase of uptake by DC cells and macrophages (slow release of antigen) -Tipically small molecules with adjuvant funtions through other mechanisms than antigen retention; -Immununogenecity: Direct stimulation of innate immune cells through interaction with PRRs TLRs,NLRs, RLRs and C-type lectins.

7 * * More used in human vaccines

8 Adjuvant action of aluminium salts Adjuvant mechanism of aluminium salts : antigen precipitaded onto insoluble particles of these salts are released slowly in the body (“depot effect”-Alexander Glenny, 1926). Studies have shown that the adjuvant activity of aluminium is more complex than this..... HOWEVER.....

9 Human macrophages pre-immunized with toxoid tetanus + aluminium hydroxide and further co-cultured with autologous T cells Increased IL-1 production and T cell proliferation Aluminium can activate antigen-specific immune response by activating APCs!

10 How aluminium salts can stimulate APCs and adaptive immune responses ? - In culture of lymph node cells from mice immunized with alum it was observed increased IL-1 and IL-4 production and proliferation of T cells. Treatment with anti-IL-4 decreased the proliferation (GRUN & MAURER, 1989) -Immunization with alum in mice enhaced only Th2 antibodies (IgG1 and IgE). However, IL-4 -/- mice presented induced IgG2a and Th1 cytokines production (BREWER et al.,1996) Aluminium hydroxide induced chemokines secretion ( CCL2/3 and 4 and CXCL-8) and inhibited CD14 expression in human monocytes and enhances MHC II and CD86 expression in DCs (monocyte activation, recruitment to blood and differentiation toward DCs.) (SEUBERT et al., 2008).

11 DCs activation by aluminium salts occurs through specific receptors??????? Deficient micefor both MyD88 and TRIF immmunized with alum + TNP-Hy showed Th2-induced IgG1 and IgE responses comparable to those observed in control mice. MyD88-deficient mice immunized with antigen and aluminum salts produced IgG1 and specially more IgE, comparing to control mice.

12 According to these observations, aluminium salts are both delivery systems and immunomodulators adjuvants, although their activities do not occur through TLRs....

13 IMMUNOMODULATORS -Are tipically small molecules that exert their adjuvant funtions through mechanisms other than antigen retention Direct stimulation of innate immune cells (monocytes, macrophages, NK and NKT cells and DCs) specially through interaction with PRRs of these cells (TLRs, NLRs, RLRs and C-type lectins PAMPs

14 However, its known that there are ENDOGENOUS compounds which are also able to be recognized by host cells, inducing the immune response even in absence of pathogens. DAMPs Damage Associated Molecular Patterns DAMPs = substances/compounds released by necrotic cells which are recognized by innate immune cells (tissue injury indicators). They activate innate immune cells according to the Danger Hypothesis.

15 Are DAMPs endogeous adjuvants ?

16 Uric acid stimulates DC maturation in vitro and stimulates CD8 + T cell response in antigen- immunized mice Dead cells co-administered with antigen induced increased antigen-specific CD4 + T cells in vivo. Moreover, dead cells could lead to DCs maturation in vitro. How DAMPs are recognized by DCs ?

17 Recognition of endogenous DAMPs by TLRs There are studies that defend the idea of TLR ligands contamination (such as LPS) in studied DAMPs, others showed that TLR knockout mice presented reduction in inflammatory response to necrotic cell death in vivo (CHEN & NUÑEZ, 2010).

18 MSU: monosodium urate Human monocytes MRU-induced peritonitis in mice Human monocytes

19

20 Mechanisms for DAMPs –induced inflammation cell recruitment

21 DAMPs and PAMPs are recognized by innate immune cells leading to further adaptive cells activation (adjuvant activity!!).

22 Aluminium adjuvants can indirectly activate APCs by causing tissue damage due to necrosis of skeletal muscle fibers (SHY et al., 2003) Uric acid is able to induce NLRP3 (MARTINON et al., 2006) Aluminium adjuvants can activate the inflammasome NALP 3?

23

24 Mice submited to OVA or OVA+alum injections i.p.

25 Nalp 3or ASC 3 or Casp 3 deficient mice injected i.p with OVA

26 Indirect activation Direct activation ex: TLRs Possible mechanisms of alum action

27

28 Giuliano Bonfá Everton dos Santos

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

30 Could silica and alum induce inflamossome activation of macrophages? Macrophages (5x10 5 /mL) Primed with LPS (1ng/mL)/3h Stimulation for 2h Macrophages (5x10 5 /mL) - Silica ( µg/mL) ; - Aluminium ( µg/mL); - ATP (1mM). ELISA (IL-1β) (culture Supernatants) Silica, alum and ATP induced inflamossome activity on LPS-primed macrophages

31 Besides of IL-1B production, could macrophages produce others inflammatory factors? Macrophages (5x10 5 /mL) Primed with LPS (1ng/mL)/3h Stimulation for 2h Macrophages (5x10 5 /mL) - Silica ( µg/mL) ; - Aluminium ( µg/mL); - ATP (1mM). ELISA (PGE2, TNF-α, IL-1β, 6, 12, 18) (culture Supernatants) These results showed that in addition to IL-1β and IL-18, macrophages also produce PGE2 in response to silica, alum and ATP.

32 Can DCs produce IL-1β and PGE2 in response stimulation for silica or alum? DCs (5x10 5 /mL) Primed with LPS (1ng/mL)/3h Stimulation for 6h - Silica (100µg/mL) ; - Aluminium (400µg/mL); ELISA (PGE2,IL-1β) (culture Supernatants) GM-CSF (10ng/mL) M-CSF (10ng/mL) BM cells Macrophages (5x10 5 /mL) DC cells (5x10 5 /mL) Macrophages (5x10 5 /mL) DCs can produce IL-1β and PGE2, but macrophages produce higher amounts of PGE2 than DCs *B6/Balb; PBMC

33 Aluminium, Silica  Activity inflammasome;  Production of IL-β  Prostaglandin E2 MacrophagesMB;Peritoneal DCMB; In short... Does is necessary the phagocytosis for production of IL-1β and prostaglandin?

34 Could the engulfment of particulates, lysosomal rupture, and release of lysosomal enzymes induce PGE2 and IL-1β production in macrophages? Macrophages (5x10 5 /mL) Primed with LPS (1ng/mL)/3h Stimulation for 2h Macrophages (5x10 5 /mL) - Silica (100µg/mL) ; -Alum (400µg/mL); -Cyt D (2μM or Cat B (10μM) ELISA (PGE2, IL-1β) (culture Supernatants) Phagocytosis, lysosomal damage and release of enzymes triggers PGE2 production in macrophages.

35 Aluminium, Silica prostaglandin E2 and IL-1β production Macrophages In short... Phagocytosis lysosomal rupture release of lysosomal enzymes Does prostaglandin production is dependent of inflammasome?

36 The production of PGE2 is dependent of activity of inflammasome? Macrophages (5x10 5 /mL) Primed with LPS (1ng/mL)/3h Stimulation for 2h Macrophages (5x10 5 /mL) - Silica (100µg/mL) ; - TiO 2 (100µg/mL); ELISA (PGE2, IL-1β) (culture Supernatants)

37 Primed with LPS (1ng/mL)/3h Stimulation for 6h -Silica (50μg/mL) --Alum (200μg/mL) ELISA (PGE2) (culture Supernatants) M-CSF (10ng/mL) BM cells Macrophages (5x10 5 /mL) WT; Nalp3, Asc, Casp1 ( -/- ) The production of PGE2 is dependent of activity of inflammasome? Aluminium, Silica  Activity inflammasome;  Production of IL-β  Prostaglandin E2 MacrophagesMB;Peritoneal PGE2 production in macrophages is independent of inflammasome

38 Could alum and silica induce the activity of cyclooxygenase (COX) and synthase PGE? Aluminium, Silica prostaglandin E2 and IL-1β production Macrophages In short... Phagocytosis lysosomal rupture release of lysosomal enzymes The PGE2 production was independent of inflammasome activity. Prostaglandin E 2 (PGE 2 ) is generated by the sequential metabolism of arachidonic acid by cyclo-oxygenase and prostaglandin E synthase (Needleman et al., 1986; Smith. 1992).

39 Could silica and alum induce activity of COX or PGE synthase enzymes? Macrophages (5x10 5 /mL) Primed with LPS (1ng/mL)/3h Stimulation for 2h Macrophages (5x10 5 /mL) - Silica (100µg/mL) ; - COX-2 inhibitor (1µM); ELISA (PGE2, IL-1β) (culture Supernatants) WT PTGES ( +/+; -/- ) Silica- and alum-induce PGE2 production in macrophages mediated by the COX-2 and PTGES.

40 Aluminium, Silica Prostaglandin E2 production Macrophages In short... Phagocytosis lysosomal rupture release of lysosomal enzymes COX-2 and PTGES Activation of the inflammasome IL-1β production Which signaling pathway is involved in this process?

41 Which signaling pathway is involved in the production of PGE2? Macrophages (5x10 5 /mL) Primed with LPS (1ng/mL)/3h Stimulation for 2h Macrophages (5x10 5 /mL) - Silica (100µg/mL) ; -Inhibitors ELISA (PGE2, IL-1β) (culture Supernatants) Absence of inhibitors were used as the 100% controls These results showed that P38 MAP kinase is involved with in the PGE2 production and the lysosomal rupture is necessary for your activation.

42 Aluminium, Silica Prostaglandin E2 production Macrophages In short... Phagocytosis lysosomal rupture release of lysosomal enzymes COX-2 and PTGES Activation of the inflammasome IL-1β production P38 MAPK phosphorilation Phospholipase A2???

43 Phosphorylation of p38 MAPK could activate phospholipase A2 after phagocytosis of silica or aluminium by macrophages? Macrophages (5x10 5 /mL) Primed with LPS (1ng/mL)/3h Stimulation for 2h Macrophages (5x10 5 /mL) - Silica (100µg/mL) ; -Inhibitors ELISA (PGE2, IL-1β) (culture Supernatants) Absence of inhibitors were used as the 100% controls Phosphorylation of p38 MAPK active phospholipase A2 The Syk can be activated by phagocytosis de silica or aluminum??

44 The Syk can be activated by phagocytosis de silica or aluminum? Macrophages (5x10 5 /mL) Primed with LPS (1ng/mL)/3h Stimulation for 2h Macrophages (5x10 5 /mL) - Silica (100µg/mL) ; -Inhibitors ELISA (PGE2, IL-1β) or blot These results suggest that lysosomal damage triggers Syk activation, and then activated Syk upregulates cPLA2 activity via the phosphorylation of p38 MAP kinase.

45 Aluminium, Silica Prostaglandin E2 production Macrophages In short... Phagocytosis lysosomal rupture release of lysosomal enzymes COX-2 and PTGES Activation of the inflammasome IL-1β production P38 MAPK phosphorilation Phospholipase A2 Syk

46 Prostaglandin synthases - PGE2 inhibits production of cytokines such as TNF-α, and IL-12 (Scales et al., 1989; Van der Pouw Kraan 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). - PGE 2 modulates the functions of cell populations, such as T cells and macrophages (Nataraj et al., 2001). Silica- and Alum-Induced Production of PGE2 by Macrophages Regulates Immune Responses In Vivo?

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

48 Aluminium, Silica Prostaglandin E2 production Macrophages Conclusion Phagocytosis lysosomal rupture release of lysosomal enzymes COX-2  PTGES Activation of the inflammasome IL-1β production P38 MAPK phosphorilation Phospholipase A2 Syk IgE A.A

49 Aim: To identify the citotoxic and adjuvant effect of alum on immune responses

50 Is alum cytotoxic on local of injection? Peritoneal lavage fluid Muscle lavage fluid Alum induce cell death and release of host DNA at sites of injection! i.m. and i.p. Cell death rate OVA + Alum i.p. days 0, 14 and 21

51 Can DNA act as an adjuvant? ELISA i.p. OVA OVA + Alum OVA + DNA days 0, 14 and 21 ELISA i.p. OVA OVA + Alum days 0 and 10 DNase I (3 and 8h)

52 Can DNA act as an adjuvant? Host DNA released by alum cytotoxicity mediates alum activity on humoral and T H 2 cell responses! i.p. OVA OVA + Alum OVA + DNA OT-II OVA-specific CD4 + CFSE DNase I (3 and 8h) Cells on the bronchial lymph nodes (BLNs) 3d WT

53 What’s the mechanism? TLR9 -/- mice develop humoral responses similar to those of their WT counterpart in response to alum immunization; Nature Reviews Immunology 10, (February 2010) | doi: /nri2690 NALP3 -/- and CASP1 -/- ?

54 What’s the mechanism? ELISA (serum) i.p. days 0, 14 and 21 NALP3 independent WT and IRF3 -/- OVA OVA + Alum OVA + DNA ELISA Peritoneal lavage fluid i.p. days 0, 14 and 21 OVA OVA + Alum OVA + DNA

55 What’s the mechanism? Alum and host genomic DNA trigger type I IFN secretion and IgE responses through activation of the TBK1-IRF3 axis! ELISA (serum) i.p. days 0, 14 and 21 TBK1 +/- /TNF -/- and TBK1 -/- /TNF -/- OVA OVA + Alum

56 Have IRF3 deficiency an effect on Th2 responses after alum imunization? IRF3 is essential for the boosting of canonical T H 2 cells by alum and genomic DNA! i.p. OVA OVA + Alum OVA + DNA OT-II OVA-specific CD4 + CFSE Cells on the bronchial lymph nodes (BLNs) 3d WT and IRF3 -/-

57 There is an effect during experimental asthma? IRF3 absence protect mice from allergic airway inflammation! Bronchoalveolar lavage fluid (BALF) i.p. OVA OVA + Alum days 0 and 14 Airway sensitization d 21 to 25 WT and IRF3 -/-

58 IRF3 deficiency changes DCs migration? IRF3 is essential for the triggering of iDC recruitment by alum! FACs (peritoneal lavage fluid and BLNs) i.p.OVA OVA + Alum OVA + DNA h WT and IRF3 -/- The recruitment of iDCs to the BLNs of alum-treated mice strongly correlated with the percentage of cell death and DNA release and that it was reduced after Dnase I treatment. iMonos (F4/80 int CD11b + Ly6C + Ly6G - cells) cDCs (MHCII + CD11c + F4/80 low Ly6C - cells) pDCs (B220 + Ly6G + CD11c int F4/80 low cells) BLNs

59 Deficiency on DCs recruitment alter the Th2 response? Deficient migration of inflammatory monocites (iMonos) impair alum-induced Th2 and IgE responses in IRF3 -/- mice! i.p. OVA OVA + Alum OT-II OVA-specific CD4 + CFSE Cells on the bronchial lymph nodes (BLNs) WT and IRF3 -/- WT immunized iMonos IgE  increase IgG1  not affected

60 IgE  attenuated IgG1  not affected IRF3 deficiency changes iDCs activation? Alum-induced iMono migration depends on IL-12p40 homodimer signaling! FACs and ELISA (peritoneal lavage fluid and serum) i.p. days 0, 14 and 21 WT and IRF3 -/- OVA OVA + Alum BLNs iMonos (F4/80 int CD11b + Ly6C + Ly6G - cells)

61 Conclusion DAMP

62 Journal Conclusion Alum NALP3 Independent NALP3 Dependent PGE 2 Dependent Host DNA (DAMP) (IRF3-TBK1 axis) Syk and p38 MAP kinase Uric acid crystals Th2 responses – IgE and IgG1


Download ppt "Alum adjuvants : Alum adjuvants : discovering their hidden secrets Ana Carolina Pagliarone Everton dos Santos Giuliano Bonfá."

Similar presentations


Ads by Google