alum adjuvants : discovering their hidden secrets
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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 ). - PowerPoint PPT PresentationTRANSCRIPT
Alum adjuvants : discovering their hidden
secrets
Ana Carolina PagliaroneEverton dos Santos
Giuliano Bonfá
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
Vaccine antigen Adjuvant
amplification of immune cells signals
Immune response
Adjuvant : additional vaccines component that enhances the immune response to antigens in vivo .
Signal 0 : antigen recognition by DC leads to cell activation and maturation
A B
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).
Signal 3 : DC secretes high amounts of cytokines that are crucial for inflammation and differentiation of CD4+ T cells.
C
CD4+ T celldifferentiation
(modified)
ADJUVANTS
DELIVERY SYSTEMS IMMUNOMODULATORS
-“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.
*
* More used in human vaccines
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 .....
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!
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).
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.
According to these observations, aluminium salts are both delivery systems and immunomodulators adjuvants, although their activities do not occur through TLRs....
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 andNKT cells and DCs) specially through interaction with PRRs of these cells (TLRs,NLRs, RLRs and C-type lectins
PAMPs
However, its known that there are ENDOGENOUS compounds which are alsoable to be recognized by host cells, inducing the immune response even in absence of pathogens.
DAMPs
DamageAssociatedMolecular 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.
Are DAMPs endogeous adjuvants ?
Uric acid stimulates DC maturation in vitro andstimulates CD8+ T cell response in antigen- immunized mice
Dead cells co-administered with antigen induced increasedantigen-specific CD4+ T cells in vivo. Moreover, dead cells could lead to DCs maturation in vitro.
How DAMPs are recognized by DCs ?
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).
MSU: monosodium urate
Human monocytes
MRU-induced peritonitis in mice
Human monocytes
Mechanisms for DAMPs –induced inflammation
cell recruitment
DAMPs and PAMPs are recognized by innate immune cells leading to furtheradaptive cells activation (adjuvant activity!!).
Aluminium adjuvants can indirectly activate APCs by causingtissue 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?
Mice submited to OVA or OVA+alum injections i.p.
Nalp 3or ASC 3 or Casp 3 deficient mice injected i.p with OVA
Indirect activation
Direct activation
ex: TLRs
Possible mechanisms of alum action
Giuliano Bonfá
Everton dos Santos
Show that silica crystal and aluminum induces
activation of MAPK beyond of NALP3
Everton dos Santos
Could silica and alum induce inflamossome activation of macrophages?
Macrophages(5x105/mL)
Primed with LPS
(1ng/mL)/3h
Stimulation for 2h
Macrophages(5x105/mL)
- Silica ( 50 -100µg/mL) ;
- Aluminium (200-400µg/mL);
- ATP (1mM).
ELISA (IL-1β)(culture Supernatants)
Silica, alum and ATP induced inflamossome activity on LPS-primed macrophages
Besides of IL-1B production, could macrophages produce others inflammatory factors?
Macrophages(5x105/mL)
Primed with LPS
(1ng/mL)/3h
Stimulation for 2h
Macrophages(5x105/mL)
- Silica ( 50 -100µg/mL) ;
- Aluminium (200-400µg/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.
Can DCs produce IL-1β and PGE2 in response stimulation for silica or alum?
DCs(5x105/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(5x105/mL)
DC cells(5x105/mL)
Macrophages(5x105/mL)
DCs can produce IL-1β and PGE2, but macrophages produce higher amounts of PGE2
than DCs *B6/Balb; PBMC
Aluminium, Silica
Activity inflammasome;
Production of IL-β
Prostaglandin E2
MacrophagesMB;
PeritonealDCMB;
In short...
Does is necessary the phagocytosis for production of IL-1β and prostaglandin?
Could the engulfment of particulates, lysosomal rupture, and release of lysosomal enzymes induce PGE2 and IL-1β production in macrophages?
Macrophages(5x105/mL)
Primed with LPS
(1ng/mL)/3h
Stimulation for 2h
Macrophages(5x105/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.
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?
The production of PGE2 is dependent of activity of inflammasome?
Macrophages(5x105/mL)
Primed with LPS
(1ng/mL)/3h
Stimulation for 2h
Macrophages(5x105/mL)
- Silica (100µg/mL) ;
- TiO2 (100µg/mL);
ELISA (PGE2, IL-1β)
(culture Supernatants)
Primed with LPS
(1ng/mL)/3h
Stimulation for 6h- Silica (50μg/mL)
- -Alum (200μg/mL)
ELISA (PGE2)
(culture Supernatants)M-CSF (10ng/mL)
BM cellsMacrophages(5x105/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
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 E2 (PGE2) is generated by the sequential metabolism of arachidonic acid by cyclo-oxygenase and prostaglandin E synthase (Needleman et al., 1986; Smith. 1992).
Could silica and alum induce activity of COX or PGE synthase enzymes?
Macrophages(5x105/mL)
Primed with LPS
(1ng/mL)/3h
Stimulation for 2h
Macrophages(5x105/mL)
- Silica (100µg/mL) ;
- COX-2 inhibitor (1µM);
ELISA (PGE2, IL-1β)
(culture Supernatants)
WTPTGES (+/+; -/-)
Silica- and alum-induce PGE2 production in macrophages mediated by the COX-2 and
PTGES.
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?
Which signaling pathway is involved in the production of PGE2?
Macrophages(5x105/mL)
Primed with LPS
(1ng/mL)/3h
Stimulation for 2h
Macrophages(5x105/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.
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 phosphorilationPhospholipase A2???
Phosphorylation of p38 MAPK could activate phospholipase A2 after phagocytosis of silica or aluminium by macrophages?
Macrophages(5x105/mL)
Primed with LPS
(1ng/mL)/3h
Stimulation for 2h
Macrophages(5x105/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??
The Syk can be activated by phagocytosis de silica or aluminum?
Macrophages(5x105/mL)
Primed with LPS
(1ng/mL)/3h
Stimulation for 2h
Macrophages(5x105/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.
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
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).
- PGE2 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?
OVA+Alum or OVA+Silica
0 7
Days
17Sera 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?
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
Aim: To identify the citotoxic and adjuvant effect of alum on immune
responses
Is alum cytotoxic on local of injection?
Peritoneal lavage fluidMuscle 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
Can DNA act as an adjuvant?
ELISA
i.p. OVAOVA + AlumOVA + DNA
days 0, 14 and 21ELISA
i.p.OVAOVA + Alum
days 0 and 10
DNase I(3 and 8h)
Can DNA act as an adjuvant?
Host DNA released by alum cytotoxicity mediates alum activity on humoral and TH2 cell responses!
i.p.OVAOVA + AlumOVA + DNA
OT-II
OVA-specific CD4+
CFSEDNase I
(3 and 8h)Cells on the bronchial lymph nodes (BLNs)
3d
WT
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, 123-130 (February 2010) | doi:10.1038/nri2690
NALP3-/- and CASP1-/-?
What’s the mechanism?
ELISA (serum)
i.p.
days 0, 14 and 21
NALP3 independent
WT and IRF3-/-
OVAOVA + AlumOVA + DNA
ELISAPeritoneal lavage
fluid
i.p.
days 0, 14 and 21
OVAOVA + AlumOVA + DNA
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 21TBK1+/-/TNF-/-
and TBK1-/-/TNF-/-
OVAOVA + Alum
Have IRF3 deficiency an effect on Th2 responses after alum imunization?
IRF3 is essential for the boosting of canonical TH2 cells by alum and genomic DNA!
i.p.OVAOVA + AlumOVA + DNA
OT-II
OVA-specific CD4+
CFSECells on the bronchial lymph nodes (BLNs)
3d
WT and IRF3-/-
There is an effect during experimental asthma?
IRF3 absence protect mice from allergic airway inflammation!
Bronchoalveolar lavage fluid (BALF)
i.p. OVAOVA + Alum
days 0 and 14
Airway sensitization
d 21 to 25
WT and IRF3-/-
IRF3 deficiency changes DCs migration?
IRF3 is essential for the triggering of iDC recruitment by alum!
FACs (peritoneal lavage fluid and BLNs)
i.p. OVAOVA + AlumOVA + DNA
24 - 48 hWT 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 (F4/80int CD11b+ Ly6C+ Ly6G- cells)• cDCs (MHCII+ CD11c+ F4/80low Ly6C- cells)• pDCs (B220+ Ly6G+ CD11cint F4/80low cells)
BLNs
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. OVAOVA + AlumOT-II
OVA-specific CD4+
CFSECells on the bronchial lymph nodes (BLNs)
WT and IRF3-/-
WT immunized iMonos
IgE increaseIgG1 not affected
IgE attenuatedIgG1 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 21WT and IRF3-/-
OVAOVA + Alum
BLNsiMonos (F4/80int CD11b+ Ly6C+ Ly6G- cells)
Conclusion
DAMPDAMP
Journal Conclusion
AlumNALP3
IndependentNALP3 Dependent
PGE2 Dependent
Host DNA (DAMP)
(IRF3-TBK1 axis)
Syk and p38 MAP kinase
Uric acid crystals
Th2 responses – IgE and IgG1