Alum adjuvants : discovering their hidden secrets

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