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Keywords:

  • Adjuvant;
  • Inflammasome;
  • Interleukin 1;
  • Vaccine

Abstract

  1. Top of page
  2. Abstract
  3. Alum and other particulate adjuvants activate the NLRP3 inflammasome
  4. Is NLRP3 required for adjuvant-induced adaptive immunity?
  5. Roles for inflammasomes in protective immunity: Lessons for future adjuvant design
  6. Concluding remarks
  7. Acknowledgements
  8. References
  9. Supporting Information

Adjuvants are essential for enhancing and directing immunity to vaccine antigens. Most adjuvants in clinical use are particulates, but how they drive innate and adaptive immune responses is unclear. A major recent advance was the demonstration that particulate adjuvants promote activation of the NLRP3 inflammasome. The mechanisms underlying this activation have been partly resolved and the role of NLRP3 in particulate adjuvant-induced adaptive immunity is currently the subject of intense interest.


Alum and other particulate adjuvants activate the NLRP3 inflammasome

  1. Top of page
  2. Abstract
  3. Alum and other particulate adjuvants activate the NLRP3 inflammasome
  4. Is NLRP3 required for adjuvant-induced adaptive immunity?
  5. Roles for inflammasomes in protective immunity: Lessons for future adjuvant design
  6. Concluding remarks
  7. Acknowledgements
  8. References
  9. Supporting Information

Most purified protein and peptide antigens are poorly immunogenic so adjuvants must be included in vaccines to activate and direct adaptive immune responses. The most commonly used adjuvants in clinical use are aluminium salts, particularly aluminium hydroxide adjuvant, which is chemically crystalline aluminium oxyhydroxide 1, 2 and comprises nanoparticles that form porous aggregates of 1–20 μm in diameter 2. Although not technically correct, aluminium-containing adjuvants are generally referred to as “alum”. Alum is not an optimal adjuvant for all protein antigens and is a relatively poor inducer of cell-mediated immunity. As a result, other particulate adjuvants, including chitosan, liposomes and biodegradable microparticles and nanoparticles are being investigated 3. However, despite the widespread use of particulates in both clinical and research settings for over 80 years, their mode of action is still not fully understood.

In 2007 it was found that aluminium-containing adjuvants enhanced the activation of caspase 1 and release of IL-1β and IL-18 from murine DC 4 and human PBMC and DC in the presence of TLR agonists 5. A number of articles then demonstrated that alum/TLR-induced inflammasome activation was dependent on NLRP3 and ASC 6, 7. Eisenbarth et al.8 demonstrated a specific requirement for uptake of alum, tubulin polymerisation and the efflux of cellular potassium for inflammasome activation. The requirement for potassium efflux is a consistent finding across all of these studies, but other aspects of inflammasome activation are more controversial. Both the generation of ROS 9 and lysosomal disruption and cathepsin B activity 7–10 have been proposed as the mediators of alum-induced NLRP3 inflammasome activation.

Activation of the NLRP3 inflammasome by various other particulates including chitosan 6 and microparticles 10 has also been demonstrated. Biodegradable poly (lactide-co-glycolide) and polystyrene microparticles and nanoparticles, in combination with TLR agonists, enhanced NLRP3-dependent IL-1β production by murine DC 10. This was dependent on particle uptake, lysosomal maturation and cathepsin B. Many of the above reports have found that while inclusion of a TLR agonist is required for adjuvant-induced IL-1β secretion in vitro, the adjuvants alone can induce IL-1β secretion in vivo, suggesting that endogenous factors can provide the second signal for inflammasome activation 10.

In addition to the induction of IL-1β and IL-18, it was suggested that silencing of ASC and NLRP3 in human cells reduced the production of IL-33 6. However, since IL-33 is active in its full-length form and its bioactivity is attenuated by apoptotic caspase-mediated proteolysis 11 and is inactivated by caspase 1 12, IL-33 is unlikely to be an effector of NLPR3 activation.

Is NLRP3 required for adjuvant-induced adaptive immunity?

  1. Top of page
  2. Abstract
  3. Alum and other particulate adjuvants activate the NLRP3 inflammasome
  4. Is NLRP3 required for adjuvant-induced adaptive immunity?
  5. Roles for inflammasomes in protective immunity: Lessons for future adjuvant design
  6. Concluding remarks
  7. Acknowledgements
  8. References
  9. Supporting Information

While the demonstration that alum and other particulate adjuvants can activate the NLRP3 inflammasome is an important discovery, the key question is whether this activation is required for the adjuvants to promote adaptive immunity. Table 1 summarises the studies that have addressed this issue to date. Eisenbarth et al.8 reported that alum-induced antigen-specific humoral and cellular immunity required NLRP3, ASC and caspase 1 while these responses were intact in MyD88 knockout mice. Previous studies also found that alum promotes antibody responses in MyD88/TRIF double knockout mice 8, 13 and Th2 priming was intact in MyD88 deficient mice 14. Since MyD88 is required for IL-1 (and IL-18) receptor signalling 15, it is difficult to assess how the proposed NLRP3-dependent effects of alum are mediated. There may be NLRP3 effectors that can signal independently of MyD88, or IL-1 may exert MyD88-independent effects 16. Li et al.6 also reported an important role for NLRP3 in the adjuvanticity of alum. In contrast Kool et al.9 found that cellular immune responses were reduced but there was no effect on IgG1 in NLRP3 deficient mice. Further studies found that NLRP3 played no role in the ability of alum or biodegradable microparticles to enhance antigen-specific antibody responses 10, 17, 18, and neither NLRP3 nor caspase 1 were required for alum to enhance endogenous CD4 or CD8 T cell priming 18. It was also reported that the immunostimulatory effects of alum were abolished when uricase was used to degrade uric acid 19. Further evidence that alum-induced adaptive immunity does not require NLRP3 or IL-1 signalling comes from studies demonstrating the development of Th2-mediated asthma induced by OVA/Alum in NLRP3-deficient mice (as summarised in 20) and alum-induced Th2 responses in IL-1R1- or IL-1β-deficient mice 21.

Table 1. Consequences of NLRP3 deficiency on innate and antigen-specific adaptive immunity in mice
AdjuvantRouteAntibody responseT-cell responseInnate immunityReference
  1. a

    OVA, ovalbumin; HSA, human serum albumin; DT, diphtheria toxoid.

Alum (Imject)• i.p OVA/alum, challenged i.n with OVA. • OVA/alum s.c • HSA/alum s.c challenged i.n with HSA[DOWNWARDS ARROW] OVA-specific IgG1[DOWNWARDS ARROW] OVA-specific IL-5[DOWNWARDS ARROW] Airway eosinophilia8
Alum• i.p HSA/alum boosted with HASNo effect on IgA, IgM, IgG or IgG subclassesNDND17
Alhydrogel, Alum (Inject)• DT/alum • OVA/alum[DOWNWARDS ARROW] Antigen-specific IgG1; [DOWNWARDS ARROW] IgE  6
Alum (Imject)• i.p OVA[DOWNWARDS ARROW] IgE; [UPWARDS ARROW] IgG2c; No effect on IgG1[DOWNWARDS ARROW] Antigen reactive T cells in LN[DOWNWARDS ARROW] Eoinophil, neutrophil, monocyte infiltration; [DOWNWARDS ARROW] IL-1β; [DOWNWARDS ARROW] DC activation9
Alhydrogel, Alum (Imject)• i.m DT/alum • OVA/alumNo effect on antigen-specific IgG1No effect on CD4+ or CD8+ T-cell priming 18
PLG microparticles (1 μm) polystyrene nanoparticles (430 nm)• i.p, OVA/micro/ • nanoparticlesNo effect of total IgG or IgG subclassesAntigen-specific IL-6 reducedReduced cell infiltration10

Furthermore, the ability of PLGA microparticles to promote antigen-specific antibody responses to OVA is intact in NLRP3 deficient mice, although antigen-specific IL-6 production was reduced 10. Thus, there is currently no consensus on the requirement for NLRP3 in particulate adjuvant-induced adaptive immune responses and further studies are required to resolve these issues (Fig. 1).

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Figure 1. The role of NLRP3 in particulate adjuvant-induced innate and adaptive immunity. A range of particulate adjuvants promote activation of the NLRP3 inflammasome and the secretion of IL-1β and IL-18. This requires a second stimulus, usually a TLR ligand in vitro (not shown in the figure) but the adjuvants can promote IL-1β secretion alone in vivo. Most studies have found a role for NLRP3 in particulate adjuvant-induced recruitment of granulocytes and monocytes but there is currently no consensus on the role of NLRP3 in adjuvant-induced adaptive immunity. These adjuvants appear to preferentially promote Th2-type responses, but the role of NLRP3 in this process is unclear. Particulate adjuvants including alum and biodegradable microparticles and nanoparticles are highly effective at promoting antigenspecific antibody responses, particularly IgG1 and a number of recent reports have found that these adjuvants can promote potent antibody responses to associated antigens in the absence of NLRP3.

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Roles for inflammasomes in protective immunity: Lessons for future adjuvant design

  1. Top of page
  2. Abstract
  3. Alum and other particulate adjuvants activate the NLRP3 inflammasome
  4. Is NLRP3 required for adjuvant-induced adaptive immunity?
  5. Roles for inflammasomes in protective immunity: Lessons for future adjuvant design
  6. Concluding remarks
  7. Acknowledgements
  8. References
  9. Supporting Information

Inflammasome activation is required for protective immunity against a number of pathogens, including Salmonella typhimurium, Listeria monocytogenes, Pseudomonas aeruginosa, Shigella flexneri and Staphylococcus aureus22. Here we look at recent evidence for the role of inflammasome activation in protective responses to three common and important pathogens; Influenza A, mycobacteria and Candida albicans in the context of vaccine development.

Influenza A

In mouse models of infection with influenza A, two recent studies have described a role for the NLRP3 inflammasome in host protective responses. Both Allen et al.23 and Thomas et al.24 demonstrated that NLRP3 directs inflammatory responses that limit lung damage and pathogenesis. However, while both studies demonstrated increased morbidity in NLRP3−/− and caspase 1−/− mice, Allen et al.23 found reduced airway inflammation in the mutants but Thomas et al.24 found increased lung pathology. Both studies demonstrated a reduction in recruitment of neutrophils and monocytes to the lungs. Moreover, while Allen et al. 23 found increased viral burdens in the mutants after 7 days, Thomas et al. 24 observed no difference after 6 days. Protective responses were dependent on both lysosome maturation and the generation of ROS, both in vitro and in vivo and correlated with increased levels of IL-1β in the lungs of infected animals 23. Ichinohe et al. found that mice deficient in ASC, caspase 1 or IL-1R, but not NLRP3, exhibited reduced CD4 and CD8 T cell responses against the virus, along with lower antigen-specific mucosal IgA and systemic IgG 24, 25 indicating the involvement of inflammasome other than NLRP3 in protective immunity to influenza. Taken together, these data support the development of inflammasome-activating adjuvants for influenza vaccines.

Mycobacteria

Mice deficient in IL-1R1 are more susceptible to pulmonary tuberculosis after infection with Mycobacterium tuberculosis, with increased mortality, defective granuloma formation and enhanced mycobacterial growth in the lungs, spleen and liver as compared with WT mice 26. Similarly, IL-1R1−/− mice infected with M. kansaii had reduced lung antibacterial responses and increased bacterial dissemination to the liver 27. Moreover, IL-1β released by monocytes infected with M. tuberculosis induced IL-8 secretion by human bronchial epithelial cells, implicating IL-1β in early host inflammatory responses to tuberculosis 28. A mycobacterial gene, zmp1, which encodes a putative Zn2+ metalloprotease, was proposed to suppress inflammasome activation in infected macrophages 29. Macrophages infected with zmp1−/−M. bovis BCG secreted more IL-1β than those infected with WT BCG. The study demonstrated that IL-1β increases maturation of mycobacteria-containing phagosomes and enhances killing of the bacilli by macrophages. Survival of zmp1−/− BCG was rescued after siRNA knockdown of caspase 1, IL-1β, ASC and IPAF 29. In another study Koo et al. found that macrophages infected with live, virulent strains of M. marinum or M. tuberculosis secreted more IL-1β than those infected with attenuated strains or heat-killed bacilli. Secretion, but not synthesis, of IL-1β and IL-18 was dependent on the mycobacterial ESX-1 secretion system and correlated with lysosome exocytosis 30. In this study, processing and secretion of IL-1β and IL-18 was dependent on caspase 1, ASC and NLRP3, but not IPAF. Thus, more than one inflammasome may be involved in the response to mycobacteria, perhaps depending on the strain/virulence of the bacilli. These data suggest that IL-1β is an important component of the early immune response to mycobacteria and thus vaccines that target inflammasome activation may have potential therapeutic benefit.

C. albicans

Recent studies have demonstrated a protective role for inflammasomes in the immune response against C. albicans. Host responses to the pathogen are dependent on signalling through IL-1R1 31, while mice deficient in IL-1α and IL-1β have a higher mortality rate compared with WT mice when infected with C. albicans32. Three recent studies have demonstrated an essential role for the NLRP3 inflammasome in protective immunity 33–35. Hise et al.34, demonstrated that TLR2 and Dectin 1 regulated IL-1β gene transcription, while NLRP3 was responsible for caspase 1 activation and cleavage of pro-IL-1β into the mature form. All of these components were essential in limiting dissemination of mucosal infection and reducing mortality 34. Gross et al.33 found that synthesis of pro-IL-1β and activation of the NLRP3 inflammasome was dependent on the tyrosine kinase Syk. In particular, syk-dependent pro-IL-1β synthesis involved CARD9, whereas inflammasome activation required K+ efflux and the generation of ROS 33. Moreover, mice deficient in NLRP3 were hyper-susceptible to Candida-induced mortality 33. Joly et al. found that activation of the NLRP3 inflammasome following infection was dependent on the ability of C. albicans to switch from the unicellular form into the filamentous form 35. This switch is essential for the pathogen to escape the phagosome, but membrane disruption by the hyphae also triggers IL-1β processing and secretion, which was inhibited by the cathepsin B inhibitor CA-074-Me 35. These data provide evidence that IL-1 secretion is essential for host protective responses against a fungal pathogen, again highlighting the potential for inflammasome-activating adjuvants in vaccine design.

Concluding remarks

  1. Top of page
  2. Abstract
  3. Alum and other particulate adjuvants activate the NLRP3 inflammasome
  4. Is NLRP3 required for adjuvant-induced adaptive immunity?
  5. Roles for inflammasomes in protective immunity: Lessons for future adjuvant design
  6. Concluding remarks
  7. Acknowledgements
  8. References
  9. Supporting Information

A number of particulate vaccine adjuvants activate the NLRP3 inflammasome and NLRP3 is clearly involved in particulate adjuvant-induced innate immunity (Fig. 1). However, there is less consensus on the importance of NLRP3 in adjuvant-induced antibody responses. A number of recent studies have found no role for NLRP3 in alum or microparticle-induced antibody responses, but the reasons for this striking discrepancy with earlier reports is unclear. Regarding the induction of T cell, particularly Th2, responses by particulate adjuvants, the role of NLRP3 is uncertain. Only a small number of studies have addressed the role of NLRP3 in particulate adjuvant-induced T-cell responses in detail and the effector responses, including cytokines measured, differ significantly between these publications. Thus, further studies are required to resolve these issues, which may be influenced by the nature of the adjuvant, the nature and purity of antigens and the vaccination route. Recent findings indicate an important role for NLRP3 in protective immunity against viral, bacterial and fungal pathogens, while other inflammasome complexes are also likely to be involved in these responses. NLRC4, for example, is required for immunity to pulmonary infection with Legionella pneumophilia36. Moreover, IL-1 is required for antigen-specific IL-17 production, which is implicated in protective immunity against a number of extracellular pathogens 37, 38. Indeed, a NLRP3 mutation that causes inflammasome hyper-activation promotes Th17 responses 39. Thus, while there are numerous issues yet to be resolved, the emerging role of inflammasomes in protective immunity provides a strong rationale for the development of inflammasome-activating vaccine adjuvants.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Alum and other particulate adjuvants activate the NLRP3 inflammasome
  4. Is NLRP3 required for adjuvant-induced adaptive immunity?
  5. Roles for inflammasomes in protective immunity: Lessons for future adjuvant design
  6. Concluding remarks
  7. Acknowledgements
  8. References
  9. Supporting Information

J. Harris is supported by Science foundation Ireland as part of the Immunology Research Centre under grant number 07/SRC/B1144). F. A. Sharp is supported by IRCSET, Ed Lavelle's work on adjuvants is also funded by grants from Science Foundation Ireland (08/RFP/BMT1363, 07/RFP/BICF537), Enterprise Ireland (IP 2007 0451) and the Meningitis Research Foundation (0610.0).

Conflict of interest: The authors declare no financial or commercial conflict of interest.

References

  1. Top of page
  2. Abstract
  3. Alum and other particulate adjuvants activate the NLRP3 inflammasome
  4. Is NLRP3 required for adjuvant-induced adaptive immunity?
  5. Roles for inflammasomes in protective immunity: Lessons for future adjuvant design
  6. Concluding remarks
  7. Acknowledgements
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Alum and other particulate adjuvants activate the NLRP3 inflammasome
  4. Is NLRP3 required for adjuvant-induced adaptive immunity?
  5. Roles for inflammasomes in protective immunity: Lessons for future adjuvant design
  6. Concluding remarks
  7. Acknowledgements
  8. References
  9. Supporting Information

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