The Vaccine Adjuvant Chitosan Promotes Cellular Immunity via DNA Sensor cGAS-STING-Dependent Induction of Type I Interferons.

The cationic polysaccharide chitosan is an attractive candidate adjuvant capable of driving potent cell-mediated immunity, but the mechanism by which it acts is not clear. We show that chitosan promotes dendritic cell maturation by inducing type I interferons (IFNs) and enhances antigen-specific T helper 1 (Th1) responses in a type I IFN receptor-dependent manner. The induction of type I IFNs, IFN-stimulated genes and dendritic cell maturation by chitosan required the cytoplasmic DNA sensor cGAS and STING, implicating this pathway in dendritic cell activation. Additionally, this process was dependent on mitochondrial reactive oxygen species and the presence of cytoplasmic DNA. Chitosan-mediated enhancement of antigen specific Th1 and immunoglobulin G2c responses following vaccination was dependent on both cGAS and STING. These findings demonstrate that a cationic polymer can engage the STING-cGAS pathway to trigger innate and adaptive immune responses.

Osteoarthritis-associated basic calcium phosphate crystals induce pro-inflammatory cytokines and damage-associated molecules via activation of Syk and PI3 kinase.

The pro-inflammatory cytokines, TNFα, IL-1 and IL-18, amplify cartilage destruction associated with osteoarthritis (OA). Current data suggest that basic calcium phosphate (BCP) crystals are potent drivers of inflammatory mediator and matrix metalloprotease expression in the OA joint. It has previously been demonstrated that synovial macrophages play a role in initiating and driving BCP-induced inflammation. However, the molecular mechanisms by which BCP crystals exert their effects remain unclear. Here we demonstrate that exposure of macrophages to BCP crystals leads to activation of Syk and PI3 kinase. Furthermore, we show that production of pro-inflammatory cytokines and phosphorylation of the downstream kinase, ERK, are suppressed following treatment with Syk and PI3 kinase inhibitors. Finally, we demonstrate that treatment of macrophages with BCP crystals induces the production of the damage-associated molecule, S100A8, in a Syk dependent manner. We therefore identify Syk and PI3 kinase as potential novel targets for the treatment of BCP-related pathologies.

The vaccine adjuvant alum inhibits IL-12 by promoting PI3 kinase signaling while chitosan does not inhibit IL-12 and enhances Th1 and Th17 responses.

Alum is the principal vaccine adjuvant for clinical applications but it is a poor inducer of cellular immunity and is not an optimal adjuvant for vaccines where Th1 responses are required for protection. The mechanism underlying the inefficiency of alum in promoting Th1 responses is not fully understood. We show that aluminium hydroxide, aluminium phosphate, and calcium phosphate adjuvants inhibit the secretion of the Th1 polarizing cytokine, IL-12 by dendritic cells (DCs). Alum selectively inhibited DC expression of the IL-12p35 subunit and the inhibitory effect results from adjuvant-induced PI3 kinase signaling. To develop a more effective adjuvant for promoting cell-mediated immunity, we investigated alternative particulates and found that in contrast to alum, the cationic polysaccharide chitosan did not inhibit IL-12 secretion. A combination of chitosan and the TLR9 agonist CpG activated the NLRP3 inflammasome and enhanced secretion of IL-12 and the other key Th1 and Th17-cell polarizing cytokines. When used as an adjuvant, CpG-chitosan induced NLRP3-dependent antigen-specific Th1 and Th17 responses. A combination of alum and CpG also enhanced Th1 and Th17 responses but was less effective than CpG-chitosan. Therefore, chitosan is an attractive alternative to alum in adjuvants for vaccines where potent cell-mediated immunity is required.

NLRP3 has a protective role in age-related macular degeneration through the induction of IL-18 by drusen components.

Age-related macular degeneration (AMD) is the leading cause of central vision loss worldwide. Drusen accumulation is the major pathological hallmark common to both dry and wet AMD. Although activation of the immune system has been implicated in disease progression, the pathways involved are unclear. Here we show that drusen isolated from donor AMD eyes activates the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome, causing secretion of interleukin-1b (IL-1b) and IL-18. Drusen component C1Q also activates the NLRP3 inflammasome. Moreover, the oxidative-stress-related protein-modification carboxyethylpyrrole (CEP), a biomarker of AMD, primes the inflammasome. We found cleaved caspase-1 and NLRP3 in activated macrophages in the retinas of mice immunized with CEP-adducted mouse serum albumin, modeling a dry-AMD­like pathology. We show that laser-induced choroidal neovascularization (CNV), a mouse model of wet AMD, is exacerbated in Nlrp3(-/-) but not Il1r1(-/-) mice, directly implicating IL-18 in the regulation of CNV development. These findings indicate a protective role for NLRP3 and IL-18 in the progression of AMD.

Pneumolysin activates the NLRP3 inflammasome and promotes proinflammatory cytokines independently of TLR4.

Pneumolysin (PLY) is a key Streptococcus pneumoniae virulence factor and potential candidate for inclusion in pneumococcal subunit vaccines. Dendritic cells (DC) play a key role in the initiation and instruction of adaptive immunity, but the effects of PLY on DC have not been widely investigated. Endotoxin-free PLY enhanced costimulatory molecule expression on DC but did not induce cytokine secretion. These effects have functional significance as adoptive transfer of DC exposed to PLY and antigen resulted in stronger antigen-specific T cell proliferation than transfer of DC exposed to antigen alone. PLY synergized with TLR agonists to enhance secretion of the proinflammatory cytokines IL-12, IL-23, IL-6, IL-1ß, IL-1α and TNF-α by DC and enhanced cytokines including IL-17A and IFN-γ by splenocytes. PLY-induced DC maturation and cytokine secretion by DC and splenocytes was TLR4-independent. Both IL-17A and IFN-γ are required for protective immunity to pneumococcal infection and intranasal infection of mice with PLY-deficient pneumococci induced significantly less IFN-γ and IL-17A in the lungs compared to infection with wild-type bacteria. IL-1ß plays a key role in promoting IL-17A and was previously shown to mediate protection against pneumococcal infection. The enhancement of IL-1ß secretion by whole live S. pneumoniae and by PLY in DC required NLRP3, identifying PLY as a novel NLRP3 inflammasome activator. Furthermore, NLRP3 was required for protective immunity against respiratory infection with S. pneumoniae. These results add significantly to our understanding of the interactions between PLY and the immune system.

Highly differentiated, resting gn-specific memory CD8+ T cells persist years after infection by andes hantavirus.

In man, infection with South American Andes virus (ANDV) causes hantavirus cardiopulmonary syndrome (HCPS). HCPS due to ANDV is endemic in Southern Chile and much of Argentina and increasing numbers of cases are reported all over South America. A case-fatality rate of about 36% together with the absence of successful antiviral therapies urge the development of a vaccine. Although T-cell responses were shown to be critically involved in immunity to hantaviruses in mouse models, no data are available on the magnitude, specificity and longevity of ANDV-specific memory T-cell responses in patients. Using sets of overlapping peptides in IFN-gamma ELISPOT assays, we herein show in 78 Chilean convalescent patients that Gn-derived epitopes were immunodominant as compared to those from the N- and Gc-proteins. Furthermore, while the relative contribution of the N-specific response significantly declined over time, Gn-specific responses remained readily detectable ex vivo up to 13 years after the acute infection. Tetramer analysis further showed that up to 16.8% of all circulating CD3(+)CD8(+) T cells were specific for the single HLA-B*3501-restricted epitope Gn(465-473) years after the acute infection. Remarkably, Gn(465-473)-specific cells readily secreted IFN-gamma, granzyme B and TNF-alpha but not IL-2 upon stimulation and showed a 'revertant' CD45RA(+)CD27(-)CD28(-)CCR7(-)CD127(-) effector memory phenotype, thereby resembling a phenotype seen in other latent virus infections. Most intriguingly, titers of neutralizing antibodies increased over time in 10/17 individuals months to years after the acute infection and independently of whether they were residents of endemic areas or not. Thus, our data suggest intrinsic, latent antigenic stimulation of Gn-specific T-cells. However, it remains a major task for future studies to proof this hypothesis by determination of viral antigen in convalescent patients. Furthermore, it remains to be seen whether Gn-specific T cells are critical for viral control and protective immunity. If so, Gn-derived immunodominant epitopes could be of high value for future ANDV vaccines.