Chitin-derived polymer deacetylation regulates mitochondrial reactive oxygen species dependent cGAS-STING and NLRP3 inflammasome activation.

Chitosan is a cationic polysaccharide that has been evaluated as an adjuvant due to its biocompatible and biodegradable nature. The polysaccharide can enhance antibody responses and cell-mediated immunity following vaccination by injection or mucosal routes. However, the optimal polymer characteristics for activation of dendritic cells (DCs) and induction of antigen-specific cellular immune responses have not been resolved. Here, we demonstrate that only chitin-derived polymers with a high degree of deacetylation (DDA) enhance generation of mitochondrial reactive oxygen species (mtROS), leading to cGAS-STING mediated induction of type I IFN. Additionally, the capacity of the polymers to activate the NLRP3 inflammasome was strictly dependent on the degree and pattern of deacetylation and mtROS generation. Polymers with a DDA below 80% are poor adjuvants while a fully deacetylated polyglucosamine polymer is most effective as a vaccine adjuvant. Furthermore, this polyglucosamine polymer enhanced antigen-specific Th1 responses in a NLRP3 and STING-type I IFN-dependent manner. Overall these results indicate that the degree of chitin deacetylation, the acetylation pattern and its regulation of mitochondrial ROS are the key determinants of its immune enhancing effects.

Type I IFN signalling is required for cationic adjuvant formulation (CAF)01-induced cellular immunity and mucosal priming.

Despite being in the midst of a global pandemic of infections caused by the pathogen Chlamydia trachomatis, a vaccine capable of inducing protective immunity remains elusive. Given the C. trachomatis mucosal port of entry, a formulation compatible with mucosal administration and capable of eliciting potent genital tract immunity is highly desirable. While subunit vaccines are considered safer and better tolerated, these are typically poorly immunogenic and require co-formulation with immune-potentiating adjuvants. However, of the adjuvants licensed for use in humans, very few drive robust cellular responses, a pre-requisite for protection against C. trachomatis infection. Recently, the cationic adjuvant formulations (CAF) have been shown to induce robust humoral and cellular immunity in pre-clinical models of chlamydia, malaria and tuberculosis (TB). Here, we demonstrate that CAF01 induces potent immune responses when combined with the major outer membrane protein (MOMP) of C. trachomatis following parenteral immunisation and also as part of a heterologous prime/boost regime. We show that a subcutaneous prime with CAF01-adjuvanted recombinant MOMP licenses antigen-specific immunity at distant mucosal sites which can be activated following oral antigen re-encounter in the absence of concomitant adjuvant stimulation. Finally, we shed light on the mechanism(s) through which CAF01 elicits robust antigen-specific immunity to co-formulated MOMP via type I interferon (IFN) signalling.

Immunomodulatory properties of chitosan polymers.

The introduction of vaccines is regarded as one of the most successful medical interventions to date. However, there is a clear need for the development of new vaccines for diseases which require the induction of a potent cellular immune response. Advancements in the field of vaccine research have resulted in a move away from the use of whole organisms and towards the use of subunit vaccines which consist of highly purified antigens with an improved safety profile. Adjuvants are immunostimulatory components that are included in subunit vaccine formulations to help direct and amplify adaptive immune responses. Chitosan is a cationic polysaccharide that has been examined in an adjuvant setting due to its biocompatible and biodegradable nature. The polysaccharide has been shown to have the capacity to induce Th1 cell responses following vaccination by injection or mucosal routes, supporting its application as an alternative to alum for vaccines that promote cell-mediated immunity. Here, we provide an overview of the physico-chemical properties of chitosan with a focus on the specific characteristics that dictate the type and scale of the immune responses induced. The potential to finely tailor chitosan polymers in order to direct specific types of immune responses can provide invaluable tools for the design of novel chitosan-based adjuvants and biomaterials.

Extracellular Neutrophil Proteases Are Efficient Regulators of IL-1, IL-33, and IL-36 Cytokine Activity but Poor Effectors of Microbial Killing.

Neutrophil granule proteases are thought to function as anti-microbial effectors, cooperatively hydrolyzing microorganisms within phagosomes, or upon deployment into the extracellular space. However, evidence also suggests that neutrophil proteases play an important role in the coordination and escalation of inflammatory reactions, but how this is achieved has been obscure. IL-1 family cytokines are important initiators of inflammation and are typically released via necrosis but require proteolytic processing for activation. Here, we show that proteases liberated from activated neutrophils can positively or negatively regulate the activity of six IL-1 family cytokines (IL-1α, IL-1ß, IL-33, IL-36α, IL-36ß, and IL-36γ) with exquisite sensitivity. In contrast, extracellular neutrophil proteases displayed very poor bactericidal activity, exhibiting 100-fold greater potency toward cytokine processing than bacterial killing. Thus, in addition to their classical role as phagocytes, neutrophils play an important immunoregulatory role through deployment of their granule proteases into the extracellular space to process multiple IL-1 family cytokines.

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.

IL-1α and inflammasome-independent IL-1ß promote neutrophil infiltration following alum vaccination.

Despite its long record of successful use in human vaccines, the mechanisms underlying the immunomodulatory effects of alum are not fully understood. Alum is a potent inducer of interleukin-1 (IL-1) secretion in vitro in dendritic cells and macrophages via Nucleotide-binding domain and leucine-rich repeat-containing (NLR) family, pyrin domain-containing 3 (NLRP3) inflammasome activation. However, the contribution of IL-1 to alum-induced innate and adaptive immune responses is controversial and the role of IL-1α following alum injection has not been addressed. This study shows that IL-1 is dispensable for alum-induced antibody and CD8 T cell responses to ovalbumin. However, IL-1 is essential for neutrophil infiltration into the injection site, while recruitment of inflammatory monocytes and eosinophils is IL-1 independent. Both IL-1α and IL-1ß are released at the site of injection and contribute to the neutrophil response. Surprisingly, these effects are NLRP3-inflammasome independent as is the infiltration of other cell populations. However, while NLRP3 and caspase 1 were dispensable, alum-induced IL-1ß at the injection site was dependent on the cysteine protease cathepsin S. Overall, these data demonstrate a previously unreported role for cathepsin S in IL-1ß secretion, show that inflammasome formation is dispensable for alum-induced innate immunity and reveal that IL-1α and IL-1ß are both necessary for alum-induced neutrophil influx in vivo.