Bile acids induce IL-1α and drive NLRP3 inflammasome-independent production of IL-1ß in murine dendritic cells.

Bile acids are amphipathic molecules that are synthesized from cholesterol in the liver and facilitate intestinal absorption of lipids and nutrients. They are released into the small intestine upon ingestion of a meal where intestinal bacteria can modify primary into secondary bile acids. Bile acids are cytotoxic at high concentrations and have been associated with inflammatory diseases such as liver inflammation and Barrett's Oesophagus. Although bile acids induce pro-inflammatory signalling, their role in inducing innate immune cytokines and inflammation has not been fully explored to date. Here we demonstrate that the bile acids, deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA) induce IL-1α and IL-1ß secretion in vitro in primed bone marrow derived dendritic cells (BMDCs). The secretion of IL-1ß was found not to require expression of NLRP3, ASC or caspase-1 activity; we can't rule out all inflammasomes. Furthermore, DCA and CDCA were shown to induce the recruitment of neutrophils and monocytes to the site of injection an intraperitoneal model of inflammation. This study further underlines a mechanistic role for bile acids in the pathogenesis of inflammatory diseases through stimulating the production of pro-inflammatory cytokines and recruitment of innate immune cells.

Soluble mediators in the function of the epidermal-immune-neuro unit in the skin.

Skin is the largest, environmentally exposed (barrier) organ, capable of integrating various signals into effective defensive responses. The functional significance of interactions among the epidermis and the immune and nervous systems in regulating and maintaining skin barrier function is only now becoming recognized in relation to skin pathophysiology. This review focuses on newly described pathways that involve soluble mediator-mediated crosstalk between these compartments. Dysregulation of these connections can lead to chronic inflammatory diseases and/or pathologic conditions associated with chronic pain or itch.

Dendritic Cell-Specific Role for Pellino2 as a Mediator of TLR9 Signaling Pathway.

Ubiquitination regulates immune signaling, and multiple E3 ubiquitin ligases have been studied in the context of their role in immunity. Despite this progress, the physiological roles of the Pellino E3 ubiquitin ligases, especially Pellino2, in immune regulation remain largely unknown. Accordingly, this study aimed to elucidate the role of Pellino2 in murine dendritic cells (DCs). In this study, we reveal a critical role of Pellino2 in regulation of the proinflammatory response following TLR9 stimulation. Pellino2-deficient murine DCs show impaired secretion of IL-6 and IL-12. Loss of Pellino2 does not affect TLR9-induced activation of NF-κB or MAPKs, pathways that drive expression of IL-6 and IL-12. Furthermore, DCs from Pellino2-deficient mice show impaired production of type I IFN following endosomal TLR9 activation, and it partly mediates a feed-forward loop of IFN-ß that promotes IL-12 production in DCs. We also observe that Pellino2 in murine DCs is downregulated following TLR9 stimulation, and its overexpression induces upregulation of both IFN-ß and IL-12, demonstrating the sufficiency of Pellino2 in driving these responses. This suggests that Pellino2 is critical for executing TLR9 signaling, with its expression being tightly regulated to prevent excessive inflammatory response. Overall, this study highlights a (to our knowledge) novel role for Pellino2 in regulating DC functions and further supports important roles for Pellino proteins in mediating and controlling immunity.

ECSIT is a critical limiting factor for cardiac function.

Evolutionarily conserved signaling intermediate in Toll pathways (ECSIT) is a protein with roles in early development, activation of the transcription factor NF-κB, and production of mitochondrial reactive oxygen species (mROS) that facilitates clearance of intracellular bacteria like Salmonella. ECSIT is also an important assembly factor for mitochondrial complex I. Unlike the murine form of Ecsit (mEcsit), we demonstrate here that human ECSIT (hECSIT) is highly labile. To explore whether the instability of hECSIT affects functions previously ascribed to its murine counterpart, we created a potentially novel transgenic mouse in which the murine Ecsit gene is replaced by the human ECSIT gene. The humanized mouse has low levels of hECSIT protein, in keeping with its intrinsic instability. Whereas low-level expression of hECSIT was capable of fully compensating for mEcsit in its roles in early development and activation of the NF-κB pathway, macrophages from humanized mice showed impaired clearance of Salmonella that was associated with reduced production of mROS. Notably, severe cardiac hypertrophy was manifested in aging humanized mice, leading to premature death. The cellular and molecular basis of this phenotype was delineated by showing that low levels of human ECSIT protein led to a marked reduction in assembly and activity of mitochondrial complex I with impaired oxidative phosphorylation and reduced production of ATP. Cardiac tissue from humanized hECSIT mice also showed reduced mitochondrial fusion and more fission but impaired clearance of fragmented mitochondria. A cardiomyocyte-intrinsic role for Ecsit in mitochondrial function and cardioprotection is also demonstrated. We also show that cardiac fibrosis and damage in humans correlated with low expression of human ECSIT. In summary, our findings identify a role for ECSIT in cardioprotection, while generating a valuable experimental model to study mitochondrial dysfunction and cardiac pathophysiology.

IL-33 Is a Negative Regulator of Vaccine-Induced Antigen-Specific Cellular Immunity.

The cytokine IL-33 is a well-established inducer of Th2 responses. However, roles for IL-33 in promoting CD8, Th1, and T regulatory cell responses have also emerged. In this study, the role of IL-33 as a regulator of particulate vaccine adjuvant-induced Ag-specific cellular immunity was investigated. We found that polymeric nanoparticles surpassed alum in their ability to enhance Ag-specific CD8 and Th1 responses. IL-33 was a potent negative regulator of both CD8+ T cell and Th1 responses following i.m. vaccination with Ag and nanoparticles, whereas the cytokine was required for the nanoparticle enhancement in Ag-specific IL-10. In contrast to the effect on cellular immunity, Ab responses were comparable between vaccinated wild-type and IL-33-deficient mice. IL-33 did not compromise alum-induced adaptive cellular immunity after i.m. vaccination. These data suggest that IL-33 attenuates the induction of cellular immune responses by nanoparticulate adjuvants and should be considered in the rational design of vaccines targeting enhanced CD8 and Th1 responses.

The vaccine adjuvant alum promotes IL-10 production that suppresses Th1 responses.

The effectiveness of many vaccines licensed for clinical use relates to the induction of neutralising antibodies, facilitated by the inclusion of vaccine adjuvants, particularly alum. However, the ability of alum to preferentially promote humoral rather than cellular, particularly Th1-type responses, is not well understood. We demonstrate that alum activates immunosuppressive mechanisms following vaccination, which limit its capacity to induce Th1 responses. One of the key cytokines limiting excessive immune responses is IL-10. Injection of alum primed draining lymph node cells for enhanced IL-10 secretion ex vivo. Moreover, at the site of injection, macrophages and dendritic cells were key sources of IL-10 expression. Alum strongly enhanced the transcription and secretion of IL-10 by macrophages and dendritic cells. The absence of IL-10 signalling did not compromise alum-induced cell infiltration into the site of injection, but resulted in enhanced antigen-specific Th1 responses after vaccination. In contrast to its decisive regulatory role in regulating Th1 responses, there was no significant change in antigen-specific IgG1 antibody production following vaccination with alum in IL-10-deficient mice. Overall, these findings indicate that injection of alum promotes IL-10, which can block Th1 responses and may explain the poor efficacy of alum as an adjuvant for inducing protective Th1 immunity.

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.

Endogenous oils derived from human adipocytes are potent adjuvants that promote IL-1α-dependent inflammation.

Obesity is characterized by chronic inflammation associated with neutrophil and M1 macrophage infiltration into white adipose tissue. However, the mechanisms underlying this process remain largely unknown. Based on the ability of oil-based adjuvants to induce immune responses, we hypothesized that endogenous oils derived from necrotic adipocytes may function as an immunological "danger signal." Here we show that endogenous oils of human origin are potent adjuvants, enhancing antibody responses to a level comparable to Freund's incomplete adjuvant. The endogenous oils were capable of promoting interleukin (IL)-1α-dependent recruitment of neutrophils and M1-like macrophages, while simultaneously diminishing M2-like macrophages. We found that endogenous oils from subcutaneous and omental adipocytes, and from healthy and unhealthy obese individuals, promoted comparable inflammatory responses. Furthermore, we also confirmed that white adipocytes in visceral fat of metabolically unhealthy obese (MUO) individuals are significantly larger than those in metabolically healthy obese individuals. Since adipocyte size is positively correlated with adipocyte death, we propose that endogenous oils have a higher propensity to be released from hypertrophied visceral fat in MUO individuals and that this is the key factor in driving inflammation. In summary, this study shows that adipocytes contain a potent oil adjuvant which drives IL-1α-dependent proinflammatory responses in vivo.

Immunomodulatory properties of the vaccine adjuvant alum.

Alum, the most common adjuvant in non-living vaccines, has a record of successful use in human vaccination where it promotes antibody-mediated protective immunity. However, alum is a poor inducer of cellular immune responses. The mechanism underlying the selective enhancement of humoral immunity is still not well understood. Here, to provide an insight into its mode of action, recent findings regarding innate immune responses induced by alum and their impact on adaptive immunity are described, with a particular emphasis on early recognition of alum, including NLRP3 and PI3 kinase activation, adjuvant-induced cell death and the release of endogenous danger signals. Expanding our knowledge of alum-induced immunomodulation will greatly enhance our capacity to rationally develop novel adjuvants with specific properties.

NLRP3 inflammasome activation and cytotoxicity induced by particulate adjuvants.

The ability of particulate materials to provoke inflammatory immune responses has been well documented. In the case of endogenous and environmental particulates, these effects can often lead to pathological disorders. In contrast, particulate adjuvants incorporated into vaccines promote immune responses, which in turn provide efficient protection against infectious diseases. In recent years, studies have revealed that the NLRP3 inflammasome plays a key role in particulate-driven inflammation and its associated cytotoxicity. Hence, this chapter covers protocols useful to (1) assess NLRP3 inflammasome activation triggered by particulate adjuvants or materials in mouse bone marrow-derived dendritic cell (BMDCs) differentiated cultures, and (2) measure particle-induced cytotoxicity. More specifically, protocols are described for the preparation and differentiation of BMDCs, their priming and stimulation using particulate NLRP3 agonists such as monosodium urate monohydrate (MSU) and the vaccine adjuvant alum. We then detail protocols to assess particulate-driven cytotoxicity via flow cytometry using annexin V-propidium iodide (PI) and novel dye LIVE/DEAD(®) aqua stain. General considerations are provided that warn against the use of endotoxin-contaminated particles and emphasize the use of experimental controls. Suggestions are also outlined for further assessment of the immunomodulatory effects of particulate materials in vivo using the mouse peritonitis model.

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.