Inflammasome-mediated pyroptotic and apoptotic cell death, and defense against infection

Cell death is an effective strategy to limit intracellular infections. Canonical inflammasomes, including NLRP3, NLRC4, and AIM2, recruit and activate caspase-1 in response to a range of microbial stimuli and endogenous danger signals. Caspase-1 then promotes the secretion of IL-1β and IL-18 and a rapid form of lytic programmed cell death termed pyroptosis. A second inflammatory caspase, mouse caspase-11, mediates pyroptotic death through an unknown non-canonical inflammasome system in response to cytosolic bacteria. In addition, recent work shows that inflammasomes can also recruit procaspase-8, initiating apoptosis. The induction of multiple pathways of cell death has probably evolved to counteract microbial evasion of cell death pathways

Canonical Inflammasomes Drive IFN-γ to Prime Caspase-11 in Defense against a Cytosol-Invasive Bacterium

SummaryThe inflammatory caspases 1 and 11 are activated in response to different agonists and act independently to induce pyroptosis. In the context of IL-1β/IL-18 secretion, however, in vitro studies indicate that caspase-11 acts upstream of NLRP3 and caspase-1. By contrast, studying infection in vivo by the cytosol-invasive bacterium Burkholderia thailandensis, we find that caspase-1 activity is required upstream of caspase-11 to control infection. Caspase-1-activated IL-18 induces IFN-γ to prime caspase-11 and rapidly clear B. thailandensis infection. In the absence of IL-18, bacterial burdens persist, eventually triggering other signals that induce IFN-γ. Whereas IFN-γ was essential, endogenous type I interferons were insufficient to prime caspase-11. Although mice transgenic for caspase-4, the human ortholog of caspase-11, cleared B. thailandensis in vivo, they did not strictly require IFN-γ priming. Thus, caspase-1 provides priming signals upstream of caspase-11 but not caspase-4 during murine defense against a cytosol-invasive bacterium

Inflammasomes Coordinate Pyroptosis and Natural Killer Cell Cytotoxicity to Clear Infection by a Ubiquitous Environmental Bacterium

Defective neutrophils in patients with chronic granulomatous disease (CGD) cause susceptibility to extracellular and intracellular infections. Microbes must first be ejected from intracellular niches to expose them to neutrophil attack, so we hypothesized that inflammasomes detect certain CGD pathogens upstream of neutrophil killing. Here, we identified one such ubiquitous environmental bacterium, Chromobacterium violaceum, whose extreme virulence was fully counteracted by the NLRC4 inflammasome. Caspase-1 protected via two parallel pathways that eliminated intracellular replication niches. Pyroptosis was the primary bacterial clearance mechanism in the spleen, but both pyroptosis and interleukin-18 (IL-18)-driven natural killer (NK) cell responses were required for liver defense. NK cells cleared hepatocyte replication niches via perforin-dependent cytotoxicity, whereas interferon-γ was not required. These insights suggested a therapeutic approach: exogenous IL-18 restored perforin-dependent cytotoxicity during infection by the inflammasome-evasive bacterium Listeria monocytogenes. Therefore, inflammasomes can trigger complementary programmed cell death mechanisms, directing sterilizing immunity against intracellular bacterial pathogens

Immune enhancement by novel vaccine adjuvants in autoimmune-prone NZB/W F1 mice: relative efficacy and safety

<p>Abstract</p> <p>Background</p> <p>Vaccines have profoundly impacted global health although concerns persist about their potential role in autoimmune or other adverse reactions. To address these concerns, vaccine components like immunogens and adjuvants require critical evaluation not only in healthy subjects but also in those genetically averse to vaccine constituents. Evaluation in autoimmune-prone animal models of adjuvants is therefore important in vaccine development. The objective here was to assess the effectiveness of experimental adjuvants: two phytol-derived immunostimulants PHIS-01 (phytanol) and PHIS-03 (phytanyl mannose), and a new commercial adjuvant from porcine small intestinal submucosa (SIS-H), relative to a standard adjuvant alum. Phytol derivatives are hydrophobic, oil-in water diterpenoids, while alum is hydrophilic, and SIS is essentially a biodegradable and collagenous protein cocktail derived from extracellular matrices.</p> <p>Results</p> <p>We studied phthalate -specific and cross-reactive anti-DNA antibody responses, and parameters associated with the onset of autoimmune disorders. We determined antibody isotype and cytokine/chemokine milieu induced by the above experimental adjuvants relative to alum. Our results indicated that the phytol-derived adjuvant PHIS-01 exceeded alum in enhancing anti-phthalate antibody without much cross reactivity with ds-DNA. Relatively, SIS and PHIS-03 proved less robust, but they were also less inflammatory. Interestingly, these adjuvants facilitated isotype switching of anti-hapten, but not of anti-DNA response. The current study reaffirms our earlier reports on adjuvanticity of phytol compounds and SIS-H in non autoimmune-prone BALB/c and C57BL/6 mice. These adjuvants are as effective as alum also in autoimmune-prone NZB/WF1 mice, and they have little deleterious effects.</p> <p>Conclusion</p> <p>Although all adjuvants tested impacted cytokine/chemokine milieu in favor of Th1/Th2 balance, the phytol compounds fared better in reducing the onset of autoimmune syndromes. However, SIS is least inflammatory among the adjuvants evaluated.</p

Novel Phytol-Derived Immunostimulants (PHIS-01) for Enhancement of Vaccine Efficacy: A Comparative Study

Adjuvants are used widely in vaccine formulations. However for humans, choices are very limited. Since they are selected empirically, it is not expected that any two adjuvants would influence immune mechanisms the same way. However they all influence host microenvironment, antigen presentation, and retention of immunological memory. This study focuses on new terpenoid adjuvants based on phytol derivatives. We previously observed that phytol and one of its derivatives PHIS-01 (a phytol-based immunostimulant, phytanol) are excellent adjuvants. To gain an understanding of the structural features important for adjuvanticity, we further studied compounds derived from a diterpene Phytol. We designed two new phytol derivatives, PHIS-02 and PHIS-03 (aminated and mannosylated compounds respectively). In this study we investigated their relative safety and efficacy compared to PHIS- 01 (phytanol) and other commonly used adjuvants that include alum, Freunds’ adjuvants and SIS (extra-cellular matrix). In addition, we examined how changes at the polar terminus affect adjuvanticity of PHIS-01, PHIS-02, PHIS-03 in term of host microenvironment and safety profile. Using these adjuvants as emulsions with different soluble protein antigens, ovalbumin and a hapten-protein conjugate phthalate-KLH, we evaluated in both autoimmune resistant and susceptible murine models. The following immunological parameters were studied: 1) effects on antibody responses in terms of titers, specificities and isotypic profiles; 2) effects on T-helper cells, cytokines, and chemokines milieu; 3) involvements of apoptotic and/or necrotic activity and inflammasome pathways as their primary modes of action. Our results indicate that: 1)modified phytol-derived adjuvants significantly augment antibody response of isotypes IgG1 and IgG2a, promote effective T cell proliferation and exhibit no adverse autoimmune anti-DNA response in either autoimmune or non autoimmune mice. 2) Phytol derivatives function by activation of antigen-presenting cells involving apoptotic/necrotic effects on target cells. 3) Phytol derivatives improve vaccine immunogenicity by promoting regulated and nonpathogenic inflammatory changes in the immediate microenvironments, as characterized by mobilization of chemo tactic factors (MCP-1, KC, MIP-1, LIX, lymphotactin, eotaxin), growth factors (MCSF, GCSF, GM-CSF), and cytokines that mobilize innate and adaptive immunity and lead to T helper polarization and a magnified antibody response 4) PHIS -01, compared to PHIS-03 and alum , is a better activator of genes in the inflammasome pathways. In conclusion, our findings also clearly highlight the importance of bonds and functional moieties in shaping the adjuvanticity of phytol derivatives. Hydrogenation of phytol generates PHIS-01 which is a very safe and superior adjuvant in terms of the quality and magnitude of the overall immune response evoked. However, modification of its polar terminus of PHIS-01 with a hydrophilic mannose moiety (PHIS-03) profoundly changes the cytokine/chemokine milieu and favors T-helper type 2 rather than the T-helper type1 induced by PHIS-01.Aachoui, YoussefGhosh, SwapanAlbig, AllanHughes, James P.Kjonaas, Richard W.Stuart, Gary W.Doctor of PhilosophyDepartment of BiologyCunningham Memorial library, Terre Haute, Indiana State University20110916-001DoctoralTitle from document title page. Document formatted into pages: contains 212p.: ill. Includes bibliography, abstract and appendix

Extracellular Matrix from Porcine Small Intestinal Submucosa (SIS) as Immune Adjuvants

Porcine small intestinal submucosa (SIS) of Cook Biotech is licensed and widely used for tissue remodeling in humans. SIS was shown to be highly effective as an adjuvant in model studies with prostate and ovarian cancer vaccines. However, SIS adjuvanticity relative to alum, another important human-licensed adjuvant, has not yet been delineated in terms of activation of innate immunity via inflammasomes and boosting of antibody responses to soluble proteins and haptenprotein conjugates. We used ovalbumin, and a hapten-protein conjugate, phthalate-keyhole limpet hemocyanin. The evaluation of SIS was conducted in BALB/c and C57BL/6 mice using both intraperitoneal and subcutaneous routes. Inflammatory responses were studied by microarray profiling of chemokines and cytokines and by qPCR of inflammasomesrelated genes. Results showed that SIS affected cytokine and chemokines microenvironments such as up-regulation of IL-4 and CD30-ligand and activation of chemotactic factors LIX and KC (neutrophil chemotactic factors), MCP-1 (monocytes chemotactic factors), MIP 1-a (macrophage chemotactic factor) and lymphotactin, as well as, growth factors like M-CSF. SIS also promoted gene expression of Nod-like receptors (NLR) and associated downstream effectors. However, in contrast to alum, SIS had no effects on pro-inflammatory cytokines (IL-6, IL-1b, TNF-a) or NLRP3, but it appeared to promote both Th1 and Th2 responses under different conditions. Lastly, it was as effective as alum in engendering a lasting and specifi

RT-PCR Microarray analysis of transcriptome profiles of inflammatory genes induced by vaccine adjuvants alone in mouse peritoneum.

<p>Eighty-four genes were assessed and those genes up-regulated (A), or down regulated (B) with an average log2 ratio ≥1.5 were selected and plotted as a Venn diagram. Results for the alum-treated group refer to our previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027083#pone.0027083-Aachoui1" target="_blank">[24]</a>.</p

Effects of porcine small intestinal submucosa (SIS) on chemokine and cytokine protein microenvironment in BALB/c peritoneal exudates.

<p>BALB/c mice were injected i.p. with SIS preparations (SIS-H or SIS-M) without the antigens and peritoneal exudates harvested after 24 hr. Control groups were treated with PBS buffer. Peritoneal fluids were assayed to determine chemokine and cytokine expression as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027083#s2" target="_blank">Materials and Methods</a>. Data are expressed as the mean relative intensity relative to positive control of each chemokine or cytokine protein detected using pooled peritoneal fluids of 3 mice per group in duplicate. The result is an average of two separate experiments.</p

Effects of small intestinal submucosa (SIS) on the murine innate immune microenvironment induced by heat-killed Staphylococcus aureus.

The use of biological scaffold materials for wound healing and tissue remodeling has profoundly impacted regenerative medicine and tissue engineering. The porcine-derived small intestinal submucosa (SIS) is a licensed bioscaffold material regularly used in wound and tissue repair, often in contaminated surgical fields. Complications and failures due to infection of this biomaterial have therefore been a major concern and challenge. SIS can be colonized and infected by wound-associated bacteria, particularly Staphylococcus aureus. In order to address this concern and develop novel intervention strategies, the immune microenvironment orchestrated by the combined action of S. aureus and SIS should be critically evaluated. Since the outcome of tissue remodeling is largely controlled by the local immune microenvironment, we assessed the innate immune profile in terms of cytokine/chemokine microenvironment and inflammasome-responsive genes. BALB/c mice were injected intra-peritoneally with heat-killed S. aureus in the presence or absence of SIS. Analyses of cytokines, chemokines and microarray profiling of inflammasome-related genes were done using peritoneal lavages collected 24 hours after injection. Results showed that unlike SIS, the S. aureus-SIS interactome was characterized by a Th1-biased immune profile with increased expressions of IFN-γ, IL-12 and decreased expressions of IL-4, IL-13, IL-33 and IL-6. Such modulation of the Th1/Th2 axis can greatly facilitate graft rejections. The S. aureus-SIS exposure also augmented the expressions of pro-inflammatory cytokines like IL-1β, Tnf-α, CD30L, Eotaxin and Fractalkine. This heightened inflammatory response caused by S. aureus contamination could enormously affect the biocompatibility of SIS. However, the mRNA expressions of many inflammasome-related genes like Nlrp3, Aim2, Card6 and Pycard were down-regulated by heat-killed S. aureus with or without SIS. In summary, our study explored the innate immune microenvironment induced by the combined exposure of SIS and S. aureus. These results have practical implications in developing strategies to contain infection and promote successful tissue repair