Non-canonical inflammasome activation mediates the adjuvanticity of nanoparticles

The non-canonical inflammasome sensor caspase-11 and gasdermin D (GSDMD) drive inflammation and pyroptosis, a type of immunogenic cell death that favors cell-mediated immunity (CMI) in cancer, infection, and autoimmunity. Here we show that caspase-11 and GSDMD are required for CD8+ and Th1 responses induced by nanoparticulate vaccine adjuvants. We demonstrate that nanoparticle-induced reactive oxygen species (ROS) are size dependent and essential for CMI, and we identify 50- to 60-nm nanoparticles as optimal inducers of ROS, GSDMD activation, and Th1 and CD8+ responses. We reveal a division of labor for IL-1 and IL-18, where IL-1 supports Th1 and IL-18 promotes CD8+ responses. Exploiting size as a key attribute, we demonstrate that biodegradable poly-lactic co-glycolic acid nanoparticles are potent CMI-inducing adjuvants. Our work implicates ROS and the non-canonical inflammasome in the mode of action of polymeric nanoparticulate adjuvants and establishes adjuvant size as a key design principle for vaccines against cancer and intracellular pathogens

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)-1a-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. © 2014 by the American Diabetes Association

A Common Variant in the Adaptor Mal Regulates Interferon Gamma Signaling

SummaryHumans that are heterozygous for the common S180L polymorphism in the Toll-like receptor (TLR) adaptor Mal (encoded by TIRAP) are protected from a number of infectious diseases, including tuberculosis (TB), whereas those homozygous for the allele are at increased risk. The reason for this difference in susceptibility is not clear. We report that Mal has a TLR-independent role in interferon-gamma (IFN-γ) receptor signaling. Mal-dependent IFN-γ receptor (IFNGR) signaling led to mitogen-activated protein kinase (MAPK) p38 phosphorylation and autophagy. IFN-γ signaling via Mal was required for phagosome maturation and killing of intracellular Mycobacterium tuberculosis (Mtb). The S180L polymorphism, and its murine equivalent S200L, reduced the affinity of Mal for the IFNGR, thereby compromising IFNGR signaling in macrophages and impairing responses to TB. Our findings highlight a role for Mal outside the TLR system and imply that genetic variation in TIRAP may be linked to other IFN-γ-related diseases including autoimmunity and cancer

Patient demographics.

<p>^a Healthcare-associated infections were defined as (i) index positive blood culture collected ≥48hrs after hospital admission, and no signs or symptoms of the infection noted at time of admission; OR (ii) index positive blood culture collected <48hrs after hospital admission if any of the following criteria are met: received intravenous therapy in an ambulatory setting in the 30 days before onset of BSI, attended a hospital clinic or haemodialysis in the 30 days before onset of BSI, hospitalised in an acute care hospital for ≥ 2 days in the 90 days prior to onset of BSI, resident of nursing home or long-term care facility.</p><p>^b<i>Staphylococcus aureus</i> bacteraemia was defined as uncomplicated if all of the following criteria were met: exclusion of endocarditis; no evidence of metastatic infection; absence of implanted prostheses; follow-up blood cultures at 2–4 days culture-negative for <i>S</i>. <i>aureus</i>; defervescence within 72 h of initiating effective therapy. Percentages shown are of entire <i>S</i>. <i>aureus</i> BSI population.</p><p>^† Three patients had chronic diabetic foot ulcers as a source of their <i>S</i>. <i>aureus</i> BSI, and in all cases the contiguous underlying bone was also found to be infected.</p><p>MRSA = methicillin-resistant <i>Staphylococcus aureus</i>. NA = not applicable. BSI = bloodstream infection.</p><p>Data are displayed as median (interquartile range) and number (percentage). <i>P</i> values are calculated by Mann-Whitney and Fisher’s exact test respectively.</p

Human <i>S</i>. <i>aureus</i> bloodstream infection is associated with increased IFNγ production.

<p>Serum from <i>S</i>. <i>aureus</i> and <i>E</i>.<i>coli</i> bloodstream infection patients was collected on day 7 ± 2 post-initial bacteraemia and assessed for IFNγ (A) and IL-17A (B) by ELISA. Results expressed as individual patient values with median indicated by bar, n = 11–24 per group. *p<0.05.</p

Human <i>S</i>. <i>aureus</i> bloodstream infection induces <i>S</i>. <i>aureus</i> antigen-specific effector memory Th1 cells and anti-ClfA antibodies.

<p>PBMCs were isolated from patients, CFSE-labelled and incubated with heat-killed <i>S</i>. <i>aureus</i> PS80 strain (1μg/ml ≈ 1x10⁷ CFU/ml) or media alone for 10 d. Proportions of <i>S</i>. <i>aureus</i> antigen-specific effector memory cells were assessed by gating on IFNγ⁺CD45RO⁺ cells in the CFSE_lo CD4⁺ population (A). For each patient, media only responses were subtracted from responses to heat-killed <i>S</i>. <i>aureus</i> to determine the antigen-specific response. n = 5–12 per group. IgG antibody binding to ClfA was measured in patient sera using a bead-based flow cytometry technique (B). n = 11–24 per group. Results shown as box-and-whiskers plots where the horizontal line indicates the median, boundaries of the box the IQR, and whiskers indicate the highest and lowest values of the results, and representative FACS plots of CD4⁺CFSE_lo cells (A). SA = <i>S</i>. <i>aureus</i>, EC = <i>E</i>. <i>coli</i>, BSI = bloodstream infection. * p<0.05.</p

Transfer of <i>S</i>. <i>aureus</i> antigen-specific peritoneal Th1 cells protects against subsequent <i>S</i>. <i>aureus</i> infection via enhanced macrophage responses.

<p>Groups of mice received transfers of 5x10⁶<i>S</i>. <i>aureus</i> specific Th1 cells originating from the peritoneal cavity of previously exposed mice via i.p. injection. Another group of mice received a transfer of 5x10⁶ naive splenic CD3⁺ cells as a control. At 3 h post transfer both groups of mice were challenged with <i>S</i>. <i>aureus</i> (5x10⁸ CFU) via i.p. injection. At 72 h post-bacterial challenge the bacterial burden was assessed in the peritoneal cavity, kidneys and spleen (A). Results expressed as log₁₀ CFU/ml with mean indicated by bars. At indicated time points post-bacterial challenge, the peritoneal cavity was lavaged with PBS to assess CXCL1 and CCL5 secretion by ELISA (B,C). Results expressed as mean ± SEM. At indicated time points post-challenge, the absolute numbers of macrophages (F4/80⁺Ly6G^-) were assessed in the peritoneal cavity by flow cytometry (D). MHCII expression by infiltrating macrophages was determined 24 h post infection (E). Absolute numbers of neutrophils (Ly6G⁺CD11b⁺) in the peritoneal cavity were assessed at the indicated time points post-challenge (F). Results expressed as mean ± SEM. n = 5–8 mice per group. Data pooled from 3 independent experiments. *p<0.05, **p<0.005.</p

Prior exposure to <i>S</i>. <i>aureus</i> increases IFNγ secretion by CD4⁺ and CD8⁺ T cells during subsequent infection.

<p>Groups of mice were exposed to <i>S</i>. <i>aureus</i> (5x10⁸ CFU) via an i.p. injection on d 0, 7 and 14. Prior exposed mice were then re-challenged with an i.p. injection of <i>S</i>. <i>aureus</i> (5x10⁸ CFU) on d 35 alongside a control group of naïve mice. At indicated time points post-challenge the peritoneal cavity was lavaged with PBS to assess IFNγ secretion by ELISA (A). n = 15 per group. At 3 h post challenge peritoneal cells were isolated to assess the proportions of IFNγ-producing CD4⁺ and CD8⁺ T cells using flow cytometry (B). Results expressed as mean ± SEM and representative FACS plots. n = 5 per group. *p<0.05, **p<0.005, ***p<0.001.</p