TRIF Is a Critical Negative Regulator of TLR Agonist Mediated Activation of Dendritic Cells In Vivo

Despite recent advances in developing and licensing adjuvants, there is a great need for more potent formulations to enhance immunogenicity of vaccines. An Eimeria tenella derived antigen (rEA) augments immune responses against several pathogens in animal models and recently was confirmed to be safe for human use. In this study, we have analyzed the molecular mechanisms underlying rEA activity in mice, and confirmed that rEA activates multiple immune cell types, including DCs, macrophages, NK, B, and T cells. The rEA adjuvant also elicits the induction of pleiotropic pro-inflammatory cytokines, responses that completely depend upon the presence of the TLR adaptor protein MyD88. Surprisingly, we also found that the TRIF adaptor protein acts as a potent negative regulator of TLR agonist-triggered immune responses. For example, IL12 production and the induction of co-stimulatory molecule expression by DCs and IFNc production by NK cells in vivo were significantly increased in rEA-treated TRIF-KO mice. Importantly, however, TRIF suppressive effects were not restricted to rEA-mediated responses, but were apparent in LPS- or ODN2006-activated DCs as well. Taken together, our findings confirm that rEA is a potent adjuvant, triggering robust activation of the innate immune system, in a manner that is augmented by MyD88 and inhibited by TRIF; thereby unveiling the potential complexities of modulating TLR activity t

Endoplasmic Reticulum Aminopeptidase-1 Functions Regulate Key Aspects of the Innate Immune Response

<div><p>Endoplasmic reticulum aminopeptidase-1 (ERAP1) is a multifunctional, ubiquitously expressed enzyme whose peptide-trimming role during antigen processing for presentation by MHC I molecules is well established, however, a role for ERAP1 in modulating global innate immune responses has not been described to date. Here we demonstrate that, relative to wild type mice, mice lacking ERAP1 exhibit exaggerated innate immune responses early during pathogen recognition, as characterized by increased activation of splenic and hepatic NK and NKT cells and enhanced production of pro-inflammatory cytokines such as IL12 and MCP1. Our data also revealed that ERAP1 is playing a critical role in NK cell development and function. We observed higher frequencies of terminally matured NK cells, as well as higher frequencies of licensed NK cells (expressing the Ly49C and Ly49I receptors) in ERAP1-KO mice, results that positively correlated with an enhanced NK activation and IFNγ production by ERAP1-KO mice challenged with pro-inflammatory stimuli. Furthermore, during pathogen recognition, ERAP1 regulates IL12 production by CD11c⁺ DCs specifically, with increases in IL12 production positively correlated with an increased phagocytic activity of splenic DCs and macrophages. Collectively, our results demonstrate a previously unrecognized, more central role for the ERAP1 protein in modulating several aspects of both the development of the innate immune system, and its responses during the initial stages of pathogen recognition. Such a role may explain why ERAP1 has been implicated by GWAS in the pathogenesis of autoimmune diseases that may be precipitated by aberrant responses to pathogen encounters.</p></div

ERAP1-KO mice exhibit dramatically enhanced capabilities to respond to rEA and release pro-inflammatory cytokines.

<p>C57BL/6 WT and ERAP1-KO mice were either mock (PBS) injected or intraperitoneally injected with 100 ng/mouse of rEA protein. n = 4 for all groups of mice. Plasma samples were collected at 6 hpi and were analyzed using a multiplexed bead array based quantitative system. Bars represent mean ± SEM. Statistical analysis was completed using two-tailed homoscedastic Student’s t-tests; *, ** - indicate values, statistically different between WT_rEA and ERAP1-KO_rEA groups, p<0.05, p<0.001, respectively.</p

Innate Immune genes.

<p>Ad5-gag induced gene expression in livers of C57BL/6 mice (fold over WT_Mock, 6 hpi). The numbers represent Mean ± SD. Statistical analysis was completed using One Way ANOVA with a Student-Newman-Keuls post-hoc test, p<0.05 was deemed a statistically significant difference. n = 4 for all Mock injected groups, n = 6 for all Ad5-HIV-gag injected groups.</p>a<p>Significant differences as compared to WT_Mock;</p>b<p>significant differences in transcriptional activation in ERAP1-KO_Ad5-HIV-gag group as compared to WT_Ad5-HIV-gag group (also indicated by boldface font).</p>*<p>denotes significant differences between WT_Mock and ERAP1-KO_Mock (baseline levels).</p

Signaling pathways.

<p>Ad5-HIV-gag induced gene expression in livers of C57BL/6 mice (fold over WT_Mock, 6 hpi). The numbers represent Mean ± SD. Statistical analysis was completed using One Way ANOVA with a Student-Newman-Keuls post-hoc test, p<0.05 was deemed a statistically significant difference. n = 4 for all Mock injected groups, n = 6 for all Ad5-HIV-gag injected groups.</p>a<p>Significant differences as compared to WT_Mock;</p>b<p>significant differences in transcriptional activation in ERAP1-KO_Ad5-HIV-gag group as compared to WT_Ad5-HIV-gag group (also indicated by boldface font).</p

Mice lacking ERAP1 exhibit increased activation of NK cells in the spleen in response to rEA stimuli.

<p>C57BL/6 WT and ERAP1-KO mice were either mock (PBS) injected or intraperitoneally injected with 100 ng/mouse of rEA protein. Splenocytes were prepared at 12 hpi, processed, stained for expression of surface markers and analyzed by FACS as described in Materials and Methods. CD69 activation NK cells is shown, Bars represent mean ± SEM. Representative plots are shown. Statistical analysis was completed using a one-way ANOVA with a Student-Newman-Keuls post-hoc test. n = 4–7 for all groups of mice. *** - indicate values, statistically different from those in mock-injected mice, p<0.0001.</p

Mice lacking ERAP1 exhibit dramatically enhanced activation of B cells, CD8⁺ and CD8⁻ T cells in response to rEA stimuli.

<p>C57BL/6 WT and ERAP1-KO mice were either mock (PBS) injected or intraperitoneally injected with 100 ng/mouse of rEA protein. Splenocytes were harvested at 6 hpi, processed, stained for expression of surface markers, and analyzed by FACS as described in Materials and Methods. CD69 activation of (<b>A</b>) CD8⁺ CD3⁺ T cells, (<b>B</b>) CD8<b>⁻</b> CD3⁺ T cells, and (<b>C</b>) B cells are shown. Bars represent mean ± SEM. Representative plots are shown. Statistical analysis was completed using a one-way ANOVA with a Student-Newman-Keuls post-hoc test. n = 4 for all groups of mice. *, ** - indicate values, statistically different from those in mock-injected mice, p<0.05, p<0.001, respectively.</p

Mice lacking ERAP1 exhibit drastically increased activation of NK and NKT cells in the liver in response to rEA stimuli.

<p>C57BL/6 WT and ERAP1-KO mice were either mock (PBS) injected or intraperitoneally injected with 100 ng/mouse of rEA protein. Liver lymphocytes were prepared at 6 hpi, processed, stained for expression of surface markers (intracellular staining was performed for IFNγ), and analyzed by FACS as described in Materials and Methods. (<b>A, B</b>) CD69 activation and (<b>C, D</b>) IFNγ release by NK cells (<b>A, C</b>) and NKT cells (<b>B, D</b>) are shown. Bars represent mean ± SEM. Representative plots are shown. Statistical analysis was completed using a one-way ANOVA with a Student-Newman-Keuls post-hoc test. n = 4 for all groups of mice. *, ** - indicate values, statistically different from those in mock-injected mice, p<0.05, p<0.001, respectively.</p