Genetic basis for variation in plasma IL-18 levels in persons with chronic hepatitis C virus and human immunodeficiency virus-1 infections

Inflammasomes are multi-protein complexes integrating pathogen-triggered signaling leading to the generation of pro-inflammatory cytokines including interleukin-18 (IL-18). Hepatitis C virus (HCV) and human immunodeficiency virus (HIV) infections are associated with elevated IL-18, suggesting inflammasome activation. However, there is marked person-to-person variation in the inflammasome response to HCV and HIV. We hypothesized that host genetics may explain this variation. To test this, we analyzed the associations of plasma IL-18 levels and polymorphisms in 10 genes in the inflammasome cascade. About 1538 participants with active HIV and/or HCV infection in three ancestry groups are included. Samples were genotyped using the Illumina Omni 1-quad and Omni 2.5 arrays. Linear regression analyses were performed to test the association of variants with log IL-18 including HCV and HIV infection status, and HIV RNA in each ancestry group and then meta-analyzed. Eleven highly correlated single-nucleotide polymorphisms (r²=0.98–1) in the IL-18-BCO2 region were significantly associated with log IL-18; each T allele of rs80011693 confers a decrease of 0.06 log pg ml⁻¹ of IL-18 after adjusting for covariates (rs80011693; rs111311302 β=−0.06, P-value=2.7 × 10⁻⁴). In conclusion, genetic variation in IL-18 is associated with IL-18 production in response to HIV and HCV infection, and may explain variability in the inflammatory outcomes of chronic viral infections

Granulocytes mediates the Fas-L-associated apoptosis during lung metastasis of melanoma that determines the metastatic behaviour

The survival of tumour cells in a new tissue environment is crucial for tumour metastasis. Factors contributing to the death of tumour cells during metastasis are not completely understood. In murine melanoma model, activation of Fas (CD95, APO-1) signal in tumour cells reduces their lung metastasis potential, which may be associated with an induction of apoptosis in tumours. To elucidate the cellular mechanism, we used a Fas-ligand (Fas-L) specific ribozyme (Fas-Lribozyme) to suppress the expression of Fas-L but not Fas or TNF-α in B16F10 melanoma cells. The Fas-Lribozyme-carrying cells grew slightly faster in vitro with better viability than controls. Suppression of Fas-L in B16F10 melanoma cells by Fas-Lribozyme enhanced lung metastasis of the cells in C57BL/6 mice, and that was correlated with reductions in both apoptotic tumour cells and granulocytic infiltration. Mice depleted of granulocytes, but not CD4+ and CD8+ cells, showed a greatly elevated susceptibility to lung metastasis. Moreover, apoptosis in tumour cells was significantly reduced in granulocyte-depleted mice during the course of tumour formation. Taken together, our findings indicate that Fas-L-associated apoptosis in tumour cells determines the metastasis behaviour of melanoma in the lung and this apoptosis is primarily mediated by the cytotoxicity of recruited granulocytes

Safety and Immunogenicity of an HIV-1 Gag DNA Vaccine with or without IL-12 and/or IL-15 Plasmid Cytokine Adjuvant in Healthy, HIV-1 Uninfected Adults

DNA vaccines are a promising approach to vaccination since they circumvent the problem of vector-induced immunity. DNA plasmid cytokine adjuvants have been shown to augment immune responses in small animals and in macaques.We performed two first in human HIV vaccine trials in the US, Brazil and Thailand of an RNA-optimized truncated HIV-1 gag gene (p37) DNA derived from strain HXB2 administered either alone or in combination with dose-escalation of IL-12 or IL-15 plasmid cytokine adjuvants. Vaccinations with both the HIV immunogen and cytokine adjuvant were generally well-tolerated and no significant vaccine-related adverse events were identified. A small number of subjects developed asymptomatic low titer antibodies to IL-12 or IL-15. Cellular immunogenicity following 3 and 4 vaccinations was poor, with response rates to gag of 4.9%/8.7% among vaccinees receiving gag DNA alone, 0%/11.5% among those receiving gag DNA+IL-15, and no responders among those receiving DNA+high dose (1500 ug) IL-12 DNA. However, after three doses, 44.4% (4/9) of vaccinees receiving gag DNA and intermediate dose (500 ug) of IL-12 DNA demonstrated a detectable cellular immune response.This combination of HIV gag DNA with plasmid cytokine adjuvants was well tolerated. There were minimal responses to HIV gag DNA alone, and no apparent augmentation with either IL-12 or IL-15 plasmid cytokine adjuvants. Despite the promise of DNA vaccines, newer formulations or methods of delivery will be required to increase their immunogenicity.Clinicaltrials.gov NCT00115960 NCT00111605

HIV and HCV Activate the Inflammasome in Monocytes and Macrophages via Endosomal Toll-Like Receptors without Induction of Type 1 Interferon

<div><p>Innate immune sensing of viral infection results in type I interferon (IFN) production and inflammasome activation. Type I IFNs, primarily IFN-α and IFN-β, are produced by all cell types upon virus infection and promote an antiviral state in surrounding cells by inducing the expression of IFN-stimulated genes. Type I IFN production is mediated by Toll-like receptor (TLR) 3 in HCV infected hepatocytes. Type I IFNs are also produced by plasmacytoid dendritic cells (pDC) after sensing of HIV and HCV through TLR7 in the absence of productive pDC infection. Inflammasomes are multi-protein cytosolic complexes that integrate several pathogen-triggered signaling cascades ultimately leading to caspase-1 activation and generation pro-inflammatory cytokines including interleukin (IL)-18 and IL-1β. Here, we demonstrate that HIV and HCV activate the inflammasome, but not Type I IFN production, in monocytes and macrophages in an infection-independent process that requires clathrin-mediated endocytosis and recognition of the virus by distinct endosomal TLRs. Knockdown of each endosomal TLR in primary monocytes by RNA interference reveals that inflammasome activation in these cells results from HIV sensing by TLR8 and HCV recognition by TLR7. Despite its critical role in type I IFN production by pDCs stimulated with HIV, TLR7 is not required for inflammasome activation by HIV. Similarly, HCV activation of the inflammasome in monocytes does not require TLR3 or its downstream signaling adaptor TICAM-1, while this pathway leads to type I IFN in infected hepatocytes. Monocytes and macrophages do not produce type I IFN upon TLR8 or TLR7 sensing of HIV or HCV, respectively. These findings reveal a novel infection-independent mechanism for chronic viral induction of key anti-viral programs and demonstrate distinct TLR utilization by different cell types for activation of the type I IFN vs. inflammasome pathways of inflammation.</p></div

Differential importance of endosomal TLRs in inflammasome sensing of HIV and HCV.

<p>Monocytes were cultured without stimulation or with HIV_BaL or HCV_{Subject 180} and IL-1β, IFNα and measured at multiple timepoints. Fold change in expression of IL-1β (black bar) and IFNα (grey bar) following HIV_BaL or HCV_{Subject 180} relative to stimulation with media alone is shown at 6 h (<b>A</b>). Functional knockdowns of the endosomal located TLRs in monocytes were generated by RNA interference. Monocytes were mock transfected or transfected with either siRNA targeting TLR7 or a non-targeting sequence (Scramble). After 24 h, cell lysates were prepared and efficacy of knockdown determined by (<b>B</b>) western blot and (<b>C</b>) qRT-PCR. (<b>D</b>) Specificity of the TLR7 siRNA was confirmed by qRT-PCR using primers for TLR3, TLR7, TLR8, and TLR9. (*) denotes comparisons with p≤0.05 compared to the mock transfected cells. Monocytes in which endosomal TLR knockdown were generated were cultured with HIV_BaL (solid bars) or HCV_{Subject 180} (hatched bars) and pro-IL-1β mRNA transcription measured at 6 h (<b>E–H</b>) and IL-18 secretion measured at 24 h (<b>I–L</b>). Shown are the relative production of pro-IL-1β mRNA and IL-18 in TLR8 (<b>E</b>, <b>I</b>), TLR7 (<b>F</b>, <b>J</b>), TLR3 (<b>G</b>, <b>K</b>) and TLR9 (<b>H</b>, <b>L</b>) knockdown monocytes normalized to mock transfected monocytes (no siRNA) stimulated with the same viruses. Bars represent the mean ± S.D. for <i>n = 6–9</i> independent transfection experiments, (**) denotes comparisons with p≤0.05 and (***) denoted p≤0.001 compared to the scramble siRNA transfected cells.</p

Monocytes produce inflammasome cytokines in response to HCV and HIV.

<p>(<b>A</b>) HIV and HCV uninfected PBMC, sorted monocytes, or <i>in vitro</i> differentiated macrophages were cultured with plasma from HCV infected subjects (V, <i>n = 15, plasma vol. creates at the final concentration of 5×10⁵ HCV RNA IU/mL in culture</i>) or plasma from the same subjects prior to infection (Pre, <i>n = 15, matched volume</i>). IL-18 and IL-1β were measured in duplicate after 24 h. Primary human hepatocytes were cultured with V and Pre plasma as well as the culture strain HCV_JFH-1 (<i>n = 5</i>). Monocytes and derived macrophages secrete IL-18 (<b>top panel</b>) and IL-1β (<b>lower panel</b>) in response to V but not Pre plasma. Hepatocytes fail to produce inflammasome cytokines. In (<b>B</b>), IL-18 and IL-1β were measured after HIV and HCV uninfected PBMC, sorted monocytes, T-cells or <i>in vitro</i> differentiated macrophages were cultured with plasma from subjects on HAART (H, <i>n = 6</i>), plasma from elite suppressors (ES, <i>n = 5</i>), or viremic HIV plasma (V, <i>n = 15</i>). Plasma with measurable HIV stimulated significant monocyte IL-18 and IL-1β secretion. IL-18 produced from cells cultured with individual plasma samples (white circles), means (horizontal dash) ± S.D. are shown.</p

Differential importance of cytoplasmic sensors in inflammasome response to HIV and HCV.

<p>Functional knockdowns of the cytoplasmic sensors NLRP3, RIG-I and AIM2 in monocytes were created as previously described for TLRs. (<b>A</b>) Knockdown monocytes were cultured with HIV_BaL, HIV_RF, HIV_IIIB, or HIV_RF and IL-18 measured after 24 h. IL-18 produced relative to that of mock transfected cells is shown. (<b>B</b>) Similarly prepared monocytes were cultured with a panel of plasma from HCV infected individuals (HCV_{Subject 117}, HCV_{Subject 54}, HCV_{Subject 16}, HCV_{Subject 180}) and IL-18 measured at 24 h. Bars represent the mean ± S.D. for <i>n = 6–9</i> independent transfection experiments.</p

HCV and HIV virions stimulate monocytes to produce IL-18.

<p>HCV viremic plasma (<b>A</b>) or HCV_JFH-1 (<b>B</b>) was separated into fractions by sucrose gradient equilibrium ultracentrifugation. HCV RNA (black circles) for each fraction was determined by RT-PCR. Monocytes are maximally stimulated to secrete IL-18 (grey bars, n = 3) when cultured with fractions in the density range 1.09–1.16 g/mL (*), which are most enriched in HCV RNA. (<b>c</b>) HIV_IIIB, HIV_MN, HIV_RF and HIV_BaL were cultured in activated CD4⁺ T-cells, p24 measured in supernatant, and supernatant transferred to monocytes. Monocytes cultured with HIV_IIIB (black circles), HIV_MN (white triangles), HIV_RF (black squares) and HIV_BaL (white diamonds) culture supernatant secrete IL-18 in a dose dependent manner. Symbols represent the mean ± S.D. of 6 experiments.</p