Synthetic double-stranded RNA induces innate immune responses similar to a live viral vaccine in humans

Adjuvants are critical for the success of vaccines. Agonists of microbial pattern recognition receptors (PRRs) are promising new adjuvant candidates. A mechanism through which adjuvants enhance immune responses is to stimulate innate immunity. We studied the innate immune response in humans to synthetic double-stranded RNA (polyinosinic:polycytidylic acid [poly IC] stabilized with poly-l-lysine [poly ICLC]), an agonist for toll-like receptor (TLR) 3, and the cytosolic RNA helicase MDA-5. Transcriptional analysis of blood samples from eight volunteers, after subcutaneous administration of poly ICLC, showed upregulation of genes involved in multiple innate immune pathways in all subjects, including interferon (IFN) and inflammasome signaling. Blocking type I IFN receptor ex vivo significantly dampened the response to poly IC. Comparative transcriptional analysis showed that several innate immune pathways were similarly induced in volunteers immunized with the highly efficacious yellow fever vaccine. Therefore, a chemically defined PRR agonist like poly ICLC can be a reliable and authentic microbial mimic for inducing innate immune responses in humans

Influence of monolayer, spheroid, and tumor growth conditions on chromosome 3 gene expression in tumorigenic epithelial ovarian cancer cell lines

<p>Abstract</p> <p>Background</p> <p>Expression microarray analyses of epithelial ovarian cancer (EOC) cell lines may be exploited to elucidate genetic and epigenetic events important in this disease. A possible variable is the influence of growth conditions on discerning candidates. The present study examined the influence of growth conditions on the expression of chromosome 3 genes in the tumorigenic EOC cell lines, OV-90, TOV-21G and TOV-112D using Affymetrix GeneChip^®HG-U133A expression microarray analysis.</p> <p>Methods</p> <p>Chromosome 3 gene expression profiles (n = 1147 probe sets, representing 735 genes) were extracted from U133A expression microarray analyses of the EOC cell lines OV-90, TOV-21G and TOV-112D that were grown as monolayers, spheroids or nude mouse xenografts and monolayers derived from these tumors. Hierarchical cluster analysis was performed to compare chromosome 3 transcriptome patterns of each growth condition. Differentially expressed genes were identified and characterized by two-way comparative analyses of fold-differences in gene expression between monolayer cultures and each of the other growth conditions, and between the maximum and minimum values of expression of all growth conditions for each EOC cell line.</p> <p>Results</p> <p>An overall high degree of similarity (> 90%) in gene expression was observed when expression values of alternative growth conditions were compared within each EOC cell line group. Two-way comparative analysis of each EOC cell line grown in an alternative condition relative to the monolayer culture showed that overall less than 15% of probe sets exhibited at least a 3-fold difference in expression profile. Less than 23% of probe sets exhibited greater than 3-fold differences in gene expression in comparisons of the maximum and minimum value of expression of all growth conditions within each EOC cell line group. The majority of these differences were less than 5-fold. There were 17 genes in common which were differentially expressed in all EOC cell lines. However, the patterns of expression of these genes were not necessarily the same for each growth condition when one cell line was compared with another.</p> <p>Conclusion</p> <p>The various alternative <it>in vivo </it>and <it>in vitro </it>growth conditions of tumorigenic EOC cell lines appeared to modestly influence the global chromosome 3 transcriptome supporting the notion that the <it>in vitro </it>cell line models are a viable option for testing gene candidates.</p

Upregulated IL-32 expression and reduced gut short chain fatty acid caproic acid in people living with HIV with subclinical atherosclerosis

Despite the success of antiretroviral therapy (ART), people living with HIV (PLWH) are still at higher risk for cardiovascular diseases (CVDs) that are mediated by chronic inflammation. Identification of novel inflammatory mediators with the inherent potential to be used as CVD biomarkers and also as therapeutic targets is critically needed for better risk stratification and disease management in PLWH. Here, we investigated the expression and potential role of the multi-isoform proinflammatory cytokine IL-32 in subclinical atherosclerosis in PLWH (n=49 with subclinical atherosclerosis and n=30 without) and HIV- controls (n=25 with subclinical atherosclerosis and n=24 without). While expression of all tested IL-32 isoforms (α, β, γ, D, ϵ, and θ) was significantly higher in peripheral blood from PLWH compared to HIV- controls, IL-32D and IL-32θ isoforms were further upregulated in HIV+ individuals with coronary artery atherosclerosis compared to their counterparts without. Upregulation of these two isoforms was associated with increased plasma levels of IL-18 and IL-1β and downregulation of the atheroprotective protein TRAIL, which together composed a unique atherosclerotic inflammatory signature specific for PLWH compared to HIV- controls. Logistic regression analysis demonstrated that modulation of these inflammatory variables was independent of age, smoking, and statin treatment. Furthermore, our in vitro functional data linked IL-32 to macrophage activation and production of IL-18 and downregulation of TRAIL, a mechanism previously shown to be associated with impaired cholesterol metabolism and atherosclerosis. Finally, increased expression of IL-32 isoforms in PLWH with subclinical atherosclerosis was associated with altered gut microbiome (increased pathogenic bacteria; Rothia and Eggerthella species) and lower abundance of the gut metabolite short-chain fatty acid (SCFA) caproic acid, measured in fecal samples from the study participants. Importantly, caproic acid diminished the production of IL-32, IL-18, and IL-1β in human PBMCs in response to bacterial LPS stimulation. In conclusion, our studies identified an HIV-specific atherosclerotic inflammatory signature including specific IL-32 isoforms, which is regulated by the SCFA caproic acid and that may lead to new potential therapies to prevent CVD in ART-treated PLWH

A Numerically Subdominant CD8 T Cell Response to Matrix Protein of Respiratory Syncytial Virus Controls Infection with Limited Immunopathology.

CD8 T cells are involved in pathogen clearance and infection-induced pathology in respiratory syncytial virus (RSV) infection. Studying bulk responses masks the contribution of individual CD8 T cell subsets to protective immunity and immunopathology. In particular, the roles of subdominant responses that are potentially beneficial to the host are rarely appreciated when the focus is on magnitude instead of quality of response. Here, by evaluating CD8 T cell responses in CB6F1 hybrid mice, in which multiple epitopes are recognized, we found that a numerically subdominant CD8 T cell response against DbM187 epitope of the virus matrix protein expressed high avidity TCR and enhanced signaling pathways associated with CD8 T cell effector functions. Each DbM187 T effector cell lysed more infected targets on a per cell basis than the numerically dominant KdM282 T cells, and controlled virus replication more efficiently with less pulmonary inflammation and illness than the previously well-characterized KdM282 T cell response. Our data suggest that the clinical outcome of viral infections is determined by the integrated functional properties of a variety of responding CD8 T cells, and that the highest magnitude response may not necessarily be the best in terms of benefit to the host. Understanding how to induce highly efficient and functional T cells would inform strategies for designing vaccines intended to provide T cell-mediated immunity

Response to intranasal Lactococcus lactis W136 probiotic supplementation in refractory CRS is associated with modulation of non-type 2 inflammation and epithelial regeneration

JustificationWe have previously documented that in individuals with chronic rhinosinusitis (CRS) refractory to surgery, intranasal application of live Lactococcus lactis W136, a probiotic bacterium, improves sinus-specific symptoms, SNOT-22, and mucosal aspect on endoscopy, accompanied by a reduction in sinus pathogens and an increase in protective bacteria. The present work explores the molecular mechanisms underpinning these observations using transcriptomics of the sinus mucosa.MethodEpithelial brushings collected prospectively as a sub-study of the L. lactis W136 clinical trial were used to probe epithelial responses to microbiome supplementation using a hypothesis-free bioinformatic analysis of gene expression analysis. Samples from twenty-four patients with CRS refractory to medical and surgical management were prospectively collected during a clinical trial assessing the effect of 14 days of BID nasal irrigation with 1.2 billion CFU of live L. lactis W136 probiotic bacteria (CRSwNP = 17, CRSsNP = 7). Endoscopically guided sinus brushings were collected as part of the initial study, with brushings performed immediately before and after treatment. Following RNA extraction, samples were assessed using the Illumina HumanHT-12 V4 BeadChip. Differential gene expression was calculated, and pathway enrichment analysis was performed to identify potentially implicated processes.ResultsDifferentially identified transcripts and pathways were assessed for the overall population and the clinical phenotypes of CRSwNP and CRSsNP. Patterns of response to treatment were similar across all groups, implicating pathways for the regulation of immunity and epithelial cell regulation. These resemble the patterns of improvement observed following successful treatment with endoscopic sinus surgery or azithromycin.ConclusionGene expression profiling following the application of live bacteria to the diseased sinus epithelium highlights the implication of multiple components of the inflammation-microbiome-epithelial barrier axis implicated in CRS. These effects appear to involve both epithelial restoration and modulation of innate and adaptive immunity, supporting the potential interest of targeting the sinus epithelium and the microbiome as potential CRS therapies

K^dM2₈₂ T cells dominate bulk cytotoxicity but D^bM₁₈₇ T cells have superior individual cytotoxicity.

<p>(<b>a)</b> Bulk cytotoxicity of D^bM₁₈₇ and K^dM2₈₂ T cells <i>in vivo</i>. The epitope peptide-loaded and fluorochrome-labeled targets, as well as OVA₂₅₇ peptide-loaded and fluorochrome-labeled controls, were co-transferred into RSV-infected mice at 7dpi, and recovered 3 hours later. The recovery ratio were assessed with flow cytometry and compared with recovery ratio from naïve recipients to calculate epitope-specific lysis. Data represent 5 independent experiments (n = 5/group/experiment). <b>(b)</b> Cytotoxicity of individual D^bM₁₈₇ and K^dM2₈₂ T cells. Ratio of specific lysis of donor targets in (a) was divided by frequency of endogenous D^bM₁₈₇ and K^dM2₈₂ T cells respectively to quantitatively express arbitrary “Killing Unit” of individual cells. Data represent 5 independent experiments (n = 5/group/experiment). All data are shown as mean with independent data point and compared by Student t-test. Each symbol represents one mouse.</p

The D^bM₁₈₇ T cells express high avidity TCR and signaling pathways promoting cytotoxic function.

<p><b>(a)</b> The TCR avidity was assessed by dissociation of D^bM₁₈₇ and K^dM2₈₂ from CD8 T cells. CD8 T cells were labeled with pMHCs and assessed cell-bound median fluorescence intensity (MFI) at indicated time point by flow cytometry. The MFI at 0 min was defined as the maximum measurement (100%). Data were analyzed with one-phase exponential decay using nonlinear regression, and shown at mean ± SEM of three independent experiments (n = 5/group/experiment). <b>(b)</b> Transcriptional expression of genes that are up-regulated after RSV infection and associated with conventional signaling pathways. The D^bM₁₈₇, K^dM2_82, and bulk CD8 T cells were sorted from spleen lymphocytes by FACS at 7 dpi. The mRNAs were isolated, amplified and labeled, then hybridized onto Illumina Mouse Chips. The quantitative gene expression were analyzed and normalized. Genes with Log₂ Fold Change (FC) > 1.3, p < 0.05 and FDR < 0.25 (listed on left side of the chat) and associated signaling pathways were shown (Pathways 1: Altered T Cell and B Cell Signaling in Rheumatoid Arthritis; 2: T Helper Cell Differentiation; 3: Dendritic Cell Maturation; 4: Type I Diabetes Mellitus Signaling; 5: Roe of NFAT in Regulation of the Immune Response; 6: Role of Pattern Recognition Receptors in Recognition of Bacteria and Viruses; 7: IL-10 Signaling; 8: Role of CHK Proteins in Cell Cycle Checkpoint Control; 9: TREM1 Signaling; 10: Communication between Innate and Adaptive Immune Cells; 11: CD40 Signaling; 12: Production of Nitric Oxide and Reactive Oxygen Species in Macrophages; 13: Cell Cycle Control of Chromosomal Replication; 14: Acute Phase Response Signaling; 15: CD28 Signaling in T Helper Cells; 16: PKC Signaling in T Lymphocytes; 17: IL-12 Signaling and Production in Macrophages.). Data were pooled from 10 or 11 individual mice in each group.</p