Reducing cell intrinsic immunity to mRNA vaccine alters adaptive immune responses in mice.

The response to mRNA vaccines needs to be sufficient for immune cell activation and recruitment, but moderate enough to ensure efficacious antigen expression. The choice of the cap structure and use of N1-methylpseudouridine (m1Ψ) instead of uridine, which have been shown to reduce RNA sensing by the cellular innate immune system, has led to improved efficacy of mRNA vaccine platforms. Understanding how RNA modifications influence the cell intrinsic immune response may help in the development of more effective mRNA vaccines. In the current study, we compared mRNA vaccines in mice against influenza virus using three different mRNA formats: uridine-containing mRNA (D1-uRNA), m1Ψ-modified mRNA (D1-modRNA), and D1-modRNA with a cap1 structure (cC1-modRNA). D1-uRNA vaccine induced a significantly different gene expression profile to the modified mRNA vaccines, with an up-regulation of Stat1 and RnaseL, and increased systemic inflammation. This result correlated with significantly reduced antigen-specific antibody responses and reduced protection against influenza virus infection compared with D1-modRNA and cC1-modRNA. Incorporation of m1Ψ alone without cap1 improved antibodies, but both modifications were required for the optimum response. Therefore, the incorporation of m1Ψ and cap1 alters protective immunity from mRNA vaccines by altering the innate immune response to the vaccine material.

The Impact of a Non-Functional Thyroid Receptor Beta upon Triiodotironine-Induced Cardiac Hypertrophy in Mice.

BACKGROUND/AIMS: Thyroid hormone (TH) signalling is critical for heart function. The heart expresses thyroid hormone receptors (THRs); THRα1 and THRß1. We aimed to investigate the regulation mechanisms of the THRß isoform, its association with gene expression changes and implications for cardiac function. METHODS: The experiments were performed using adult male mice expressing TRßΔ337T, which contains the Δ337T mutation of the human THRB gene and impairs ligand binding. Cardiac function and RNA expression were studied after hypo-or hyperthyroidism inductions. T3-induced cardiac hypertrophy was not observed in TRßΔ337T mice, showing the fundamental role of THRß in cardiac hypertrophy. RESULTS: We identified a group of independently regulated THRß genes, which includes Adrb2, Myh7 and Hcn2 that were normally regulated by T3 in the TRßΔ337T group. However, Adrb1, Myh6 and Atp2a2 were regulated via THRß. The TRßΔ337T mice exhibited a contractile deficit, decreased ejection fraction and stroke volume, as assessed by echocardiography. In our model, miR-208a and miR-199a may contribute to THRß-mediated cardiac hypertrophy, as indicated by the absence of T3-regulated ventricular expression in TRßΔ337T mice. CONCLUSION: THRß has important role in the regulation of specific mRNA and miRNA in T3-induced cardiac hypertrophic growth and in the alteration of heart functions.

Toxicity of surface-modified PLGA nanoparticles toward lung alveolar epithelial cells.

In vitro cytotoxicity and inflammatory response following exposure to nanoparticles (NPs) made of poly(lactide-co-glycolide) (PLGA) have been investigated on A549 human lung epithelial cells. Three different PLGA NPs (230 nm) were obtained using different stabilizers (polyvinyl alcohol, chitosan, or Pluronic(®) F68) to form respectively neutral, positively or negatively charged NPs. Polystyrene NPs were used as polymeric but non-biodegradable NPs, and titanium dioxide (anatase and rutile) as inorganic NPs, for comparison. Cytotoxicity was evaluated through mitochondrial activity as well as membrane integrity (lactate dehydrogenase release, trypan blue exclusion, propidium iodide staining). The cytotoxicity of PLGA-based and polystyrene NPs was lower or equivalent to the one observed after exposure to titanium dioxide NPs. The inflammatory response, evaluated through the release of the IL-6, IL-8, MCP-1, TNF-α cytokines, was low for all NPs. However, some differences were observed, especially for negative PLGA NPs that led to a higher inflammatory response, which can be correlated to a higher uptake of these NPs. Taken together, these results show that both coating of PLGA NPs and the nature of the core play a key role in cell response.

Thyroid hormone regulation of Sirtuin 1 expression and implications to integrated responses in fasted mice.

Sirtuin 1 (SIRT1), a NAD(+)-dependent deacetylase, has been connected to beneficial effects elicited by calorie restriction. Physiological adaptation to starvation requires higher activity of SIRT1 and also the suppression of thyroid hormone (TH) action to achieve energy conservation. Here, we tested the hypothesis that those two events are correlated and that TH may be a regulator of SIRT1 expression. Forty-eight-hour fasting mice exhibited reduced serum TH and increased SIRT1 protein content in liver and brown adipose tissue (BAT), and physiological thyroxine replacement prevented or attenuated the increment of SIRT1 in liver and BAT of fasted mice. Hypothyroid mice exhibited increased liver SIRT1 protein, while hyperthyroid ones showed decreased SIRT1 in liver and BAT. In the liver, decreased protein is accompanied by reduced SIRT1 activity and no alteration in its mRNA. Hyperthyroid and hypothyroid mice exhibited increases and decreases in food intake and body weight gain respectively. Food-restricted hyperthyroid animals (pair-fed to euthyroid group) exhibited liver and BAT SIRT1 protein levels intermediary between euthyroid and hyperthyroid mice fed ad libitum. Mice with TH resistance at the liver presented increased hepatic SIRT1 protein and activity, with no alteration in Sirt1 mRNA. These results suggest that TH decreases SIRT1 protein, directly and indirectly, via food ingestion control and, in the liver, this reduction involves TRß. The SIRT1 reduction induced by TH has important implication to integrated metabolic responses to fasting, as the increase in SIRT1 protein requires the fasting-associated suppression of TH serum levels.

Thyroid hormone beta receptor mutation causes renal dysfunction and impairment of ClC-2 chloride channel expression in mouse kidney.

BACKGROUND/AIMS: Mutations in the thyroid hormone receptor beta (TR-beta) gene result in resistance to thyroid hormone (RTH). Mutation Delta337T in the TR-beta gene has been shown to have the characteristics of RTH syndrome in mice. The aim of this work was to study the possible involvement of TR-beta receptor in thyroid modulation of ClC-2 in mouse kidney. METHODS: Expression of mouse (Delta337T and normal C57BL/6) renal RNA and protein expression were studied by reverse transcriptase-polymerase chain reaction and Western blot, respectively, in mice with hyper- or hypothyroidism. Renal function was studied by analysis of urinary electrolyte excretion. Studies of the ClC-2 promoter region were performed in immortalized renal proximal tubule (IRPT) cells. RESULTS: In RTH syndrome mice (Delta337T), renal dysfunction was found to be associated with changes in the fractional excretion of sodium (FE(Na)) and chloride (FE(Cl)). ClC-2 chloride channel mRNA and protein expression were found to be decreased by 40% in heterozygous and homozygous mutant mouse kidneys and high levels of plasma thyroid hormone were detected in both groups. Hypothyroidism induced by methimazole decreased the renal expression of ClC-2 in normal mice but not in Delta337T mutant mice. In in vitro studies performed on IRPT cells subjected to thyroid hormone treatment, the promoter region of the ClC-2 chloride channel was stimulated in a dose-dependent manner. CONCLUSIONS: This work emphasizes the importance of thyroid hormone in electrolyte handling along the nephron and suggests its participation in renal ClC-2 gene transcription via the TR-beta receptor pathway.