Lipid nanoparticle-encapsulated mRNA therapy corrects serum total bilirubin level in Crigler-Najjar syndrome mouse model

Crigler-Najjar syndrome is a rare disorder of bilirubin metabolism caused by uridine diphosphate glucuronosyl transferase 1A1 (UGT1A1) mutations characterized by hyperbilirubinemia and jaundice. No cure currently exists; treatment options are limited to phototherapy, whose effectiveness diminishes over time, and liver transplantation. Here, we evaluated the therapeutic potential of systemically administered, lipid nanoparticle-encapsulated human UGT1A1 (hUGT1A1) mRNA therapy in a Crigler-Najjar mouse model. Ugt1 knockout mice were rescued from lethal post-natal hyperbilirubinemia by phototherapy. These adult Ugt1 knockout mice were then administered a single lipid nanoparticle-encapsulated hUGT1A1 mRNA dose. Within 24 h, serum total bilirubin levels decreased from 15 mg/dL (256 μmol/L) to <0.5 mg/dL (9 μmol/L), i.e., slightly above wild-type levels. This reduction was sustained for 2 weeks before bilirubin levels rose and returned to pre-treatment levels by day 42 post-administration. Sustained reductions in total bilirubin levels were achieved by repeated administration of the mRNA product in a frequency-dependent manner. We were also able to rescue the neonatal lethality phenotype seen in Ugt1 knockout mice with a single lipid nanoparticle dose, which suggests that this may be a treatment modality appropriate for metabolic crisis situations. Therefore, lipid nanoparticle-encapsulated hUGT1A1 mRNA may represent a potential treatment for Crigler-Najjar syndrome

Optimal expression of TCRβ is associated with coexpression of both CD14 and F4/80 on CD11b^high cells.

<p><i>A</i>: C57BL/6 (n = 6) and Balb/c (n = 6) mice were infected with 10⁶<i>Plasmodium berghei</i> ANKA parasites. CD14 and F4/80 expression was then measured on gated CD11b^high splenocytes on days 2, 4, and 6 post-infection and in naïve mice (data not shown) and the absolute number of CD11b^high, CD11b^highCD14⁺, CD11b^highF4/80⁺, and CD11b^highCD14⁺F4/80⁺ cells was enumerated at each time point. <i>B</i>: The effect of CD14 and F4/80 on TCRβ expression on CD11b^high splenocytes was also determined in C57BL/6 and Balb/c mice by comparing the expression of TCRβ on CD11b^high, CD11b^highCD14⁺, CD11b^highF4/80⁺, and CD11b^highCD14⁺F4/80⁺ cells. The percentage of cellular subsets that were TCRβ⁺CD3ε⁻ and the TCRβ-FITC mean fluorescence intensity (MFI) were calculated on days 2, 4, and 6 post-<i>Pb−A</i> infection. Data presented is representative of three independent experiments. The absolute number of cellular subsets that were TCRβ⁺CD3ε⁻ can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201043#pone.0201043.s003" target="_blank">S3 Fig</a>.</p

TCRβ-expressing macrophages induced by a pathogenic murine malaria correlate with parasite burden and enhanced phagocytic activity

<div><p>Macrophages express a wide array of invariant receptors that facilitate host defense and mediate pathogenesis during pathogen invasion. We report on a novel population of CD11b^highCD14⁺F4/80⁺ macrophages that express TCRβ. This population expands dramatically during a <i>Plasmodium berghei</i> ANKA infection and sequesters in the brain during experimental cerebral malaria. Importantly, measurement of TCRβ transcript and protein levels in macrophages in wildtype versus nude and <i>Rag1</i> knockout mice establishes that the observed expression is not a consequence of passive receptor expression due to phagocytosis or trogocytosis of peripheral T cells or nonspecific antibody staining to an Fc receptor or cross reactive epitope. We also demonstrate that TCRβ on brain sequestered macrophages undergoes productive gene rearrangements and shows preferential Vβ usage. Remarkably, there is a significant correlation in the proportion of macrophages that express TCRβ and peripheral parasitemia. In addition, presence of TCRβ on the macrophage also correlates with a significant increase (1.9 fold) in the phagocytosis of parasitized erythrocytes. By transcriptional profiling, we identify a novel set of genes and pathways that associate with TCRβ expression by the macrophage. Expansion of TCRβ-expressing macrophages points towards a convergence of the innate and adaptive immune responses where both arms of the immune system cooperate to modulate the host response to malaria and possibly other infections.</p></div

TCRβ expression correlates with enhanced phagocytosis of parasites by macrophages.

<p><i>A</i>: Splenocytes and parasitized red blood cells (pRBCs) were isolated from C57BL/6 mice on day 3 post-infection with <i>Plasmodium berghei</i> ANKA. A 1:1 ratio of splenocytes (labeled with macrophage and T lymphocyte markers) and pRBCs (labeled with CellTrace^®) were then incubated for 90 minutes to assess the effect of TCRβ expression by Ly6G⁻CD11b^highF4/80⁺ macrophages on phagocytosis in an <i>in vitro</i> assay. <i>B</i>: Expression of TCRβ correlates with enhanced phagocytosis of pRBCs by macrophages. n = 3 for each phagocytosis assay. Results are presented as mean ± standard deviation and are a replicate of three independent experiments. Significant difference between TCRβ⁺ versus TCRβ⁻ macrophages is indicated as **P < 0.01. P−value was calculated using the Mann Whitney <i>U</i> test.</p

Comparison of TCRβ expression on macrophages in C57BL/6 wildtype verses nude and <i>rag1</i> knockout mice.

<p><i>A</i>: Wildtype (WT), nude, and <i>rag1</i> knockout mice were infected with 10⁶ <i>Plasmodium berghei</i> ANKA (<i>Pb−A</i>) parasites and TCRβ expression was measured on gated Ly6G⁻CD11b^highCD14⁺F4/80⁺ macrophages from spleen tissue of the three types of mice. <i>B</i>: The percentage of macrophages that were TCRβ⁺CD3ε⁻ and the absolute number of TCRβ⁺CD3ε⁻ macrophages was compared in naïve and <i>Pb−A</i> infected (day 6) WT, nude, and <i>rag1</i> knockout mice. Results are presented as mean ± standard deviation and are representative of two independent experiments. Significant difference between WT versus nude mice is indicated as *P < 0.05. P−value was calculated using the Mann Whitney <i>U</i> test.</p

TCRβ expression by the macrophage correlates with <i>Plasmodium berghei</i> ANKA parasite burden.

<p>On day 3 post-infection, the correlation between the percentage of Ly6G⁻CD11b^highCD14⁺F4/80⁺ macrophages that are TCRβ⁺CD3ε⁻ and peripheral parasitemia (parasitized erythrocytes/total erythrocytes x 100) in five individual mice was determined. Results shown are representative of four independent experiments. Pearson r = 0.96, P<0.01.</p

Brain sequestered macrophages express high levels of TCRβ.

<p><i>A</i>: Brain sequestered leukocytes (BSLs) purified from perfused brain tissue of moribund mice (n = 4) with experimental cerebral malaria on day 6 post-infection with <i>Plasmodium berghei</i> ANKA were stained for flow cytometric analysis. An FMO control (green) was used for gating purposes to determine the proportion of brain sequestered macrophages that express TCRβ (blue). <i>B</i>: CD11b^highCD14⁺F4/80⁺ macrophages that were TCRαβ⁺CD3ε<i>−</i> and CD4<i>−</i>CD8<i>−</i> were gated from live singlet BSLs purified from perfused brain tissue of moribund mice on day 6 post-infection and colocalization of TCRβ with the CD11b, CD14, and F4/80 monocyte/macrophage lineage markers was visualized on individual cells by AMNIS imaging. In addition, measurement of cell size by AMNIS imaging demonstrates that TCRβ expressing CD11b^highCD14⁺F4/80⁺ cells (n = 314) are significantly larger than CD4⁺TCRβ⁺CD3ε⁺ (n = 102) and CD8⁺TCRβ⁺CD3ε⁺ (n = 586) lymphocytes. <i>C</i>: Brain sequestered leukocytes (BSLs) purified from perfused brain tissue of naïve or moribund C57BL/6 mice on day 6 post-<i>Pb−A</i> infection were stained with the pan-macrophage marker F4/80 (red) and TCRβ (green) and counterstained with DAPI (blue). Cellular colocalization of TCRβ with F4/80 was observed by confocal microscopy on BSLs from moribund but not naïve mice. Importantly, isotype controls for F4/80 (rat IgG2a, κ) and TCRβ (Armenian hamster IgG) stained negative in both moribund and naïve mice. Significant differences between macrophage versus CD4⁺ and CD8⁺ T lymphocyte cell size are indicated as ****P < 0.001. P<i>−</i>values were calculated using the Student <i>t</i> test. Error bars indicate standard deviation.</p

Subsets of brain sequestered leukocytes during experimental cerebral malaria.

<p>C57BL/6 mice were infected with 10⁶ <i>Plasmodium berghei</i> ANKA parasites and brain sequestered leukocytes <b>(</b>BSLs) were purified from perfused brain tissue of moribund mice (n = 5). <i>A</i>: CD4⁺ T cells, CD8⁺ T cells and macrophages were then quantitated by flow cytometric analysis. FMO controls for CD14 (purple) and F4/80 (light blue) were used to determine the proportion of CD11b^high brain sequestered leukocytes that express CD14 and F4/80. <i>B</i>: Phenotypic analysis of stained BSLs was performed to compare expression of the CD4⁺ and CD8⁺ T cell coreceptors and the CD11b, CD14, and F4/80 monocyte/macrophage lineage markers on TCRβ⁺CD3ε⁺ versus TCRβ⁺CD3ε^- BSLs. Fluorescence minus one controls (dashed line) were used for gating purposes for CD11b, CD14, and F4/80 antibody staining. Data presented is representative of three independent experiments.</p

Brain sequestered macrophages preferentially express Vβ3, Vβ7, and Vβ8.1/2 during experimental cerebral malaria.

<p><i>A</i>: C57BL/6 mice were infected with 10⁶<i>Plasmodium berghei</i> ANKA parasites and T cell receptor Vβ usage was evaluated in splenic CD11b^highCD14⁺F4/80⁺ macrophages of moribund mice (n = 5) on day 6 post-infection using a flow cytometry based mouse Vβ TCR Screening Panel. <i>B</i>: The type of and preference for Vβ TCR was also assessed in brain sequestered CD11b^highCD14⁺F4/80⁺ macrophages during the cerebral phase of experimental cerebral malaria. Results shown depict the proportion of macrophages that express each Vβ chain and the total number of macrophages that express each Vβ chain after normalization for cell volume and are representative of three independent experiments.</p