Journal: Clinical and Translational Medicine

Article Title: The IL‐21‐TET2‐AIM2‐c‐MAF pathway drives the T follicular helper cell response in lupus‐like disease

doi: 10.1002/ctm2.781

Figure Lengend Snippet: Absent in melanoma 2 (AIM2) is highly expressed in human T follicular helper (T FH ) cells and in vitro differentiated T FH cells. (A–F) Naïve CD4 + T cells from the peripheral blood of healthy controls were used for the differentiation of T FH cells in vitro for 5 days ( n = 10, detailed information of the donors is shown in Table ). Heatmaps of mRNA‐seq analysis of (A) global genes and (B) T FH cell signature genes, as well as (C) volcano plots of upregulated (red) and downregulated (blue) genes, are shown. (D) Real‐time PCR was performed to detect the Aim2 mRNA levels of naïve CD4 + T cells and T FH cells (d5) ( n = 6). (E) AIM2 accumulation during T FH cell differentiation is shown. (F) Representative images of AIM2, c‐Maf and Bcl6 in naïve CD4 + T cells and T FH cells as determined by confocal microscopy. (G) Data plots of AIM2 expression in CD4 + T‐cell subsets from human tonsils by flow cytometry ( n = 8, detailed information of the donors is shown in Table ). (H) Gating strategy and representative flow cytometric plots of AIM2 percentages among various CD4 + T‐cell subsets from human tonsils in (G). (I) Representative images of AIM2 + T FH cells from human tonsils by multicolour immunohistochemistry staining. Bars show the mean ± SEM. * p < .05, ** p < .01, *** p < .001, **** p < .0001

Article Snippet: The following antibodies for Western blotting were used: anti‐c‐MAF (Abcam, ab230928, 1:1000) and anti‐human AIM2 (Abcam, ab204995, 1:1000) antibodies.

Techniques: In Vitro, Real-time Polymerase Chain Reaction, Cell Differentiation, Confocal Microscopy, Expressing, Flow Cytometry, Immunohistochemistry, Staining

Journal: Clinical and Translational Medicine

Article Title: The IL‐21‐TET2‐AIM2‐c‐MAF pathway drives the T follicular helper cell response in lupus‐like disease

doi: 10.1002/ctm2.781

Figure Lengend Snippet: Absent in melanoma 2 (AIM2) is highly expressed in T follicular helper (T FH )‐like cells from peripheral blood and skin lesions in systemic lupus erythematosus (SLE) patients. (A) AIM2 expression in CD4 + T‐cell subsets in peripheral blood from normal controls and SLE patients was assessed by flow cytometry ( n = 19 or 29). (B) Detection of AIM2 and Bcl6 in peripheral CD4 + T cells from normal controls and SLE patients was performed by confocal microscopy ( n = 3). (C) AIM2 accumulation in peripheral CD4 + T cells from SLE patients and normal controls is shown ( n = 4). (A–C) Detailed information of the donors is shown in Table . (D) Real‐time PCR was performed to assess the mRNA levels of Aim2 and T FH ‐related genes in skin lesions from lupus patients and normal controls ( n = 19). (E) Representative images of AIM2 expression in skin lesion T FH ‐like cells (CD4 + Bcl6 + PD1 + ) from patients with discoid lupus erythematosus (DLE), SLE, or psoriasis and normal controls determined by multicolour immunochemistry staining ( n = 4). (F) Quantified percentage of AIM2 + T FH cells in (E). (D–F) Detailed information of the donors is shown in . Bars show the mean ± SEM. * p < .05, ** p < .01, *** p < .001, **** p < .0001

Article Snippet: The following antibodies for Western blotting were used: anti‐c‐MAF (Abcam, ab230928, 1:1000) and anti‐human AIM2 (Abcam, ab204995, 1:1000) antibodies.

Techniques: Expressing, Flow Cytometry, Confocal Microscopy, Real-time Polymerase Chain Reaction, Staining

Journal: Clinical and Translational Medicine

Article Title: The IL‐21‐TET2‐AIM2‐c‐MAF pathway drives the T follicular helper cell response in lupus‐like disease

doi: 10.1002/ctm2.781

Figure Lengend Snippet: Deficiency of CD4 + T‐cell‐intrinsic absent in melanoma 2 (AIM2) impairs T follicular helper (T FH ) cell differentiation and IgG production after NP‐keyhole limpet haemocyanin (KLH) immunisation. (A) Schematic of the KLH and NP‐KLH mouse model. (B–F) Aim2 fl/fl and CD4 cre Aim2 fl/fl mice were challenged with KLH for 15 days ( n = 11). (B) Volcano plot of mRNA‐seq analysis of CD4 + T cells from Aim2 fl/fl and CD4 cre Aim2 fl/fl mice. Upregulated (red) and downregulated (blue) genes are shown ( n = 3). (C) Heatmaps of global gene expression and T FH cell signature gene expression in CD4 + T cells from Aim2 fl/fl and CD4 cre Aim2 fl/fl mice by mRNA‐seq. (D) The pathway enrichment analysis of RNA‐seq. Representative flow cytometric profiles and data plots of (E) T FH (CD4 + CXCR5 + PD1 + ) cells and (F) germinal centre (GC) B (B220 + GL7 + Fas + ) cells in draining lymph nodes (dLNs) ( n = 11). (G) Serum levels of anti‐NP IgG and IgM in Aim2 fl/fl and CD4 cre Aim2 fl/fl mice challenged with NP‐KLH for 28 days ( n = 5). (H) Naïve CD4 + T cells from Aim2 fl/fl and CD4 cre Aim2 fl/fl mice were used for in vitro T FH cell differentiation for 3 days. Representative flow cytometric profiles and data plots of T FH cells (CD4 + CXCR5 + PD1 + ) are shown ( n = 3). Bars show the mean ± SEM. * p < .05, ** p < .01

Article Snippet: The following antibodies for Western blotting were used: anti‐c‐MAF (Abcam, ab230928, 1:1000) and anti‐human AIM2 (Abcam, ab204995, 1:1000) antibodies.

Techniques: Cell Differentiation, Gene Expression, RNA Sequencing, In Vitro

Journal: Clinical and Translational Medicine

Article Title: The IL‐21‐TET2‐AIM2‐c‐MAF pathway drives the T follicular helper cell response in lupus‐like disease

doi: 10.1002/ctm2.781

Figure Lengend Snippet: Absent in melanoma 2 (AIM2) deficiency in CD4 + T cells ameliorates lupus development in mice. A pristane‐induced lupus mouse model was generated in Aim2 fl/fl and CD4 cre Aim2 fl/fl mice. (A) Schematic of pristane‐induced lupus model. (B) Proteinuria and (C) serum levels of anti‐dsDNA antibody and ANA in Aim2 fl/fl and CD4 cre Aim2 fl/fl mice were assessed ( n = 5). (D) Representative images of haematoxylin and eosin (H&E)‐stained kidneys and immunofluorescence staining with anti‐C3 and anti‐IgG antibodies in the kidney glomeruli are shown. Representative profiles of flow cytometric and data plots of (E) T follicular helper (T FH ) (CXCR5 + PD1 + CD4 + ) cells and (F) germinal centre (GC) B (GL7 + Fas + B220 + ) cells ( n = 8). Bars show the mean ± SEM. * p < .05, ** p < .01

Article Snippet: The following antibodies for Western blotting were used: anti‐c‐MAF (Abcam, ab230928, 1:1000) and anti‐human AIM2 (Abcam, ab204995, 1:1000) antibodies.

Techniques: Generated, Staining, Immunofluorescence

Journal: Clinical and Translational Medicine

Article Title: The IL‐21‐TET2‐AIM2‐c‐MAF pathway drives the T follicular helper cell response in lupus‐like disease

doi: 10.1002/ctm2.781

Figure Lengend Snippet: Absent in melanoma 2 (AIM2) regulates T follicular helper (T FH ) cell differentiation and C‐Maf expression. (A and B) Antisense oligonucleotides of AIM2 and the control were transfected into naïve CD4 + T cells during T FH cell differentiation for 5 days ( n = 3). (A) Representative flow cytometric profiles and data plots of T FH cells and (B) mRNA levels of AIM2 , CXCR5 , PD1 , IL21 and c‐MAFmaf detected by real‐time PCR are shown. (C) Real‐time PCR was performed to assess AIM2 , IL21 and c‐MAF mRNA levels in peripheral CD4 + T cells from systemic lupus erythematosus (SLE) patients and normal controls ( n = 10 or 23, detailed information of the donors is shown in Table ). (D) Correlations of mRNA expression of Il21 and Aim2 and of Cmaf and Aim2 in (C). (E) Co‐immunoprecipitation of AIM2 with c‐Maf in naïve T and T FH cells ( n = 2). (F) The binding domains of c‐Maf‐AIM2. Bars show the mean ± SEM. * p < .05, ** p < .01, *** p < .001

Article Snippet: The following antibodies for Western blotting were used: anti‐c‐MAF (Abcam, ab230928, 1:1000) and anti‐human AIM2 (Abcam, ab204995, 1:1000) antibodies.

Techniques: Cell Differentiation, Expressing, Control, Transfection, Real-time Polymerase Chain Reaction, Immunoprecipitation, Binding Assay

Journal: Clinical and Translational Medicine

Article Title: The IL‐21‐TET2‐AIM2‐c‐MAF pathway drives the T follicular helper cell response in lupus‐like disease

doi: 10.1002/ctm2.781

Figure Lengend Snippet: IL‐21 positively regulates absent in melanoma 2 (AIM2) expression by promoting ten‐eleven translocation 2 (TET2) enrichment in the Aim2 promoter region of T follicular helper (T FH ) cells. (A) DNA methylation map of naïve CD4 + T cells and T FH cells ( n = 3). Bisulphite sequencing PCR was performed to assess DNA methylation levels in the Aim2 promoter of (B) naïve CD4 + T cells and T FH cells ( n = 8) and (D) peripheral CD4 + T cells from systemic lupus erythematosus (SLE) patients and normal controls ( n = 10). Chromatin immunoprecipitation was performed to assess TET2 enrichment in the Aim2 promoter of (C) naïve CD4 + T cells and T FH cells ( n = 4 or 6) and (E) peripheral CD4 + T cells from SLE patients and normal controls ( n = 3 or 4). Representative flow cytometric profiles and data plots of (F) T FH cells and (G) germinal centre (GC) B cells in draining lymph nodes (dLNs) from Tet2 fl/fl and CD4 cre Tet2 fl/f l mice immunised with keyhole limpet haemocyanin (KLH) ( n = 9 or 10). (H) AIM2 accumulation in CD4 + T cells in wild‐type (WT) and CD4 cre Tet2 fl/fl mice ( n = 3). AIM2 accumulation in response to treatments with T FH ‐related cytokines assessed by (I) Western blot and (J) real‐time PCR ( n = 3). (K) Chromatin immunoprecipitation was performed in human CD4 + T cells with or without IL‐21 treatment after TET2 pull‐down ( n = 3). Bars show the mean ± SEM. * p < .05, ** p < .01, *** p < .001, **** p < .0001

Article Snippet: The following antibodies for Western blotting were used: anti‐c‐MAF (Abcam, ab230928, 1:1000) and anti‐human AIM2 (Abcam, ab204995, 1:1000) antibodies.

Techniques: Expressing, Translocation Assay, DNA Methylation Assay, Bisulfite Sequencing, Chromatin Immunoprecipitation, Western Blot, Real-time Polymerase Chain Reaction

Journal: Micromachines

Article Title: Optimization of PLGA Nanoparticle Formulation via Microfluidic and Batch Nanoprecipitation Techniques

doi: 10.3390/mi16090972

Figure Lengend Snippet: Inlet geometries of the microfluidic chips (scalebar: 1 mm) and the schematic representation of the workflow for the microfluidic preparation and characterization of PLGA NPs.

Article Snippet: A commercial microfluidic platform (Tamara, Inside Therapeutics) supplied with microfluidic chips featuring herringbone and baffle mixers were used for the preparation and optimization of PLGA NPs.

Techniques:

Journal: Nanomaterials (Basel, Switzerland)

Article Title: Multivariate Analysis of Protein-Nanoparticle Binding Data Reveals a Selective Effect of Nanoparticle Material on the Formation of Soft Corona.

doi: 10.3390/nano13212901

Figure Lengend Snippet: Figure 1. Binding curves (τ versus protein concentration) of protein–nanoparticle mixtures. The top panels show the adsorption isotherms obtained by mixing SA (a), TF (b) and PT (c) with silica nanoparticles. The bottom panels show SA (d), TF (e) and PT (f) mixtures with gold nanoparticles. The dashed curves on each panel represent the fits of the experimental data to the Langmuir–Hill equation, from which the parameters in Table 1 have been computed. Error bars represent the standard error of the mean over four repeats. Circle markers identify SA, square markers identify TF and triangle markers identify PT. Blue markers indicate data obtained for silica nanoparticles, while orange markers identify gold nanoparticles.

Article Snippet: A quantity of 100 nm silica nanoparticles was purchased from Polysciences as a 5% solid colloidal suspension in water, while 100 nm gold nanoparticles were purchased from NanoComposix as a 0.05 mg/mL colloidal suspension in 2 mM sodium citrate.

Techniques: Binding Assay, Protein Concentration, Adsorption

Journal: Nanomaterials (Basel, Switzerland)

Article Title: Multivariate Analysis of Protein-Nanoparticle Binding Data Reveals a Selective Effect of Nanoparticle Material on the Formation of Soft Corona.

doi: 10.3390/nano13212901

Figure Lengend Snippet: Figure 3. The effects of protein type on KD (a) and τmax (b) in mixtures of silica (blue bars) and gold (orange bars) nanoparticles. Error bars represent the standard error of estimates and asterisks identify groups that are statistically different from the others (* p < 0.05).

Article Snippet: A quantity of 100 nm silica nanoparticles was purchased from Polysciences as a 5% solid colloidal suspension in water, while 100 nm gold nanoparticles were purchased from NanoComposix as a 0.05 mg/mL colloidal suspension in 2 mM sodium citrate.

Techniques:

Journal: Nanomaterials (Basel, Switzerland)

Article Title: Multivariate Analysis of Protein-Nanoparticle Binding Data Reveals a Selective Effect of Nanoparticle Material on the Formation of Soft Corona.

doi: 10.3390/nano13212901

Figure Lengend Snippet: Figure 4a shows that the KD of TF and PT is much lower, indicating higher affinity, for gold nanoparticles compared to silica nanoparticles. However, in the case of SA, there is no significant difference in KD between the two nanoparticle materials. On the other hand, while the τmax of TF and PT is similar for both nanoparticle materials, SA forms a significantly thinner protein corona on silica nanoparticles compared to gold (Figure 4b). In Figure 4b, the τmax values were compared to the expected thickness τex for a protein monolayer. The exact arrangement and space occupied by the adsorbed proteins is unknown, but the measured thickness was compared to a monolayer formed by proteins approximated to spheres packed at the maximum density on the nanoparticle surface. In this configuration, τex is equivalent to the diameter of the sphere approximating SA, TF and PT, which was calculated based on existing models, resulting in values of 5.4, 5.7 and 5.5 nm for SA, TF and PT, respectively [29,30]. The data in Figure 4b show that τmax is thicker than τex in all cases, except for SA on silica nanoparticles, suggesting that the protein soft corona of the single-protein mixtures presents multiple layers of protein in all cases, except for SA on silica nanoparticles, where the estimated τmax value is compatible with a monolayer.

Article Snippet: A quantity of 100 nm silica nanoparticles was purchased from Polysciences as a 5% solid colloidal suspension in water, while 100 nm gold nanoparticles were purchased from NanoComposix as a 0.05 mg/mL colloidal suspension in 2 mM sodium citrate.

Techniques:

Journal: Nanomaterials (Basel, Switzerland)

Article Title: Multivariate Analysis of Protein-Nanoparticle Binding Data Reveals a Selective Effect of Nanoparticle Material on the Formation of Soft Corona.

doi: 10.3390/nano13212901

Figure Lengend Snippet: Figure 4. The effects of nanoparticle material on KD (a) and τmax (b) in SA, TF and PT mixtures. Blue bars indicate silica nanoparticles, while orange bars indicate gold nanoparticles. Error bars represent the standard error of estimates and asterisks identify groups that are statistically different from the others (* p < 0.05). The dashed lines in (b) represent the value of τex, which is the expected thickness for a monolayer of the relevant protein.

Article Snippet: A quantity of 100 nm silica nanoparticles was purchased from Polysciences as a 5% solid colloidal suspension in water, while 100 nm gold nanoparticles were purchased from NanoComposix as a 0.05 mg/mL colloidal suspension in 2 mM sodium citrate.

Techniques: