Journal: Advanced Science

Article Title: Type I Interferon Signaling Augments Autoimmunity in Neuromyelitis Optica Spectrum Disorder

doi: 10.1002/advs.202500942

Figure Lengend Snippet: IFN‐I facilitated the development of CD4 + T cells in NMOSD. A) UMAP plot depicting the subclusters of CD4 + T cells derived from all samples of scRNA‐seq data. B) The gene‐set scores of different T helper subsets (Th1, Th2, and Th17 gene signatures) in CD4 + T cells. C) The gene scores of different helper T cell subsets (characterized by Th1, Th2, and Th17 gene signatures) were compared between the control group and patients with NMOSD. D,E) UMAP plot showing pseudotime trajectory of CD4 + T cells based on Slingshot demonstrated three distinct trajectories (Th1, Th17, and Tfh). F) The density plot of CD4 + T cell differentiation trajectories showing the changes in the intensity of different T cell subtypes during the differentiation process between the NMOSD group and the control group. G) Heat map illustrating the normalized log count of gene expression associated with IFN‐I response in CD4 + T cells during the trajectory of differentiation and maturation. H) The expression levels of representative genes driving CD4 + T cells development along Th1 and Th17 trajectories. I) The differences in IFN‐I‐related pathway scores between the NMOSD group and the control group during the differentiation process of CD4 + T cells.

Article Snippet: The cells were cultured under three conditions: The culture groups comprised the control group: Baseline culture with CD3/CD28 co‐stimulation alone, Th17 polarization conditions: AQP4 p201‐220 , 10 μg ml −1 ; IL‐23, 20 ng ml −1 ; and IL‐6, 20 ng ml −1 , and Th17 polarization conditions with additional IFN‐I stimulation: IFNA1 (MCE, HY‐P700183AF), IFN‐β (MCE, HY‐P73130), both at 100 U ml −1 .

Techniques: Derivative Assay, Control, Cell Differentiation, Gene Expression, Expressing

Journal: Advanced Science

Article Title: Type I Interferon Signaling Augments Autoimmunity in Neuromyelitis Optica Spectrum Disorder

doi: 10.1002/advs.202500942

Figure Lengend Snippet: Enhanced cGAS‐STING‐IFN‐I activity in NMOSD Mouse Models. A) Schematic representation of NMO‐IgG injection model. B) Heatmap illustrating the expression levels of cGAS‐STING‐IFN‐I, disease‐associated microglia (DAM), and chemokine genes derived from bulk RNA‐seq of microglia sorted from WT mice, Control‐IgG treated mice, and NMO‐IgG treated mice (n=3 per group). C) Quantitative PCR analysis revealed altered expression for cGAS‐STING‐IFN‐I and microglia activation‐related genes in MACS‐sorted CD11b + microglia from WT mice, Control‐IgG‐treated mice, and NMO‐IgG‐treated mice during the peak of disease ( n = 5 per group). D,E) Representative transmission electron microscopy images and quantitative analysis of damaged mitochondrial alterations in microglia of control‐IgG‐treated mice and NMO‐IgG‐treated mice ( n = 4 per group). F,G) Representative immunofluorescence staining images and quantitative analysis of the changes in DNA mean fluorescence intensity within IBA1 + microglia in WT mice, control‐IgG‐treated mice, and NMO‐IgG‐treated mice ( n = 4 per group). H) Flow chart depicting the acquisition and in vitro culture procedure of AQP4‐specific T cells. I) Representative flow cytometry and statistical analysis demonstrated that IFN‐I promoted an increase in the proportion of I‐A b ‐AQP4(205‐215) tetramer‐positive T cells within CD4 + T cells ( n = 6–8). J) Representative flow cytometry and statistical analysis revealed that under Th17 polarization conditions, IFN‐I promoted the increase in the proportion of IFN‐γ and IL‐17A positive T cells in CD4 + T cells ( n = 6). K) Representative flow cytometry and statistical analysis demonstrated that IFN‐I promoted an increase in the proportion of chemokine receptors CCR5, CCR6, and CXCR3 positive T cells in CD4 + T cells ( n = 6). Data presented as Mean ± s.e.m; * p < 0.05, ** p < 0.01, *** p < 0.001, P values were calculated by the one‐way ANOVA test, Kruskal‐Wallis test, or Mann‐Whitney test.

Article Snippet: The cells were cultured under three conditions: The culture groups comprised the control group: Baseline culture with CD3/CD28 co‐stimulation alone, Th17 polarization conditions: AQP4 p201‐220 , 10 μg ml −1 ; IL‐23, 20 ng ml −1 ; and IL‐6, 20 ng ml −1 , and Th17 polarization conditions with additional IFN‐I stimulation: IFNA1 (MCE, HY‐P700183AF), IFN‐β (MCE, HY‐P73130), both at 100 U ml −1 .

Techniques: Activity Assay, Injection, Expressing, Derivative Assay, RNA Sequencing, Control, Real-time Polymerase Chain Reaction, Activation Assay, Transmission Assay, Electron Microscopy, Immunofluorescence, Staining, Fluorescence, In Vitro, Flow Cytometry, MANN-WHITNEY

Journal: Advanced Science

Article Title: Type I Interferon Signaling Augments Autoimmunity in Neuromyelitis Optica Spectrum Disorder

doi: 10.1002/advs.202500942

Figure Lengend Snippet: Attenuated glia damage and IFN‐I activation in AQP4 specific Sting ‐/‐ deficiency mouse model. A) Schematic representation of Th17‐AQP4 specific T cells passive transfer model. B) Clinical scores of the Th17‐AQP4 model were compared between WT and Sting ‐/‐ mice ( n = 8 per group). C) Representative H&E and LFB staining images of Th17‐AQP4 model between WT and Sting ‐/‐ mice, scale=500 µm. D) Quantitative analysis revealed a significant decrease in inflammatory infiltration and demyelination level among Sting ‐/‐ mice ( n = 6 per group). E) Representative immunofluorescence images and quantitative analysis were conducted to assess the presence of TMEM119 + /C1q + microglia of WT mice and Sting ‐/‐ mice within spinal cord lesions at the peak of the disease, indicating a significant reduction in microglial activation in Sting ‐/‐ mice compared to WT mice ( n = 6 per group). F) Representative immunofluorescence images and quantitative analysis of the GFAP + /C3 + activated astrocytes in spinal cord lesions, showing lower levels of astrocyte activation in Sting ‐/‐ mice during the peak of the disease ( n = 5 per group). G) Representative immunofluorescence images and quantitative analysis of pTBK1 mean fluorescence intensity were compared between IBA1 + microglia within spinal cord lesions of Sting ‐/‐ mice and WT mice ( n = 5 per group). H) Representative immunofluorescence images and quantitative analysis of the MFI of pIRF3 were compared between IBA1 + microglia within the spinal cord lesions of Sting ‐/‐ mice and WT mice ( n = 5 per group). I) qPCR analysis showed decreased expression of Ifna1 , Ifnar1 , Irf3 , Cxcl10 , Il‐1b , and C1qa genes in the microglia of Sting ‐/‐ mice during the peak of disease ( n = 5 per group). Statistical analysis for clinical scores was performed using one‐way ANOVA with Tukey's post‐test, while unpaired student's t‐test or Mann‐Whitney test was employed for histology and flow cytometry comparisons; data presented as Mean ± s.e.m.; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The cells were cultured under three conditions: The culture groups comprised the control group: Baseline culture with CD3/CD28 co‐stimulation alone, Th17 polarization conditions: AQP4 p201‐220 , 10 μg ml −1 ; IL‐23, 20 ng ml −1 ; and IL‐6, 20 ng ml −1 , and Th17 polarization conditions with additional IFN‐I stimulation: IFNA1 (MCE, HY‐P700183AF), IFN‐β (MCE, HY‐P73130), both at 100 U ml −1 .

Techniques: Activation Assay, Staining, Immunofluorescence, Fluorescence, Expressing, MANN-WHITNEY, Flow Cytometry

Journal: Advanced Science

Article Title: Type I Interferon Signaling Augments Autoimmunity in Neuromyelitis Optica Spectrum Disorder

doi: 10.1002/advs.202500942

Figure Lengend Snippet: STING inhibitor H‐151 effectively improved the AQP4‐specific T cells model. A) Schematic representation of Th17‐AQP4 model treated with STING inhibitor H‐151. B) Clinical symptom scores compared between control group mice and mice treated with H‐151 in the Th17 mouse model ( n = 8 per group). C) Representative immunofluorescence images depicting changes in TMEM119 + /C1q + microglia within the spinal cord lesion of WT mice and H‐151‐treated mice at the peak of disease. D) Quantitative analysis of TMEM119 + /C1q + microglia ( n = 5 per group). E) Representative immunofluorescence images demonstrating alterations in GFAP + /C3 + astrocytes within the spinal cord lesion of WT mice and H‐151‐treated mice at the peak of disease. F) Quantitative analysis revealing changes in GFAP + /C3 + astrocyte ( n = 5 per group). G) Flow cytometry analysis to quantify CNS‐infiltrated cells in both WT and H‐151‐treated mice at the peak of disease. The gating strategy was consistent with that in Figure ( n = 5 per group). Statistical analyses: Clinical scores were assessed using one‐way ANOVA with Tukey's post‐test. Unpaired student's t‐test or Mann‐Whitney test was used to compare results from flow cytometry and immunofluorescence studies. Data were presented as Mean ± s.e.m, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The cells were cultured under three conditions: The culture groups comprised the control group: Baseline culture with CD3/CD28 co‐stimulation alone, Th17 polarization conditions: AQP4 p201‐220 , 10 μg ml −1 ; IL‐23, 20 ng ml −1 ; and IL‐6, 20 ng ml −1 , and Th17 polarization conditions with additional IFN‐I stimulation: IFNA1 (MCE, HY‐P700183AF), IFN‐β (MCE, HY‐P73130), both at 100 U ml −1 .

Techniques: Control, Immunofluorescence, Flow Cytometry, MANN-WHITNEY

Journal: Molecular immunology

Article Title: LKB1 isoform expression modulates T cell plasticity downstream of PKCθ and IL-6.

doi: 10.1016/j.molimm.2023.03.020

Figure Lengend Snippet: Fig. 1. The short isoform of LKB1 is predominantly expressed in Th17 cells CD4 T cells isolated from were differ entiated under Th17, iTreg, or non- polarizing (NP) conditions for 7 days prior to harvesting. We utilized flow cytometry to assess a) the percent of LKB1 positive cells within each cell subset and b) LKB1 protein expression, as measured by median fluorescence intensity (MFI). c) We visualized LKB1S expression by immunoblotting and quantified LKB1S expression relative to NP. Vinculin is shown as a loading control. We amplified the d) short and e) long splice variants of Stk11 using qRT-PCR, as well as f) the ratio of Stk11S to Stk11L transcript expression. Stk11 transcripts were quantified using the 2-ΔΔCt method, normalized to ActB (β-actin), and are expressed relative to NP. Protein band intensity was quanti fied using ImageJ. Data are the mean of three independent experiments. * p < 0.05; * * p < 0.01; * ** p < 0.001; unpaired, two-tailed Student’s t-test.

Article Snippet: For some experiments, CD4 T cells were polarized under iTreg or Th17 polarizing conditions and on days 5 and 6 of the differentiation process were treated with 10 μM of the specific Stat3 inhibitor, Stattic (Santa Cruz Biotechnology, Dallas, TX), according to the manufacturer’s D. Mohan et al. Molecular Immunology 157 (2023) 129–141 recommendations.

Techniques: Isolation, Flow Cytometry, Expressing, Fluorescence, Western Blot, Control, Amplification, Quantitative RT-PCR, Two Tailed Test

Journal: Molecular immunology

Article Title: LKB1 isoform expression modulates T cell plasticity downstream of PKCθ and IL-6.

doi: 10.1016/j.molimm.2023.03.020

Figure Lengend Snippet: Fig. 2. HnRNPLL associates with Stk11S in Th17 cells and requires PKCθ for its expression CD4 T cells isolated from WT mice were differentiated under Th17, iTreg, or non-polarizing (NP) conditions for 7 days prior to harvesting. a) We assessed hnRNPLL expression by immunoblotting and b) Hnrnpll levels by qRT-PCR. Vinculin is shown as a loading control. c) For RNA-IP, hnRNPLL was immunoprecipitated from whole cell lysate with RNase inhibitor to preserve RNA content. RNA was extracted from the lysate after immunoprecipitation, and we used qRT-PCR to amplify Stk11S transcripts. Hnrnpll transcripts were quantified using the 2-ΔΔCt method, normalized to ActB (β-actin), and are expressed relative to the WT NP sample. Stk11S is expressed as bound units (BU) relative to Stk11S bound to hnRNPLL immunoprecipitated from NP samples. Protein band intensity was quantified using ImageJ. Data are the mean ± S.E.M of three independent experiments. * p < 0.05; * * p < 0.01; * ** p < 0.001; * ** * p < 0.0001; two-way ANOVA with Bonferroni correction applied.

Article Snippet: For some experiments, CD4 T cells were polarized under iTreg or Th17 polarizing conditions and on days 5 and 6 of the differentiation process were treated with 10 μM of the specific Stat3 inhibitor, Stattic (Santa Cruz Biotechnology, Dallas, TX), according to the manufacturer’s D. Mohan et al. Molecular Immunology 157 (2023) 129–141 recommendations.

Techniques: Expressing, Isolation, Western Blot, Quantitative RT-PCR, Control, Immunoprecipitation

Journal: Molecular immunology

Article Title: LKB1 isoform expression modulates T cell plasticity downstream of PKCθ and IL-6.

doi: 10.1016/j.molimm.2023.03.020

Figure Lengend Snippet: Fig. 3. PKCθ regulates Stk11 splice variant and LKB1 isoform expression upstream of hnRNPLL CD4 T cells isolated from WT or PKCθ-/- mice were differentiated under Th17, iTreg, or non-polarizing (NP) conditions for 7 days prior to harvesting. We used qRT-PCR to quantify a) Prkcq and b) Hnrnpll transcript levels in WT Th17 and iTreg cells, as well as the levels of c) Stk11S and Stk11L. Relative gene expression was determined using the 2-ΔΔCT method. For Il17f and Prkcq, For Prckq and Hnrnpll, results are presented as fold expression of the gene of interest normalized to the housekeeping gene, ActB (β-actin), for each sample, and expressed relative to the For Stk11 splice variants, the results are presented as percent of the variant expressed relative to total Stk11 (common) for each sample. d) We used immunoblotting to assess differences in LKB1 isoform in differentiated CD4 T cells from WT and PKCθ-/- mice. Vinculin is shown as a loading control. Protein band intensity was quantified using ImageJ. Data are the mean ± S.E.M of three independent experiments. Statistical analyses shown in gray refer to comparisons between conditions for cells derived from PKCθ-/- mice. * p < 0.05; * * p < 0.01; * ** * p < 0.0001; two-way ANOVA with Bonferroni correction applied.

Article Snippet: For some experiments, CD4 T cells were polarized under iTreg or Th17 polarizing conditions and on days 5 and 6 of the differentiation process were treated with 10 μM of the specific Stat3 inhibitor, Stattic (Santa Cruz Biotechnology, Dallas, TX), according to the manufacturer’s D. Mohan et al. Molecular Immunology 157 (2023) 129–141 recommendations.

Techniques: Variant Assay, Expressing, Isolation, Quantitative RT-PCR, Gene Expression, Western Blot, Control, Derivative Assay

Journal: Molecular immunology

Article Title: LKB1 isoform expression modulates T cell plasticity downstream of PKCθ and IL-6.

doi: 10.1016/j.molimm.2023.03.020

Figure Lengend Snippet: Fig. 4. HnRNPLL regulates Stk11S expression CD4 T cells isolated from WT or PKCθ-/- mice were differentiated under Th17, iTreg, or non-polarizing (NP) conditions for 7 days prior to har vesting. On day 3 of the differentiation process, Th17 polarized cells were transfected with 50 μM of Hnrnpll siRNA (siLL) or with a scrambled control (siSCR). At the end of the differentiation period, we used qRT-PCR to quantify a) Stk11S and b) Stk11L. c) LKB1 isoform expression was assessed by immuno blotting. Vinculin is shown as a loading control. We also quantified d) Il17f and e) Prkcq by qRT-PCR. Relative gene expression was determined using the 2-

Article Snippet: For some experiments, CD4 T cells were polarized under iTreg or Th17 polarizing conditions and on days 5 and 6 of the differentiation process were treated with 10 μM of the specific Stat3 inhibitor, Stattic (Santa Cruz Biotechnology, Dallas, TX), according to the manufacturer’s D. Mohan et al. Molecular Immunology 157 (2023) 129–141 recommendations.

Techniques: Expressing, Isolation, Transfection, Control, Quantitative RT-PCR, Gene Expression

Journal: Molecular immunology

Article Title: LKB1 isoform expression modulates T cell plasticity downstream of PKCθ and IL-6.

doi: 10.1016/j.molimm.2023.03.020

Figure Lengend Snippet: Fig. 5. IL-6 signaling acts upstream of PKCθ-regulated Stk11 splicing CD4 T cells isolated from WT or PKCθ-/- mice were differentiated under Th17, iTreg, or non-polarizing (NP) conditions for 7 days prior to harvesting. iTregs were dosed with 20 ng/ml of IL-6 on day 5 of the differentiation process, then har vested 48 h later. We used qRT-PCR to quantify a) Prkcq, b) Hnrnpll, and d) Rorc and relative gene expression was determined using the 2-ΔΔCt method. The results are presented as fold expression of the gene of interest normalized to the housekeeping gene, ActB (β-actin), for each sample, and is expressed relative to the WT NP sample. c) Stk11S is expressed as a percent of the total Stk11 (common) for each sample. e) In vitro differentiated iTregs from WT or PKCθ-/- mice were labeled with Red650 and mixed with Ultra Green labeled responder cells (CD4 T cells) at three different ratios. Percent suppres sion was determined as described in Materials & Methods. Data are the mean ± S.E.M of three independent experi ments. Statistical analyses shown in gray refer to comparisons between conditions for cells derived from PKCθ-/-

Article Snippet: For some experiments, CD4 T cells were polarized under iTreg or Th17 polarizing conditions and on days 5 and 6 of the differentiation process were treated with 10 μM of the specific Stat3 inhibitor, Stattic (Santa Cruz Biotechnology, Dallas, TX), according to the manufacturer’s D. Mohan et al. Molecular Immunology 157 (2023) 129–141 recommendations.

Techniques: Isolation, Quantitative RT-PCR, Gene Expression, Expressing, In Vitro, Labeling, Derivative Assay

Journal: Molecular immunology

Article Title: LKB1 isoform expression modulates T cell plasticity downstream of PKCθ and IL-6.

doi: 10.1016/j.molimm.2023.03.020

Figure Lengend Snippet: Fig. 6. Stk11 splicing proceeds through a Stat3-mediated pathway CD4 T cells isolated from WT or PKCθ-/- mice were differentiated under Th17, iTreg, or non- polarizing (NP) conditions for 7 days prior to harvesting. iTregs were dosed with 20 ng/ml of IL-6 on day 5 of the differentiation process. For some cultures, iTregs were also treated with 10 μM of the specific STAT3, Stattic, on days 5 and 6 of polarization. All cells were harvested on day 7. We used qRT-PCR to quantify a) Prkcq and b) Hnrnpll using the 2-ΔΔCt method. The results are presented as fold expression of the gene of interest normalized to the housekeeping gene, ActB (β-actin), for each sample, and is expressed relative to levels in WT NP sample. We used flow cytometry to assess the percentage of c) IL-17-expressing or e) Foxp3-expressing cells generated for each treatment condition and the median fluorescence intensity (MFI) of d) IL-17 or f) Foxp3 expressed by each population. Data are the mean ± S.E.M of three independent experiments. Statistical analyses shown in gray refer to comparisons between conditions for cells derived from PKCθ-/- mice. Statistical analyses represented by symbols refer to comparisons between cells derived from WT and PKCθ-/- mice, subjected to identical treatments. $ or * p < 0.05; & or * * p < 0.01; * ** p < 0.001; # or * ** * p < 0.0001; two-way ANOVA with Bonferroni correction applied.

Article Snippet: For some experiments, CD4 T cells were polarized under iTreg or Th17 polarizing conditions and on days 5 and 6 of the differentiation process were treated with 10 μM of the specific Stat3 inhibitor, Stattic (Santa Cruz Biotechnology, Dallas, TX), according to the manufacturer’s D. Mohan et al. Molecular Immunology 157 (2023) 129–141 recommendations.

Techniques: Isolation, Quantitative RT-PCR, Expressing, Flow Cytometry, Generated, Fluorescence, Derivative Assay

Journal: Molecular immunology

Article Title: LKB1 isoform expression modulates T cell plasticity downstream of PKCθ and IL-6.

doi: 10.1016/j.molimm.2023.03.020

Figure Lengend Snippet: Fig. 7. Stk11s expression is sufficient to induce Th17 programming CD4 T cells were isolated from WT or PKCθ-/- mice. Prior to differentiation, cells to be differ entiated into Th17 cells were transduced with empty vector (Th17 +EV) or with Stk11L (Th17 +Stk11L), while cells to be differentiated into iTreg cells were transduced with empty vector (iTreg+EV) or with Stk11S (iTreg+Stk11S). At the end of the 7-day polarization process a) LKB1 expression was visualized by immunoblotting. Vinculin as a loading control. We quantified b) Foxp3, c) Rorc, and d) Il17f by qRT-PCR. Relative gene expression was determined using the 2-ΔΔCt method. The results are presented as the fold expression of the gene of interest normalized to the housekeeping gene, ActB (β-actin), for each sample and is expressed relative to the WT NP sample. Protein band intensity was quantified using ImageJ. Data are the mean ± S.E.M of three independent experiments. Statistical analyses shown in gray refer to comparisons between conditions for cells derived from PKCθ-/- mice. Statistical analyses represented by symbols refer to comparisons between cells derived from WT and PKCθ-/- mice, subjected to identical treatments. $ or * p < 0.05; & or * * p < 0.01; * ** p < 0.001; # or * ** * p < 0.0001; two-way ANOVA with Bonferroni correction applied.

Article Snippet: For some experiments, CD4 T cells were polarized under iTreg or Th17 polarizing conditions and on days 5 and 6 of the differentiation process were treated with 10 μM of the specific Stat3 inhibitor, Stattic (Santa Cruz Biotechnology, Dallas, TX), according to the manufacturer’s D. Mohan et al. Molecular Immunology 157 (2023) 129–141 recommendations.

Techniques: Expressing, Isolation, Transduction, Plasmid Preparation, Western Blot, Control, Quantitative RT-PCR, Gene Expression, Derivative Assay