Journal: Inflammation

Article Title: KANK4 Regulates CXCL16 Glycosylation Through TMEM260 to Modulate Microglial Activation in Sepsis-associated Encephalopathy

doi: 10.1007/s10753-026-02481-y

Figure Lengend Snippet: KANK4 regulated CXCL16 glycosylation and microglial activation via TMEM260. Microglia were isolated from the hippocampus of mice in Sham, SAE, SAE + AAV-NC, and SAE + AAV-KANK4 groups, followed by the following analyses: (A) IHC was performed to detect CXCL16 protein expression. Microglia were isolated from the Sham and SAE groups for the following assays: (B) Western blot analysis was conducted to assess CXCL16 glycosylation levels ( n = 3). (C) Co-IP was carried out to examine the interaction between TMEM260 and CXCL16. BV2 microglial cells were treated with LPS to establish a cell model. Control and LPS groups were subjected to the following analyses: (D) Western blot analysis was applied to detect CXCL16 expression using both anti-CXCL16 and anti-His-CXCL16 antibodies ( n = 3). (E) Co-IP was conducted to validate the interaction between TMEM260 and CXCL16. (F) Western blot was used to assess O-mannosylation of CXCL16 protein in LPS-treated microglia ( n = 3). (G) Effect of CXCL16 glycosylation site mutations on its glycosylation level. Wild-type (WT) and site-specific mutant (S137A, S117A, S139A) CXCL16 plasmids were transfected into microglia. CXCL16 glycosylation was assessed by Western blot ( n = 3). (H) Impact of CXCL16 glycosylation site mutation (S117A) on the expression of inflammatory factors. In an LPS-induced microglial inflammation model, cells were transfected with either WT or glycosylation-site mutant (S117A) CXCL16 plasmid. The levels of inflammatory cytokines IL-1β, IL-6, and TNF-α in the cells were measured by ELISA. BV2 cells were transfected for TMEM260 overexpression or knockdown. The groups included Control, si-NC, si-TMEM260, NC-OE, and TMEM260-OE, and the following analyses were performed: (I) Western blot analysis was used to detect the expression of TMEM260 and CXCL16 proteins ( n = 3). (J) CHX assay was performed to evaluate the stability of CXCL16 protein. LPS-treated BV2 cells were used to conduct KANK4 overexpression and TMEM260 knockdown rescue experiments, with six groups: Control, LPS, LPS + OE-NC, LPS+KANK4-OE, LPS+KANK4-OE + si-NC, and LPS+KANK4-OE + si-TMEM260. The following analysis was conducted: (K) Western blot analysis was performed to measure CXCL16 protein expression in BV2 cells ( n = 3). For overexpression of TMEM260 and CXCL16 in LPS-treated BV2 cells, the groups included: Control, LPS, LPS + OE-NC, LPS+TMEM260-OE, LPS+TMEM260-OE + OE-NC, and LPS+TMEM260-OE+CXCL16-OE. The following analyses were carried out: (L) Western blot analysis was conducted to detect TMEM260 and CXCL16 protein levels in each group ( n = 3). (M) CCK-8 assay was applied to evaluate microglial cell viability across groups. (N) IF staining was performed to assess the expression of Iba-1 (green) and CD11b (red), indicating microglial activation status (Scale bar = 50 μm). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. LPS/LPS + OE-NC/LPS+TMEM260-OE + OE-NC; ns, no significant difference vs. LPS/Control

Article Snippet: To assess the stability of CXCL16 protein, CHX (MCE, HY-12320) was used to block new protein synthesis, and a protein stability tracking experiment was conducted.

Techniques: Glycoproteomics, Activation Assay, Isolation, Expressing, Western Blot, Co-Immunoprecipitation Assay, Control, Mutagenesis, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Over Expression, Knockdown, CCK-8 Assay, Staining

Journal: The Journal of Experimental Medicine

Article Title: Nasal CD4 + tissue-resident memory T cells provide cross-protective immunity to influenza

doi: 10.1084/jem.20251793

Figure Lengend Snippet: CXCR6–CXCL16 axis promotes NT CD4 TRM establishment. (A) Box plot showing the expression of Cxcr6 mRNA among CD4 TRM of the NT and lungs. Data are presented as median and interquartile range. NS, not significant; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05 by unpaired two-tailed t test. (B and C) Box plot showing the expression of Cxcr6 mRNA among different cell clusters of lungs and NT. Data are presented as median and interquartile range. NS, not significant; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05 by Wilcoxon rank sum test with Benjamini–Hochberg correction. (C) Volcano plot for differential expressed genes in the NT in comparison with the lungs of the Th17 cluster. The dotted lines indicate fold change and adjusted P value cutoffs. (D) Bar plot with individual data points for the expression of CXCR6 (each median fluorescence intensity [MFI] normalized to mean MFI from CD4 TEM iv + of NT) in CD4 TRM and iv + CD4 TEM from the lungs and NT of naïve mice and PR8 IAV-infected mice (30 dpi) as indicated. The experiment was repeated thrice, and the results (mean ± SEM) are pooled. NS, not significant; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05 by two-way ANOVA, with Tukey’s multiple comparison test. (E and F) Expression of CXCR6 on OT-II CD4 TRM of NT and lung on day 30 following PR8-OVA infection as indicated. (E) Representative histograms for CXCR6 expression from OT-II CD4 TRM of NT and lung are shown. (F) Bar plot with individual data points showing the expression of CXCR6 (each MFI normalized to mean MFI of NT OT-II CD4 TRM). The experiment was repeated thrice, and the results (mean ± SEM) are pooled. NS, not significant; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05 by unpaired two-tailed t test. (G–K) Frequency of different cell populations within different organs derived from WT and Cxcr6 −/− BM chimeric mice on day 30 after infection with PR8 IAV as indicated. (G) Schematic representation of the experimental setup. (H) Representative flow cytometry plots showing the percentage of WT and Cxcr6 −/− CD4 TRM in NT and lungs. (I) Bar plot with individual data points for the percentage of WT and Cxcr6 −/− CD4 TRM in NT and lungs as indicated. (J) Bar plot with individual data points for the percentage of I-A b NP 306–322 tetramer-specific WT and Cxcr6 −/− CD4 TRM in NT and lungs as indicated. Samples that had <7 OT-II CD4 TRM were excluded from the analysis. The experiment was repeated thrice, and the results (mean ± SEM) are pooled. NS, not significant; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05 by unpaired two-tailed t test. (K) Bar plot with individual data points showing the percentage of Cxcr6 −/− and WT CD4 T cells in different organs of the BM chimera on day 30 after infection with PR8. The experiment was repeated thrice and the results (mean ± SEM) were pooled. NS, not significant; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05 by unpaired two-tailed t test. (L) A representative microscopic image of CXCL16 expression (magenta) and OT-II CD4 T cells (red and green) in the olfactory epithelium of the murine NT. NT is isolated on day 30 after PR8-OVA infection from mice that received OT-II CD4 T cells. Scale bar: 100 μm for main image and 50 μm for the inset. (M–O) Antigen-specific CD4 TRM in the lungs and NT of mice treated with isotype control or anti-CXCL16 antibody on day 10 after PR8 IAV infection. (M) Schematic representation of the experimental setup. (N) Representative flow cytometry plots indicating the percentage of I-A b NP 306–322 tetramer-specific CD4 TRM are shown. (O) Bar plot with individual data points indicating the absolute number of I-A b NP 306–322 tetramer-specific CD4 TRM. The experiment was performed twice, and the results (mean ± SEM) are pooled. NS, not significant; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05 by unpaired two-tailed t test.

Article Snippet: Mouse CXCL16 antibody (MAB503) was purchased from R&D Systems and was dissolved in 1× PBS.

Techniques: Expressing, Two Tailed Test, Comparison, Fluorescence, Infection, Derivative Assay, Flow Cytometry, Olfactory, Isolation, Control

Journal: The Journal of Experimental Medicine

Article Title: Nasal CD4 + tissue-resident memory T cells provide cross-protective immunity to influenza

doi: 10.1084/jem.20251793

Figure Lengend Snippet: Additional characterization of experimental models, related to Figs. 6, 7, and 8. (A) Percentage of CD45 + donor cells (both Cxcr6 −/− and WT) and recipient cells in the blood of BM chimera on day 67 following BM transplantation. Left panel: Bar plot with individual data points for the percentage of donor cells and recipient cells. The experiment was repeated thrice, and the results (mean ± SEM) were pooled. NS, not significant; ****P < 0.0001; ***P < 0.001 by one-way ANOVA, with Tukey’s multiple comparison test. Right panel: A representative flow cytometry plot showing the percentage of donor cells and recipient cells. (B) Bar plot with individual data points showing the percentage of donor and recipient cells among CD4 T cells in different organs in the BM chimera on day 30 after infection with PR8. The experiment was repeated thrice, and the results (mean ± SEM) were pooled. NS, not significant; ****P < 0.0001; ***P < 0.001 by two-way ANOVA, with Tukey’s multiple comparison test. (C) Representative microscopic image of CXCL16 expression (magenta) in the olfactory epithelium of the murine NT is shown. Two negative controls for CXCL16 staining showing NT stained (1) with α-rabbit secondary IgG Texas red only and (2) stained with isotype control and α-rabbit secondary IgG Texas red. Hoechst is indicated in grey. NT is isolated on day 30 after PR8-OVA infection from mice that received OT-II CD4 T cells. Top images are stitched to show the whole NT. Scale bar: 500 μm for top panels and 100 μm for bottom. (D) Gating strategy to identify CD4 TEM (CD4 + CCR7 − CD45RA − ), CD4 TCM (CD4 + CCR7 + CD45RA − ), naïve CD4 Tc (CD4 + CCR7 + CD45RA + ), CD4 TEMRA(CD4 + CCR7 − CD45RA + ), and CD4 TRM (CD4 + CCR7 − CD45RA − CD69 + ) in NT and blood. (E) RORγt + CD4 + T cells in the PPs of WT C57BL/6 mice, CD4 cre Rorc fl/wt , and CD4 cre Rorc fl/fl mice. Left panel: Bar plot with individual data points showing the frequency of RORγt + CD4 + T cells among all CD3 + T cells. The experiment was done twice and the results (mean ± SEM) were pooled. NS, not significant; ****P < 0.0001; ***P < 0.001 by unpaired two-tailed t test. Right panel: A representative flow cytometry plot for the percentage of RORγt + CD4 + T cells in different groups. (F) Microscopy images (magnified) for TUNEL + cells in the nasal septum (respiratory region) from CD4 cre Rorc fl/wt and CD4 cre Rorc fl/fl mice (same as ). Scale bar: 50 μm. (G and H) Microscopy for TUNEL + cells and viral titer (TCID 50 /g) from organs of mice infected with PR8 and reinfected with X31 IAV on day 30 following PR8 IAV infection. The mice were treated with isotype or IL-17a/f antibody. (G) Bar plot with individual data points showing number of TUNEL + cells in the nasal septum (respiratory region) on day 4 after X31 IAV infection. The experiment was repeated twice. NS, not significant; ****P < 0.0001; ***P < 0.001 by unpaired two-tailed t test. (H) Viral titer (TCID 50 /g) from NT and lungs on day 3 after X31 IAV infection. NS, not significant; ****P < 0.0001; ***P < 0.001 by unpaired two-tailed t test. TEMRA, terminally differentiated effector memory T cells re-expressing CD45RA.

Article Snippet: Mouse CXCL16 antibody (MAB503) was purchased from R&D Systems and was dissolved in 1× PBS.

Techniques: Transplantation Assay, Comparison, Flow Cytometry, Infection, Expressing, Olfactory, Staining, Control, Isolation, Two Tailed Test, Microscopy, TUNEL Assay

Journal: Inflammation

Article Title: KANK4 Regulates CXCL16 Glycosylation Through TMEM260 to Modulate Microglial Activation in Sepsis-associated Encephalopathy

doi: 10.1007/s10753-026-02481-y

Figure Lengend Snippet: KANK4 regulated CXCL16 glycosylation and microglial activation via TMEM260. Microglia were isolated from the hippocampus of mice in Sham, SAE, SAE + AAV-NC, and SAE + AAV-KANK4 groups, followed by the following analyses: (A) IHC was performed to detect CXCL16 protein expression. Microglia were isolated from the Sham and SAE groups for the following assays: (B) Western blot analysis was conducted to assess CXCL16 glycosylation levels ( n = 3). (C) Co-IP was carried out to examine the interaction between TMEM260 and CXCL16. BV2 microglial cells were treated with LPS to establish a cell model. Control and LPS groups were subjected to the following analyses: (D) Western blot analysis was applied to detect CXCL16 expression using both anti-CXCL16 and anti-His-CXCL16 antibodies ( n = 3). (E) Co-IP was conducted to validate the interaction between TMEM260 and CXCL16. (F) Western blot was used to assess O-mannosylation of CXCL16 protein in LPS-treated microglia ( n = 3). (G) Effect of CXCL16 glycosylation site mutations on its glycosylation level. Wild-type (WT) and site-specific mutant (S137A, S117A, S139A) CXCL16 plasmids were transfected into microglia. CXCL16 glycosylation was assessed by Western blot ( n = 3). (H) Impact of CXCL16 glycosylation site mutation (S117A) on the expression of inflammatory factors. In an LPS-induced microglial inflammation model, cells were transfected with either WT or glycosylation-site mutant (S117A) CXCL16 plasmid. The levels of inflammatory cytokines IL-1β, IL-6, and TNF-α in the cells were measured by ELISA. BV2 cells were transfected for TMEM260 overexpression or knockdown. The groups included Control, si-NC, si-TMEM260, NC-OE, and TMEM260-OE, and the following analyses were performed: (I) Western blot analysis was used to detect the expression of TMEM260 and CXCL16 proteins ( n = 3). (J) CHX assay was performed to evaluate the stability of CXCL16 protein. LPS-treated BV2 cells were used to conduct KANK4 overexpression and TMEM260 knockdown rescue experiments, with six groups: Control, LPS, LPS + OE-NC, LPS+KANK4-OE, LPS+KANK4-OE + si-NC, and LPS+KANK4-OE + si-TMEM260. The following analysis was conducted: (K) Western blot analysis was performed to measure CXCL16 protein expression in BV2 cells ( n = 3). For overexpression of TMEM260 and CXCL16 in LPS-treated BV2 cells, the groups included: Control, LPS, LPS + OE-NC, LPS+TMEM260-OE, LPS+TMEM260-OE + OE-NC, and LPS+TMEM260-OE+CXCL16-OE. The following analyses were carried out: (L) Western blot analysis was conducted to detect TMEM260 and CXCL16 protein levels in each group ( n = 3). (M) CCK-8 assay was applied to evaluate microglial cell viability across groups. (N) IF staining was performed to assess the expression of Iba-1 (green) and CD11b (red), indicating microglial activation status (Scale bar = 50 μm). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. LPS/LPS + OE-NC/LPS+TMEM260-OE + OE-NC; ns, no significant difference vs. LPS/Control

Article Snippet: Protein A/G agarose beads (Beyotime) and anti-CXCL16 (Proteintech, 60123-1-Ig, 1:1000) or anti-His (Abcam, ab9108, 1:1000) antibodies were incubated overnight at 4 °C to immunoprecipitate CXCL16 and His-CXCL16 protein complexes, respectively.

Techniques: Glycoproteomics, Activation Assay, Isolation, Expressing, Western Blot, Co-Immunoprecipitation Assay, Control, Mutagenesis, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Over Expression, Knockdown, CCK-8 Assay, Staining

Journal: Inflammation

Article Title: KANK4 Regulates CXCL16 Glycosylation Through TMEM260 to Modulate Microglial Activation in Sepsis-associated Encephalopathy

doi: 10.1007/s10753-026-02481-y

Figure Lengend Snippet: KANK4 regulated CXCL16 glycosylation and microglial activation via TMEM260. Microglia were isolated from the hippocampus of mice in Sham, SAE, SAE + AAV-NC, and SAE + AAV-KANK4 groups, followed by the following analyses: (A) IHC was performed to detect CXCL16 protein expression. Microglia were isolated from the Sham and SAE groups for the following assays: (B) Western blot analysis was conducted to assess CXCL16 glycosylation levels ( n = 3). (C) Co-IP was carried out to examine the interaction between TMEM260 and CXCL16. BV2 microglial cells were treated with LPS to establish a cell model. Control and LPS groups were subjected to the following analyses: (D) Western blot analysis was applied to detect CXCL16 expression using both anti-CXCL16 and anti-His-CXCL16 antibodies ( n = 3). (E) Co-IP was conducted to validate the interaction between TMEM260 and CXCL16. (F) Western blot was used to assess O-mannosylation of CXCL16 protein in LPS-treated microglia ( n = 3). (G) Effect of CXCL16 glycosylation site mutations on its glycosylation level. Wild-type (WT) and site-specific mutant (S137A, S117A, S139A) CXCL16 plasmids were transfected into microglia. CXCL16 glycosylation was assessed by Western blot ( n = 3). (H) Impact of CXCL16 glycosylation site mutation (S117A) on the expression of inflammatory factors. In an LPS-induced microglial inflammation model, cells were transfected with either WT or glycosylation-site mutant (S117A) CXCL16 plasmid. The levels of inflammatory cytokines IL-1β, IL-6, and TNF-α in the cells were measured by ELISA. BV2 cells were transfected for TMEM260 overexpression or knockdown. The groups included Control, si-NC, si-TMEM260, NC-OE, and TMEM260-OE, and the following analyses were performed: (I) Western blot analysis was used to detect the expression of TMEM260 and CXCL16 proteins ( n = 3). (J) CHX assay was performed to evaluate the stability of CXCL16 protein. LPS-treated BV2 cells were used to conduct KANK4 overexpression and TMEM260 knockdown rescue experiments, with six groups: Control, LPS, LPS + OE-NC, LPS+KANK4-OE, LPS+KANK4-OE + si-NC, and LPS+KANK4-OE + si-TMEM260. The following analysis was conducted: (K) Western blot analysis was performed to measure CXCL16 protein expression in BV2 cells ( n = 3). For overexpression of TMEM260 and CXCL16 in LPS-treated BV2 cells, the groups included: Control, LPS, LPS + OE-NC, LPS+TMEM260-OE, LPS+TMEM260-OE + OE-NC, and LPS+TMEM260-OE+CXCL16-OE. The following analyses were carried out: (L) Western blot analysis was conducted to detect TMEM260 and CXCL16 protein levels in each group ( n = 3). (M) CCK-8 assay was applied to evaluate microglial cell viability across groups. (N) IF staining was performed to assess the expression of Iba-1 (green) and CD11b (red), indicating microglial activation status (Scale bar = 50 μm). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. LPS/LPS + OE-NC/LPS+TMEM260-OE + OE-NC; ns, no significant difference vs. LPS/Control

Article Snippet: Protein A/G agarose beads (Beyotime) and anti-CXCL16 (Proteintech, 60123-1-Ig, 1:1000) or anti-His (Abcam, ab9108, 1:1000) antibodies were incubated overnight at 4 °C to immunoprecipitate CXCL16 and His-CXCL16 protein complexes, respectively.

Techniques: Glycoproteomics, Activation Assay, Isolation, Expressing, Western Blot, Co-Immunoprecipitation Assay, Control, Mutagenesis, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Over Expression, Knockdown, CCK-8 Assay, Staining