Journal: The Journal of Biological Chemistry

Article Title: The lipid transporter Mfsd2a maintains pulmonary surfactant homeostasis

doi: 10.1016/j.jbc.2022.101709

Figure Lengend Snippet: Mfsd2a deficiency results in AT2 hypertrophy and changes to immune response and stress pathways. A , immunohistochemical staining on lung sections indicate Mfsd2a ( red ) expression in AT2 cells of 2a fl/fl mice but absent in AT2aKO. Pdpn ( green ) stains AT1 cells. 2a fl/fl , n = 6; AT2aKO, n = 4. The scale bar represents 50 μm. Inset in images is the enlarged area indicated by white box . The scale bar represents 12.5 μm. B , H&E-stained lung section of AT2aKO and 2a fl/fl control 4 weeks post-tamoxifen. n = 6 per genotype. Masson's trichome-stained lung section of AT2aKO and 2a fl/fl control 12 weeks post-tamoxifen. 2a fl/fl , n = 3; AT2aKO, n = 7. AT2 cells are indicated by black arrows . The scale bar represents 20 μm. Inset in images is the enlarged area indicated by black box . The scale bar represents 10 μm. C , quantification of AT2 cell diameter from AT2aKO and 2a fl/fl controls at 4 and 12 weeks post-tamoxifen represented as a scatterplot. Unpaired t test with Welch's correction, ∗∗∗∗ p < 0.0001. Biological replicates as indicated in B . D , RNA-Seq analysis of AT2aKO and 2a fl/fl AT2 cells. Bubble plot represents top 20 significantly upregulated or downregulated Gene Ontology terms. E , heatmap highlighting inflammatory and fibrosis markers that were upregulated in AT2aKO relative to 2a fl/fl controls. Color bar indicates z -score transformation on median ratio normalized counts. n = 4 per genotype. Wald test, ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. AT1, alveolar epithelial type 1; AT2, alveolar epithelial type 2; Mfsd2a, major facilitator superfamily domain containing 2a.

Article Snippet: Tamoxifen-inducible AT2-specific knockout mice (AT2aKO) were generated by crossing 2a fl/fl with Sftpc-CreER T2 driver (B6.129S-Sftpc tm1(cre/ERT2)Blh /J; The Jackson Laboratory) ( ).

Techniques: Immunohistochemical staining, Staining, Expressing, Control, RNA Sequencing, Transformation Assay

Journal: The Journal of Biological Chemistry

Article Title: The lipid transporter Mfsd2a maintains pulmonary surfactant homeostasis

doi: 10.1016/j.jbc.2022.101709

Figure Lengend Snippet: Mfsd2a mediates the uptake of LPCs into AT2 at the apical surface. A , orthoview of z-stack of lung sections stained for Mfsd2a ( red ) and the AT2 basolateral membrane marker Na + /K + ATPase α1 ( green ). The scale bar represents 20 μm. B , 3D reconstruction of confocal images in ( A ) indicated enrichment of Mfsd2a ( red ) at the apical side of AT2 cells. 2a fl/fl , n = 6; AT2aKO, n = 4. The scale bar represents 50 μm. C , intratracheal instillation of LPC-NBD. Immunohistochemical staining of Sftpc ( red ) and F4/80 ( gray ) on lung sections indicates accumulation of LPC-NBD ( green ) only in 2a fl/fl AT2 cells but absent in AT2aKO AT2. LPC-NBD accumulation in macrophages is observed in both AT2aKO and 2a fl/fl AT2. The scale bar represents 20 μm. Inset in images is the enlarged area indicated by white box . The scale bar represents 10 μm. n = 4 per genotype. D , intravenous instillation of LPC-NBD. No LPC-NBD uptake was observed in AT2aKO or 2a fl/fl AT2 cells. Sftpc ( red ) stains AT2 cells. n = 4 per genotype. The scale bar represents 50 μm. E , LPC-NBD ( green ) was taken up by AT2 cells isolated from 2a fl/fl but not AT2aKO mice in vitro . Sftpc ( red ) denotes AT2 cells. Hoechst ( blue ) denotes nuclei. n = 5 per genotype. The scale bar represents 20 μm. AT2, alveolar epithelial type 2; LPC, lysophosphatidylcholine; Mfsd2a, major facilitator superfamily domain containing 2a; NBD, [12-(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]-dodecanoyl.

Article Snippet: Tamoxifen-inducible AT2-specific knockout mice (AT2aKO) were generated by crossing 2a fl/fl with Sftpc-CreER T2 driver (B6.129S-Sftpc tm1(cre/ERT2)Blh /J; The Jackson Laboratory) ( ).

Techniques: Staining, Membrane, Marker, Immunohistochemical staining, Isolation, In Vitro

Journal: The Journal of Biological Chemistry

Article Title: The lipid transporter Mfsd2a maintains pulmonary surfactant homeostasis

doi: 10.1016/j.jbc.2022.101709

Figure Lengend Snippet: Uptake of LPC by AT2 cells is dependent on lung phospholipase A 2 (PLA 2 ) activity. A , intratracheal instillation of Red/Green BODIPY PC-A2. Both red LPC and green FA are taken up by 2a fl/fl AT2 as seen by colocalization with Sftpc (indicated by white arrows in insets of area denoted by white squares ), whereas only green FA is taken up by AT2aKO AT2. B , red LPC is also taken up by macrophages as seen by colocalization with F4/80 (indicated by white arrows in insets of area denoted by white squares ) in both AT2aKO and 2a fl/fl AT2. C , quantification of red and green fluorescence in lung sections. 2a fl/fl , n = 5; AT2aKO, n = 4. The scale bar represents 50 μm; the scale bar ( inset ) represents 12.5 μm. Unpaired t test with Welch's correction, ∗ p < 0.05; ∗∗ p < 0.01. AT2, alveolar epithelial type 2; FA, fatty acid; LPC, lysophosphatidylcholine.

Article Snippet: Tamoxifen-inducible AT2-specific knockout mice (AT2aKO) were generated by crossing 2a fl/fl with Sftpc-CreER T2 driver (B6.129S-Sftpc tm1(cre/ERT2)Blh /J; The Jackson Laboratory) ( ).

Techniques: Activity Assay, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: The lipid transporter Mfsd2a maintains pulmonary surfactant homeostasis

doi: 10.1016/j.jbc.2022.101709

Figure Lengend Snippet: Assessment of sPLA 2 activity in BAL. sPLA 2 activity was measured in BAL isolated from AT2aKO or 2a fl/fl using Red/Green BODIPY PC ( A and B ) or Bis-BODIPY FL C11-PC ( C ), and lipids were extracted and resolved by TLC. sPLA 2 activity was comparable between both genotypes. LPC and FA were indicated by red and blue arrows , respectively. Unhydrolyzed PC substrates (no BAL, black arrows ) are fluorescent in organic solvent used for TLC analysis as these substrates are no longer ordered and quenched as they are in BAL in vivo . 2a fl/fl , n = 5; AT2aKO, n = 3. D , quantification of sPLA 2 activity is represented as the mean ± SE of the ratio of the sum of products (LPC and FA) over the sum of products and unhydrolyzed substrate. 2a fl/fl , n = 5; AT2aKO, n = 3. BAL, bronchioalveolar lavage; FA, fatty acid; FL, full-length; LPC, lysophosphatidylcholine; PC, phosphatidylcholine; sPLA 2 , secretory PLA 2 .

Article Snippet: Tamoxifen-inducible AT2-specific knockout mice (AT2aKO) were generated by crossing 2a fl/fl with Sftpc-CreER T2 driver (B6.129S-Sftpc tm1(cre/ERT2)Blh /J; The Jackson Laboratory) ( ).

Techniques: Activity Assay, Isolation, Solvent, In Vivo

Journal: The Journal of Biological Chemistry

Article Title: The lipid transporter Mfsd2a maintains pulmonary surfactant homeostasis

doi: 10.1016/j.jbc.2022.101709

Figure Lengend Snippet: Mfsd2a deficiency leads to reductions in total BAL phospholipids (PLs). Lipidomic analysis of AT2aKO and 2a fl/fl BAL 4 weeks post-tamoxifen treatment. A , BAL lipid species represented as a volcano plot. Dots above dashed horizontal line indicate lipid species that are significantly different between AT2aKO and 2a fl/fl AT2. Most of the lyso-PL and PL species are reduced in AT2aKO BAL relative to 2a fl/fl control (PL, SL, and NL). Total PC 32:0 ( B ) and PL species containing DHA (PL-DHA) ( C ) or AA (PL-AA) ( D ) were reduced in AT2aKO BAL relative to 2a fl/fl . BAL PLs ( E ) and lyso-PLs ( F ) represented as a heatmap. Color bar indicates z -score transformation on μmol/μl BAL PLs. Data for B – D are represented as mean μmol/μl BAL ± SE, with individual points denoting biological replicates. Fatty acid identity is designated as the number of carbons:number of double bonds. For all panels in this figure, 2a fl/fl , n = 6; AT2aKO, n = 5. Unpaired t test with Welch's correction, ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. AA, arachidonic acid; AT2, alveolar epithelial type 2; BAL, bronchioalveolar lavage; DHA, docosahexaenoic acid; Mfsd2a, major facilitator superfamily domain containing 2a; NL, neutral lipid; PC, phosphatidylcholine; SL, sphingolipid.

Article Snippet: Tamoxifen-inducible AT2-specific knockout mice (AT2aKO) were generated by crossing 2a fl/fl with Sftpc-CreER T2 driver (B6.129S-Sftpc tm1(cre/ERT2)Blh /J; The Jackson Laboratory) ( ).

Techniques: Control, Transformation Assay

Journal: The Journal of Biological Chemistry

Article Title: The lipid transporter Mfsd2a maintains pulmonary surfactant homeostasis

doi: 10.1016/j.jbc.2022.101709

Figure Lengend Snippet: Mfsd2a deficiency alters the AT2 cell lipidome reflective of changes in BAL. Lipidomic analysis of AT2aKO and 2a fl/fl BAL 2 weeks post-tamoxifen treatment. Total PC 32:0 ( A ) and phospholipid (PL) species containing DHA (PL-DHA) ( B ) or AA (PL-AA) ( C ) were reduced in AT2aKO BAL relative to 2a fl/fl . D , lipidomic analysis of AT2aKO and 2a fl/fl AT2 cells 2 weeks post-tamoxifen treatment. Total PC 32:0 was reduced in AT2aKO AT2 cells relative to 2a fl/fl . E , mol% lipid species represented as a volcano plot. Dots above dashed line indicate lipid species that are significantly different between AT2aKO and 2a fl/fl AT2 (PL, SL, and NL. F , PLs with monounsaturated fatty acids (PL-MUFA) species were increased in AT2aKO AT2 cells relative to 2a fl/fl . G , PC–DHA species were reduced in AT2aKO AT2 cells relative to 2a fl/fl , similar to changes in the BAL lipidome. For A – D , data are represented as mean μmol/μl BAL ± SE, with individual points denoting biological replicates. 2a fl/fl , n = 13; AT2aKO, n = 12. For F – H , data represented as mol% ± SE of PLs, with individual points denoting biological replicates. n = 7 per genotype. Unpaired t test with Welch's correction, ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. H , model to show Mfsd2a is required for surfactant homeostasis. AT2 cells synthesize surfactants, which are secreted as DPPC-rich lamellar bodies at the apical surface that unravel into large surfactant aggregates that make up the alveolar lining fluid. Spent surfactant is released as small surfactant aggregates that are hydrolyzed by sPLA 2 in the alveolar space to produce lysophosphatidylcholines (LPCs). LPC are then taken up by Mfsd2a expressed at the apical surface of AT2 cells and serve as a precursor for the synthesis of PC–DHA that are then remodeled into the main surfactant PL DPPC. Some LPCs can also be cleared by alveolar macrophages. In the absence of Mfsd2a, uptake of LPCs is limited resulting in reduction in PC–DHA leading to reduced resynthesis of DPPC. Moreover, reduced sPLA 2 activity in the absence of Mfsd2a might be a result of altered surfactant composition.

Article Snippet: Tamoxifen-inducible AT2-specific knockout mice (AT2aKO) were generated by crossing 2a fl/fl with Sftpc-CreER T2 driver (B6.129S-Sftpc tm1(cre/ERT2)Blh /J; The Jackson Laboratory) ( ).

Techniques: Activity Assay

Journal: Nature Communications

Article Title: SEPTIN2 suppresses an IFN-γ-independent, proinflammatory macrophage activation pathway

doi: 10.1038/s41467-023-43283-2

Figure Lengend Snippet: a Survival of Sept2 fl/fl Lyz2 -Cre mice ( n = 15) and their littermates ( Sept2 fl/fl , n = 13) after intraperitoneal infection with 1 × 10 7 PFU VSV. Daily injection of αIFN-γ (12 mg/kg) from 1 day before infection to the end of the experiments was performed to block IFN-γ signaling. b , c H&E staining ( b ) and the pathology scores ( c ) of lung lesions in mice at 7 dpi. Scale bar = 400 μm. n = 6 in each group ( b , c ). d ELISA analysis of proinflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-12 p40) in mice BALF at 7 dpi. n = 6 in each group ( d ). e Flow cytometry analysis of innate immune cell populations in mice lungs at 7 dpi. n = 6 in each group ( e ). f Immunohistochemistry analysis of iNOS in mice lungs at 7 dpi. Scale bar = 400 μm. n = 6 in each group ( f ). g , i PMs obtained from Sept2 fl/fl Lyz2 -Cre and Sept2 fl/fl mice were infected with VSV (MOI = 1). The iNOS, CD80, and CD86 levels were detected by flow cytometry ( g ), and secretion of proinflammatory cytokines was detected by ELISA ( i ) at 0.5, 6, and 12 h post-infection. The gating of iNOS high , CD80 high , and CD86 high populations was determined against those of the uninfected control. n = 3 in each group ( g ). n = 6 in each group ( i ). h , j . PMs obtained from Sept2 fl/fl Lyz2 -Cre and Sept2 fl/fl mice were infected with VSV at MOI of 0.1, 1, or 5, respectively. The iNOS, CD80, and CD86 levels were detected by flow cytometry ( h ), and secretion of proinflammatory cytokines were detected by ELISA ( j ) at 12 hours post-infection. n = 3 in each group ( h ). n = 6 in each group ( j ). Data are shown as Kaplan-Meier curves ( a ) and the mean ± s.e.m. ( c , d , e , i , j ). Log-rank (Mantel–Cox) test ( a ) and one-way ANOVA followed by Bonferroni post hoc test ( c – e , i , j ) were used for data analysis.UI uninfected. Source data are provided as a Source Data file.

Article Snippet: To generate myeloid-cell-specific SEPT2-deficient ( Sept2 fl/fl Lyz2 -Cre) mice and tamoxifen-inducible SEPT2 conditional knockout ( Sept2 fl/fl Lyz2 -Cre-ERT2) mice, we inter-crossed mice that contained loxP sequence flanking the 4-5 exons of SEPT2 ( Sept2 fl/fl ) with Lyz2 -Cre mice or C57BL/6JSmoc- Lyz2 em1(2A-CreERT2-WPRE-pA)Smoc mice (Shanghai Model Organisms Center, Inc., Shanghai, China), respectively.

Techniques: Infection, Injection, Blocking Assay, Staining, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Immunohistochemistry, Control

Journal: Nature Communications

Article Title: SEPTIN2 suppresses an IFN-γ-independent, proinflammatory macrophage activation pathway

doi: 10.1038/s41467-023-43283-2

Figure Lengend Snippet: a Heatmap of upregulated (red) or downregulated (blue) ER stress-related genes in Sept2 fl/fl Lyz2 -Cre mice PMs after being infected with VSV (MOI = 1) or HSV-1 (MOI = 5) for 12 h. b , c Immunoblot analysis of UPR-related signaling pathways in Sept2 fl/fl Lyz2 -Cre and Sept2 fl/fl PMs after being infected with VSV (MOI = 1) for the indicated times ( b ). Quantitative data are graphed in ( c ). n = 3 in each group ( b , c ). d NTPAN-MI probe was used to determine the accumulation of unfolded proteins in Sept2 fl/fl Lyz2 -Cre and Sept2 fl/fl PMs after being infected with VSV (MOI = 1). e The mean fluorescence intensity (MFI) was quantitated and shown as IntDen/Area. Scale bar = 20 μm. n = 3 in each group ( d , e ). f , g iNOS activity ( f ) and proinflammatory cytokines ( g ) in Sept2 fl/fl and Sept2 fl/fl Lyz2 -Cre PMs after being infected with VSV (MOI = 1) in the absence or presence of 4-PBA (5 mM) for 12 h. n = 6 in each group ( f , g ). h , i . Sept2 fl/fl and Sept2 fl/fl Lyz2 -Cre PMs were transfected with siRNAs targeting PERK, IRE1 or ATF6 for 24 h, followed by infection of VSV (MOI = 1) for 12 h. The iNOS activity ( h ) and proinflammatory cytokines ( i ) were detected. n = 6 in each group ( h , i ). Data are shown as the mean ± s.e.m. ( c , e – i ). One-way ANOVA followed by Bonferroni post hoc test ( c , e – i ) was used for data analysis. UI uninfected. Source data are provided as a Source Data file.

Article Snippet: To generate myeloid-cell-specific SEPT2-deficient ( Sept2 fl/fl Lyz2 -Cre) mice and tamoxifen-inducible SEPT2 conditional knockout ( Sept2 fl/fl Lyz2 -Cre-ERT2) mice, we inter-crossed mice that contained loxP sequence flanking the 4-5 exons of SEPT2 ( Sept2 fl/fl ) with Lyz2 -Cre mice or C57BL/6JSmoc- Lyz2 em1(2A-CreERT2-WPRE-pA)Smoc mice (Shanghai Model Organisms Center, Inc., Shanghai, China), respectively.

Techniques: Infection, Western Blot, Protein-Protein interactions, Fluorescence, Activity Assay, Transfection

Journal: Nature Communications

Article Title: SEPTIN2 suppresses an IFN-γ-independent, proinflammatory macrophage activation pathway

doi: 10.1038/s41467-023-43283-2

Figure Lengend Snippet: a , b . Immunoblot analysis of UPR-related genes in PMs after being infected with VSV (MOI = 1) ( a ). Quantitative data are graphed in ( b ). n = 3 in each group ( a , b ). c , d Flow cytometry ( c ) and qRT-PCR ( d ) analysis of HSPA5 in iBMDMs after being infected with VSV (MOI = 1) and treated with DOX (1 µg/mL). The quantitative flow cytometry data are graphed in ( c ). n = 3 in each group ( c ). The dotted line in ( d ) indicated the uninfected control. n = 6 in each group ( d ). e Quantitative flow cytometry data of HSPA5 in iBMDMs after being infected with VSV (MOI = 1) and treated with CHX (50 µg/mL) and DOX (1 µg/mL). n = 3 in each group ( e ). f , g TUBE analysis of HSPA5 in PMs after being infected with VSV (MOI = 1) for 12 h ( f ). Quantitative data are graphed in ( g ). h Quantitative flow cytometry data of HSPA5 in iBMDMs after being infected with VSV (MOI = 1), treated with DOX (1 µg/mL) and transfected with SCNN1B siRNA. i The ubiquitination of HSPA5 in an in vitro ubiquitination system was analyzed by immunoblots. j , k PMs were transfected with SCNN1B siRNA, followed by VSV infection (MOI = 1) for 12 h. The ubiquitination of HSPA5 was detected by K48-TUBE analysis ( j ). Quantitative data are graphed in ( k ). n = 3 in each group ( f – k ). l HPLC-MS/MS analysis of HSPA5 ubiquitination sites in Sept2 fl/fl Lyz2 -Cre PMs after being infected with VSV (MOI = 1) for 12 h. m The ubiquitination of HSPA5 and the HSPA5-SCNN1B interaction in an in vitro ubiquitination system were analyzed by immunoblots. n = 3 in each group ( m ). MG132 (10 μg/mL) was used to inhibit the proteasomal degradation ( f , g , j – l ). Data are shown as the mean ± s.e.m. ( b – e , g , h , k ). One-way ANOVA followed by Bonferroni post hoc test ( b , c , e , g , h , k ) was used for data analysis. UI uninfected. NC negative control. Source data are provided as a Source Data file.

Article Snippet: To generate myeloid-cell-specific SEPT2-deficient ( Sept2 fl/fl Lyz2 -Cre) mice and tamoxifen-inducible SEPT2 conditional knockout ( Sept2 fl/fl Lyz2 -Cre-ERT2) mice, we inter-crossed mice that contained loxP sequence flanking the 4-5 exons of SEPT2 ( Sept2 fl/fl ) with Lyz2 -Cre mice or C57BL/6JSmoc- Lyz2 em1(2A-CreERT2-WPRE-pA)Smoc mice (Shanghai Model Organisms Center, Inc., Shanghai, China), respectively.

Techniques: Western Blot, Infection, Flow Cytometry, Quantitative RT-PCR, Control, Transfection, Ubiquitin Proteomics, In Vitro, Tandem Mass Spectroscopy, Negative Control

Journal: Nature Communications

Article Title: SEPTIN2 suppresses an IFN-γ-independent, proinflammatory macrophage activation pathway

doi: 10.1038/s41467-023-43283-2

Figure Lengend Snippet: a , b . Immunoblot analysis of acetylated HSPA5 in PMs after being infected with VSV (MOI = 1) for the indicated times ( a ). Quantitative data are graphed in ( b ). n = 3 in each group ( a , b ). c , d iBMDMs were transfected with SEPT2 siRNA, followed by treatment of DIC (1×) and infection of VSV (MOI = 1) for 12 h. Afterward, iNOS activities ( c ) and secretion of proinflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-12 p40) ( d ) were detected. n = 6 in each group ( c , d ). e Immunoprecipitation analysis of the interaction between ATAT1, SCNN1B, HSPA5, and SEPT2 in PMs after being infected with VSV (MOI = 1) for 12 h. f The acetylation of HSPA5 in an in vitro acetylation system was analyzed by immunoblots. g , h Atat1 +/+ and Atat1 −/− iBMDMs were infected with VSV (MOI = 1) for the indicated times, followed by immunoprecipitation analysis of acetylated HSPA5 using an Ac-Lysine antibody ( g ). Quantitative data are graphed in ( h ). n = 3 in each group ( e – h ). i , j iNOS activities ( i ) and secretion of proinflammatory cytokines ( j ) in iBMDMs after being infected with VSV (MOI = 1) for 12 h. n = 6 in each group ( i , j ). k – o Sept2 fl/fl Lyz2 -Cre PMs were transfected with V5-tagged ATAT1 or HA-tagged SEPT2, followed by VSV infection (MOI = 1) for 12 h. k The expression of V5-ATAT1 and HA-SEPT2 was detected by western blotting. l NTPAN-MI probe was used to determine the accumulation of unfolded proteins. Scale bar = 20 μm. m The MFI was quantitated and shown as IntDen/Area. n , o iNOS activities ( n ) and secretion of proinflammatory cytokines ( o ) were detected. n = 3 ( k – m ) or n = 6 ( n , o ) in each group. MG132 (10 μg/mL) was used to inhibit the proteasomal degradation of HSPA5 ( a , b , g , h ). Data are shown as the mean ± s.e.m. ( b – d , h – j , m – o ). One-way ANOVA followed by Bonferroni post hoc test ( b – d , h – j , m – o ) was used for data analysis. UI uninfected. EV empty vector. Source data are provided as a Source Data file.

Article Snippet: To generate myeloid-cell-specific SEPT2-deficient ( Sept2 fl/fl Lyz2 -Cre) mice and tamoxifen-inducible SEPT2 conditional knockout ( Sept2 fl/fl Lyz2 -Cre-ERT2) mice, we inter-crossed mice that contained loxP sequence flanking the 4-5 exons of SEPT2 ( Sept2 fl/fl ) with Lyz2 -Cre mice or C57BL/6JSmoc- Lyz2 em1(2A-CreERT2-WPRE-pA)Smoc mice (Shanghai Model Organisms Center, Inc., Shanghai, China), respectively.

Techniques: Western Blot, Infection, Transfection, Immunoprecipitation, In Vitro, Expressing, Plasmid Preparation

Journal: Nature Communications

Article Title: SEPTIN2 suppresses an IFN-γ-independent, proinflammatory macrophage activation pathway

doi: 10.1038/s41467-023-43283-2

Figure Lengend Snippet: a Schematic diagram of HSPA5 K327Q plasmid in vivo transfection. b The in vivo transfection efficiency of HSPA5 WT and HSPA5 K327Q was detected in lungs at day 7 without VSV infection. n = 3 in each group ( b ). c PMs were transfected with HSPA5 K327Q plasmids and infected with VSV (MOI = 1). CHX (50 µg/mL) was used to inhibit the protein synthesis. The expression of HSPA5 K327Q was detected. n = 3 in each group ( c ). d Survival of Sept2 fl/fl (HSPA5 WT, n = 15. HSPA5 K327Q, n = 13) and Sept2 fl/fl Lyz2 -Cre (HSPA5 WT, n = 12. HSPA5 K327Q, n = 14) mice intraperitoneally infected with 1 × 10 7 PFU VSV. e ELISA analysis of proinflammatory cytokines in BALF at 7 dpi. n = 6 in each group ( e ). f , g H&E staining ( f ) and the pathology scores ( g ) of lung lesions at 7 dpi. Scale bar = 400 μm. n = 6 in each group ( f , g ). h – n Sept2 fl/fl and Sept2 fl/fl Lyz2 -Cre PMs were transfected with HSPA5 WT or HSPA5 K327Q plasmids and then infected with VSV (MOI = 1) for 12 h. h – j The ubiquitination level of HSPA5 was detected by PLA ( h ) and TUBE analysis ( i ). Scale bar = 20 μm ( h ). Quantitative data of the TUBE analysis are graphed in ( j ). k , l NTPAN-MI probe was used to determine the accumulation of unfolded proteins ( k ). Scale bar = 20 μm. The MFI was quantitated and shown as IntDen/Area ( l ). m , n The expression of iNOS, CD80, and CD86 was detected by flow cytometry ( m ). The uninfected controls were shown as the blank peaks. Quantitative data are graphed in ( n ). n = 3 in each group ( h – n ). MG132 (10 μg/mL) was used to inhibit the proteasomal degradation of HSPA5 ( h-j ). Data are shown as Kaplan–Meier curves ( d ) and the mean ± s.e.m. ( c , e , g , j , l , n ). Log-rank (Mantel–Cox) test ( d ) and one-way ANOVA followed by Bonferroni post hoc test ( e , g , j , l , n ) was used for data analysis. UI uninfected. Source data are provided as a Source Data file.

Article Snippet: To generate myeloid-cell-specific SEPT2-deficient ( Sept2 fl/fl Lyz2 -Cre) mice and tamoxifen-inducible SEPT2 conditional knockout ( Sept2 fl/fl Lyz2 -Cre-ERT2) mice, we inter-crossed mice that contained loxP sequence flanking the 4-5 exons of SEPT2 ( Sept2 fl/fl ) with Lyz2 -Cre mice or C57BL/6JSmoc- Lyz2 em1(2A-CreERT2-WPRE-pA)Smoc mice (Shanghai Model Organisms Center, Inc., Shanghai, China), respectively.

Techniques: Plasmid Preparation, In Vivo, Transfection, Infection, Expressing, Enzyme-linked Immunosorbent Assay, Staining, Ubiquitin Proteomics, Flow Cytometry