Journal: Molecular cell

Article Title: Deciphering functional tumor-immune crosstalk through highly multiplexed imaging and deep visual proteomics.

doi: 10.1016/j.molcel.2024.12.023

Figure Lengend Snippet: Figure 3. T cell subsets have distinctive spatial proteome signatures in colorectal tumor microenvironment (A and B) Ranking of protein quantified in cytotoxic T cells (CTLs) (A) and helper T cells (TH) (B), ranked by median transformed intensity values detected by mass spectrometry, with cell-type-specific markers highlighted. (C and D) Volcano plots illustrating proteomic comparisons of CTLs (C) and TH (D) between the lamina propria and tumor epithelial regions, with T cell-activity- related markers highlighted. Significantly enriched proteins are denoted as yellow or green dots (two-sided t test, false discovery rate [FDR] < 0.05, S0 = 0.1, n = 3). (E and F) Gene Ontology (GO) term analysis of significantly altered proteins in CTLs (E) and TH (F) residing in the lamina propria and tumor epithelial regions.

Article Snippet: For CD4 + ARG1 staining, the sections were incubated overnight at 4 C with primary antibodies (anti-CD4-FITC, 1:100, R&D FAB8165G; anti-ARG1-PE, 1:200,Miltenyi Biotec 130-127-839) in a humidified chamber.

Techniques: Transformation Assay, Mass Spectrometry, Activity Assay

Journal: Molecular cell

Article Title: Deciphering functional tumor-immune crosstalk through highly multiplexed imaging and deep visual proteomics.

doi: 10.1016/j.molcel.2024.12.023

Figure Lengend Snippet: Figure 5. Cytotoxic T cells (CTLs) exhibit close proximity to tumors with a suppressive impact on tumor proliferation (A) Density gradient in the background indicates distance to tumor islands, with brighter yellow representing greater distance from tumor cells. Left panel only has the gradient whereas the middle, zoomed panel additionally depicts yellow masks of the CTLs and the right panel red dots of helper T cells (TH), respectively. (B) Representative images showcasing markers for T cell phenotyping. (C) T cell gating based on the expression of CD3, CD8, CD45RA, CD45RO, and PD1 to analyze the corresponding T cell subpopulations. (D) Representative images showing CD3- and CD8-positive CTLs in tumor parenchyma (TIL-CTLs) with absent CD45RA and CD45RO expression. (E) Distance heatmap depicting proximity and interactions between tumor cells and T and B lymphocyte subtypes, with lower values indicating higher proximity as in Figure 4E. (F) Histogram of Ki67/Ki67+ tumor cell number as a function of distance from RARO CTLs. Data represent three technical replicates. Statistical significance was determined using one-way ANOVA. ***p < 0.001, **p < 0.01. (G) Representative image displaying masks of tumor cells at varying proximities to RARO TIL-CTLs.

Article Snippet: For CD4 + ARG1 staining, the sections were incubated overnight at 4 C with primary antibodies (anti-CD4-FITC, 1:100, R&D FAB8165G; anti-ARG1-PE, 1:200,Miltenyi Biotec 130-127-839) in a humidified chamber.

Techniques: Expressing

Journal: Clinical and Experimental Pharmacology & Physiology

Article Title: Mechanisms of IL‐17A Neutralisation in Alleviating Renal Fibrosis and Inflammation in Spontaneously Hypertensive Rats

doi: 10.1111/1440-1681.70116

Figure Lengend Snippet: Effects of IL‐17A neutralisation on macrophage polarisation in SHR renal tissues. (A) Representative IHC staining of M1 macrophage markers (iNOS and CD86) with quantitative analysis of their positive areas. (B) Representative IHC staining of M2 macrophage markers (Arg‐1 and CD163) with quantitative analysis of their positive areas. (C) Representative immunoblots and relative expression levels of iNOS, CD86, Arg‐1, and CD163 proteins. (D) Proportion of CD86 + and CD163 + cells among CD68 + macrophages. (E) mRNA expression levels of iNOS, CD86, Arg‐1, and CD163. Data are presented as mean ± SD ( n = 6).

Article Snippet: Sections were then incubated overnight at 4°C with primary antibodies against: E‐cadherin (Boster, China), Collagen III (Boster, China), inducible nitric oxide synthase (iNOS) (Boster, China), CD86 (Boster, China), arginase‐1 (Arg‐1) (Boster, China), CD163 (Boster, China), α‐smooth muscle actin (α‐SMA) (Cell Signalling Technology, USA).

Techniques: Immunohistochemistry, Western Blot, Expressing

Journal: bioRxiv

Article Title: Alternative Macrophage Activation Requires Granulocyte-Derived Lipids

doi: 10.1101/2024.02.17.580795

Figure Lengend Snippet: ( A ) Species-specific synergistic effect of fetal bovine serum vs. adult bovine, adult human, and adult mouse serums on the expression of mRNA for Arginase 1 (Arg1) in interleukin - 4 (IL-4) - treated mouse bone marrow-derived macrophages (BMDM). ( B ) Comparison of effects of fetal bovine serum (FBS) and adult bovine serum (ABS) on the kinetics (day 1, 4, 8, and 10) of expression of Arginase 1 on mRNA ( Arg1 , left panel, real-time RT-PCR) and protein (ARG1, middle panel; quantitative western blotting) levels, and arginase enzymatic activity (right panel) in IL-4 treated or untreated murine BMDM (see Methods and Figure S1A , Supplemental Material ). ( C ) Identification of more than 100 kDa (>100 kDa) high molecular weight fractions of FBS and ABS (FBS-hi and ABS-hi, respectively) as an active component for cofactor for IL-4 to induce Arg1 in BMDM. ( D-G ) Comparisons of effects ABS, ABS-lo (<100 kDa), and ABS-hi (>100 kDa) serum fractions on the expression of M2 markers Arg1 and Chitinase-like protein 3 (Chil3) on mRNA ( D ) and protein ( E, F ) levels, as well as arginase enzymatic activity ( G ) in IL-4 - treated or untreated BMDM. For ( A-D ), ( F ), ( G ): *p<0.05, **, p<0.01, ****, p<0.0001, data represented as mean ± s.e.m.; see Table S1 for detailed statistics. Abbreviations : ABS, adult bovine serum; AHS, adult human serum; AMS, adult mouse serum; FBS, fetal bovine serum; FBS-lo, low molecular weight (>100 kDa) fraction of FBS; Untr., untreated. See also Table S1 and Figure S1, Supplemental Material .

Article Snippet: The antibodies to the following antigens were used for western blot analysis: β-Actin (Cell Signaling 4967), ARG1 (Cell Signaling 9819), CHIL3 (Biolegend 853803), NOS2 (Cell Signaling 2982), and APOA1 (Biomatik CAC08238).

Techniques: Expressing, Derivative Assay, Comparison, Quantitative RT-PCR, Western Blot, Activity Assay, High Molecular Weight, Molecular Weight

Journal: bioRxiv

Article Title: Alternative Macrophage Activation Requires Granulocyte-Derived Lipids

doi: 10.1101/2024.02.17.580795

Figure Lengend Snippet: ( A ) Schematic diagram for ex-vivo preparations of adult mouse serum (AMS), citrate mouse plasma (CMP), and AMS with subsequently added sodium citrate (AMS + SC) substances (see Methods). ( B-E ) Comparisons of effects of AMS, CMP, and AMS+SC substances on the expressions of M2 markers Arg1 and Chil3 on mRNA ( B ) and protein ( C, D ) levels and arginase enzymatic activity (E) in IL-4-treated or untreated murine BMDM (see Methods). ( F-I ) Comparison of effects of AMS and CMP on oxidative respiration ( F, H ) and glycolysis ( G, I ) rates in IL-4-treated or untreated murine BMDM (see Methods). Representative real-time kinetics of oxygen consumption ( F ) and extracellular acidification ( G ) rates (OCR and ECAR, respectively) are shown in ( F, G ). Quantifications of basal (left panel) and maximal (right panel) levels of OCR ( H ) and ECAR ( I ) are shown in ( H, I ). For ( B ), ( D ), ( E ), ( H ), ( I ): **, p<0.01; data represented as mean ± s.e.m.; see Table S1 for detailed statistics. Abbreviations : AMS, adult mouse serum; AA, antimycin A, CMP, citrate mouse plasma; ECAR, extracellular acidification rate; FBS-lo, low molecular weight (>100 kDa) fraction of FBS; FCCP, Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone; Gluc, glucose; OCR, oxygen consumption rate; R, rotenone; SC, sodium citrate; OM, oligomycin; Untr., untreated. See also Table S1 and Figure S2, Supplemental Material .

Article Snippet: The antibodies to the following antigens were used for western blot analysis: β-Actin (Cell Signaling 4967), ARG1 (Cell Signaling 9819), CHIL3 (Biolegend 853803), NOS2 (Cell Signaling 2982), and APOA1 (Biomatik CAC08238).

Techniques: Ex Vivo, Clinical Proteomics, Activity Assay, Comparison, Molecular Weight

Journal: bioRxiv

Article Title: Alternative Macrophage Activation Requires Granulocyte-Derived Lipids

doi: 10.1101/2024.02.17.580795

Figure Lengend Snippet: ( A ) Schematic diagram of isolation LDL, HDL, and non-lipoprotein ‘protein’ fractions from adult murine or bovine serums. ( B ) Comparisons of effects of unfractionated ABS or AMS vs. LDLs, HDLs, and non-lipoprotein ‘protein’ fractions on Arg1 expression in IL-4-treated macrophage cell line (see Methods). ( C ) Schematic images of structures of HDLs and artificial liposomes, and the experimental design for enzymatic treatment of murine HDLs from AMS with proteases and glycosidases, as well as plan for isolation of mouse serum lipids and the creation of artificial liposomes (see Methods). ( D ) Effects of enzymatic treatments of mouse HDLs with proteinases (proteinase K and trypsin), glycosidases (galactosidase and neuraminidase), addition of artificial liposomes containing AMS-derived lipids on Arg1 expression in IL-4-treated macrophages. ( E ) Experimental design of isolation of platelets, mononuclear leukocytes (‘lymphocytes’), granulocytes, and erythrocytes (red blood cells, RBCs) by separation of platelet-rich plasma by low-speed centrifugation followed by two-step Percoll density gradient centrifugation. ( F ) Blood-derived cell populations were activated with thrombin (TB) or lysed with water to make water lysate (WL), their supernatants were mixed with CMP (as a source of lipoproteins) and then added to macrophages for analysis of Arg1 expression (refer to Figure S5A-C and Methods). Supernatants that contained granulocyte-derived microparticles (GMPs) or WL, which were mixed with CMP (but not without CMP), exhibited significant upregulation of Arg1 . For ( B ), ( D ), ( F ): **, p<0.01; ns, not significant; data represented as mean ± s.e.m.; see Table S1 for statistics). Abbreviations : β-Gal., β-galactosidase; Gr., granulocytes; GMPs, granulocyte-derived microparticles; NA, neuraminidase; Plt., platelets; Prot.K, proteinase K; Tryp., trypsin; TB, thrombin; WL, water lysate; Untr., untreated. See also Table S1 and Figure S5, Supplemental Material.

Article Snippet: The antibodies to the following antigens were used for western blot analysis: β-Actin (Cell Signaling 4967), ARG1 (Cell Signaling 9819), CHIL3 (Biolegend 853803), NOS2 (Cell Signaling 2982), and APOA1 (Biomatik CAC08238).

Techniques: Isolation, Expressing, Liposomes, Derivative Assay, Clinical Proteomics, Centrifugation, Gradient Centrifugation

Journal: bioRxiv

Article Title: Alternative Macrophage Activation Requires Granulocyte-Derived Lipids

doi: 10.1101/2024.02.17.580795

Figure Lengend Snippet: ( A ) Schematic diagram of inhibition of secretion of granulocyte-derived microparticles (GMPs) by granulocytes activated with thrombin (TB) using two inhibitors (‘Inhibitor 1’ and ‘Inhibitor 2’) of neutral sphingomyelinase (nSMase, see Methods). ( B ) Flow cytometry analysis of percentages of GMPs in untreated granulocytes or the cells pre-treated with two nSMase inhibitors to prevent the formation of GMPs (Inh1 and Inh2, see also Figure S5D-G, Supplemental Material and Methods). ( C ) Schematic image of secretion of GMPs by activated granulocytes, which contain various amounts of lipid droplets with triglycerides (TG). ( D, E ) Quantitative analysis of GMP ( D ) and triglyceride ( E ) releases in untreated granulocytes or the cells pre-treated with two nSMase inhibitors (Inh1 and Inh2) to prevent the formation of TG-containing GMPs. ( F, G ) In vitro ( F ) and in vivo ( G ) effects of nSMase inhibitors ( F, G : Inh1 or Inh 2), or platelet ( G : ‘-Plt.’), or platelet and granulocyte ( G : ‘-Plt./Gr.’) depletions on the ability of GMP-containing supernatant ( F ) or AMS ( G ) to cause upregulation of Arg1 in macrophage cell line. For ( D-G ): **, p<0.01; data represented as mean ± s.e.m.; see Table S1 for statistics. Abbreviations : Gr., granulocytes; GMPs, granulocyte-derived microparticles; nSMase, neutral sphingomyelinase; Plt., platelets; TB, thrombin; TG, triglycerides; Untr., untreated. See also Table S1 and Figure S5, Supplemental Material .

Article Snippet: The antibodies to the following antigens were used for western blot analysis: β-Actin (Cell Signaling 4967), ARG1 (Cell Signaling 9819), CHIL3 (Biolegend 853803), NOS2 (Cell Signaling 2982), and APOA1 (Biomatik CAC08238).

Techniques: Inhibition, Derivative Assay, Flow Cytometry, In Vitro, In Vivo

Journal: bioRxiv

Article Title: Alternative Macrophage Activation Requires Granulocyte-Derived Lipids

doi: 10.1101/2024.02.17.580795

Figure Lengend Snippet: ( A ) Global untargeted lipidomics analysis of lipids in granulocyte-derived microparticles compared to citrate mouse plasma (GMPs vs. CMP). The volcano plot is shown on the left panel (x-axis: -log(P-value); y-axis: log(fold change)), while the distribution of fatty acids in GMP-specific triglycerides (marked in red in the upper right quadrant of the volcano plot) is shown on the right panel. ( B ) Global untargeted lipidomics analysis of lipids in adult mouse serum compared to citrate mouse plasma (AMP vs. CMP). The volcano plot is shown in the left panel (x-axis: -log(P-value); y-axis: log(fold change)), while the distribution (left panel) and composition (right panel) of lipids enriched in AMS vs. CMP (FAs, marked in orange color; lysolipids, marked in green color) are shown in the upper right quadrant of volcano plot, and the pie chart, respectively. ( C-E ) Targeted metabolomics analysis of free FAs in GMPs ( C ), and saturated ( D ) vs. unsaturated ( E ) FAs in AMS (blue color) vs. CMP (grey color) indicates a 1.3-2-fold increase in free FA concentrations and substantial decrease in TG concentration in AMS compared to CMP. ( F ) ‘ Gain-of-function’ analysis of AMS- and GMP-derived lipids and various free FAs for their ability to induce Arg1 in the macrophages. ( G, H ) The role of external lipases on functional activity of HDLs to induce Arg1 expression in macrophages. ( G ) Scheme of the proposed model of external lipolysis of GMP-derived triglycerides and phospholipids with triglyceride- and phospholipases that are present in granulocyte lipid droplets (e.g. ATGL), serum (e.g. LPL), or associated with lipoproteins such as HDLs (e.g. Lp-PLA 2 ). ( H ) Effects of external lysolipids with PLA 2 or the addition of external fatty acids to HDLs on the ability of these HDLs to induce Arg1 expression in the macrophage cell line. For ( C-F ), ( H ): *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; data represented as mean ± s.e.m.; see Table S1 for detailed statistics. Abbreviations : AA, arachidonic acid; ATGL, adipose triglyceride lipase; FA, fatty acids; Gran., granulocytes; GMPs, granulocyte-derived microparticles; LA, linoleic acid; αLA, α-linolenic acid (ω3); γLA, γ-linolenic acid (ω6); DγLA, Dihomo-γ-linolenic acid; LPL, lipoprotein lipase; Lp-PLA2, lipoprotein-associated phospholipase A2; Lyso-PL, lysophospholipids; MA, myristic acid; OA, oleic acid; PA, palmitic acid; PenA, pentadecanoic acid; SA, stearic acid. See also Table S1 and Figure S6, Supplemental Material .

Article Snippet: The antibodies to the following antigens were used for western blot analysis: β-Actin (Cell Signaling 4967), ARG1 (Cell Signaling 9819), CHIL3 (Biolegend 853803), NOS2 (Cell Signaling 2982), and APOA1 (Biomatik CAC08238).

Techniques: Derivative Assay, Clinical Proteomics, Concentration Assay, Functional Assay, Activity Assay, Expressing

Journal: bioRxiv

Article Title: Alternative Macrophage Activation Requires Granulocyte-Derived Lipids

doi: 10.1101/2024.02.17.580795

Figure Lengend Snippet: ( A ) Analysis of Arg1 expression in macrophages with inactivated SR-B1 and/or SR-B3 receptors using blocking antibodies (left panel) and siRNA cocktails for receptor knockdown (right panel). ( B ) Experimental design (left panel) and experimental results (right panel) of induction of IL-4/IL-13-mediated type 2 inflammation in peritoneally cavity using i.p chitin administration with or without inhibition of blood coagulation with heparin (H, 30 and 50 U per mouse), or platelet (-Plt.), or platelet and granulocyte (-Plt./Gr.) depletions, and subsequent isolation of peritoneal macrophages for gene expression analysis of Arg1 and Chil3 . ( C ) Experimental design (left panel) and observed results (right panel) of induction type 2 inflammation in the peritoneal cavity using chitin with or without the addition of granulocyte-derived microparticles (GMPs), and gene expression analysis of Arg1 and Chil3 expression in peritoneal macrophages 48h after co-administration of chitin and GMPs ( D ) Intraperitoneal injection of AMS results in 5-8-fold upregulation of Arg1 and Chil3 in peritoneal macrophages. The experimental design is shown in the upper panel, and the observed results are shown on the bottom panel. ( E ) Proposed model of regulation of type 2 inflammation by platelet- and granulocyte-derived FAs associated with microparticles that become loaded on HDLs during blood coagulation when platelets and granulocytes become activated to work synergistically with IL-4 or IL-13 produced by Th2, ILC2, eosinophils, or tissue-resident mast cells. For ( A )-( D ): *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; data represented as mean ± s.e.m.; see Table S1 for detailed statistics. Abbreviations : AMS, adult mouse serum; Cont., control; FAs, fatty acids; GMPs, granulocyte-derived microparticles; Gr., granulocytes; H, heparin; PBS, phosphate buffered saline; Plt., platelets; PMPs, platelet-derived microparticles; TG, triglycerides; Unmanop., unmanipulated. See also Table S1 and Figure S7, Supplemental Material .

Article Snippet: The antibodies to the following antigens were used for western blot analysis: β-Actin (Cell Signaling 4967), ARG1 (Cell Signaling 9819), CHIL3 (Biolegend 853803), NOS2 (Cell Signaling 2982), and APOA1 (Biomatik CAC08238).

Techniques: Expressing, Blocking Assay, Knockdown, Inhibition, Coagulation, Isolation, Gene Expression, Derivative Assay, Injection, Produced, Control, Saline

Journal: Scientific Reports

Article Title: Abnormal M1/M2 macrophage phenotype profiles in the small airway wall and lumen in smokers and chronic obstructive pulmonary disease (COPD)

doi: 10.1038/s41598-017-13888-x

Figure Lengend Snippet: Arginase-1 expression in the small airway wall of ( a ) NC compared to ( b ) COPD-CS. A significant increase in Arginase-1 expression was observed in both ( c ) epithelium and ( d ) sub-epithelium. Data are presented as median and range; group comparisons with Mann-Whitney two-tailed t-test; p < 0.05 was considered significant.

Article Snippet: To characterize M2 macrophages, CD163s and Arginase-1 staining were performed using mouse anti-CD163 (EDHu-1, AbD Serotec, MCA1853, 1/100 dilution) anti-Arg1 (BD Biosciences, 610708, 1/100 dilution) antibodies, for 90 minutes.

Techniques: Expressing, MANN-WHITNEY, Two Tailed Test

Journal: Scientific Reports

Article Title: Abnormal M1/M2 macrophage phenotype profiles in the small airway wall and lumen in smokers and chronic obstructive pulmonary disease (COPD)

doi: 10.1038/s41598-017-13888-x

Figure Lengend Snippet: Expression patterns of alveolar macrophages (AMs) in the alveolar spaces in resected tissue of COPD patients (200x), ( a ) M1 AMs dual stained for iNOS (brown) and CD68 (blue), counterstained with nuclear fast red (pink), M2 phenotype macrophages stained brown with ( b ) CD163 and ( c ) arginase-1(ARG-1), counterstained with nuclear-stained hematoxylin (blue).

Article Snippet: To characterize M2 macrophages, CD163s and Arginase-1 staining were performed using mouse anti-CD163 (EDHu-1, AbD Serotec, MCA1853, 1/100 dilution) anti-Arg1 (BD Biosciences, 610708, 1/100 dilution) antibodies, for 90 minutes.

Techniques: Expressing, Staining

Journal: Scientific Reports

Article Title: Abnormal M1/M2 macrophage phenotype profiles in the small airway wall and lumen in smokers and chronic obstructive pulmonary disease (COPD)

doi: 10.1038/s41598-017-13888-x

Figure Lengend Snippet: Representative pictures (400x) of M1 AMs dual stained for iNOS (brown) and CD68 (blue); ( a ) Normal control, and ( b ) COPD-CS, counterstained with nuclear fast red (pink). M2 phenotype macrophages stained brown with CD163 and arginase-1(ARG-1), ( c , e ) Normal controls and ( d , f ) COPD-CS respectively, with nuclear-stained hematoxylin (blue).

Article Snippet: To characterize M2 macrophages, CD163s and Arginase-1 staining were performed using mouse anti-CD163 (EDHu-1, AbD Serotec, MCA1853, 1/100 dilution) anti-Arg1 (BD Biosciences, 610708, 1/100 dilution) antibodies, for 90 minutes.

Techniques: Staining