Fig 2: Immunophenotyping of Tgm2 CRISPR-Silenced BMMs.Bone marrow was isolated from C57BL/6 J mice and cultured in the presence of M-CSF for BMMs differentiation for 4 days. BMMs were then transfected with CRISPR targeting Tgm2 and evaluated 72 h post-transfection. A Western blot analysis confirming Tgm2 silencing in sgControl (n = 9) or sgTGM2 (n = 9) transfected BMMs. B Evaluation of TGM2 secretion in CRISPR silenced BMMs (n = 8/group), C Transglutaminase enzymatic activity in Tgm2 CRISPR silenced BMMs (n = 7/group) and D Cytokine profile in harvested supernatant from CRISPR targeted BMMs treated with LPS for 24 h (n = 9–10/group). E Gating strategy for flow cytometric analysis of CRISPR silenced BMMs (n = 10/group). Flow cytometric data analysis from CRISPR silenced BMMs polarized into E M0, F M1 (50 ng/mL IFNy and 10 ng/mL LPS in DMEM) or G M2 (M2 = 20 ng/mL IL-4 in DMEM), polarizing stimuli, on day 7 of differentiation. Cells were assessed 24 h post-polarization. Data are shown as bar graphs with SEM of seven or ten mice per control or treated group and are representative of three independent experiments. For all graphs, data normality status was used to determine statistical analysis. Normal data statistical significance was determined by parametric student’s unpaired t-test, while not normally distributed data was analyzed using non-parametric Mann-Whitney test. ns = P > 0.05, **=P < 0.05, ** =P < 0.01, ***P < 0.001.

Fig 3: In vivo silencing of Tgm2 in myeloid cells: effects on male mouse metabolic health.Tgm2 male floxed homozygous mice injected with pLV.Control or pLV.CD11b-Cre lentiviruses at 5 weeks of age were placed on a 60%HFD at 6 weeks of age. A weekly weight monitoring (n = 5/group). B DEXA scanning evaluating changes in C lean and D fat mass (n = 5/group). E gross eWAT and F liver weights post-study termination (n = 5/group). G eWAT H&E histology and H histological analysis evaluating number of adipocytes and adipocyte area in pLV.Control vs. pLV.CD11b-cre treated mice (n = 5/group). Whole animal metabolic health assessment by I GTT and J ITT evaluations (n = 5/group). Data are shown as bar graphs with SEM of five mice per control or treated group and are representative of two independent experiments. For body weight, GTT (group: P = 0.4298, time: P = < 0.0001 and group × time interaction: P = < 0.0889) and ITT (group: P = 0.0004, time: P = < 0.0001 and group × time interaction: P = < 0.0534) statistical significance was determined by repeated measure Two-way ANOVA post-hoc. For bar graphs, data normality status was used to determine statistical analysis. Normal data statistical significance was determined by parametric student’s unpaired t test, while not normally distributed data were analyzed using non-parametric Mann-Whitney test. ns = P > 0.05, *=P < 0.05, ** =P < 0.01, ***P < 0.001.

Fig 4: TGM2 co-expression with adipose tissue macrophages (ATMs) in diet-induced obesity.C57BL/6 J mice were placed on chow diet (CD) or 60% high fat diet (HFD) at 6 weeks of age and remained on diet treatment until 16 weeks of age. A Total transglutaminase activity assay in eWAT from CD (n = 8) vs. HFD (n = 8) mice. B qPCR evaluation of Tgm2 mRNA expression in eWAT from CD (n = 10) or HFD (n = 10) mice. C Western blot for TGM2 protein expression in eWAT from CD control (n = 11) or HFD-treated mice (n = 16) normalized to B-ACTIN or VINCULIN expression. eWAT-derived single-nucleus RNA-seq from C57BL/6 J mice placed on a 60% HFD for 18 weeks (n = 3 pooled mice) showing Tgm2 expression in adipose tissue D global tissue populations or E immune cells in CD vs. HFD diet-treatment groups, where we observed a greater number of cells in the HFD sub-group as evidenced by the larger number of dots in the violin plot. Next, eWAT- derived from CD (n = 3) or HFD-treated mice (n = 3) was cryosectioned or paraffin sectioned and fluorescently stained for F Perilipin, F4/80 and intracellular TGM2 (or corresponding isotype control) for microscopy analysis. G CD (n = 12) vs. HFD (n = 12) eWAT-derived SVF was analyzed by flow cytometry for H number of macrophages and I Number of macrophages expressing TGM2 in CD vs. HFD diet treatment groups. I CD (n = 5) vs. HFD (n = 5) eWAT-derived SVF sorted preadipocytes, macrophages or monocytes evaluated for Tgm2 expression by qPCR assessment. J snRNAseq assessment of 60%HFD Tgm2- vs. Tgm2 + ATMs differential gene expression profile. Data are shown as bar graphs with SEM of five or twelve mice per control or treated group and are representative of two -four independent experiments. For all graphs, data normality status was used to determine statistical analysis. Normal data statistical significance was determined by parametric student’s unpaired t test, while not normally distributed data were analyzed using non-parametric Mann-Whitney test. ns = P > 0.05, *= P < 0.05, ** = P < 0.01, ***P < 0.001.

Fig 5: Macrophage-derived TGM2 effects on adipose tissue leukocyte inflammatory profile.CD C57BL/6 J mice’s long bones were used for preparation of bone marrow-derived macrophages (BMDM) ex vivo. Next, bone marrow-derived macrophages were transfected with CRISPR sgTGM2 or sgCtrl to silence Tgm2 expression on day 4 of the ex vivo culture timeline and co-cultured with eWAT-derived SVF in the presence of T cell activating anti-CD3/28 magnetic beads. A Study timeline. B Flow cytometric gating strategy and analysis for C–D myeloid cells (MHC Class II + F4/80+ gated) and E T cells (CD45 + TCRb + CD25 + CD4 + gated) in the co-cultured SVF cells (n = 7/group), and F ELISA for IL-10 in supernatant from co-cultured sgTGM2 or sgCtrl BMMs and SVF cells (n = 10/group). For all graphs, data normality status was used to determine statistical analysis. Normal data statistical significance was determined by parametric student’s unpaired t test, while not normally distributed data were analyzed using non-parametric Mann-Whitney test. ns = P > 0.05, *= P < 0.05, ** = P < 0.01, ***P < 0.001.