Lipocalin-2 promotes adipose-macrophage interactions to shape peripheral and central inflammatory responses in experimental autoimmune encephalomyelitis

Abstract

Objective: Accumulating evidence suggests that dysfunctional adipose tissue (AT) plays a major role in the risk of developing multiple sclerosis (MS), the most common immune-mediated and demyelinating disease of the central nervous system. However, the contribution of adipose tissue to the etiology and progression of MS is still obscure. This study aimed at deciphering the responses of AT in experimental autoimmune encephalomyelitis (EAE), the best characterized animal model of MS.

Results and methods: We observed a significant AT loss in EAE mice at the onset of disease, with a significant infiltration of M1-like macrophages and fibrosis in the AT, resembling a cachectic phenotype. Through an integrative and multilayered approach, we identified lipocalin2 (LCN2) as the key molecule released by dysfunctional adipocytes through redox-dependent mechanism. Adipose-derived LCN2 shapes the pro-inflammatory macrophage phenotype, and the genetic deficiency of LCN2 specifically in AT reduced weight loss as well as inflammatory macrophage infiltration in spinal cord in EAE mice. Mature adipocytes downregulating LCN2 reduced lipolytic response to inflammatory stimuli (e.g. TNFα) through an ATGL-mediated mechanism.

Conclusions: Overall data highlighted a role LCN2 in exacerbating inflammatory phenotype in EAE model, suggesting a pathogenic role of dysfunctional AT in MS.

Keywords: Adipocyte; Adipose tissue; Cachexia; Immune cells; Lipid metabolism; Macrophages; Mitochondria.

Figure 1

EAE immunization induces AT loss, inflammation and fibrosis. (A) Body weight was measured at 15 days post EAE induction (n = 9 Ctrl and n = 15 EAE mice). (B–D) Representative photograph (B), hematoxylin/eosin (C) and May Grunwald Giemsa (D) in sWAT of Ctrl and EAE mice 15 days post immunization (n = 5 Ctrl and n = 5 EAE mice). (E) Col1A1, Col3A1 and Pparγ mRNA expression in sWAT of Ctrl and EAE mice 15 days post immunization (n = 5 Ctrl and n = 5 EAE mice). (F) Representative immunoblots of α-SMA and PPARγ proteins in sWAT of Ctrl and EAE mice 15 days post immunization (n = 5 Ctrl and n = 5 EAE mice). HSL was used as loading control. (G) Tnfα and Il1β mRNA expression in sWAT of Ctrl and EAE mice 15 days post immunization (n = 6 Ctrl and n = 4 EAE mice). (H–J) Flow cytometry plots of total macrophages (CD45^highF4/80⁺CD11b⁺CD11c⁻) (H), M1-like macrophages (I) and M2-like macrophages (J) in sWAT of CTRL and EAE mice 15 days post immunization (n = 6 Ctrl and n = 6 EAE mice). Data was reported as mean percentages of positive cells or mean fluorescence intensities $\pm$ SD. Student's T test ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 EAE vs Ctrl.

Figure 2

LCN2 is induced in AT of EAE, cancer- and sepsis-inducing cachectic mice. (A) Venn diagram including the up-regulated genes in choroid plexus, dura mater and spinal cord of EAE mice. (B, C) Lcn2 mRNA (B) and protein (C) expression. HSL was used as loading control. HSL was the same as shown in Figure 1F because it is part of the same western blot. (D, E) Tumor mass progression (D), % body weight loss (E) in mice during LLC post inoculation (n = 17 PBS and n = 11 LLC mice). (F) sWAT weight 21 days post inoculation (n = 17 PBS and n = 11 LLC mice). Representative immunofluorescence of Phalloidin (green), LCN2 (red), Dapi (blue) in sWAT of PBS and LLC mice 21 days post inoculation (n = 5 PBS and n = 4 LLC mice). Representative immunoblots of LCN2 in in sWAT of PBS and LLC mice 21 days post inoculation (n = 5 PBS and n = 4 LLC mice). VINCULIN was used as loading control. (H) Volcano plot and functional enrichment analysis for biological processes of up-regulated genes (Log2FC > 1.5; pAdj<0.05) in sWAT of EAE and LCC mice (n = 5 mice/group). (H) Single cell RNA-sequencing (PRJNA626597) of visceral white adipose tissue isolated from mice 1 month after sepsis induction. (J) Fibroblast annotation for fibroblast adipocyte precursors (FAP: Podh, Col5a3) and adipose stem cells (ASC: Dpp4, Sema3c). (K) Lcn2 expression in fibroblast of control and sepsis mice. Data was reported as mean $\pm$ SD. Student's T test ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 treated vs Ctrl.

Figure 3

Inflammatory or hypoxic stimuli induce Lcn2 expression through redox-dependent mechanism. (A) Differentially expressed genes in mature 3T3-L1 adipocytes treated with TNFα (GSE62635). (B) Lcn2 mRNA and protein expression in TNFα- (10 ng/mL: 6 h in serum-free media), CoCl2-treated (0.5 mM: 6 h in serum-free media) or untreated mature adipocytes. Mature adipocytes were preconditioned with DMF (50 μM) for 24 h. Ponceau staining was used as loading control. (C) Mature 3T3-L1 adipocytes were labelled with MitoSox for 30 min and then stimulated with TNFα (10 ng/mL: 6 h in serum-free media). DMF (50 μM) was added 24 h prior TNFα stimulation. (D) Representative immunoblots of LCN2 in cell culture medium. Mature 3T3-L1 adipocytes treated with TNFα (10 ng/mL: 6 h in serum-free media) and N-acetyl cysteine (NAC: 2 mM) was added 1-hour prior TNFα stimulation. Ponceau staining was used as loading control. Data was reported as mean $\pm$ SD. ANOVA test ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

Figure 4

Adipose-derived LCN2 promote macrophage inflammatory activation. (A) Representative immunoblots of LCN2 in undifferentiated (day 0) and fully differentiated (day 8) cells and culture medium. VINCULIN was used as loading control. (B) TNFα, Nos2, Il1β and Arg1 mRNA expression in RAW264.7 co-cultured with adipocytes downregulating LCN2 (Lcn2 KD). TNFα (10 ng/mL: 6 h in serum-free media) was added to co-culture system. (C) Total body weigh 15-day post immunization in Flox and Lcn2 KO mice specifically in adipocytes (Adipo-Lcn2 KO) (n = 9/15 mice/group). (D) Gene expression markers of macrophages in the spinal cord of flox and Lcn2 KO mice specifically in adipocytes (Adipo-Lcn2 KO). (E, F) Flow cytometry plots of infiltrated leukocytes (CD45^highCD11b^+/−) and macrophages (CD45^highF4/80⁺CD11b⁺CD11c⁻) in the spinal cord of Flox and Lcn2 KO mice specifically in adipocytes (Adipo-Lcn2 KO) (n = 8/9 mice/group). Data was reported as mean percentages of positive cells $\pm$ SD. Student's T test or ANOVA test ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

Figure 5

LCN2 participates in the uncanonical lipolysis of adipocytes. (A) Representative immunoblots of ATGL, HSLpSer660 in adipocytes downregulating LCN2 (Lcn2 KD) treated with TNFα (10 ng/mL: 6 h in serum-free media) (left panel). HSL and VINCULIN were used as loading controls. Lcn2 mRNA expression in adipocytes downregulating LCN2 (Lcn2 KD) treated with TNFα (10 ng/mL: 6 h in serum-free media) (right panel). (B) Glycerol release from adipocytes downregulating LCN2 (Lcn2 KD) treated with TNFα (10 ng/mL: 6 h in serum-free media). (C) Representative immunoblots of LCN2 in cell culture medium of adipocytes treated with TNFα (10 ng/mL: 6 h in serum-free media). Atglistatin (200 μM) was added 1 h prior to TNFα treatment. Ponceau staining was used as loading control. (D) Representative immunoblots of LCN2 (left panel) and mRNA expression (right panel) in adipocytes treated with TNFα (10 ng/mL: 6 h in serum-free media). Atglistatin (200 μM) was added 1 h prior to TNFα treatment. TUBULIN was used as loading control. (E) Glycerol release from adipocytes treated with IBMX (500 μM) or TNFα (10 ng/mL) for 8 h. (F) Representative immunoblots of LCN2 in cell culture medium of 3T3-L1 adipocytes treated with IBMX (500 μM) or TNFα (10 ng/mL) for 8 h. Ponceau staining was used as loading control. (G) Nos2, Arg1 mRNA expression and Nos2/Arg1 mRNA ratio in RAW264.7 downregulating LCN2 receptor (LCN2r KD) co-cultured with TNFα-treated adipocytes. TNFα (10 ng/mL: 6 h in serum-free media) was added to co-culture system. Data was reported as mean percentages of positive cells $\pm$ SD. Student's T test or ANOVA test ∗p < 0.05; ∗∗p < 0.01.