Transcriptional Profiling of Polarized Macrophages using RNA-Sequencing

Adipose tissue macrophages (ATMs) are pivotal regulators for adipose tissue function, specifically contributing to the homeostasis of the adipose niche. Significantly increased ATMs and their altered activation patterns are causal factors to the pathogenesis of adipose tissue inflammation, and subsequently, obesity associated cardiovascular risks, type II diabetes and other metabolic syndromes. Macrophages primarily display an anti-inflammatory M2 status in lean adipose tissues whereas a pro-inflammatory M1 state in adipose tissues of obese individuals. Modulatory networks governing ATMs polarized activation have been investigated but the full picture remains vague. To understand the genome wide signaling networks in controlling ATM polarization, we generated transcriptome profiles from macrophages with various activation statuses- M0, M1 and M2. Among 23400 aligned unique loci from the RNA-sequencing results, around 3500 displayed differential expression pattern during macrophage polarization. The most enriched Gene Ontology terms in the category of KEGG pathways are allograft rejection and Type I diabetes mellitus pathways in M1 macrophages. IFNg was found to be one of the top upstream regulator in M1 playing pivotal role in different functional pathways. In addition, the anti-inflammatory regulator miR-223 was found to be one of top upstream regulator in M2 datasets and playing role in important functional pathways. Understanding of the complex network of interactions among different factors involved in state of polarization of macrophages would be of great advantage in finding solutions to major health issues

miR-150 regulates obesity-associated insulin resistance by controlling B cell functions

Adipose tissue resident B cells account for more than 20% of stromal cells within visceral adipose tissues; however, their functions in the adipose tissue niche are poorly elucidated. Here we report that miR-150 modulates adipose tissue function by controlling activation of B cells and their interactions with other immune cells. miR-150KO mice displayed exacerbated obesity-associated tissue inflammation and systemic insulin resistance, which is recapitulated by adoptive transfer of B cells, but not purified immunoglobulin, into obese B(null) mice. Using purified cell populations, we found that enhanced proinflammatory activation of adipose tissue T cells and macrophages was due to miR-150KO B cells action but not cell-autologous mechanisms. miR-150KO B cells displayed significantly enhanced antigen presentation upon stimulation, ultimately leading to elevated inflammation and insulin resistance, compared to wild type B cells. Knockdown of identified miR-150 target genes, Elk1, Etf1 or Myb attenuated B cell action by altering B cell receptor pathways and MHCII cell surface presentation. Our results demonstrate a critical role for miR-150 in regulating B cell functions in adipose tissue which ultimately regulate both metabolic and immunologic homeostasis in the adipose tissue niche

Interferon Tau Alleviates Obesity-Induced Adipose Tissue Inflammation and Insulin Resistance by Regulating Macrophage Polarization

Chronic adipose tissue inflammation is a hallmark of obesity-induced insulin resistance and anti-inflammatory agents can benefit patients with obesity-associated syndromes. Currently available type I interferons for therapeutic immunomodulation are accompanied by high cytotoxicity and therefore in this study we have examined anti-inflammatory effects of interferon tau (IFNT), a member of the type I interferon family with low cellular toxicity even at high doses. Using a diet-induced obesity mouse model, we observed enhanced insulin sensitivity in obese mice administered IFNT compared to control mice, which was accompanied by a significant decrease in secretion of proinflammatory cytokines and elevated anti-inflammatory macrophages (M2) in adipose tissue. Further investigations revealed that IFNT is a potent regulator of macrophage activation that favors anti-inflammatory responses as evidenced by activation of associated surface antigens, production of anti-inflammatory cytokines, and activation of selective cell signaling pathways. Thus, our study demonstrates, for the first time, that IFNT can significantly mitigate obesity-associated systemic insulin resistance and tissue inflammation by controlling macrophage polarization, and thus IFNT can be a novel bio-therapeutic agent for treating obesity-associated syndromes and type 2 diabetes

IFNT modulates macrophage polarization and cytokine profiles.

<p>The surface makers CD69, CD80, and CD86 of BMDMs were analyzed using flow cytometry after 48-hIL-4 (20 ng/mL; A) or lipopolysaccharide (LPS, 100 ng/mL; B) stimulation (n = 3). BMDMs were treated with IFNT at 5,000 antiviral units (AVU)/mL. The expression of cytokines IL-1β (C), TNF-α (D) and IL-10 (E) and peroxisome proliferator-activated receptor γ (PPARγ) (F) in BMDMs activated in the presence of IFNT (black bars) were analyzed by qRT-PCR (normalized to β-actin, n = 3) and compared to activated BMDMs with no IFNT treatment (white bars). Data are presented as mean ± SEM. *<i>P</i><0.05, **<i>P</i><0.001, ***<i>P</i><0.0001. MFI, medium fluorescence intensity.</p

IFNT induces STAT1 and STAT3 activation in BMDMs.

<p>(A) The phosphorylation of signal transducer and activator of transcription 3 (pSTAT3) and (B) pSTAT1 in BMDMs was measured by flow cytometry after 90-min of LPS (100 ng/mL) or IL-4 (20 ng/mL) stimulation (n = 3). (C) The expression of interferon regulatory factor 9 (IRF9) was analyzed by qRT-PCR after 48-h LPS (100 ng/mL) or IL4 stimulation (20 ng/mL); n = 3. Data are presented as mean ± SEM. *<i>P</i><0.05, **<i>P</i><0.001.</p

IFNT administration altered the cytokine profile and insulin signaling.

<p>(A) Gene expression of cytokines. Cytokines, chemokine (C-C motif) ligand 2 (CCL2) and adipokineadiponectin in VAT of HFD mice measured using quantitative reverse transcriptase-PCR (qRT-PCR; normalized to β-actin). IL, interleukin; TNF-α, tumor necrosis factor-α. (B) Concentration of IL-1β, IL-6, TNF-α, CCL2, and IL-10 in plasma of HFD mice were measured using the Bio-Plex™Cytokine Assay (Bio-Rad). (C) Adipose tissue insulin signaling. After 16-h fasting, mature adipocytes were collected from VAT and treated with insulin (100 nM) for 15 min. Total AKT (tAKT) and phosphorylated AKT (pAKT) protein in adipocytes were measured using Bio-Plex Cell Signaling Magnetic Assays (Bio-Rad). Data are presented as mean ± SEM. *<i>P</i><0.05.</p

Interferon tau (IFNT) alleviates high-fat diet (HFD)-induced insulin resistance.

<p>Body weight (A) and food intake (B) of mice were monitored during a 12-week feeding period (n = 9–10). (C) Concentrations of glucose and insulin in plasma of control or IFNT-treated mice fed a HFD or low-fat diet (LFD), or fasted for 16 h. (D) Glucose tolerance test (n = 6). (E) Insulin tolerance test (n = 6). Data are presented as mean ± SEM. *<i>P</i><0.05, **<i>P</i><0.001, ***<i>P</i><0.0001.</p

IFNT reduces obesity-associated adipose tissue inflammation.

<p>(A) Visceral adipose tissues (VAT) weight and adiposity of mice after 12-week HFD feeding. (B) Nuclear factor-κB (NFκB) activation in VAT of HFD mice. Western blotting was performed with antibodies against p65 and phosphorylated p65 (Pp65; n = 3). C, control; T, IFNT. (C) Activation of c-Jun N-terminal kinase (JNK) signaling pathway in VAT of HFD mice. Fluorescent-labeled beads conjugated with antibodies against total JNK (tJNK) and phosphorylated JNK (phospho JNK) in VAT were measured using the Bio-Plex® MAGPIX™ multiplex reader. MFI, medium fluorescence intensity. Data are presented as mean ± SEM. *<i>P</i><0.05.</p