Adipocyte-derived inflammatory molecules induce senescent B cells through metabolic pathways

Abstract

Objective.

To demonstrate that an adipocyte tissue-derived conditioned medium (ACM) contains inflammatory molecules that induce senescence in B cells.

Methods.

We incubated blood-derived B cells from lean donors with the ACM obtained from the adipose tissue (AT) of adult female donors with obesity undergoing weight reduction surgery, or with medium as control. After 24 hrs, cells were harvested and the expression of transcripts for pro-inflammatory cytokines (TNF/IL-6), chemokines (IL-8), and for markers of the senescence-associated secretory phenotype (SASP) was measured by qPCR. B cells were also stained with the marker of immunosenescence β-Galactosidase, and their metabolic status was evaluated in Seahorse using the mitostress test.

Results.

We show that the incubation of B cells from lean donors with the ACM induces the expression of transcripts for inflammatory and SASP transcripts, increases the amount of β-Galactosidase staining and induces a metabolic phenotype characterized by higher basal and maximal oxygen consumption, spare respiratory capacity (difference between maximal and basal respiration), non-mitochondrial oxygen consumption, ATP production and proton leak.

Conclusions.

These results demonstrate that B cells from lean individuals, after incubation with the ACM, become inflammatory and senescent, and this occurs through metabolic pathways needed to support their secretory phenotype.

Introduction

Obesity, indicated by body-mass index (BMI) ≥ 30 kg/m², is an inflammatory condition associated with persistent local and systemic low-grade inflammation (1), and dysfunctional humoral immunity, i.e. reduced antibody responses to infections and vaccines (2, 3) and increased autoimmunity (4, 5).

Adipocytes from the adipose tissue (AT) of individuals with obesity secrete multiple pro-inflammatory cytokines and chemokines (6, 7) involved in the recruitment of pro-inflammatory immune cells to the tissue (8, 9), which also secrete inflammatory mediators. Adipocytes and immune cells contribute together to local and systemic inflammation.

In this study, we wanted to evaluate if adipocytes from the human obese AT induce B cell immunosenescence. Results show that the incubation of blood-derived B cells from lean individuals with an AT-derived conditioned medium (ACM) obtained from the AT of adult female donors with obesity induces intrinsic B cell inflammation measured by the expression of multiple inflammatory markers and of markers associated with the senescence-associated secretory phenotype (SASP). This occurs through an ACM-induced metabolic shift that generates B cells characterized by high oxidative phosphorylation (OXPHOS), and high intrinsic B cell inflammation (5, 10). These findings provide additional mechanisms that can further elucidate the effects of obesity-induced inflammation on immune dysfunction.

Methods

Subjects

We obtained fresh discarded subcutaneous AT from adult female donors with obesity (BMI≥30, age 40±4 years) undergoing breast reduction surgery at the Division of Plastic and Reconstructive Surgery of the University of Miami Hospital. We obtained PBMC from adult female donors (n=15), all lean (BMI<25, age 39±4 years). Donors of AT were hispanic white, donors of PBMC were hispanic white (n=8) and black (n=7). PBMC were collected using Vacutainer CPT tubes (BD 362761) and cryopreserved.

Study participants provided written informed consent. The study was reviewed and approved by the Institutional Review Board (IRB, protocols #20070481 and #20160542), which reviews all human research conducted under the auspices of the University of Miami. All donors were screened for diseases known to alter the immune response or for consumption of medications that could alter the immune response. We excluded subjects with autoimmune, cardiovascular, renal or hepatic diseases, as well as with chronic infectious diseases, cancer, or under substance and/or alcohol abuse.

Preparation of adipocyte tissue-derived conditioned medium (ACM)

A piece of freshly harvested subcutaneous AT, obtained from adult female donors with obesity undergoing weight reduction surgery, was cultured in Dulbecco’s modified Eagle’s Medium (DMEM), supplemented with 15 mM HEPES, 1 mM Sodium Pyruvate, 100 U/mL Penicillin-Streptomycin, 1% BSA and 200 nM Adenosine, for 24 hrs at the concentration of 1 g/100 μL, as previously described (11). Three different ACM were used to stimulate total B cells isolated from the peripheral blood of lean female donors. Their cytokine content, evaluated by the Cytometric Bead Array (CBA) human TH1/TH2/TH17 kit (BD 560484), is in the legend of Fig. 1.

Fig. 1. ACM induces the expression of transcripts for inflammatory and SASP markers.

Top. Scheme of the experiment. Center and bottom. B cells (10⁶/ml), sorted from the blood of 15 lean individuals, were stimulated for 24 hrs in the presence of 3 different ACM (ACM#1, round symbols; ACM#2, square symbols; ACM#3, triangle symbols), or with DMEM as control, to detect RNA expression of TNF/IL6/IL8 (center) and p16/p21/p53 (bottom). ACM cytokine content was as follows. ACM#1: IL-6, 317±40 pg/mL; IL-10, 14±2 pg/mL; IL-17A, 72±8 pg/mL; TNF-α, 11±1 pg/mL; IFN-γ, 212±15 pg/mL. ACM#2: 351±27 pg/mL; IL-10, 16±3 pg/mL; IL-17A, 75±11 pg/mL; TNF-α, 13±3 pg/mL; IFN-γ, 224±21 pg/mL. ACM#3: 402±33 pg/mL; IL-10, 17±3 pg/mL; IL-17A, 84±7 pg/mL; TNF-α, 18±3 pg/mL; IFN-γ, 243±19 pg/mL. Results show qPCR values (2^−ΔCt) of RNA expression. Mean comparisons between groups were performed by Mann Whitney U test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

B cell isolation and stimulation

Total B cells were isolated from PBMC using magnetic CD19 Microbeads (Miltenyi 130-050-301), following manufacturer’s instructions. Cell preparations were typically >98% pure. B cells were stimulated with ACM at the concentration of 10⁶ cells/mL of ACM from age-matched donors for 24 hrs. Cell proliferation was measured by 3H-thymidine incorporation as previously described (5).

Flow cytometry

After culture with ACM or DMEM, B cells were stained for 20 minutes at room temperature with a Live/Dead detection kit (InVitrogen 1878898), anti-CD45 (Biolegend 368540), anti-CD19 (BD 348794) antibodies, and anti-HLA-DR (Biolegend 307617) to evaluate immune activation. After membrane staining, B cells were washed with FACS buffer. Then they were stained for an additional 20 minutes at room temperature with β-Galactosidase, using the Cellular Senescence detection kit SPiDER β-Gal (Dojindo Molecular Technologies SG04). Working concentrations were recommended by the manufacturer. In every experiment we acquired up to 10⁵ events in the B cell gate on a LSR-Fortessa (BD). Results were analyzed using FlowJo 10.5.3 software. Single color controls were included in every experiment for compensation. Isotype controls were also used in every experiment to set up the gates.

RNA extraction and quantitative (q)PCR

After culture with ACM or DMEM, B cells were resuspended in TRIzol (ThermoFischer Scientific 15596026) at the concentration of 10⁶ cells/500 μL. Then RNA was extracted for quantitative (q)PCR. Total RNA was isolated according to the manufacturer’s protocol, eluted into 10 μL distilled water and stored at −80°C.

Reverse Transcriptase (RT) reactions were performed in a Mastercycler Eppendorf Thermocycler to obtain cDNA, as previously described (4).

Reagents and primers for qPCR amplification, all from ThermoFisher Scientific, were the following: GAPDH, Hs99999905_m1; TNF, Hs01113624_g1; IL6, Hs00985639_m1; IL8, Hs00174103_m1; p16^INK4 (CDKN2A), Hs00923894_m1; p21^CIP1/WAF1, Hs00355782_m1; p53, Hs01034249_m1.

Mitostress test

We used a mitostress test (Agilent 103010) to evaluate oxygen consumption rates (OCR), a measure of oxidative phosphorylation, and mitochondrial fitness. The test was conducted in a Seahorse XFp extracellular flux analyzer (Agilent). Briefly, we seeded B cells incubated 24 hrs without or with ACM in a plate coated with CellTAK (Corning 354240). After culture with ACM or DMEM, cells were counted and plated at the concentration of 2x10⁵/well and ran in triplicates in XF DMEM medium (Agilent 1035750 supplemented with glutamine (Agilent 103579), glucose (Agilent 103577) and pyruvate (Agilent 103578), 200 μL of each reagent in 20 mL of medium. Maximal respiratory capacity was measured by the addition of the following compounds: Oligomycin (1 μM), inhibitor of mitochondrial complex V, to block ATP production, and then FCCP (fluoro-carbonyl cyanide phenylhydrazone, 5 μM), an uncoupling agent, to dissipate proton gradients and allow electron transport and oxygen consumption to operate at maximal rate. Maximal respiration was then suppressed by the addition of Rotenone/Antimycin (1 μM), inhibitors of mitochondrial complex I and III, respectively, showing that respiration is mitochondrial.

Statistical analyses

To examine differences between groups (ACM-treated versus DMEM-treated), Mann Whitney U test was employed. AUC, area under the curve, was employed to compare Seahorse mitostress profiles. GraphPad Prism version 10.1.0 software was used to construct all graphs.

Results

Results in Fig. 1 show that the incubation of blood-derived total B cells from 15 lean donors with 3 different ACM preparations induces B cells with a senescent-like phenotype as indicated by expression of transcripts for pro-inflammatory cytokines (TNF/IL6), chemokines (IL8) and SASP markers (p16/p21/p53). Other transcripts for cytokines (IL4, IL5, IL10) were not affected in ACM- versus DMEM-treated B cell cultures (data not shown).

In line with the expression of SASP transcripts, B cells from lean individuals stimulated with ACM are positively stained with the marker of immunosenescence β-Galactosidase (Fig. 2), showing for the first time the role of ACM-derived inflammatory products on the generation of B cells with a senescent-like phenotype. This pathway involves immune activation, evaluated by membrane expression of HLA-DR, also associated with intrinsic inflammation as we have previously demonstrated (4). Moreover, cell proliferation does not increase over time in cultures with ACM versus DMEM, as expected based on cell cycle arrest associated with the establishment of a senescent-like phenotype. We do not currently know which other inflammatory molecules in the ACM may be involved in this process.

Fig. 2. ACM induces B cells with a senescent-like phenotype.

B cells (10⁶/ml), sorted from the blood of lean individuals, were stimulated for 24 hrs in the presence of ACM or DMEM and then stained with with β-Galactosidase and with anti-HLA-DR, a marker of immune activation. B cells were also evaluated for cell proliferation measured by 3H-thymidine incorporation. Top (from left to right). Gating strategies to detect β-Galactosidase positive B cells after 24 hrs in DMEM or ACM; frequencies of β-Galactosidase positive B cells; MFI from one representative experiment in which one sample of B cells in DMEM (grey) and one sample of B cells in ACM (black) are shown; MFI results from all individuals. Negative controls (cells in DMEM or cells in ACM stained with Bafilomycin A only) are indicated by a grey or black dotted line, respectively. Center. MFI of HLA-DR expression from one representative experiment (left) in which one sample of B cells in DMEM (grey) and one sample of B cells in ACM (black), or from all the experimental samples (right), are shown. Negative controls (cells in DMEM or cells in ACM left unstained) are indicated by a grey or black dotted line, respectively. Bottom. Cell proliferation was measured after 24-72 hrs in DMEM or ACM. Results show means±SE of 3H-thymidine incorporation (cpm). Mean comparisons between groups were performed by Mann Whitney U test. *p<0.05, ****p<0.0001.

Previously published observations have clearly shown that metabolic reprogramming supports specific cell functions, including secretion of SASP products, by providing energy for optimal responses. Therefore, we evaluated the metabolic status of B cells from lean individuals incubated with the ACM as compared to those incubated with DMEM. Results in Fig. 3 show that ACM induces higher OXPHOS in peripheral blood-derived B cells from lean donors, with higher OCR measures, such as basal and maximal respiration, spare respiratory capacity (difference between maximal and basal respiration), non-mitochondrial oxygen consumption, ATP production and proton leak. These results demonstrate that B cells from lean individuals, after incubation with the ACM, show higher OXPHOS because they need more energy to support their secretory phenotype.

Fig. 3. ACM induces B cells with high OXPHOS.

B cells (10⁶/ml), sorted from the blood of lean individuals, were stimulated for 24 hrs in the presence of ACM or DMEM (control). B cells were seeded into the wells of an extracellular flux analyzer at the concentration of 2x10⁵/well in triplicate and run in a mitostress test. Top, left. Scheme of the mitostress test. Top, right OCR results from a representative experiment in which one sample of B cells in DMEM (white symbols) and one sample of B cells in ACM (grey symbols) are shown. Calculations of area under the curve showed significant differences between mitostress performed on B cells cultured with DMEM versus B cells cultured with ACM (2078±26 versus 2917±59, respectively, p<0.05). Center and bottom. Mitochondrial measures in 7 independent experiments. Each point, which is the mean of triplicate measures in the mitostress test, represents one sample of B cells in DMEM or B cells in ACM. For each measure, results are mean±SE. Mean comparisons between groups were performed by Mann Whitney U test. *p<0.05, **p<0.01.

Discussion and conclusions

Results herein clearly indicate for the first time that ACM-derived inflammatory cytokines induce B cells with a senescent-like phenotype, characterized by higher OXPHOS. ACM contains all factors secreted by non immune (adipocytes, endothelial cells, adipose-derived stem cells) and immune cells in the AT, and therefore represents a mixture of secretomes of different cell types. ACM-derived factors include adipokines (12, 13), cytokines (6, 7), growth factors (14) and exosomes (15).

Many studies have shown beneficial effect of ACM obtained from the AT of lean individuals in processes like angiogenesis, wound healing and tissue regeneration, as reviewed in (16), suggesting the potential use of ACM as a commercial off-the-shelf product that can benefit various patients. However, ACM obtained from the AT of individuals with obesity is primarily enriched in pro-inflammatory mediators that can activate pathogenic processes such as the generation of antibodies with autoimmune specificity, as we have previously shown (8), impair myogenesis through secretion of the metabolic stressor resistin (17), reduce the response of cancer cells to anti-cancer therapeutic drugs (18). Therefore, more comprehensive analyses of inflammatory signaling pathways are needed in order to develop innovative and safe therapeutics to prevent immunosenescence in the obese AT.

Study importance.

What is already known?

• Adipocytes from the adipose tissue (AT) of individuals with obesity are highly inflammatory, and secrete several pro-inflammatory cytokines and chemokines responsible for the recruitment of immune cells to the tissue.

• The immune cells recruited to the AT also contribute to local inflammation. The inflammatory mediators that they secrete, once released in the circulation, support systemic low-grade chronic inflammation.

What does this study add?

• This study provides the first evidence that an adipocyte-conditioned medium (ACM) obtained from the AT of adult female donors with obesity induces the expression of transcripts for senescence-associated markers in B cells from adult lean individuals.

• This study demonstrates that B cells from lean individuals that acquire a senescent-like phenotype are supported by a metabolic reprogramming, needed to support their inflammatory phenotype and secretory function.

How might these results change the direction of research or the focus of clinical practice?

• Our findings highlight the importance of better understanding the contribution of the obese AT to the generation of dysfunctional B cells.

• Although we have previously shown that the increased inflammatory phenotype of B cells is associated with increased secretion of pathogenic antibodies, there is still so much to learn. This area of research could lead to the identification of senescent and metabolic pathways to target to improve humoral immunity in individuals with obesity.

Data sharing statement

The data and materials that support the findings of this study are available upon request to the corresponding author.