Abstract

Background

   Mycobacterium tuberculosis has co-evolved with the human host, adapting
   to exploit the immune system for persistence and transmission. While
   immunity to tuberculosis (TB) has been intensively studied in the lung
   and lymphoid system, little is known about the participation of adipose
   tissues and non-immune cells in the host-pathogen interaction during
   this systemic disease.

Methods

   C57BL/6J mice were aerosol infected with M. tuberculosis Erdman and
   presence of the bacteria and the fitness of the white and brown adipose
   tissues, liver and skeletal muscle were studied compared to uninfected
   mice.

Findings

   M. tuberculosis infection in mice stimulated immune cell infiltration
   in visceral, and brown adipose tissue. Despite the absence of
   detectable bacterial dissemination to fat tissues, adipocytes produced
   localized pro-inflammatory signals that disrupted adipocyte lipid
   metabolism, resulting in adipocyte hypertrophy. Paradoxically, this
   resulted in increased insulin sensitivity and systemic glucose
   tolerance. Adipose tissue inflammation and enhanced glucose tolerance
   also developed in obese mice after aerosol M. tuberculosis infection.
   We found that infection induced adipose tissue Akt signaling, while
   inhibition of the Akt activator mTORC2 in adipocytes reversed
   TB-associated adipose tissue inflammation and cell hypertrophy.

Interpretation

   Our study reveals a systemic response to aerosol M. tuberculosis
   infection that regulates adipose tissue lipid homeostasis through
   mTORC2/Akt signaling in adipocytes. Adipose tissue inflammation in TB
   is not simply a passive infiltration with leukocytes but requires the
   mechanistic participation of adipocyte signals.

   Keywords: Tuberculosis, Adipose tissue, Inflammation, mTORC2, Akt,
   Insulin resistance
     __________________________________________________________________

Research in context.

Evidence before the study

   Tuberculosis (TB) is a chronic infectious disease that mainly affects
   the lungs, but advanced TB is marked by cachexia that was historically
   called consumption. Previous studies identified bacterial dissemination
   to liver and, with high dose infection, to adipose tissues but the
   impact of TB disease on the metabolic function of those organs has not
   been investigated.

Added value of this study

   Modeling M. tuberculosis infection in mice, we found that TB was
   associated with increased systemic glucose tolerance and insulin
   signaling in adipocytes. Surprisingly, the infection promoted adipocyte
   hypertrophy and adipose tissue inflammation, which are typically
   associated with insulin resistance. Mice on a high fat diet had higher
   adipose tissue inflammation after infection, but better glucose
   tolerance than the uninfected mice. Adipose tissue inflammation
   depended on mTORC2/Akt signaling in adipocytes, revealing a previously
   unknown role for adipocytes in the host response to TB.

Implications of all the available evidence

   This study uncovers a paradoxical metabolic adaptation of the host in
   TB. We provide insight on the mechanistic signaling that causes adipose
   tissue inflammation and glucose and lipid metabolism dysregulation
   during TB and possible targets for therapeutic intervention.

   Alt-text: Unlabelled Box

1. Introduction

   Tuberculosis (TB) remains the leading cause of death by infectious
   diseases worldwide. According to the World Health Organization, an
   estimated 1.6 million people died from M. tuberculosis infection in
   2017 [[43]1]. Lacking an environmental reservoir, M. tuberculosis has
   adapted to the human host over some 70,000 years of coevolution [[44]2]
   to ensure its perpetuation that depends on low host mortality and
   immune pathology that enables aerosol transmission. The bacillus may
   persist in a lifelong latent state in ~90% of infected individuals and
   when active TB develops, the average duration of untreated disease
   prior to death or non-sterilizing recovery is estimated to be 2–3 years
   [[45]3]. The chronicity of TB compared to most acute infectious
   diseases imposes unique and incompletely understood systemic effects in
   the host-pathogen interaction. Alveolar macrophages are the first host
   cells infected with inhaled M. tuberculosis, but dissemination is
   universal even in clinical latency [[46]4]. Upon recognition of the
   bacteria, alveolar macrophages induce the release of chemokines that
   initiates the migration of secondary myeloid cells like dendritic cells
   and neutrophils to the lung [[47]5]. These cells engulf and disseminate
   the bacteria to lymphoid tissues where T cells are primed at around
   9–10 days in mice and after exposure to the pathogen [[48]6]. Antigen
   specific T cells are found in the lung as early as 18–20 days after
   infection, at a point where macrophages are activated, and the
   bacterial growth plateaus. Common sites for disseminated TB include
   lymphoid tissues, bones, the gastrointestinal tract, and pleura, but
   virtually any tissue may be targeted including adipose tissue
   [[49]7,[50]8]. Presence of the bacteria in adipocytes has been shown in
   vitro and in adipose tissue biopsies from TB patients [[51][9],
   [52][10], [53][11]]. In mice, bacteria were found in visceral adipose
   tissue after infection with higher aerosol doses (~200 CFU) or
   different routes of infection (intravenously at 8 × 10^4 CFU and
   intranasal at 320 CFU) [[54][10], [55][11], [56][12]].

   The adipose tissue is an endocrine organ that communicates with other
   organs to regulate systemic metabolic fitness [[57]13]. Adipose tissue
   has also been implicated in immunity, participating in a signaling
   network that involves the endothelium, lungs, and bone marrow [[58]14].
   It has been well described that M. tuberculosis modifies the lipid and
   glucose metabolism at a cellular level, by differentiating macrophages
   into foamy cells [[59]15] and by triggering switch from oxidative
   phosphorylation to glycolytic metabolism in lung T cells and
   macrophages [[60]16], but any organ and systemic metabolic regulation
   are completely unknown. Here, we report increased leukocyte
   infiltration and cytokine expression in adipose tissues of lean mice
   and mice fed a high fat diet (HFD) following low-dose aerosol infection
   with M. tuberculosis. This inflammatory response occurred without
   detectable bacteria in adipose tissues, it was followed by adipocyte
   hypertrophy, and it was paradoxically associated with increased
   systemic glucose tolerance. This response was associated with increased
   adipose tissue Akt S473 phosphorylation, while adipocyte-specific
   depletion of the mTOR complex 2 (mTORC2) subunit Rictor, which is
   essential for Akt S473 phosphorylation, abrogated adipose tissue
   inflammation, cytokine expression, and adipocyte hypertrophy in mice
   with TB. Our data suggest an alternative to the classical
   metaflammation model by showing that M. tuberculosis infection
   uncouples adipose tissue inflammation and adipocyte hypertrophy from
   increased lipolysis, insulin resistance, and glucose intolerance. These
   results demonstrate a novel host-pathogen interaction linking immunity
   and metabolism in TB.

2. Materials and methods

2.1. Mice

   Age matched (6–8 wk. old) male C57BL/6J mice were obtained from Jackson
   Laboratory (Bar Harbor, ME) and Adiponectin-Cre;Rictor^fl/fl mice were
   supplied by Dr. Guertin (UMMS). Mice were housed in the Animal Medicine
   facility at UMMS where experiments were performed under protocols
   approved by the Institutional Animal Care and Use Committee and
   Institutional Biosafety Committee.

   Mice were aerosol infected with M. tuberculosis Erdman using a Glas-Col
   Inhalation Exposure System (Terre Haute, IN) set to deliver ~50 CFU to
   the lung. Mice were fed a HFD (60% fat content, isocaloric) (Research
   Diets, New Brunswick, NJ). Mice were weighed weekly, blood glucose
   measurements were performed with a BD Logic glucometer (Becton
   Dickinson, Franklin Lakes, NJ) biweekly and blood samples and tissues
   were taken after 2, 4, 8 and 20 weeks of infection.

2.2. Pre-adipocyte isolation and differentiation

   Stromal vascular fraction (SVF) cells were isolated from the inguinal
   white adipose tissue (iWAT) as previously described and using a
   gentleMACS Dissociator (Miltenyi Biotec Inc., Auburn, CA) with
   collagenase [[61]17]. Cells were seeded in 48 well plates and infected
   with M. tuberculosis Erdman at a MOI10 for 24, 48 or 72 h. Gene
   expression of cytokines by quantitative PCR and cell volume using a
   Beckman Coulter Z2 cell counter (Beckman Coulter Life Sciences,
   Indianapolis, IN) were measured in these cells.

2.3. Flow cytometry

   Cells were isolated from the tissues and stained with Zombie Aqua
   Viability Kit and for CD45 APC-Cy7, CD3 PE-Cy7, CD64 PE-594, CD11c PE,
   CD11b PercP-Cy5.5, Ly6C A700, Ly6G FITC and MHC-II Bv421 (Biolegend,
   San Diego, CA). Other staining panels were used: CD3 PercPCy5.5, CD8
   FITC, CD4 PE, CD40L PE-Cy7 and CD69 A700. Data was acquired on an
   LSR-II (BD Biosciences, San Jose, CA) and analyzed with FlowJo v10.
   Gating strategy is detailed in Supplementary Figs. S1A and S2.

2.4. Quantitative PCR

   Zymo RNA extraction kit (Zymo Research Corp, Irvine, CA) was used to
   isolate total RNA from tissue or cells. Equal amounts of RNA were
   retro-transcribed with High Capacity Reverse Transcription Kit (Applied
   Biosystems). Real-time PCR for Ccl2, Ccl5, Cxcl1, Cxcl2, Il1b, Tnfa,
   Il6, Il4, Il10, Il13, Lpl, Cd36, Pnpla2, Lipe, Cebpa, Ppara, Pparg,
   Bmp4 and Ebf1 was performed at 60 °C of annealing temperature and using
   Applied Biosystems SYBR Green PCR mix. Tpb,Gapdh and ActB were used as
   housekeeping genes and primer sequences were designed by Primer3 input
   ([62]Supplementary Table S1). Results were calculated as fold change to
   the control group by the delta-delta Ct method.

2.5. Bacterial growth

   Whole tissue was homogenized in PBS containing 0.05% Tween 80 and cells
   from the SVF fraction were lysed in 10% Triton X-100. Homogenates were
   plated on 7H11 agar plates and colonies were counted after 3 weeks.

2.6. M. tuberculosis chromosome equivalent quantitative PCR

   Bacterial genomic DNA was extracted and the qPCR for SigF DNA fragment
   was performed following the protocol from Munoz-Elias et al. [[63]18].
   Briefly, mycobacterial genomic DNA was extracted after homogenizing the
   tissue and using phenol-chloroform-isoamyl alcohol solution (25:24:1)
   and several centrifugation steps. Quantitative PCR was performed using
   TaqMan Universal Master Mix II (Applied Biosystems).

2.7. RNAsequencing

   RNA was isolated from tissue followed by a Qiagen RNeasy Micro clean-up
   procedure (Qiagen, Hilden, Germany) according to the manufacturer's
   protocol. RNAs were analyzed on Perkin Elmer Labchip GX system (Perkin
   Elmer, Waltham, MA, USA) for quality assessment with RNA Quality
   Score ≥ 7.9. cDNA libraries were prepared using 2 ng of total RNA and
   1 μl of a 1:50,000 dilution of ERCC RNA Spike in Controls (Ambion®
   Thermo Fisher Scientific, Waltham, MA, USA) using SMARTSeq v2 protocol
   [[64]19] except for the following modifications: 1. Use of 20 μM TSO,
   2. Use of 250 pg of cDNA with 1/5 reaction of Illumina Nextera XT kit
   (Illumina, San Diego, CA, USA). The length distribution of the cDNA
   libraries was monitored using DNA High Sensitivity Reagent Kit on the
   Perkin Elmer Labchip. All samples were subjected to an indexed PE
   sequencing run of 2 × 51 cycles on an Illumina HiSeq 2000.

   Reads were mapped to MM10 with STAR v2.2.3 and gencode M9 annotation.
   Gene counts were determined with feature counts, differential gene
   expression with edgeR in R v 3.1.2 [[65]20]. Data analysis was
   performed in pipeline pilot ([66]www.accelrys.com) and plots generated
   in Spotfire ([67]www.tibco.com).

2.8. Morphological analysis

   Images of the perigonadal white adipose tissue (pgWAT), iWAT and
   interscapular brown adipose tissue (iBAT) were taken on a bright field
   microscope at 4× magnification. Using ImageJ 1.46r (NIH, Bethesda, MD)
   cell area was quantified, for pgWAT and iWAT, and lipid droplet area,
   for iBAT in ~200 cells. Cells were distributed in different bins
   according to their size and percentage was calculated.

   Area of lung lesion was measured in proportion to total lung area using
   ImageJ 1.46r.

2.9. Biochemical analysis

   Glycerol (Sigma-Aldrich, St Louis, MO), NEFA (Cell Biolabs, Inc., San
   Diego, CA), insulin, adiponectin, leptin (R&D Systems, Minneapolis,
   MN), HDL, LDL and total cholesterol (Cell Biolabs) were analyzed in
   plasma or media following the manufacturer's guidelines. Triglycerides
   (TG) (Cayman Chemicals, Ann Arbor, MI) were measured in tissue
   homogenates from pgWAT, liver and quadriceps (quad).

2.10. Immunoblotting

   Total protein was obtained from adipose tissue, quantified by BCA assay
   (Pierce Biotechnology, Rockford, IL) and SDS-PAGE separated. Antibodies
   rabbit anti-mouse for P-HSL, HSL, P-IR, IR, S473Akt, T408Akt, total Akt
   and β-actin (Cell signaling Technology Inc., Danvers, MA) were used and
   bands were quantified by ImageJ 1.46r.

2.11. Metabolic analysis

   The insulin tolerance test (ITT) and glucose tolerance test (GTT) were
   performed after 6 h or overnight fasting, respectively. Mice were
   injected with 0·75 U/kg insulin for ITT or 1 mg/kg lucose for GTT and
   blood glucose was measured before and at several time points after
   injection.

2.12. Bone marrow transfer

   Bone marrow cells were isolated from WT or KO mice and 10^6 cells were
   transferred via tail vein injection into lethally irradiated WT or KO
   mice. After 6–8 wk., mice were infected with M. tuberculosis Edrman by
   aerosol and tissues were harvested 4 or 8 wk. post infection (p.i.).

2.13. Statistical analysis

   GraphPad Prism 6.0 was used to perform statistical analysis. When
   normality was confirmed, data were analyzed using a Student's t-test or
   by the Mann-Whitney U test if otherwise. A p value of <0·05 was
   considered statistically significant.

2.14. Data and software availability

   The data generated from the RNAsequencing (RNAseq) experiment can be
   found at:
   [68]https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE106611

3. Results

3.1. Aerosol M. tuberculosis infection promotes adipose tissue inflammation

   To investigate the involvement of the tissues linked to glucose and
   lipid metabolism and the progression towards insulin resistance
   [[69]13,[70]21] (adipose tissue, liver and skeletal muscle) in TB
   pathogenesis, we tracked tissue-associated leukocytes in lean C57BL/6 J
   mice infected with 50 CFU of M. tuberculosis Erdman by aerosol. SVF
   cells isolated from pgWAT, iWAT, and iBAT at 2, 4, and 8 wk. p.i. were
   characterized by flow cytometry as follows: macrophages (MΦ;
   CD11b^+CD11c^−), inflammatory macrophages (IMΦ; CD11c^+),
   polymorphonuclear cells or neutrophils (PMN; Ly6G^+), dendritic cells
   (DC; CD64^−CD11c^+ MHCII^+) and CD3^+ T cells (gating strategy shown in
   Supplementary Fig. S1a). The number of total SVF cells and the number
   of IMΦ, DC and T cells were significantly increased in pgWAT at 4 wk.
   p.i. ([71]Fig. 1a and [72]Supplementary Fig. S1b) while all of them
   were increased at 8 wk. p.i. in both pgWAT and liver ([73]Fig. 1a and
   [74]Supplementary Fig. S1c). Only the number of MΦ, IMΦ and T cells
   were significantly increased in iBAT at 8 wk. p.i. and MΦ, PMN and T
   cells in quad at 8 wk. p.i. ([75]Fig. 1c and [76]Supplementary Fig.
   S1d). Just a few populations were significantly increased in iBAT
   ([77]Fig. 1c), liver and quad ([78]Supplementary Fig. S1c-d) at 4 wk.
   p.i. while iWAT did not show any differences at all the time points
   ([79]Fig. 1b). These results demonstrate that low dose aerosol
   infection with virulent M. tuberculosis is associated with a
   generalized leukocyte inflammatory response occurring within weeks in
   visceral and brown adipose tissues and liver while only a few
   populations are increased in quadriceps.

Fig. 1.

   [80]Fig. 1
   [81]Open in a new tab

   Adipose tissue inflammation following aerosol M. tuberculosis
   infection. Number of macrophages (MΦ), inflammatory macrophages (IMΦ),
   polymorphonuclear cells (PMN), dendritic cells (DC) and T cells in
   perigonadal white adipose tissue (pgWAT) (a), inguinal white adipose
   tissue (b) and interscapular brown adipose tissue (c) from uninfected
   mice or mice infected for 2, 4 and 8 wk. (d) Number of Ly6C^− and
   Ly6C^+ populations in MΦ and IMΦ from pgWAT from uninfected mice or
   mice infected for 8 wk. (e) Gene expression of Ccl2, Ccl5, Cxcl1,
   Cxcl2, Il1b, Tnf1, and Il6 in pgWAT, iWAT and iBAT expressed as
   fold-change in tissues of infected vs uninfected mice 8 wk. after
   aerosol challenge. (f) Bone marrow cells from WT or TLR2/4 DKO mice
   were adoptively transferred to lethally irradiated WT or TLR2/4 DKO
   recipient mice. Number of MΦ, IMΦ, PMN, DC and T cells in pgWAT from
   uninfected mice or mice infected for 8 wk. Data are expressed as
   mean ± SD (n = 6–8). All experiments were repeated at least twice.
   *P < 0·05 and **P < 0·01. See also Supplementary Figs. S1 and S2. (For
   interpretation of the references to colour in this figure legend, the